Compositions and methods for potent protein synthesis inhibitors for toxoplasmosis treatment

Abstract

Among the various aspects of the present disclosure is the provision of compositions and methods for potent protein synthesis inhibitors for toxoplasmosis treatment. The present disclosure includes a method of treatment or prophylaxis of Toxoplasma gondii infection or infection caused by a parasite closely related to Toxoplasma gondii in a subject in need thereof. Also disclosed are small molecule compositions of bicyclic pyrrolidine inhibitors of Toxoplasma gondii phenylalanine t-RNA synthetase (PheRS).

Description

COMPOSITIONS AND METHODS FOR POTENT PROTEIN SYNTHESIS INHIBITORS FOR TOXOPLASMOSIS TREATMENT CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 552,438, filed February 12, 2024, the contents of which are incorporated by reference herein in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AI143857 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF INVENTION

[0003] The present disclosure generally relates to compositions of potent inhibitors of protein synthesis and their methods of use in toxoplasmosis. BACKGROUND OF INVENTION

[0004] Toxoplasma gondii is a parasite capable of infecting most warm-blooded animals and it causes the disease toxoplasmosis. The only known definitive host of the parasite are cats, which shed oocysts into the environment and these can then readily infect wild and domesticated herbivorous animals. Both wild and domestic cats are susceptible to infection, and although cats normally do not suffer from severe disease, they remain a risk for humans due to their potential to transmit the infection. Upon subject infection, the parasites undergo a brief acute phase during which the parasite propagates as fast-growing tachyzoites that disseminate to all organs of the body. Following the acute phase, the parasite differentiates into slow-growing bradyzoites residing in long-lived tissue cysts forming in differentiated cells such as neurons and muscle cells. Infections can be passed vertically, resulting in congenital infection, or through oral ingestion of tissue cysts due to omnivorous or carnivorous feeding. Humans often become infected from water or food-borne contamination or via congenital infection. Although most infections are benign, a large proportion of the world's human population is chronically infected, thereby putting this infected population at risk of reactivation upon a decline in immune surveillance of the host. Additionally, in some regions 1 CORE / 3510075.0137 / 196268526.1of South America, toxoplasmosis is associated with severe outcomes including more severe ocular disease even in healthy adults.

[0005] The population structure of T. gondii is comprised of 6 major clades, each of which contains several related haplotypes. In North America and Europe, animal and human infections are predominantly caused by type 1 (reference strains GT-1, RH (Clade A), type 2 (reference strain ME49 (Clade D)) and type 3 (reference strains VEG, CTG (Clade C) strains. The majority of human cases of toxoplasmosis in Europe and North America are due to type 2 strains. Type 1 strains are also reasonably abundant in North America, and they are of interest as they are more pathogenic in many hosts including immunocompromised humans. Type 3 strains are much less pathogenic in animals and also rarely found to infection humans, a trait that may result from their enhanced susceptibility to clearance by macrophages. In contrast to North America and Europe, strains in South America are dominated by highly pathogenic lines such as type 4 / 8 (reference strains MAS, CtBr5 (Clade B), type 5 (reference strain RUB (Clade F), and type 10 (reference strain VAND (Clade F). The VAND and RUB strains were isolated from severe human cases of toxoplasmosis that are characteristic of the Amazon region. In addition, type 6 (reference strain FOU (Clade A)) is broadly distributed.

[0006] Current therapies for the treatment of toxoplasmosis rely on inhibition of the folate pathway in the parasite, although macrolide antibiotics have also been used with some success. Although these treatments are designed to block DNA replication and protein synthesis in the parasite, respectively, they are not effective in eliminating the tissue cyst forms that are responsible for chronic infection. The effectiveness of these treatments is often augmented by the strong Th1 immune response in infected subjects, which contributes to the control of acute infection. However, these treatment modalities cannot eradicate chronic infections, and suffer from toxicity and allergic reactions. Moreover, such treatments relying on the Th1 immune response have less effect on immunocompromised patients. Serological studies suggest that between 1-2 billion people worldwide are chronically infected with T. gondii and harbor tissue cysts in their organs and tissues, and thus are at risk of reactivation should their immune system decline.

[0007] Related parasites such as Sarcocystis neurona, which causes equine protozoal myeloencephalitis in horses, and Neospora caninum, which causes neosporosis in cattle and dogs, have inadequate treatment modalities as well.

[0008] It is therefore an object of the disclosure to provide compositions and methods of treatment for infections caused by T. gondii and closely related parasites thereto. 2 CORE / 3510075.0137 / 196268526.1SUMMARY OF INVENTION

[0009] Among the various aspects of the present disclosure is the provision compositions and methods for potent protein synthesis inhibitors for toxoplasmosis treatment.

[0010] An aspect of the disclosure is the description of compounds of Formula 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0011] wherein R1is alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, or alkaryl; L1 is a bond, alkylene, alkenylene, or alkynylene; R2 is aryl or substituted aryl; and R3 is cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, or alkaryl.

[0012] Additionally, the compounds of Formula 1 can further have a structure corresponding to Formula 2, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0013] wherein R1 is C1-C6 alkyl, C3-C6 cycloalkyl, substituted cycloalkyl, 5- or 6- membered ring aryl, substituted 5- or 6-membered ring aryl, or alkaryl; L1is a bond, C2-C4alkylene, C2-C4 alkenylene, or C2-C4 alkynylene; E2 is N or C(H); E3 is N or C(H); E4 is N or C(R32); E5 is N or C(H); E6 is N or C(R32); R21 and R22 are independently hydrogen or halo; R31is hydrogen, alkyl, alkoxy, substituted alkoxy, halo, or morpholino; and R32is hydrogen or halo.

[0014] The compounds of Formula 2 can also correspond to a compound having a structure corresponding to Formula 3, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: 3 CORE / 3510075.0137 / 196268526.1

[0015] wherein R13, R14, and R15are independently hydrogen, alkyl, hydroxyl, halo, or cyano; E1 is N or C(H); E2 is N or C(H); E3 is N or C(H); E4 is N or C(R32); E5 is N or C(H); E6is N or C(R32); R21and R22are independently hydrogen or halo; R31is hydrogen, alkyl, alkoxy, substituted alkoxy, halo, or morpholino; and R32is hydrogen or halo.

[0016] Another aspect of the disclosure is a method of treatment of a Toxoplasma gondii infection or infection caused by a parasite closely related to Toxoplasma gondii in a subject in need thereof, the method comprising administering a therapeutic amount of a compound of any one of Formula 1, 2, or 3 to the subject.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0018] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF THE DRAWINGS

[0019] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. 4 CORE / 3510075.0137 / 196268526.1

[0020] FIG. 1A and Fig. 1B are graphs of the correlation of enzyme inhibition and parasite growth inhibition. Points represent means from N=3 experiments. The values are presented in the table below that is the Comparison of EC50, IC50 and ΔTm for PheRS enzymes.

[0021] FIG.2A, 2B, and 2C represent the time dependent inhibition of tachyzoite and bradyzoite growth of T. gondii by bicyclic pyrrolidines. FIG. 2A is a graph of the time dependent inhibition of T. gondii tachyzoites with top analogs. The compounds were contacted with TgMe49-Fluc in vitro, cultured on a HFF plate and either washed out after 4 hours or allowed to contact the T. gondii tachyzoites for 72 hours. FIG. 2B is a graph of the time dependent inhibition of ex vivo purified bradyzoites. The purified bradyzoites were contacted with acid / pepsin for 10 minutes at 37ºC, neutralized with acid, and then the top analogs were added and either washed out at 4 hours or allowed to treat for 72 hours. FIG.2C is a graph of the time dependent inhibition of bradyzoites within intact cysts. The bradyzoites were contacted with acid / pepsin for 10 minutes at 37ºC, neutralized with acid, and then the top analogs were added and either washed out at 4 hours or allowed to treat for 72 hours. Graphs indicate means from two or more experiments (mean ± S.D., N= 2,3 experiments shown as individual data points).

[0022] FIG.3 is a graph of the plasma levels of Compound 12 after single oral dose of compound suspended in 75% PEG 300, 25% DSW. n=3 animals per time point. 10X EC90 estimated from in vitro activity.

[0023] FIG.4 depicts in vivo efficacy trials with Compound 12. In trial 1, C57 / BL68 group mice received intraperitoneal injection of Tg49-Fluc 300 Tachyzoite per mouse and Compound 12 was administered once per day or twice per day orally from day 4 post infection to day 10 post infection. The surviving mic at 30 days post infection had tests for serum-Tg antibody and brain homogenate (in vitro culture / HFF and plaque assay) FIG.4A show a graph of % survival vs. days post infection (DPI) to result in survival curves for trial 1, FIG.4B shows a graph of % body weight versus days post infection (DPI) for trial 1. In trial 2, C57 / BL68 group mice received intraperitoneal injection of Tg49-Fluc 1000 Tachyzoite per mouse and Compound 12 was administered twice per day orally from day 4 post infection to day 10 post infection. FIG.4C shows a graph of % survival vs. days post infection (DPI) to result in survival curves for trial 2, FIG.4D shows a graph of % body weight versus days post infection (DPI) for trial 2. DPI, days post infection, po, per oral, ip, intraperitoneal, Rx, treatment.

[0024] FIG. 5 shows ELISA serology testing results. FIG. 5A shows serum ELISA responses from all surviving mice in Trial 1 compared to two naive mice as negative control 5 CORE / 3510075.0137 / 196268526.1and two T. gondii chronically infected mice as positive controls. FIG.5B shows serum ELISA response from all surviving mice in Trial 2, compared to four naive mice as negative controls and six T. gondii chronically infected mice as positive controls. Each dot represents an individual mouse surviving after day 30 post-infection.

[0025] FIG. 6 shows the time dependent inhibition of T. gondii bradyzoite growth within intact cysts. The purified bradyzoites were contacted with acid / pepsin for 10 minutes at 37ºC, neutralized with acid, and then the top analogs were added. Purified cysts were treated with bicyclic pyrrolidines for 4 h. Bars indicate means from two experiments.

[0026] FIG.7 is a graph of the concentration (ng / mL) versus time of the exposure of Compound 7 in the plasma (circles) and brain (squares).

[0027] FIG.8 is a graph of the concentration (ng / mL) versus time of the exposure of Compound 10 in the plasma (circles) and brain (not detected).

[0028] FIG.9 is a graph of the concentration (ng / mL) versus time of the exposure of Compound 15 in the plasma (circles) and brain (squares).

[0029] FIG.10 is a graph of the concentration (ng / mL) versus time of the exposure of Compound 19 in the plasma (circles) and brain (squares).

[0030] FIG.11 is a graph of the concentration (ng / mL) versus time of the exposure of Compound 20 in the plasma (circles) and brain (squares).

[0031] FIG.12 is a graph of the concentration (ng / mL) versus time of the exposure of Compound 16 in the plasma (circles) and brain (squares).

[0032] FIG.13 is a graph of the concentration (ng / mL) versus time of the exposure of Compound 12 in the plasma (circles) and brain (squares).

[0033] FIG.14 is a graph of the concentration (ng / mL) versus time of the exposure of Compound 12 in the plasma at 3mg / kg (circles), in the plasma at 10 mg / kg (squares), in the brain at 10mg / kg (upward pointing triangles), in the plasma at 30 mg / kg (downward pointing triangles), and in the plasma at 10 mg / kg (diamonds).

[0034] FIG. 15 shows that the core-scaffold isomerism provides an opportunity for expansion of the chemical space for the cPheRS inhibitors. Chemical structure of Pf cPheRS inhibitors Compound 63 and Compound A [Kato 2016, Maetani 2017] and proposed designed of an isomeric analog, Compound 1. B. Superimposition of calculated molecular structures of Compound A (dark) and Compound 1 (light). The atoms indicated with red labels were used to quantify the similarity of the positions of key molecular appendages (atom-pair RMSD 0.5813 Å). Ligand geometry optimized using DFT (B3LYP-D3 theory level, 6-31G** basis 6 CORE / 3510075.0137 / 196268526.1set, PBF solvation [water]) on Jaguar Version 12.1, Schrödinger Maestro Version 13.7.125, MMshare Version 6.3.125, Release 2023-3, Platform Darwin-x86_64. DETAILED DESCRIPTION OF INVENTION

[0035] The present disclosure is based, at least in part, on the discovery of new chemical matter - small molecules that inhibit an essential enzyme called phenylalanine tRNA synthetase (PheRS) in the parasite Toxoplasma gondii. As shown herein, bicyclic pyrrolidine inhibitors of Toxoplasma gondii phenylalanine t-RNA synthetase with antiparasitic potency in vitro and brain exposure are described.

[0036] One aspect of the present disclosure provides for small molecule compositions that inhibit an essential enzyme called phenylalanine tRNA synthetase (PheRS) in the parasite Toxoplasma gondii. In an exemplary aspect, the new chemical scaffold composition described herein specifically inhibits the parasite PheRS enzyme. In some aspects, the inhibitors can block protein synthesis and rapidly arrest growth leading to parasite death.

[0037] Detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the disclosure that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the disclosure is intended to be illustrative, and not restrictive. Compounds

[0038] The present disclosure provides for compounds and pharmaceutical compositions useful for the treatment or prophylaxis of toxoplasmosis caused by Toxoplasma gondii or parasites closely related thereto. The disclosure also provides methods of using these compounds and compositions.

[0039] An aspect of this disclosure is a compound of Formula 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0040] wherein R1is alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, or alkaryl; L1 is a bond, alkylene, alkenylene, or alkynylene; R2 is aryl or substituted aryl; and R3 is cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, or alkaryl. 7 CORE / 3510075.0137 / 196268526.1

[0041] The compounds of Formula 1 disclosed herein can have R1be propyl, phenyl, substituted phenyl, cyclohexenyl, cyclohexyl, substituted cyclohexyl, benzyl, cyclopropyl, pyridinyl, substituted pyridinyl, pyrimidinyl, pyridazinyl, or pyrazinyl; preferably, R1 can be phenyl, substituted phenyl, cyclohexenyl, cyclohexyl, substituted cyclohexyl, or substituted pyridyl; most preferably, R1 is substituted phenyl.

[0042] Further, the compounds of Formula 1can have R1 be substituted phenyl and the phenyl is substituted with halo, hydroxyl, cyano, alkylene-OH, or alkyl; preferably, R1can be substituted phenyl and the phenyl is substituted with fluoro, chloro, hydroxyl, cyano, C1-C3alkylene-OH, or C1-C3 alkyl; more preferably, R1 can be substituted phenyl and the phenyl is substituted with fluoro or hydroxyl.

[0043] The compounds of Formula 1 disclosed herein can have L1be C2-C4alkylene, C2-C4 alkenylene, or C2-C4 alkynylene; preferably, L1 can be ethylene, ethenylene, or ethynylene; more preferably, L1 can be ethynylene.

[0044] The compounds of Formula 1 can further have R2be phenyl, pyridinyl, substituted pyridinyl, or pyridazinyl; more preferably, R2 can be phenyl.

[0045] Additionally, the compounds of Formula 1 can have R3 be phenyl, substituted phenyl, benzyl, cyclohexyl, cyclopropyl, substituted pyridinyl, substituted pyrizinyl, or substituted pyrimidinyl; more preferably, R3 can be phenyl, substituted phenyl, substituted pyridinyl, substituted pyrizinyl, or substituted pyrimidinyl; even more preferably, R3 can be substituted phenyl or substituted pyridinyl; most preferably, R3can be substituted phenyl.

[0046] The compounds of Formula 1, can further have a structure corresponding to Formula 2, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0047] wherein R1 is C1-C6 alkyl, C3-C6 cycloalkyl, substituted cycloalkyl, 5- or 6- membered ring aryl, substituted 5- or 6-membered ring aryl, or alkaryl; L1is a bond, C2-C4alkylene, C2-C4alkenylene, or C2-C4alkynylene; E2is N or C(H); E3is N or C(H); E4is N or C(R32); E5 is N or C(H); E6 is N or C(R32); R21 and R22 are independently hydrogen or halo; R31is hydrogen, alkyl, alkoxy, substituted alkoxy, halo, or morpholino; and R32is hydrogen or halo. 8 CORE / 3510075.0137 / 196268526.1

[0048] The compounds of Formula 2 can have R1be phenyl or substituted phenyl; preferably, R1 can be substituted phenyl.

[0049] The compounds of Formula 2 can have L1 be ethylene, ethenylene, or ethynylene; preferably, L1can be ethynylene.

[0050] The compounds of Formula 2 can have E2 be N or E2 can be C(H).

[0051] Also, the compounds of Formula 2 can have E3 be C(H).

[0052] The compounds of Formula 2 can have E4be C(R32).

[0053] For the compounds of Formula 2, when E4is C(R32), R32can be hydrogen.

[0054] Also, for the compounds of Formula 2, when E4 is C(R32), R32 can be fluoro.

[0055] The compounds of Formula 2 can have E5be C(H).

[0056] Alternatively, the compounds of Formula 2 can have E5is N.

[0057] The compounds of Formula 2 can also have E6 be N.

[0058] The compounds of Formula 2 can have E6 be C(R32).

[0059] When the compounds of Formula 2 have E6of C(R32), wherein R32is fluoro.

[0060] Additionally, the compounds of Formula 2 can have R21 of hydrogen.

[0061] Also, the compounds of Formula 2 can have R22 of hydrogen.

[0062] The compounds of Formula 2 can alternatively have R22of fluoro.

[0063] The compounds of Formula 2 can also have R31 be C1-C3 alkoxy; preferably, R31 is methoxy.

[0064] The compounds of Formula 2 can correspond to the compound having a structure corresponding to Formula 3, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0065] wherein R13, R14, and R15are independently hydrogen, alkyl, hydroxyl, halo, or cyano; E1is N or C(H); E2is N or C(H); E3is N or C(H); E4is N or C(R32); E5is N or C(H); 9 CORE / 3510075.0137 / 196268526.1E6is N or C(R32); R21and R22are independently hydrogen or halo; R31is hydrogen, alkyl, alkoxy, substituted alkoxy, halo, or morpholino; and R32 is hydrogen or halo.

[0066] The compounds of Formula 3 can have E1 be N.

[0067] The compounds of Formula 3 can have E1be C(H).

[0068] The compounds of Formula 3 can have E2 be N.

[0069] Also, the compounds of Formula 3 can have E2 be C(H).

[0070] The compounds of Formula 3 can have E3be C(H).

[0071] The compounds of Formula 3 can also have E4be C(R32) and R32is hydrogen or fluoro.

[0072] The compounds of Formula 3 can have E5be C(H).

[0073] Additionally, the compounds of Formula 3 can have E5be N.

[0074] The compounds of Formula 3 can have E6 be N.

[0075] The compounds of Formula 3 can also have E6 be C(R32) and R32 is fluoro.

[0076] Further, the compounds of Formula 3 can have R21of hydrogen.

[0077] The compounds of Formula 3 can have R22 be hydrogen.

[0078] The compounds of Formula 3 can also have R22 of fluoro.

[0079] The compounds of Formula 3 can further have R31of C1-C3alkoxy; preferably, R31 is methoxy.

[0080] Another aspect of the disclosure is a method of treatment of a Toxoplasma gondii infection or infection caused by a parasite closely related to Toxoplasma gondii in a subject in need thereof, the method comprising administering a therapeutic amount of a compound of any one of Formula 1, 2, or 3 to the subject.

[0081] The methods of treatment described herein can be directed to human subject.

[0082] The methods of treatment can also be administered to subjects of a mouse, rat cat, dog, chicken, turkey, rabbit, non-human primate, lizard, gecko, cow, calf, sheep, goat, lamb, horse, foal, pig, or piglet.

[0083] The methods of treatment can have the compound of Formula 1, 2, or 3 be coadministered with one or more antibiotics selected from pyrimethamine, sulfamethoxazole, sulfadiazine, or clindamycin.

[0084] Further, the methods of treatment can have the infection be caused by a parasite closely related to Toxoplasma gondii selected from Sarcocystis neurona or Neospora caninum.

[0085] The methods of treatment can have the infection be caused by Toxoplasma gondii. 10 CORE / 3510075.0137 / 196268526.1

[0086] A method of treatment or prophylaxis of a Toxoplasma gondii infection or infection caused by a parasite closely related to Toxoplasma gondii in a subject in need thereof, the method comprising administering a therapeutic amount of a compound to the subject, the compound comprising the structure:, wherein R1and R2are selected from the table ID R1R228 29 30 31 32 33 34 353637 38 11 CORE / 3510075.0137 / 196268526.139 40   41   42   43   44   45   46   47   2   48   49   50   51   52   53   54   54   .

[0087] The compound can comprise the structure selected from: 12 CORE / 3510075.0137 / 196268526.113 CORE / 3510075.0137 / 196268526.1

[0088] The compounds can be used to treat a Toxoplasma gondii infection in a subject in need thereof, by administering a therapeutic amount of the compound to the subject, wherein the subject is a mouse, rat, cat, dog, chicken, turkey, rabbit, non-human primate, lizard, gecko, cow, calf, sheep, goat, lamb, horse, foal, pig, or piglet.

[0089] The compound can be co-administered with one or more antibiotics selected from pyrimethamine, sulfamethoxazole, sulfadiazine, and clindamycin.

[0090] For the methods of treatment described herein the infection can be caused by a parasite closely related to Toxoplasma gondii selected from Sarcocystis neurona or Neospora caninum. Preferably, for the methods of treatment described herein, the infection is caused by Toxoplasma gondii.

[0091] Also disclosed are small molecule compositions comprising a bicyclic pyrrolidine inhibitor of Toxoplasma gondii phenylalanine t-RNA synthetase (PheRS).

[0092] The small molecule compositions can have the PheRS is from Toxoplasma gondii.

[0093] The small molecule compositions can comprise a compounds having any one of the structures described herein.

[0094] The compounds of the present disclosure include the compounds themselves, as well as their salts and their prodrugs, if applicable. A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt can also be formed between a cation and a negatively charged substituent (e.g., carboxylate) on a compound described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethyl ammonium ion. Examples of prodrugs include C1-6alkyl esters of carboxylic acid groups, which, upon administration to a subject, are capable of providing active compounds.

[0095] Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from pharmaceutically acceptable inorganic and organic acids and bases. As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the addition of a pharmaceutically acceptable acid or base to a compound disclosed herein. As used herein, the phrase "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient. 14 CORE / 3510075.0137 / 196268526.1

[0096] Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the present disclosure and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium, and N-(alkyl)4 salts. The present disclosure also encompasses the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Salt forms of the compounds of any of the formulae herein can be amino acid salts of carboxyl groups (e.g., L-arginine, -lysine, -histidine salts).

[0097] Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418; Journal of Pharmaceutical Science, 66, 2 (1977); and "Pharmaceutical Salts: Properties, Selection, and Use A Handbook; Wermuth, C. G. and Stahl, P. H. (eds.) Verlag Helvetica Chimica Acta, Zurich, 2002 [ISBN 3- 906390-26-8] each of which is incorporated herein by reference in their entireties.

[0098] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise, the salts are equivalent to the parent form of the compound for the purposes of the present disclosure.

[0099] In addition to salt forms, the present disclosure provides compounds that are in a prodrug form. Prodrugs of the compounds described herein are those compounds that undergo chemical changes under physiological conditions to provide the compounds of the present disclosure. Additionally, prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present disclosure when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because, 15 CORE / 3510075.0137 / 196268526.1in some situations, they may be easier to administer than the parent drug. They may, for instance, be more bioavailable by oral administration than the parent drug. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. A wide variety of prodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. An example, without limitation, of a prodrug would be a compound of the present disclosure which is administered as an ester (the "prodrug"), but then is metabolically hydrolyzed to the carboxylic acid, the active entity. Additional examples include peptidyl derivatives of a compound of the present disclosure.

[0100] The present disclosure also includes various hydrate and solvate forms of the compounds.

[0101] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I), or carbon-14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure. Methods

[0102] The compounds described herein are useful in the methods provided herein and, while not bound by any particular theory, are believed to exert their desirable effects through their ability to inhibit the growth of or kill T. gondii tachyzoites which are responsible for acute infections. Such compounds may also inhibit the growth of T. gondii bradyzoites, which are responsible for chronic infection. In some embodiments, the compounds may also inhibit the growth of T. gondii sporozoites which are responsible for infection following ingestion of oocysts. In some embodiments, the treatment includes causative prophylaxis, such as preventing the spread of T. gondii. In some embodiments, the treatment of infections caused by T. gondii refers to treatment intended to achieve cure, e.g., treatment for radical cure (i.e., clearing tachyzoites and / or bradyzoites from the subject, including eliminating chronic infection).

[0103] In other embodiments, the compounds described herein may be useful in the treatment or prophylaxis of infections caused by parasites closely related to Toxoplasma gondii. In some embodiments, the parasites closely related to Toxoplasma gondii are Sarcocystis neurona and Neospora caninum. In certain embodiments, the compounds may be 16 CORE / 3510075.0137 / 196268526.1useful in the treatment or prophylaxis of protozoal myeloencephalitis. In some embodiments, the compositions may be useful in the treatment or prophylaxis of neosporosis. Pharmaceutical Compositions 1. Formulations

[0104] For use in the methods described herein, the compounds can be formulated as pharmaceutical or veterinary compositions. The formulation selected can vary depending on the subject to be treated, the mode of administration, and the type of treatment desired (e.g., prevention, prophylaxis, or therapy). A summary of formulation techniques is found in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference. Exemplary routes of administration and formulations are described as follows.

[0105] In the practice of the disclosed methods, the compounds (or pharmaceutically acceptable salts thereof) or compositions can be administered by any of the usual and acceptable routes and methods known in the art. The compounds or compositions can thus be administered, for example, by the enteral or gastrointestinal route (e.g., orally or rectally), topically (e.g., to the skin or an accessible mucous membrane (e.g., an intraoral (e.g., sublingual or buccal), intranasal, intrarectal, or genitourinary surface)), parenterally (e.g., by intramuscular, intravenous, subcutaneous, intraarticular, intravesicular, intrathecal, epidural, ocular, or aural application or injection), transdermally, or by inhalation (e.g., by aerosol).

[0106] The compositions can be in the form of a solid, liquid, or gas, as determined to be appropriate by those of skill in the art. Thus, as general examples, the pharmaceutical compositions may be in the form of tablets, capsules, syrups, pills, enterically coated or other protected formulations, sustained release formulations, elixirs, powders, granulates, suspensions, emulsions, solutions, gels (e.g., hydrogels), pastes, ointments, creams, plasters, transdermal patches, drenches, suppositories, enemas, injectables, implants, sprays, or aerosols.

[0107] The compositions, in general, include an effective amount of a compound described herein and one or more pharmaceutically acceptable carriers or excipients, as is well known in the art. The compositions can thus include one or more diluents, buffers, preservatives, salts, carbohydrates, amino acids, carrier proteins, fatty acids, lipids, etc. The compounds described herein may be present in amounts totaling, for example, 0.1-95% by 17 CORE / 3510075.0137 / 196268526.1weight of the total weight of the composition (e.g., 0.1-1 % by weight of the composition, 1- 10% by weight of the composition, 10-20% by weight of the composition, 20-30% by weight of the composition, 30-40% by weight of the composition, etc.).

[0108] For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions, as solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients for these formulations include, for example, water, saline, dextrose, and glycerol. Such compositions can also contain nontoxic auxiliary substances, such as wetting or emulsifying agents, and pH buffering agents, such as sodium acetate, sorbitan monolaurate, and so forth.

[0109] Formulations for oral use include tablets containing a compound in a mixture with one or more non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, and buffering agents.

[0110] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus, or spray drying equipment.

[0111] Dissolution or diffusion controlled release can be achieved by an appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled-release coating may 18 CORE / 3510075.0137 / 196268526.1include one or more of the coating substances mentioned above and / or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbon.

[0112] The liquid forms in which the compounds and compositions can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0113] The pharmaceutical composition may also be formulated as a veterinary composition, intended for use with subjects other than humans. The veterinary compositions according to the present disclosure can be in any appropriate forms to suit the requested administration modes, for instance nasal, oral, intradermic, cutaneous, or parenteral. In certain embodiments, the composition is in a form intended for oral administration and, for instance when the domestic animal eating, either mixed to the food ration, or directly into the mouth after a meal. The veterinary compositions of the disclosure are in the form of a nasal, oral, or injectable liquid suspension or solution, or in solid or semi-solid form, powders, pellets, capsules, granules, sugar-coated pills, gelules, sprays, cachets, pills, tablets, pastes, implants or gels. In a particular embodiment, the compositions are in the form of an oral solid form such as tablets. In some embodiments, the veterinary compositions may have an effective amount of the compound for a specific species of animal (e.g., cow, lamb, goat, horse, dog, poultry such as chicken, or turkey, etc.). In certain embodiments, the veterinary composition is formulated for the treatment or prophylaxis of Toxoplasma gondii, Sarcocystis neurona, and / or Neospora caninum. The veterinary composition may be formulated for the treatment or prophylaxis of protozoal myeloencephalitis in horses. In some embodiments, the veterinary composition may be formulated for the treatment or prophylaxis of neosporosis in cattle and dogs. 19 CORE / 3510075.0137 / 196268526.1

[0114] In specific embodiments, the compositions of the disclosure are formulated in pellets or tablets for oral administration. According to this type of formulation, they comprise lactose monohydrate, cellulose microcrystalline, crospovidone / povidone, aroma, compressible sugar, and magnesium stearate as excipients. When the compositions are in the form of pellets or tablets, they are for instance 1 mg, 2 mg, or 4 mg torasemide pellets or tablets. Such pellets or tablets are divisible so that they can be cut to suit the posology according to the disclosure in one or two daily takes. In certain embodiments, the compositions may be formulated in injectable solutions or suspensions for a parenteral administration. The injectable compositions are produced by mixing therapeutically efficient quantity of torasemide with a pH regulator, a buffer agent, a suspension agent, a solubilization agent, a stabilizer, a tonicity agent and / or a preservative, and by the transformation of the mixture into an intravenous, sub- cutaneous, intramuscular injection or perfusion according to a conventional method. Possibly, the injectable compositions may be lyophilized according to a conventional method. Examples of suspension agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, xanthan gum, sodic carboxymethylcellulose, and polyethoxylated sorbitan monolaurate. Examples of solubilization agents include polyoxy ethylene-solidified castor oil, polysorbate 80, nicotinamide, polyethoxylated sorbitan monolaurate, macrogol, and ethyl ester of caste oil fatty acid. Moreover, the stabilizer includes sodium sulfite, sodium metal sulfite, and ether, while the preservative includes methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol. An example of a tonicity agent is mannitol. When preparing injectable suspensions or solutions, it is desirable to make sure that they are blood isotonic. 2. Dosage

[0115] The dose of a compound depends on a number of factors, such as the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such an amount of the compound, as determined by the attending physician or veterinarian, is referred to herein, and in the claims, as a "therapeutically effective amount." For example, the dose of a compound disclosed herein is typically in the range of about 1 to about 1000 mg per day. The therapeutically effective amount may be, for example, an amount from about 1 mg to about 500 mg per day.

[0116] Administration of each drug, as described herein, can, independently, be one to four times daily for one day to one year, and may even be for the life of the subject. Chronic, long-term administration may be indicated. 20 CORE / 3510075.0137 / 196268526.13. Kits

[0117] The compounds and compositions can be packaged in a kit, optionally with one or more other pharmaceutical agents (see below). Non-limiting examples of the kits include those that contain, e.g., two or more pills, a pill and a powder, a suppository and a liquid in a vial, or two topical creams. The kits can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, or inhalers. Additionally, the unit dose kits can contain instructions for the preparation and administration of the compositions. The kits can be manufactured as a single-use unit dose for one subject, multiple uses for a particular subject (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kits can contain multiple doses suitable for administration to multiple subjects ("bulk packaging"). The kit components can be assembled in cartons, blister packs, bottles, and tubes.

[0118] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to a PheRS inhibitor, a solubilizer, and at least one solvent. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing the activity of the components.

[0119] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil- lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having 21 CORE / 3510075.0137 / 196268526.1a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0120] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit. 4. Combination Therapies

[0121] The compounds and pharmaceutical compositions can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account the compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder, or they may achieve different effects (e.g., control of any adverse effects). In some embodiments, the combination therapy may comprise the administration of one or more of compounds having the structure of formula (I), (II), (III), and / or (IV) with another antibiotic for the treatment of T. gondii infection. For example, the antibiotic may be selected from pyrimethamine, sulfadiazine, sulfamethoxazole, clindamycin, and mirincamycin. In some embodiments, the combination therapy may comprise the administration of one or more of the compounds described above.

[0122] Phenylalanine tRNA Synthetase (PheRS) Modulation Agents

[0123] As described herein, PheRS expression has been implicated in various diseases, disorders, and conditions. As such, modulation of PheRS (e.g., modulation of PheRS in Toxoplasma gondii) can be used for the treatment of such conditions. A PheRS modulation agent can modulate PheRS response or induce or inhibit PheRS. PheRS modulation can comprise modulating the expression of PheRS in cells, modulating the quantity of cells that express PheRS, or modulating the quality of the PheRS-expressing cells.

[0124] PheRS modulation agents can be any composition or method that can modulate PheRS expression in cells (e.g., a small molecule inhibitor for Toxoplasma gondii 22 CORE / 3510075.0137 / 196268526.1PheRS). For example, a PheRS modulation agent can be an activator, an inhibitor, an agonist, or an antagonist. As another example, the PheRS modulation can be the result of gene editing.

[0125] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0126] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled- release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of the agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0127] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition. Therapeutic Methods

[0128] Also provided is a process of treating, preventing, or reversing toxoplasmosis in a subject in need of administration of a therapeutically effective amount of a 23 CORE / 3510075.0137 / 196268526.1phenylalanine tRNA synthetase (PheRS) inhibitor, so as to block protein synthesis and rapidly arrest growth, leading to parasite death.

[0129] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing toxoplasmosis. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0130] Generally, a safe and effective amount of a PheRS inhibitor is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a PheRS inhibitor described herein can substantially inhibit toxoplasmosis, slow the progress of toxoplasmosis, or limit the development of toxoplasmosis.

[0131] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0132] When used in the treatments described herein, a therapeutically effective amount of a PheRS inhibitor can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to treat toxoplasmosis.

[0133] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses. 24 CORE / 3510075.0137 / 196268526.1

[0134] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0135] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single-dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0136] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical 25 CORE / 3510075.0137 / 196268526.1or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

[0137] Administration of a PheRS inhibitor can occur as a single event or over a time course of treatment. For example, a PheRS inhibitor can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0138] Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for toxoplasmosis.

[0139] A PheRS inhibitor can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a PheRS inhibitor can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a PheRS inhibitor, an antibiotic, an anti- inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a PheRS inhibitor, an antibiotic, an anti- inflammatory, or another agent. A PheRS inhibitor can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, a PheRS inhibitor can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent. Administration

[0140] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0141] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, 26 CORE / 3510075.0137 / 196268526.1intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0142] Agents and compositions described herein can be administered in a variety of methods well-known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 µm), nanospheres (e.g., less than 1 µm), microspheres (e.g., 1-100 µm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0143] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0144] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier- based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; 27 CORE / 3510075.0137 / 196268526.1decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product. Screening

[0145] Also provided are methods for screening.

[0146] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.

[0147] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0148] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals etc.).

[0149] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about - 28 CORE / 3510075.0137 / 196268526.12 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl.24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0150] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0151] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of compounds during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0152] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8Å to about 15Å.

[0153] A control sample or a reference sample as described herein can be a sample from a healthy subject. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0154] The methods and algorithms of the invention may be enclosed in a controller or processor. Furthermore, methods and algorithms of the present invention can be embodied as a computer-implemented method or methods for performing such computer- implemented method or methods, and can also be embodied in the form of a tangible or non- 29 CORE / 3510075.0137 / 196268526.1transitory computer-readable storage medium containing a computer program or other machine-readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer programs include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and / or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general-purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.

[0155] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P.1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif.41(1), 207–234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0156] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present 30 CORE / 3510075.0137 / 196268526.1disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0157] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0158] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0159] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device 31 CORE / 3510075.0137 / 196268526.1that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0160] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0161] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0162] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0163] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples. Definitions

[0164] It is to be understood that the terminology employed herein is for the purpose of describing particular embodiments, and is not intended to be limiting.

[0165] All terms used herein are intended to have their ordinary meaning in the art unless otherwise provided. All concentrations are in terms of percentage by weight of the 32 CORE / 3510075.0137 / 196268526.1specified component relative to the entire weight of the topical composition unless otherwise defined.

[0166] As used herein, the articles "a" and "an" refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0167] As used herein, all ranges of numeric values include the endpoints and all possible values disclosed between the disclosed values. The exact values of all half integral numeric values are also contemplated as specifically disclosed and as limits for all subsets of the disclosed range. For example, a range of from 0.1 % to 3% specifically discloses a percentage of 0.1 %, 1%, 1.5%, 2.0%, 2.5%, and 3%. Additionally, a range of 0.1 to 3% includes subsets of the original range including from 0.5% to 2.5%, from 1 % to 3%, from 0.1 % to 2.5%, etc. It will be understood that the sum of all weight % of individual components will not exceed 100%.

[0168] The term "hydrocarbon" refers to a radical or group containing carbon and hydrogen atoms. Examples of hydrocarbon radicals include, without limitation, alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl, and any combination thereof (e.g., alkyl-aryl-alkyl, etc.). As used herein, unless otherwise indicated, hydrocarbons may be monovalent or multivalent (e.g., divalent, trivalent, etc.) hydrocarbon radicals. A radical of the form -(CH2 )n-, including a methylene radical, i.e., -CH2-, is regarded as an alkyl radical if it does not have unsaturated bonds between carbon atoms. Unless otherwise specified, all hydrocarbon radicals (including substituted and unsubstituted alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl, etc.) will have from 1-20 carbon atoms. In other embodiments, hydrocarbons will have from 1-12 or from 1-8 or from 1-6 or from 1-4 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms.

[0169] A "substituted" hydrocarbon may have as a substituent one or more hydrocarbon radicals, substituted hydrocarbon radicals, or may comprise one or more heteroatoms. Examples of substituted hydrocarbon radicals include, without limitation, heterocycles, such as heteroaryls. Unless otherwise specified, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-20 heteroatoms. In other embodiments, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-12 or from 1-8 or from 1-6 or from 1-4 or from 1-3 or from 1-2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorous, halogen (e.g., F, Cl, Br, I, etc.), boron, silicon, etc. In some embodiments, heteroatoms will be selected from the group 33 CORE / 3510075.0137 / 196268526.1consisting of oxygen, nitrogen, sulfur, phosphorous, and halogen (e.g., F, Cl, Br, I, etc.). In some embodiments, a heteroatom or group may substitute a carbon. In some embodiments, a heteroatom or group may substitute a hydrogen. In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms in the backbone or chain of the molecule (e.g., interposed between two carbon atoms, as in "oxa"). In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms pendant from the backbone or chain of the molecule (e.g., covalently bound to a carbon atom in the chain or backbone, as in "oxo").

[0170] In addition, the phrase "substituted with a[n]," as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is "substituted with an unsubstituted C1 -C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl," the group may contain one or more unsubstituted C1 -C20 alkyls, and / or one or more unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as "R-substituted." Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different.

[0171] The term "substituent" refers to a group "substituted" on, e.g., an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group, replacing one or more hydrogen atoms therein. In one aspect, the substituent(s) on a group are independently any one single, or any combination of two or more of the permissible atoms or groups of atoms delineated for that substituent. In another aspect, a substituent may itself be substituted with any one of the above substituents. Further, as used herein, the phrase "optionally substituted" means unsubstituted (e.g., substituted with an H) or substituted. It is understood that substitution at a given atom is limited by valency. Common substituents include halo (e.g., F), C1-12straight chain or branched chain alkyl, C2-12alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C6-12 aryl, C3-12 heteroaryl, C3-12 heterocyclyl, C1-12 alkylsulfonyl, nitro, cyano, -COOR, ----C(O)NRR', -OR, -SR, -NRR', and oxo, such as mono- or di- or tri- substitutions with moieties such as halogen, fluoroalkyl, perfluoroalkyl, perfluroalkoxy, trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl, piperazinyl, pyrazoyl, imidazoyl, and the like, each optionally containing one or more heteroatoms such as halo, N, 0, S, and P. R and R' are independently hydrogen, C1-12alkyl, C1-12haloalkyl, C2-12alkenyl, C2-12alkynyl, C3-12cycloalkyl, C4-24cycloalkylalkyl, C6-12aryl, C7-24aralkyl, C3-12 heterocyclyl, C3-24 heterocyclylalkyl, C3-12 heteroaryl, or C4-24 heteroarylalkyl. Unless otherwise noted, all groups described herein optionally contain one or more common 34 CORE / 3510075.0137 / 196268526.1substituents, to the extent permitted by valency. Further, as used herein, the phrase "optionally substituted" means unsubstituted (e.g., substituted with H) or substituted. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced by a substituent (e.g., a common substituent). It is understood by one of ordinary skill in the chemistry art that substitution at a given atom is limited by valency. The use of a substituent (radical) prefix name such as alkyl without the modifier "optionally substituted" or "substituted" is understood to mean that the particular substituent is unsubstituted. However, the use of "haloalkyl" without the modifier "optionally substituted" or "substituted" is still understood to mean an alkyl group, in which at least one hydrogen atom is replaced by a halo.

[0172] It will be understood that the description of compounds herein is limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding with regard to valencies, etc., and to give compounds that are not inherently unstable. For example, any carbon atom will be bonded to two, three, or four other atoms, consistent with the four valence electrons of carbon.

[0173] As used herein, the term "alkyl," alone or in combination with other groups, refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of one to twenty carbon atoms (e.g., one to sixteen carbon atoms, one to twelve carbon atoms, one to ten carbon atoms, or one to six carbon atoms, etc.). In some embodiments, the alkyl group may be substituted with 1, 2, 3, or 4 substituent groups as defined herein.

[0174] The term "alkylene" as used herein, represents a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms and is exemplified by methylene, ethylene, and isopropylene. In some embodiments, the alkylene may be substituted with 1, 2, 3, or 4 substituent groups as defined herein.

[0175] The term "aryl" refers to an aromatic mono- or polycyclic radical of 6 to 12 carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalyl, 1,2-dihydronaphthalyl, indanyl, and 1H-indenyl. In some embodiments, the aryl may be substituted with 1, 2, 3, or 4 substituent groups as defined herein.

[0176] The "arylalkyl" group, which as used herein, represents an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. Exemplary unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 35 CORE / 3510075.0137 / 196268526.1or from 7 to 20 carbons, such as C6-10aryl C1-6alkyl, C6-10aryl C1-10alkyl, or C6-10aryl C1-20alkyl). In some embodiments, the aryl-alkyl may be substituted with 1, 2, 3, or 4 substituent groups as defined herein.

[0177] The terms "carbocyclic" and "carbocyclyl," as used herein, refer to an optionally substituted non-aromatic C3-12 monocyclic, bicyclic, or tricyclic structure in which the rings are formed by carbon atoms. Carbocyclic structures include cycloalkyl, cycloalkenyl, and cycloalkynyl groups. In some embodiments, the carbocycle may be substituted with 1, 2, 3, or 4 substituent groups as defined herein.

[0178] The term "cycloalkyl" refers to a monovalent mono- or polycarbocyclic radical of three to ten (e.g. three to six) carbon atoms. This term is further exemplified by radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, adamantyl, and indanyl. In certain embodiments, the "cycloalkyl" moieties can optionally be substituted with one, two, three, or four substituents. Each substituent can independently be, alkyl, alkoxy, halogen, amino, hydroxyl, or oxygen unless otherwise specifically indicated. Examples of cycloalkyl moieties include, but are not limited to, optionally substituted cyclopropyl, optionally substituted cyclobutyl, optionally substituted cyclopentyl, optionally substituted cyclopentenyl, optionally substituted cyclohexyl, and optionally substituted cycloheptyl, or those which are specifically exemplified herein. In some embodiments, the cycloalkyl may be substituted with 1, 2, 3, or 4 substituent groups as defined herein.

[0179] In some embodiments, hydrocarbons containing one or more heteroatoms (e.g., heteroalkyl, heteroaryl, heterocycle, alkoxy, etc.) may be substituted with 1, 2, 3, or 4 substituent groups as defined herein. Examples of heteroaryl groups are pyridyl, benzooxazolyl, benzoimidazolyl, and benzothiazolyl. Examples of heterocyclyl groups include, but are not limited to, oxetanyl, morpholinyl, thiomorpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, and 1,3-dioxanyl. The heterocyclyl groups may be unsubstituted or substituted, and attachment may be through their carbon frame or through their heteroatom(s) where appropriate.

[0180] Compounds provided herein can have one or more asymmetric carbon atoms and can exist in the form of optically pure enantiomers, mixtures of enantiomers such as racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates. The optically active forms can be obtained for example by resolution of the racemates, by asymmetric synthesis, or by asymmetric chromatography (chromatography with a chiral adsorbent or eluant). That is, 36 CORE / 3510075.0137 / 196268526.1certain of the disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. "Enantiomer" means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the configuration of substituents around one or more chiral carbon atoms. Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well-known techniques and methods, such as chiral chromatography and separation methods based thereon. The appropriate technique and / or method for separating an enantiomer of a compound described herein from a racemic mixture can be readily determined by those of skill in the art. "Racemate" or "racemic mixture" means a mixture containing two enantiomers, wherein such mixtures exhibit no optical activity; i.e., they do not rotate the plane of polarized light. "Geometric isomer" means isomers that differ in the orientation of substituent atoms (e.g., to a carbon-carbon double bond, to a cycloalkyl ring, to a bridged bicyclic system, etc.). Atoms (other than H) on each side of a carbon-carbon double bond may be in an E (substituents are on opposite sides of the carbon-carbon double bond) or Z (substituents are oriented on the same side) configuration. "R," "S," "S*," "R*," "E," "Z," "cis," and "trans," indicate configurations relative to the core molecule. Certain of the disclosed compounds may exist in atropisomeric forms. Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the isolation of the conformers. The compounds disclosed herein may be prepared as individual isomers by either isomer-specific synthesis or resolved from an isomeric mixture. Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods. 37 CORE / 3510075.0137 / 196268526.1

[0181] When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9%) by weight relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60% 70% 80%, 90%, 99%, or 99.9% by weight optically pure.' When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60% 70% 80% 90%, 99%, or 99.9% by weight pure. Percent optical purity is the ratio of the weight of the enantiomer or over the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer or over the weight of all the diastereomers. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70% 80% 90% 99%, or 99.9% by mole fraction pure relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60% 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure. Percent purity by mole fraction is the ratio of the moles of the enantiomer or over the moles of the enantiomer plus the moles of its optical isomer. Similarly, percent purity by moles fraction is the ratio of the moles of the diastereomer or over the moles of the diastereomer plus the moles of its isomer. When a disclosed compound is named or depicted by a structure without indicating the stereochemistry, and the compound has at least one chiral center, it is to be understood that the name or structure encompasses either enantiomer of the compound free from the corresponding optical isomer, a racemic mixture of the compound or mixtures enriched in one enantiomer relative to its corresponding optical isomer. When a disclosed compound is named or depicted by a structure without indicating the stereochemistry and has two or more chiral centers, it is to be understood that the name or structure encompasses a diastereomer free of other diastereomers, a number of diastereomers free from other diastereomeric pairs, mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomer(s) or mixtures of diastereomers in which one or more diastereomer is enriched relative to the other diastereomers. The disclosure embraces all of these forms.

[0182] The term "effective amount" or "therapeutically effective amount" of an agent, as used herein, is that amount sufficient to effect beneficial or desired results such as clinical results, and, as such, an "effective amount;' depends upon the context in which it is 38 CORE / 3510075.0137 / 196268526.1being applied. For example, in the context of administering an agent that is an antibiotic agent, an effective amount of an agent is, for example, an amount sufficient to achieve alleviation or amelioration or prevention or prophylaxis of one or more symptoms or conditions; diminishment of the extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition (e.g., preventing the spread of T. gondii infection; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable, as compared to the response obtained without administration of the agent.

[0183] The term "pharmaceutical composition," as used herein, represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gel cap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein (see below).

[0184] Useful pharmaceutical carriers for the preparation of the compositions hereof can be solids, liquids, or gases. Thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g., binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, and aerosols. The carrier can be selected from the various oils including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, and sesame oil. Water, saline, aqueous dextrose, and glycols are examples of liquid carriers, particularly (when isotonic with the blood) for injectable solutions. For example, formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or 39 CORE / 3510075.0137 / 196268526.1emulsifying agents, salts for adjusting osmotic pressure, and buffers. Suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for administration to the recipient.

[0185] As used herein, the term "pharmaceutically acceptable salt" refers to salts of any of the compounds described herein that within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1- 19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCR, 2008. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, dichloroacetate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glutamate, glycerophosphate, hemisulfate, heptonate, hexanoate, hippurate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, mucate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative basic salts include alkali or alkaline earth metal salts including sodium, lithium, potassium, calcium, and magnesium, aluminum salts, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, caffeine, and ethylamine.

[0186] As used herein, the term "subject" refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, dogs, non-human primates, and humans, lizards, geckos, etc.). The subject may be domesticated animals (e.g., cows, calves, sheep, goats, lambs, 40 CORE / 3510075.0137 / 196268526.1horses, poultry, foals, pigs, piglets, etc.), or animals in the family Muridae (e.g., rats, mice, etc.), or animals in the family Felidae. A subject may seek or be in need of treatment, require treatment, be receiving treatment, may be receiving treatment in the future, or a human or animal that is under care by a trained professional for a particular disease or condition. In some embodiments, the subject may be a domesticated animal.

[0187] As used herein, and as well understood in the art, "to treat" a condition or "treatment" of the condition (e.g., the conditions described herein such as toxoplasmosis) is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition (e.g., preventing the spread of T. gondii infection); delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. "Palliating" a disease, disorder, or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or the time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.

[0188] The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with any suitable pharmaceutical excipient or excipients. Exemplary, non- limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gel cap, and syrup (also see below).

[0189] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0190] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated 41 CORE / 3510075.0137 / 196268526.1Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc. may also be used.

[0191] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption-delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0192] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 ºC and about 60 ºC, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years. EXAMPLES

[0193] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the preceding description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0194] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0195] For all of the examples, N.D. indicates "not determined."

[0196] For all of the synthesis examples, the compound numbering system restarts for each example but is consistent within individual examples. 42 CORE / 3510075.0137 / 196268526.1EXAMPLE 1: Exemplary Compounds and Data Table 1. Distal Aromatic R1, Proximal Phenyl R2 and Aniline R3 Compounds43 CORE / 3510075.0137 / 196268526.144 CORE / 3510075.0137 / 196268526.145 CORE / 3510075.0137 / 196268526.1Compound ID AlogD Tg EC50 (µM) Tg EC90 (µM) THP-1 CC50 (µM) HEPG2 CC50 (µM) 1 4.0 0.291 ± 0.034 0.439 ± 0.043 14.5 ± 0.133 18.450 ± 0.67 2 4.5 0.202 ± 0.091 0.377 ± 0.021 12.9 ± 0.658 11.57 ± 4.39 3 4.3 0.233 ± 0.013 0.587 ± 0.17 22.67 ± 1.12 15.94±3.43 4 4.5 5.01 ± 0.525 8.21 ± 1.12 nd nd 5 4.1 1.26 ± 0.15 1.83 ± 0.515 >20 > 20 6 3.4 1.33 ± 0.168 1.99 ± 0.557 nd nd 7 3.7 0.0165 ± 0.0025 0.032 ± 0.016 3.95 ± 0.385 > 20 8 3.8 0.013 ± 0.002 0.043 ± 3 9.97 ± 2.93 > 20 9 4.0 0.004 ± 0.001 0.010 2.27 ± 1.06 > 20 10 4.4 0.0105 ± 0.0025 0.0335 ± 0.0005 6.42 ± 2.77 > 20 11 3.8 7.69 ± 1.11 12.42 ± 0.679 nd nd 12 3.8 0.0008 ± 0.0002 0.0023 ± 0.0005 0.183 ± 0.115 1.12 ± 0.271 13 3.6 0.359± 0.66 0.95 ± 0.08 14.13±0.66 >20 14 4.1 0.0045±0.0025 0.0075±0.0035 0.0895 ±0.0095 2.69 ± 1.29 15 3.2 0.0026 ± 0.008 0.004 ± 0.0007 0.509 ± 0.302 4.660 ± 1.430 16 3.4 0.0016 ± 0.0017 0.0036 ± 0.0032 0.072 ± 0.031 1.69 ± 0.55 17 3.1 0.012 ± 0.0005 0.019 ± 0.005 1.48 ± 0.083 11± 2.59 18 3.0 0.092 ± 0.004 0.155 ± 0.036 4.44 ± 0.91 >20 19 3.7 0.0031± 0.0012 0.0062 ± 0.0019 0.209 ± 0.08 1.18 ± 0.22 20 3.2 0.005 ± 0.001 0.011 ± 0.003 0.317 ± 0.209 1.35 ± 0.02 46 CORE / 3510075.0137 / 196268526.121 3.1 0.007 ± 0.001 0.016 ± 0.0005 2.720± 2.55 4.16 ± 0.285 22 2.7 0.025 ±0.008 0.036 ± 0.007 1.5 ± 0.377 11.8 ± 1.74 23 3.0 0.0027 ± 0.0008 0.0053 ± 0.0015 0.074 ± 0.024 14± 0.565 24 4.3 0.827 ± 0.206 1.140± 0.085 >20 >20 25 3.7 0.0165 ± 0.0005 0.061 ± 0.007 2.18±0.18 9.05 ± 0.05 26 4.1 0.0015 ± 0.0005 0.004 ± 0.000 0.005 ± 0.001 0.495 ± 0.115 27 2.7 8.528 ± 2.238 15.728 ± 6.636 nd nd Results and Discussion

[0197] Previous studies have described two 5,7 bicyclic pyrrolidines that are included here for reference: Compound 1 which has phenyl groups in both R and R1of the biaryl alkyne, and 2, which has a proximal phenyl at R2and a cyclohexene at R1. Although these compounds retain activity and selectivity for PheRS, they are less potent than earlier 4,8 bicyclic azetidine analogs. To improve on the 5,7 bicyclic pyrrolidine series, we began by probing SAR on the distal phenyl ring analogs. Introducing various ortho-substituents, including fluorine (3), methyl (4), cyano (5), to alter the electronics of the aromatic ring either had no effect or reduced the potency. Intriguingly, a meta-phenol analog 8 enhanced the activity by over 20-fold (EC50= 0.013 ^M), and potency was further enhanced in the 2-fluor-meta- phenol in compound 9. Adding N to the ring to generate the pyridone (10) or extension of the meta-phenyl methanol (6) offered no further improvements in potency. Further investigation of the phenol SAR reveals that the ortho-phenol, 12, offers substantial improvement and this compound and exhibits nano molar potency (EC50= 0.0008 ^M). In comparison, this gain in potency was not observed in aliphatic alcohol analog 7, although it has improved potency compared to the previous leads. Additionally, the catechol 13 shows much lower potency than either of the meta or ortho single substituted phenols. In contrast to the improved potency of the meta and ortho-phenols, a dramatic loss of potency was observed with the para-phenol 11 (EC50=7.69 ^M). In comparison, the 5-fluoro ortho-phenol in Compound 14 retained almost the full potency of the simple ortho-phenol 12. Collectively, these observations suggest proper 47 CORE / 3510075.0137 / 196268526.1orientation of the hydroxyl moiety is important for formation of hydrogen bond donor in the L-Phe binding pocket of the enzyme to stabilize the ligand.

[0198] Next, to investigate the proximal phenyl ring SAR to improve physicochemical properties, we synthesized several heterocyclic analogs, among which pyridine replacements at C2 of the proximal phenyl ring in compounds 15 and 16 have similar activity as leading compound 12. In contrast, addition of N at C3 in Compound 17 or inclusion of a pyridazine in compound 18 lead to reduced potency.

[0199] We also attempted to replace the p-methoxyaniline, due to its known association with toxicity, with less nucleophilic amino-pyridines and other heterocycles. These derivatives were based on the favorable distal ortho-phenol identified in 12. Most derivatives showed slightly lower activity than Compound 12, including the pyridine containing compound 19 and two pyrazine containing compounds 21 and 22, although this later compound was less potent. Modification of the aniline to a pyridine was also tolerated with the proximal phenyl R2 ring containing a 2 pyridyl as 23 demonstrated good potency. Importantly, all of these modifications would be expected to eliminate the potential toxicity of the aniline.

[0200] Not all modifications to R3 were favorable. For example, the 2-amino pyrimidine analog 27, and addition of a 3-fluoro in compound 24 resulted in loss of activity against parasites. We also explore further extensions to the para position of the aniline, since the existing crystal structures of Plasmodium PheRS suggest additional room is available in the ancillary pocket12, 13. Addition of a morpholine ring in compound 20 did not compromise potency compared to the pyridine containing compound 19. Aliphatic extensions such as the fluoroethoxy pyridine in Compound 25 decreased activity and capping this extension with a cyclopropyl lead to increased activity against HepG2 cells. Although these anlogs failed to exploit differences in the anciallry binding site, future strucure guided studies may enable design of new analogs to take advantage of this feature. 48 CORE / 3510075.0137 / 196268526.1Values in ^M, average of three or more biological replicates. Compound TgM49aTgME4 TgL497I Tg68cTg68cHepG2 SI THP-1 ID EC509aEC50 / EC50EC5CC50CC50SSI 90arentalb0EC P (Alkaline (Glutami I EC50) ne) (FR)acnLuc expressing Nano luciferase FR = EC50 fold resistance between Compound 63 resistant parasite line TgL497I and parental parasite line TgRH∆∆. SI = selectivity index (Host cell CC50 / EC50 TgME49). Bicyclic pyrrolidines are selective for parasite PheRS

[0201] Based on the initial assessment of potency and selectivity, we focused on a core set of eight 5,7 bicyclic pyrrolidine compounds for further analysis. These compounds contain a range of modifications that add heteroatoms within or appended to the ring structures in R1, R2 and R3 (Scheme 1). For comparison, we included the previously described 4,8 bicyclic azetidine 6311, as well as two 5,7 bicyclic pyrrolidines 1 and 214. Initially, we tested the compounds for on-target activity based on known mutations in PheRS that render the enzyme resistant to inhibition by the 4,8 series of inhibitor11as well as early leads of the 5,7 series14. We used the point mutation L497I in the background of the type I RH strain to test the top compounds in the 5,7 bicyclic pyrrolidine series. Although the RH strain grows faster in vitro, previous studies indicate that it has comparable sensitivity to the bicyclic pyrrolidines 1 and 2 when compared to the ME49 strain used for the determination of EC50 / EC90values14. Among the most active analogs, the fold resistance in comparing EC50growth inhibition of the 49 CORE / 3510075.0137 / 196268526.1mutant to the parental RH∆∆ parasite ranged from ~ 10 to > 300-fold. Of note, Compound 12 was intermediate in this range with a resistance change of ~ 50-fold. We also tested the compounds for growth inhibition of mammalian hepatic HepG2 cells and differentiated mononuclear phagocyte THP-1 cells. HepG2 cells proved to be insensitive to the compounds and comparing the CC50 of host cell proliferation to EC50 of parasite growth revealed selectivity indexes (SI) ranging from 35 to > 2,000. THP-1 cells were somewhat more sensitive to growth inhibition, but still showed SI that ranged from ~20 to > 400. These findings are consistent with the compounds being selective inhibitors of TgPheRS, as further supported below. Correlation of growth inhibition and enzyme selectivity

[0202] To further support the specificity of the inhibitors for PHeRS, we expressed the parasite (TgcPheRS) and host (HscPheRS) enzymes in E. coli and tested the top hits from the 5,7 bicyclic pyrrolidine series inhibition for inhibition of enzyme activity in vitro. Initially, we tested them using a thermal shift, melting assays where tighter compound binding to the active site results in a greater difference in the temperature of melting (∆Tm). Incubation of the TgcPheRS enzyme with inhibitors led to ∆Tm values ranging from 10-14oC, while this value was typically < 6oC for the human enzyme. We observed a strong inverse correlation between the ∆Tm of compounds with TgcPheRS and their potency in inhibiting parasite growth in vitro (Figure 1A, Table below). We also observed a very strong positive correlation between the EC50based on parasite growth inhibition vs. IC50against parasite enzyme activity (Figure 1A, Table below). In contrast, IC50values for inhibition of the host enzyme were 50-100 times higher (Table below). These findings further support the conclusion that the 5,7 compounds are potent and selective inhibitors of the parasite PheRS enzyme and that it is the primary target underlying parasite growth inhibition in vitro. Table. Comparison of EC50, IC50 and ∆Tm for PheRS enzymes. Compound ID TgME49- TgcPheRS ΔTmcTgcPheRS IC50aHscFRS Δ TmdHscFRS Fluc EC50aIC50b630.041 ± 0.007 12.0±0.10 0.320±0.017nd nd10.305 ± 0.046 10.8±0.32 0.421±0.0103.5 13.0420.331 ± 0.099 10.6±0.26 0.229±0.0173.6 2.54120.001 ± 0.0003 14.1±0.23 0.002±0.0004.1 0.5270.037 ± 0.023 11.3±0.15 0.009±0.001nd nd150.003 ± 0.001 13.5±0.25 0.008±0.0042.6 3.06190.003 ± 0.001 13.8±0.25 0.001±0.0012.5 1.58230.003 ± 0.001 12.1±0.21 0.003±0.002nd nd50 CORE / 3510075.0137 / 196268526.1Compound ID TgME49- TgcPheRS ΔTmcTgcPheRS IC50aHscFRS Δ TmdHscFRS Fluc EC50aIC50b160.004 ± 0.0004 13.0±0.21 0.002±0.002nd nd170.007 ± 0.005 12.5±0.25 0.030±0.0042.8 nd220.003 ± 0.001 12.7±0.15 0.005±0.0022.8 16.3210.007 ± 0.001 12.9±0.31 0.007±0.0012.6 4.15200.004±0.001 13.2±0.25 0.003±0.0012.1 1.61100.019±0.01 11.5±0.06 0.016±0.003nd 10.6580.022±0.008 11.8±0.26 0.083±0.0113.04 ndavalues are presented in µM, as mean ± SD of three replicates. b values are presented in µM, c values are presented in °C, as mean ± SD of three replicates. d values are presented in °C, nd, not determined The majority of bicyclic pyrrolidines are fast-acting and irreversible

[0203] To determine if the 5,7 bicyclic pyrrolidine series compounds act rapidly or if they require a prolonged incubation for inhibition, we compared treatment for 4 h followed by washout and re-culture in the absence of compound to continuous treatment for 72 h. Most of the bicyclic pyrrolidines were highly potent even when used for only 4 h and showed comparable EC50values to continuous treatment (Figure 2A). Notably, compound 12, which is featured below, show equal potency when treated or 4 h vs continuously. Notable exceptions included compounds 10, 22, 7 and 15 that showed > 10-fold increase in EC50 values when only treated for 4 vs.72 h (Figure 2A). The reason some compounds required extended treatments is not evident from comparing either their relative potencies, or predicted hydrophobicity, but may result from differences in uptake and retention in cells. In contrast to the rapid action of most bicyclic pyrrolidines, atovaquone, which targets the mitochondrial bc1 complex15, and pyrimethamine, which xinhibits DHFR16, 17, were only effective when used for 72 h treatment (Figure 2A). Bicyclic pyrrolidines act on in vitro differentiated and ex vivo derived bradyzoites.

[0204] Although the bicyclic pyrrolidines showed potent activity against tachyzoite stages of T. gondii in vitro, bradyzoites are thought to be much harder to inhibit due to their semi-dormant state. We tested the top active bicyclic pyrrolidines against in vitro differentiated bradyzoites using two methods for differentiation including high pH shock and reduced glycose supplemented with glutamine, as recently described18. For these assays, we used a Nano luciferase expressing strain called Tg68 (Tg68nLuc), which is permissive for 51 CORE / 3510075.0137 / 196268526.1bradyzoite induction in vitro19. The bicyclic pyrrolidines performed well against bradyzoites in both assays with EC50 values that were comparable in most cases including Compound 12 and elevated by 3 to 5-fold in other cases (e.g. compound 2, 63), when compared to tachyzoite conditions. Several compounds showed enhanced activity against bradyzoites induced using glutamine-rich media, including Compounds 12 and 16.

[0205] We also tested the top active compounds against bradyzoites purified from tissue cysts isolated from the brains of chronically infected mice. Initially, we tested the effects of compounds on bradyzoites after trypsin-mediated digestion of the cyst wall. Compounds were added at 3X EC90 for 4 h and then washed out and re-cultured on HFF monolayers without compound vs. continual treatment during outgrowth, during which the parasite converts to tachyzoites and forms plaques in the monolayer (Figure 2B). Most compounds acted rapidly and only 4 h treatment was necessary to observe a substantial reduction in subsequent outgrowth (Figure 2B). However, a subset of compounds, including several that showed slower inhibition kinetics on tachyzoites in vitro (e.g.10, 15, and 7), were only active when used continuously (Figure 2B). Consistent with the results on tachyzoite growth inhibition, atovaquone and pyrimethamine were only active when used continuously (Figure 2B).

[0206] We used a similar protocol to test the effect of bicyclic pyrrolidines on intact cysts purified from the brains of chronically infected mice. In this assay, compounds were added at 3X EC90to intact cysts for 4 h or 24 h then removed by centrifugation, three times wash with PBS and medium replacement. Bradyzoites were then liberated by trypsin treatment and plated on HFF monolayers in the absence (washout) vs. presence (constant) of compounds (Figure 3C). None of the compounds was able to inhibit outgrowth after only 4 h of treatment (Figure 6); however, several of the compounds showed up to 50% inhibition when used for 24 h of treatment followed by washing and plating in the absence of compounds (e.g. 63, 2, 16, 17, 10) (Figure 2C). The properties that determine these differences are not evident from examining the physiochemical properties of the molecules nor their potency or time- dependent killing ability on tachyzoites or liberated bradyzoites (Figure 2A,B). The differential activity on intact cysts may reflect their ability to cross the cyst wall, which is comprised of a dense meshwork of glycoproteins with poorly defined permeability20, 21. Notably, atovaquone, which has previously been shown to reduce the burden of cysts in chronically infected mice22, and delay the reactivation of chronic CNS infection in immunocompromised mice23, was among the most effective compounds in this assay (Figure 2C). Although the properties that 52 CORE / 3510075.0137 / 196268526.1determine the uptake and activity of compounds on intact cysts are unknown at present, this assay provides a useful surrogate for how compounds might act in vivo. ADME properties of top active compounds

[0207] To further evaluate the top 5,7 bicyclic pyrrolidine compounds, we profiled them for physiochemical properties as well as in several assays designed to estimate their ADME properties. Most compounds were highly hydrophobic, like the previously described compounds used here for reference including, Compound 63 (ALogD = 4.4), 1 (AlogD = 4.0), and 2 (AlogD = 4.5). However, some improvement was seen with the new analogs, especially with Compound 22 (AlogD = 2.7), which contains an ortho hydroxyl at R1and a methoxypyrazine in the R3moiety (Table below). The reference compounds showed high plasma protein and brain homogenate binding, and this property was shared by the new analogs, although we only tested a few representative ones. The reference compounds showed only moderate stability in mouse liver microsomes in vitro; however, this property was considerably improved with analogs 7, 12, and 22. Low absorption (permeability A-B) and high levels of efflux (permeability B-A) were generally found for most compounds, except for the previously described Compound 2. Some improvement in the uptake rate (permeability A- B) was seen with Compound 22, which contains an ortho hydroxyl at R1 and a methoxypyrazine in the R3 moiety. Finally, hERG activity has previously been reported with 4,8 bicyclic azetidine compounds such as Compound 639. Relatively high inhibition of hERG was also observed for the new compounds, although a promising trend was seen in comparing Compound 12 (hERG inhibition 0.83 ^M), which as a distal orthro hydroxyl at R1, to Compound 19 (hERG = 2.33 ^M), which shares the ortho hydroxyl at R1 and contains an ethoxy pyridyl at R3, and Compound 20 (hERG = 2.51 ^M), which has the orthro hydroxyl at R1 and a pyridyl, morpholine at R3. Collectively, these trends suggest that further modifications to introduce heteroatoms into the various saturated rings of the 5,7 bicyclic pyrrolidine core could further reduce hydrophobicity, decrease efflux, and further reduce inhibition of hERG. Table. ADME profiles for top compounds. Plasma Plasma MDCK- MD in Brain Microsoma CK- protein prote l hERG Homogenat stability inhibit MDR1 MDR1 Compoun Alog Alog Tg EC (uM) bindin binding io e binding m Permeabilit Permeabilit d ID D P90g % % ouse n CHO %(CD-1 (µ y A-B y B-A (CD-1 (human L / min / mg cell IC50Papp(1- -mouse) ) (µ 0 Papp(10 mouse) ) M)6cm / s)6cm / s) 53 CORE / 3510075.0137 / 196268526.163 4.4 6.4 0.134 ± 0.085 99.6 99.56 99.98 15.41 0.54 0.28 9.62 1 4.0 6.1 0.439±0.043 >99.9 nd 35.82 % nd 0.23 0.45 1.28 2 4.5 6.5 0.377±0.021 >99.9 99.99 4 % 99.8 9.8 1.97 0.02 0.66 73.7 5.6 0.032±0.016 98.8 ndnd 147.91nd 0.87 64.1912 3.8 5.8 0.0023±0.000 99.9 137.8 5 99.9 99.4 0.83 0.61 11.64 15 3.2 5.1 0.004±0.0007 nd ndnd nd0.2 0.65 40.4716 3.4 5.3 0.0036±0.003 n nd 73.59 2 d nd 0.56 nd nd 19 3.7 5.6 0.0062±0.001 nd 99 nd nd 9 .58 2.33 0.38 15.04 20 3.2 5.1 0.011±0.003 nd 99.24nd nd2.51 0.92 16.3321 3.1 5.0 0.016±0.0005 nd ndnd nd0.36 nd nd22 2.7 4.6 0.036±0.007 99.6 nd99.7 nd0.8 3.01 19.2517 3.1 5.0 0.019±0.005 nd ndnd nd0.11 2.2 36.8623 3.0 4.9 0.0053±0.001 5 nd 99.1 nd nd nd 0.06 58.47 24 4.3 6.3 1.14±0.0855 nd 99.7 nd nd 1.08 nd nd nd: not determined Pharmacodynamic (PK) studies

[0208] To evaluate the pharmacodynamic properties of the compound in vivo, we monitored plasma and brain exposure after dosing po with compounds in several different vehicles designed to ensure a uniform suspension (Table below). Previous compounds such as 63, 1, and 2 showed good Cmax exposure when adjusted for dose, modest AUClastadjusted for dose, and good brain exposure. However, we have previously encountered issues with increased exposure and toxicity with repeated dosing of Compound 6311, likely due to its high tissue distribution (e.g. Vss 29 l / kg9). Also, the compounds 1 and 2 were less potent against the parasite in vitro and hence the exposure achieved in vivo would be unlikely to reach sufficient levels for efficacy. Hence, we explored the PK of selected new compounds, particularly Compound 12 which shows very potent inhibition of the parasite in vitro. Compound 12 showed modest oral bioavailability of ~ 10%, relatively low CMax, and modest AUClast values. Compound 12, which has an ortho phenol at R1, showed good exposure in plasma and at 30 mg / kg, Compound 12 exceeded the 10X EC90 total levels in plasma for 24 hr (Figure 4), while exposure dropped below this value by ~15 hr for 10 mg / kg and by 8 hr for 3 54 CORE / 3510075.0137 / 196268526.1mg / kg (Figure 4). Notably, Compound 12 had a prolonged half-life T½of 10-15 h. The brain- to-plasma ratio of Compound 12 was 0.884 at 8 h following 10 mg / kg oral dosing. In contrast, the meta phenol at R1 found in 8 had very poor absorption and was barely detectable in plasma. Among the other analogs, none showed marked improvement in any of the PK parameters and most failed to achieve extended plasma coverage above 10X EC90 (e.g. 19, 10). Compounds 15, 20, and 16 exhibited good plasma exposure but had poor CNS penetration with low brain / plasma ratios. Table. Comparison of pharmacokinetic parameters for top compounds. Plasma Plasm Plasma Brain Compoun Dose(mg / kg Formulatio Plasm AUCla / D Ora Brain Brai AUC / D Mouse d ID ) n C / D a a tstmL) tmax(h) (h) (hr*ng / l C / lastmax 1 / 2 maxD n tmaxB / P (ng / mL (hr*ng / mL ) F% (ng / mL) (h) ) (time) 63 10 (PO) 1(solution) 40.6 ± 10.4 8 na 220.9 ND 179±60.7 8 807 4.4(8h) 1 10 (PO) 1(solution) 32.5±5 3 na 195.1 ND 304±132 8 2121 9.13(3h) 2 10 (PO) 1(solution) 11.2±2.2 1 na 26.8 ND 33.9±7.6 1 108.1 3.03(1h) 10(PO) 1(solution)3 na 193 nd 5.42±0.1 1 24.3 0.26(8h) 8 10 (PO) 1(solution) 0.5±0.2 1 na 1.3 nd na na na na 9 10 (PO) 1(solution) na nd na na nd na nd na na 10 10 (PO) 2(solution) 0.6±0.7 1 na 1.3 nd na nd na na 12 2 (IV) 2(solution) 209±68 0.5 3.73 482 - nd nd nd nd 12 10 (PO) 2(solution) 10.4±5.3 1 3.54 45.1 9.4 nd nd nd nd 3 (PO) 1(solution)0.5 15.5 43.3 9 nd nd nd nd 12 10 (PO) 1(solution) 5.6±6.3 3 nd 25.8 5.3 28.3 ± 16.2 1h 16.8 1.58(8h) 30 (PO) 1(solution)2 0.5 10.5 69.2 3 nd nd nd nd 15 100 (PO) 2(solution) 43.9±15. 1 1.37 93 0.154(8h) 7 .6 nd nd nd nda19 10 (PO) 2(solution) 13.4±9.1 1 na 34.8 nd 1.45±0.1 2 3 9.8 0.614(8h) 20 10 (PO) 1(solution) 2.75±0.2 3 na 16.8 nd nd nd na na 16 10 (PO) 1(solution) 3.66± 1 5.4 21 nd na nd na 0.005(1h) Formulation: 1-75% PEG300, 25% D5W; 2-20% PEG 400, 10% Vitamin E TPGS, 70% sodium acetate buffer (50mM, pH = 4.1); 3-0.2% MC, 0.2%Tween80 nd: not determined na: not detecteda-only terminal b / p available at 8h Compound 12 provides protection against infection in vivo

[0209] Based on the combination of properties described above, we choose the compound 12 to test in vivo for efficacy against toxoplasmosis in the murine model. We used the ME49 strain, which has intermediate virulence in the mouse, and injected 300 tachyzoites 55 CORE / 3510075.0137 / 196268526.1i.p. to simulate an LD50dose (Figure 4A). We waited 4 days to allow the infection to establish and then began treatment by oral gavage of compounds given daily for 7 days. Based on the PK properties described above, we choose doses of 10 mg / kg QD and 3 mg / kg BID for Compound 12. In addition, we included a group given the previous 4,8 compound 63 at 10 mg / kg QD, and a group given sulfadiazine in the drinking water at 0.25 mg / ml (equals an average daily dose of ~1.5 mg / animal). As expected, we observed ~ 50% death in the vehicle control group, although the time to death was delayed, suggesting a lower level of infection than anticipated (Figure 4B). Vehicle control mice also experienced high levels of weight loss, which began to increase in surviving animals after 3 weeks (Figure 4C). In comparison, mice given sulfadiazine, 63 and 3 mg / kg BID Compound 12 experience little weight loss and minimal mortality (Figure 4B,C). Animals given 10 mg / kg Compound 12 QD experienced considerable weight loss during the period of treatment, suggesting this high dose was toxic.

[0210] Given the high rate of survival in this trial, we also examined the animals by ELISA performed on serum collected at ~ 30 days post-infection24. All the animals were seropositive, indicating they did become infected (Figure 5A). We also isolated the brain from surviving animals and examined it for the presence of tissue cysts, using standard protocols for staining with Dolichos biflorus lectin24. Consistent with the lower intensity of infection, we did not detect tissue cysts in brain homogenates from any of the animals, although the sensitivity of this assay is limited to > 50 cysts / brain. To determine if the surviving mice harbored chronic cyst stages, we also inoculated mouse brain homogenates onto monolayers of HFF cells in 6 well plates to performing plaquing assays and into T25 flasks containing HFF cells to isolate any surviving parasites. These results indicate that all 4 mice surviving in the vehicle control group harbored chronic infection (Table below), as well as 2 of 8 mice given Compound 12 at 10 mg / kg QD, 1 of 8 surviving mice given 63 at 10 mg / kg QD, and 7 of 8 mice given sulfadiazine. In contrast, none of the mice given Compound 12 at 3 mg / ml BID showed evidence of chronic infection.

[0211] Based on the outcome of the initial trial, we repeated the efficacy test with several changes. First, we increased the inoculum of parasites to 1,000 tachyzoites i.p. to assure greater intensity of infection (Figure 4D). Second, we discontinued testing at 10 mg / kg out of concern for possible toxicity. Instead, we compared doses of Compound 12 at 1 mg / kg and 3mg / kg BID given over a similar time frame from day 4 to 11 postinfection (Figure 4D). This second trial led to greater mortality in the vehicle control group while only a single animal succumbed at 1 mg / kg BID Compound 12. Unlike the other two groups, animals given 3 mg / kg 56 CORE / 3510075.0137 / 196268526.1BID Compound 12 did not lose weight and all animals survived infection (Figure 4E,F). All the surviving animals were seropositive, indicating they all initially got infected (Figure 5B). Comparison of the outgrowth of parasites from brain homogenates indicated that all surviving mice given sulfadiazine or Compound 12 at 1 mg / kg BID harbored chronic infection. However, at a dose of 3 mg / kg Compound 12 animals showed no sign of residual chronic infection. We also examined plasma samples from mice treated in parallel with similar doses of Compound 12 for evidence of liver or kidney toxicity using serval commonly sampled enzymes (second Table below). We did not observe any significant increase in any of the indicator enzymes, suggesting a lack of toxicity at the doses of Compound 12 used in the efficacy trial. Collectively, these findings indicate that at modestly low doses, Compound 12 protects against lethal infection and prevents establishment of chronic infection in immunocompetent animals. Table. Outcome of efficacy treatment as established by outgrowth of brain homogenate samples from surviving mice Plaque Culture Experiment 1 Experiment 2 Survivors EC50cpositiveapositivebControl (Vehicle) BID (n=8) 4 4 4 nd Cmpd 12, 3 mg / kg BID (n=8) 7 0 0 nd Cmpd 12, 10 mg / kg 8 2 2 No QD (n=8) change Cmpd 63, 10 mg / kg No QD (n=8) 8 1 1 change Sulfadiazine 0.25 mg / ml (n=8) 8 7 6 nd Control (Vehicle) BID (n=8) 2 2 2 nd Cmpd 12, 1 mg / kg No BID (n=8) 7 2 7 change Cmpd 12, 3 mg / kg BID (n=8) 8 0 0 ndaPositive in plaquing assay after 14 daysbPositive by outgrowth in HFF culturecEC50determination using TgME49 expressing firefly luciferase nd: not determined Table. Analysis of plasma enzyme levels in mice treated with Compound 12. Vehicle Control Compound Compound Analyte BID 12, 1 mg / kg BID 12, 3 mg / kg BID n=7 n=8 n=8 ALT (U / L)a20.14 ± 1.95 20.75 ± 1.98 22.25 ± 3.01 57 CORE / 3510075.0137 / 196268526.1AST (U / L)b49.43 ± 10.37 48.88 ± 9.52 62.25 ± 21.09 BUN (mg / dL)c20.86 ± 1.77 21.13 ± 1.81 19.13 ± 3.27 Creatinine (mg / dL) 0.31 ± 0.03 0.33 ± 0.02 0.33 ± 0.05 Total Protein (g / dL) 5.73 ± 0.33 5.69 ± 0.15 5.59 ± 0.38 Glucose (mg / dL)d124.57 ±33.17 146.63 ± 17.05 142.88 ± 29.02 Values represent mean ± S.D.aAspartate amino transferasebAlanine amino transferasecBlood urea nitrogendnon-fasting Conclusions

[0212] We have identified, via structure-activity relationship studies on a recently reported 5,7 bicyclic pyrrolidine series of T. gondii PheRS inhibitors, analogs that feature increased in vitro antiparasitic potency in addition to reduced hydrophobicity and toxicity, and increased bioavailability. We further show that these series analogs inhibit the parasite enzyme with selectivity over the human ortholog, and that they are capable of curing infection in vivo. Lead compound 12 shows promise for the development of new therapeutic agents that could eliminate infection with T. gondii. Compound 12 contains an ortho hydroxyl in the distal ring of the biaryl acetylene extension from the core. The placement of this hydroxyl proved to be crucial to in vitro antiparasitic activity as the meta and para isomers were far less active and altogether inactive, respectively. The basis for this difference in potency is presently unknown but would not be predicted from physicochemical properties. We therefore speculate that the ortho hydroxyl establishes favorable interactions within the L-Phe binding site of the target enzyme. The pharmacokinetic profile of this lead compound suggests a reasonable bioavailability, Cmax, and T1 / 2 that provides extended coverage in both plasma and brain, and yet allows clearance over time. Compared to our previous lead, the 4,8 bicyclic azetidine 63, this profile is predicted to afford greater safety as it is unlikely to accumulate with repeated dosing. Although Compound 12 does show some liability in inhibition of hERG, its potent activity against the parasite may overcome this deficiency. Finally, Compound 12 performed better than both Compound 63 and sulfadiazine in preventing lethal infection and preventing the development of chronic infection in immunocompetent mice. Example 1A: Methods In vitro Growth Inhibition and Cytotoxicity assays 58 CORE / 3510075.0137 / 196268526.1

[0213] Compounds were obtained from Calibr (Scripps Research, La Jolla, CA 92037, USA) as lyophilized powder or 10 mM stocks in 100% DMSO and stored at −80°C. Human foreskin fibroblasts (HFF) and parasite cultures were cultured in a medium consisting of 10% DMEM (Dulbecco's Modified Eagle Medium), supplemented with 10 mM glutamine, 10 μg / mL gentamycin, and 10% fetal bovine serum. The cultures were incubated at 37°C with 5% CO2 and routinely confirmed negative for mycoplasma contamination using the e-Myco Plus kit from Intron Biotechnology.

[0214] Luciferase-based growth assays were conducted using the previously described TgME49-Fluc strain25. To reduce variability, only the inner 60 wells of white 96- well plates (Costar) were used to generate a 3-fold series of compounds. Each well received 5×103freshly harvested tachyzoites cultured in confluent HFF in 100 µL volumes, achieving a final compound concentration (200 µL / well, 0.1% DMSO in 10% DMEM). Plates were incubated for 72 h at 37°C before analysis, following the Promega Luciferase Assay System protocol. In a similar setup, plates were incubated for 4 h at 37°C, washed three times with PBS, and then incubated for an additional 68 h at 37°C. After incubation, culture media were replaced with 50 µL of 1x Cell Culture Lysis Reagent (Promega). After 10 min at room temperature, 100 µL of Luciferase Assay reagent was added, and luciferase activity was measured using a BioTek Cytation 3 multi-mode imager with Gen5 software.

[0215] To evaluate the on-target mechanism of inhibitors, inhibitors were tested against a resistant strain of T. gondii named TgRH-PheRS[L497I]-FLuc that was generated in the parental TgRHΔΔ-FLuc (type I) strain, described previously11. To test inhibitors on bradyzoites, a permissive bradyzoite-inducible strain of T. gondii, Tg68pBAG1:nLuc,DHFR (type II) was used, as previously documented18, 19. Freshly harvested Tg68pBAG1:nLuc,DHFR parasites (3×10^3) in a 100 µL volume were added to the inner 60 wells of a black-bottom, black 96-well plates (Costar) containing monolayers of HFF cells and incubated for 2 h at 37°C in a 5% CO2 incubator to allow parasite invasion. Afterwards, the culture medium was replaced with either 200 µL / well of alkaline medium (RPMI 1640 containing 1% FBS and 50 mM HEPES, adjusted to pH 8.2) or glutamine medium (glucose- free RPMI 1640 containing 1% FBS, 50 mM HEPES, and 10 mM glutamine, adjusted to pH 7.2). The cultures were incubated at 37°C in ambient CO2and maintained for 10 days, with media changes on days 3 and 6. On day 6, the plates were aspirated, and 150 µL / well of fresh media was added, followed by 50 µL / well of a dilution series containing 4X concentrations of compound. On day 10, the plates were equilibrated to room temperature for 30 min prior to the 59 CORE / 3510075.0137 / 196268526.1assay readout. The medium was aspirated, 50 µL of Promega Nano-Glo reagent was added to each well, and the plates were covered with a black lid. After 10 min at room temperature the reaction was read using a BioTek Cytation 3 equipped with Gen5 software.

[0216] Human hepatocellular carcinoma cells (HepG2, ATCC-HB-8065) and the human monocytic tumor line (THP-1, ATCC-TIB-202) were used to assess toxicity against host cells, as described previously18. For toxicity screening, compounds were diluted to 120 µM (2x concentration in 10% DMEM, 0.4% DMSO) and then serially diluted in a 10-dose, 3- fold series in 10% DMEM. Host cells were seeded at a density of 10×103cells / well in 100 µL volumes, allowing sub-confluent monolayers to expand during the 72 h growth assay. THP-1 cells were treated with 10 ng / mL phorbol 12-myristate 13-acetate (PMA) for 24 h to differentiate into macrophages before compound addition. For HepG2 cells, compounds were added 6 h post-seeding into plates containing host cells (200 µL final volume, 0.2% DMSO) and incubated at 37°C with 5% CO2. After 72 h, the culture media were aspirated to 80 µL, an equal volume of CellTiter-Glo® Luminescent Cell Viability Assay reagent (Promega) was added, and luciferase activity was measured using a BioTek Cytation 3 equipped with Gen5 software (v3.08). Protein Expression and Purification

[0217] The full-length cytosolic parasitic TgcPheRS and HscPheRS genes (^ and ^) were synthesized and codon optimized for optimal expression in E. coli cells. The genes were cloned together in a pETM11 vector respectively using Nco1 and Kpn1 restriction sites as described previously1,2. This composite vector was transformed in the BL21 strain of E. coli; cultures were grown briefly at 37 °C until OD600reached 0.6–0.8, and protein expression was induced by adding 0.6 mM (IPTG) at 18 °C. After 18–20 h, cells were harvested via centrifugation at 5,000 g for 20 min, resuspended and lysed in binding buffer comprising 50 mM Tris–HCl (pH 8), 200 mM NaCl, 4 mM β-mercaptoethanol (βMe), 10% (v / v) glycerol, 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 0.1 mg / mL lysozyme by sonication. After centrifugation at 20,000 g for 45 min, the supernatant was loaded on a prepacked Ni-NTA column (GE Healthcare) and washed with 20 column volumes of binding buffer supplemented with 20 mM imidazole to eliminate impurities. The bound proteins were eluted using a concentration gradient of imidazole (0 to 1 M) using the AKTA-FPLC system (GE Healthcare). The eluted fractions containing protein were concentrated using 30-kDa cut-off centrifugal devices (Millipore) and purified by size exclusion chromatography using the HiLoad 60 / 600 Superdex 200 pg column (GE Healthcare) column in a buffer comprising 25 mM Tris (pH 8), 60 CORE / 3510075.0137 / 196268526.1200 mM NaCl, 4 mM β-mercaptoethanol. Purity was confirmed via SDS PAGE gel, followed by pooling and storing of fractions at −80 °C for further use. Thermal Shift Assays (TSA)

[0218] TSA experiments were conducted by diluting the purified TgcPheRS and HscPheRS enzyme in a buffer comprising 50 mM Tris (pH 7.5), 200 mM NaCl, 5 mM MgCl2 and 2 X SYPRO orange dye (Life Technologies) as described earlier2, 3. The purified enzyme (2 uM) was incubated with 20 ^M of the test compounds at room temperature for 10min. The samples were heated from 25°C TO 99°C at a rate of 1°C min-1and the fluorescence signals of SYPRO® orange dye were monitored using StepOnePlusTMquantitative real-time PCR system (Life Technologies). Mean values of derivative Tm from triplicates are presented, and analysis was done using Protein Thermal shift software (v1.3, Thermofisher). Enzyme Assays

[0219] Enzyme inhibition assays were assessed using malachite green dye- based aminoacylation assays as previously described1, 2, 3, 4. Reactions were carried out with 50nM of purified TgcPheRS and HscPheRS enzyme in the presence of 50 µM L-phenylalanine and 100 µM ATP, and 2 U / mL E. coli inorganic pyrophosphatase in a buffer containing 30 mM HEPES (pH 7.5), 50 mM MgCl2,150 mM NaCl, 30 mM KCl, 1 mM DTT. For Ic50determination, the test compounds were serially diluted (10-fold dilution) in 96-well clear, flat- bottom plates. The plates containing the reaction mixtures were incubated at 37°C for 2 h and terminated using malachite green dye. Absorbance at 620 nm were measured using SpectraMax M2 (Molecular Devices). Three independent replicates were performed and data sets were analyzed using graph pad Prism6. Ex-vivo Bradyzoite differentiation assay.

[0220] CBA / CaJ mice (Strain 517#000654) from Jackson Laboratory were housed at Washington University School of Medicine in an approved facility. All animal studies adhered to ethical guidelines approved by the Institutional Animal Care and Use Committee. Production of chronically infected mice and isolation of tissue cysts was conducted using previously described protocols24. In brief, CBA / CaJ mice were infected via oral gavage with 5−10 TgME49-EW strain26tissue cysts from the brain homogenate of previously infected mice. At 1−2 months post-infection, brains of mice infected with the strain were homogenized, and tissue cysts were isolated using Percoll gradients. To release bradyzoites, purified tissue 61 CORE / 3510075.0137 / 196268526.1cysts were treated with an acid-pepsin solution (170 mM NaCl and 60 mM HCl) and a freshly prepared pepsin solution (0.1 mg / mL in 1x PBS) for 10 min at 37°C. The reaction was stopped by adding a neutralization buffer (94 mM Na2CO3). Liberated bradyzoites were distributed into duplicate 6-well plates (technical replicates, one for constant compound treatment and other for 4 h treatment followed my compound free media named washout), each containing 5 mL of culture media. Each plate included a negative control (media with 0.1% DMSO) and pyrimethamine (2.0 μM). Compounds were tested at 3xEC90concentrations based on their in vitro tachyzoite growth inhibition (TgEC50) assays. After a 4 h treatment, compounds were removed from one set of plates by washing 3 times with PBS and replacing with compound- free D10 medium. Plates were then incubated at 37°C with 5% CO2for 12−14 days to form plaques. Plaques were fixed in 100% ethanol for 5 min, stained with 0.1% crystal violet for 10 min, rinsed with water, and air-dried. Plaque quantification was performed using a Nikon Eclipse TS2 microscope with a 4X objective, and the number of plaques was normalized to DMSO control as a percentage.

[0221] To test the effect of the compounds on intact cysts, replicate samples of Percoll-purified cysts (10 cysts / compound) were treated with 3xEC90 concentrations in a 1 mL Eppendorf tube at 37°C for 4 or 24 h. Following treatment, cysts were washed three times with PBS, bradyzoites were released using an acid-pepsin solution, and the reaction was stopped with a neutralization buffer. Bradyzoites were then distributed into parallel 6-well plates in media with 3xEC90concentration compounds (Constant) or media without compounds (Washout). Plates were incubated at 37°C with 5% CO2for 12−14 days to form plaques. Plaques were quantified as described above. In vivo efficacy studies.

[0222] Female C57 / BL6 mice (Strain 517#000654) were purchased from Jackson Laboratory and housed at Washington University School of Medicine in an approved facility. All procedures followed ethical guidelines approved by the Institutional Animal Care and Use Committee. The compounds 12 and 63 were formulated at 2 mg / mL in a freshly prepared compound resuspension buffer (20% PEG 400, 10% Vitamin E TPGS, 70% sodium acetate buffer (50 mM, pH 4.1), and 0.25 mg / mL sulfadiazine in deionized water (filtered sterilized). In the first trial, five cohorts of 8 mice were infected with 300 TgME49Fluc tachyzoite parasites via intraperitoneal (IP) injection. Four days post-infection, one cohort, received 3 mg / kg Compound 12 twice daily (BID), one cohort received 10 mg / kg Compound 12 once daily (QD), one cohort received 10 mg / kg 63 QD, one cohort received sulfadiazine 62 CORE / 3510075.0137 / 196268526.1(0.25 mg / mL in drinking water), and a control cohort received an equal volume of vehicle BID. In the 2ndtrial, three cohorts of 8 mice were infected with 1000 TgME49Fluc tachyzoite parasites via IP injection. Four days post-infection, one cohort received 1 mg / kg Compound 12 BID, one cohort received 3 mg / kg Compound 12 BID and control cohort received an equal volume of vehicle BID. All mice were monitored for survival and weight loss every 2-3 days for 30 days post-infection. Blood samples (50-100 uL) were collected from surviving mice to test for seroconversion using a previously described ELISA assay based on whole tachyzoite lysate24. Animals were sacrifices, and the brain was excised and homogenization in 1 mL of 1x PBS. Brain homogenates (2×100 µL / well) were seeded in 6-well plates containing confluent HFF in D10 media and monitored for 10-14 days for parasite growth and plaque formation. Plaques were fixed in 100% ethanol for 5 min, stained with 0.1% crystal violet for 10 min, rinsed with water, and air-dried. Presence of any plaque was performed using a Nikon Eclipse TS2 microscope with a 4X objective. Any parasite growth observed was further tested for resistance to the respective compound using Luciferase-based growth inhibition TgEC50assay defined earlier. Pharmacokinetics in Mouse.

[0223] Female C57 / BL6 mice (Strain 517#000654) were purchased from Jackson Laboratory and housed at Washington University School of Medicine in an approved facility. All procedures followed ethical guidelines approved by the Institutional Animal Care and Use Committee. The Compound 12 were prepared as define above. Three cohorts of 8 mice were used in this experiment, one cohort received 1 mg / kg Compound 12 BID, one cohort received 3 mg / kg Compound 12 BID and control cohort received an equal volume of vehicle BID for 7 days. Blood samples were collected at 24 h postdosing and analyzed by Washington University Division of Comparative Medicine Diagnostic Laboratory using the ACE Axcel®Clinical Chemistry System. Statistical Analysis

[0224] Statistical analyses were performed using Prism 10 (GraphPad Software, Inc.). Dose-response inhibition curves for parasite and host cell toxicity screens (EC50and CC50values) were generated using the "Log(inhibitor) vs. normalized response— Variable slope" function. Values are reported as means of three or more biological replicates. EXAMPLE 1C: Pharmacokinetic Studies 63 CORE / 3510075.0137 / 196268526.1

[0225] General methods. For (IV or PO) pharmacokinetic studies described herein, three fasted animals per study group were administered the test article as a solution or suspension formulation. Male CD-1 mice were used. Blood and brain samples were collected at 1, 3, 8 if not specified. The blood samples were centrifuged to obtain the plasma; the plasma was stored at below −20 °C until analysis. Plasma concentrations were determined by liquid chromatography-tandem mass spectrometry (LC−MS / MS). The PK parameters were determined by noncompartmental methods using WinNonLin (v6.1 or higher version, Certara Inc.). See also Figure 7. The formulation was 2 mg / mL in 75% PEG 300: 25% D5W, clear solution.Compound TissueDose Dosing CmaxTmaxT1 / 2AUClastMRT0-last(mg / kg) Route volume (ng / mL) (hr) (hr) (hr*ng / mL) (h) Cl / F(mL / min / kg) Plasma 448 3 ND 1925 3.06 ND 7 10 PO 5 mL / kg Brain 54.2 1 1 243 3.30 578 ND-Not detected NA-Not available (Below the quantifiable limit)

[0226] In connection with 10 pharmacokinetics, please see the table below and Figure 8. The formulation was PEG400200uL / mL (20% w / v); Vitamin E TPGS 100uL / mL (10% w / v); pH 4.1 Acetate buffer, USP(50mM) 700uL / mL (70% w / v), 2mg / mL, clear solution.Compound TissueDose Route Dosing CmaxTmaxT1 / 2AUC (hr*ng / m MRT0-(mg / kg) volume (ng / mL) (hr) (hr)lastL) last(h) Cl / F(mL / min / kg) Plasma 5.81 1 ND 13.1 2.93 ND 10 10 PO 5 mL / kg Brain NA NA NA NA NA NAaOnly measured terminal brain concentration at 8h

[0227] In connection with Compound 15 pharmacokinetics, please see the table below and Figure 9. The formulation was PEG400200uL / mL (20% w / v); Vitamin E TPGS 100uL / mL (10% w / v); pH 4.1 Acetate buffer, USP(50mM) 700uL / mL (70% w / v), 20 mg / mL, clear solution.Compound TissueDose Rout Dosing CmaxTmaxT1 / 2MRT0-(mg / kg) e volume (ng / mL) (hr) (hr) AUClast(hr*ng / mL) last(h) Cl / F(mL / min / kg) Plasma 4387 1 1.37 9360 2.30 180 15 100 PO 5 mL / kg BrainaND ND ND ND ND ND 64 CORE / 3510075.0137 / 196268526.1

[0228] In connection with Compound 19 pharmacokinetics, please see the table below and Figure 10. The formulation was PEG400200uL / mL (20% w / v); Vitamin E TPGS 100uL / mL (10% w / v); pH 4.1 Acetate buffer, USP(50mM) 700uL / mL (70% w / v), 2mg / mL, clear solution.Compound TissueDose Dosing CmaxT T AUC MRT (mg / kg) Routemax 1 / 2 last 0-lastvolume (ng / mL) (hr) (hr) (hr*ng / mL) (h) Cl / F(mL / min / kg) Plasma 134 1 ND 348 2.95 ND 19 10 PO 5 mL / kg Brain 54.2 3 ND 98 ND ND

[0229] In connection with Compound 20 pharmacokinetics, please see the table below and Figure 11. The formulation was 2 mg / mL in 75% PEG 300: 25% D5W, clear solution.Compound TissueDose Dosing CmaxTmaxT1 / 2AUClastMRT0-last(mg / kg) Route volume (ng / mL) (hr) (hr) (hr*ng / mL) (h) Cl / F(mL / min / kg) Plasma 27.5 3 ND 168 4.25 ND 20 10 PO 5 mL / kg Brain ND ND ND ND ND NDaOnly measured terminal brain concentration at 8h

[0230] In connection with Compound 16 pharmacokinetics, please see the table below and Figure 12. The formulation was 2 mg / mL in 75% PEG 300: 25% D5W, clear solution.Compound TissueDose Rou Dosing CmaxTmaxT1 / 2AUClastMRT0-last(mg / kg) te volume (ng / mL) (hr) (hr) (hr*ng / mL) (h) Cl / F(mL / min / kg) Plasma 36.6 1 5.4 210 3.72 502 16 10 PO 5 mL / kg Brain 5.75 1 ND ND ND ND

[0231] In connection with Compound 12 pharmacokinetics, please see the table below and Figure 13. The formulation was PEG400200uL / mL (20% w / v); Vitamin E TPGS 100uL / mL (10% w / v); pH 4.1 Acetate buffer, USP(50mM) 700uL / mL (70% w / v), 0.4 mg / mL, clear solution. 65 CORE / 3510075.0137 / 196268526.1Compound Tissue Dose R Dosing CmaxTmaxT1 / 2AUC0-8hAUClastMRT0-(mg / kg) oute volume (ng / mL) (hr) (hr) (hr*ng / mL) (hr*ng / mL)last(h) Cl / F(mL / min / kg) 12 Plasma 2 IV 5 mL / kg 36.6 0.5 3.73 964 1083 3.26 30.6

[0232] In further connection with Compound 12 pharmacokinetics, please see the table below and Figure 14. The formulations were as follows: A - 0.6 mg / mL in 75% PEG 300: 25% D5W, clear solution; B- 2 mg / mL in 75% PEG 300: 25% D5W, clear solution; C- 6 mg / mL in 75% PEG 300: 25% D5W, clear solution; and D- PEG400200uL / mL (20% w / v); Vitamin E TPGS 100uL / mL (10% w / v); pH 4.1 Acetate buffer, USP(50mM) 700uL / mL (70% w / v), 0.4 mg / mL, clear solution. Compound Experiment Dose Dosing CmaxTmaxT AUC MRT kg) T1 / 2 last 0-(mg / issue Route volume (ng / mL) (hr) (hr) (hr*ng / mL)last(h) Cl / F(mL / min / kg) A 3 Plasma PO 5 mL / kg 38.3 0.5 15.5 130 3.51 169 Plasma PO 55.5 3 ND 258 3.90 ND B 10 5 mL / kg 12 Brain PO 28.3 1 ND 168.35 ND ND C 30 Plasma PO 5 mL / kg 320 0.5 10.5 2077 8.55 199 D 10 Plasma PO 5mg / kg 104 1 3.54 451 3.61 235 EXAMPLE 2: Additional Antiparasitic Activity Data 66 CORE / 3510075.0137 / 196268526.1

[0233] We recently found that potent small-molecule inhibitors of Plasmodium cytosolic phenylalanyl tRNA synthetase (cPheRS), typified by the bicyclic azetidine 63 (Figure 15),5 also potently inhibit the closely related Toxoplasma gondii and Cryptosporidium orthologs resulting in antiparasitic activity in vitro and efficacy in vivo.6, 7 This antiparasitic mechanism of action, previously unknown and yet to see clinical introduction in any indication, has garnered attention in the malaria field 8. Crystallographic structures of bicyclic azetidines in complex with Plasmodium falciparum and Plasmodium vivax cPheRS demonstrated that two molecular appendages interacted with deep pockets in the enzyme target: the biarylacetylene motif was found to occupy the L-Phe site, while the methoxyphenyl urea motif occupied a distinct site of unknown function, so-called auxiliary site 9, 10. As part of structure- activity relationship (SAR) studies, we envisioned that alternative core scaffolds could mimic the critical exit vectors of the bicyclic azetidine lead series while providing an opportunity to expand the cPheRS inhibitor chemical space. Relying on molecular modeling, we specifically hypothesized that the bicyclic pyrrolidine scaffold in Compound 1 (Figure 15) could position the key appendages in close proximity to those of its bicyclic azetidine isomer Compound A. 67 CORE / 3510075.0137 / 196268526.1The modular synthesis11of Compound A was subsequently adapted to access Compound 1 in short order (vide infra Scheme 1).

[0234] We were encouraged to find that Compound 1 inhibited T. gondii growth at submicromolar concentrations (EC500.39 µM, Table 1), with a significant loss in potency observed in the TgcPheRS L497I mutant, corroborating on-target mechanism. Additional evidence of cPheRS binding and inhibition in T. gondii was obtained via thermal-shift and aminoacylation assays, respectively (∆Tm 10.4 ºC, IC500.42 µM). Of note, Compound 1 showed significantly weaker binding and inhibition of the human ortholog (∆Tm 3.5 ºC, IC5016 µM), suggesting high selectivity for the parasite target. Despite loss of activity with respect to the bicyclic azetidine lead 63 (Tg growth EC500.053 µM, TgcPheRS ∆Tm 11.6 ºC, aminoacylation IC500.035 µM, Table below)12, we deemed the bicyclic pyrrolidine series worthy of further investigation. Table. Inhibition of TgcPheRS mediates the antiparasitic activity of bicyclic pyrrolidines. Compound 63 1 2 TgRH Fluc 0cEC50a.053 0.39 0.60 TgL497I Fluc 1cEC50a.17 3.98 4.98 Tg cPheRS ∆Tmb11.6 10.4 10.5 Hs cPheRS ∆Tmb4.5 3.5 3.6 Tg cPhe RS IC50a0.035c0.42 0.24 Hs cPheRS IC50a18 15.58 3.58avalues in µMbdifference in melting temperature (Tm, ºC)cvalues originally reported in Radke et al. (ref. [Radke 2022])

[0235] This work describes the process of developing the new compound 2 using 1 as a model system. Compound 2 shows potential to be a promising lead against T. gondii and other parasitic infections because of its potency and levels of brain exposure. It also demonstrates the opportunity for saturation and diversity in the distal ring position.

[0236] Previous reports based on testing of a limited set of bicyclic azetidines suggested that an aromatic distal ring in the biaryl alkyne was more potent than analogs bearing an aliphatic distal ring. However, sp3systems introduce molecular complexity without significantly increasing molecular weight and can adjust molecular shape to optimize receptor / ligand compatibility. Aliphatic systems usually have higher selectivity and solubility compared to flat aromatic compounds and have been shown to correlate with more success in 68 CORE / 3510075.0137 / 196268526.1clinical testing.136Therefore, we explored additional aliphatic rings not examined previously and it was exciting to see that the aliphatic compound 2 maintained the potency seen in 1.

[0237] The core changes that were explored in synthesis of 1. Compound 55 56, and 57; these compounds were synthesized to test the importance of the linkage between the proximal and distal ring systems. Unfortunately, these compounds were inactive. It appears that the alkyne imparts essential rigidity to the system and extends the distal ring into an active position. Lactams 58 and 59 lack the urea appendage that is expected, based on Plasmodium structural data9, 10, to interact with the target by occupying the so-called auxiliary site; consistent with this model, these compounds were found to be inactive. Trans ring systems were also synthesized but the potency was severely inhibited in each case. Compound 63 is a 4,8-bicyclic azetidine system that was an early lead in T. gondii studies from the HTS at the Broad institute.6This compound has good potency and exposure, but high toxicity. The PK of this and other compounds will be discussed later in this paper. It is clear the alkyne linker, urea segment, and the stereochemistry of the core is essential for potency against T. gondii. These requirements are consistent with what is known about the docking of the previous 4,8 bicyclic azetidines in the active site of parasite PheRS enzymes: the biaryl alkyne group occupies the phenylalanine binding pocket while the methoxyphenyl that emanates from the urea occupies and auxiliary site that is unique to the parasite enzyme9, 10. ID R R EC (µM) 28 4.88 29 3.87 30 >10 31 4.17 69 CORE / 3510075.0137 / 196268526.132 9.69 ID R R EC (µM) 42 5.42 33 >10 43 4.63 ± 0.08 34 3.63 ± 0.13 44 >10 35 >10 45 4.78 36 >10 46 1.8 ± 0.41 37 0.71 ± 0.22 47 1.21 ± 0.09 38 5.6 2 0.38 ± 0.02 39 >10 48 3.09 ± 0.19 40 4.82 ± 0.13 49 3.07 ± 0.33 41 1.26 ± 0.08 50 0.46 ± 0.04 51 3.89 ± 0.32 52 2.10 ± 1.10 53 0.52 ± 0.06 54 4.88 ± 0.73

[0238] Distal ring substituents of our targets could be explored with the core structure established. Substituting the distal phenyl ring with heteroaromatic rings improved the CNS MPO score modestly but was detrimental to potency. For example, Compounds 28, 29, and 31 are slightly active, with Compound 30 being inactive. Compound 32 was inactive, while the pyrazine (33) and pyridazine (34) were only slightly active. Compounds 35 and 36 were synthesized to see if a smaller aliphatic group could be tolerated but both were found to be inactive. Encouragingly, the larger aliphatic system of Compound 37 maintained a similar 70 CORE / 3510075.0137 / 196268526.1potency to 1. Potency suffered in both the meta-tolyl substitution in Compound 38 and the unconjugated system of Compound 39.

[0239] Next, we explored the different aniline substitutions that could be made on 1. Unfortunately, Compounds 40, 41, and 42 did not reach the potency of 1. Replacement of R2 aromatic substituents with alicyclic substituents, as demonstrated by Compounds 43, 44, and 45, also resulted in a stark decrease in potency.

[0240] With the success of Compound 37 in mind, we turned back to the idea of an sp3system at the distal ring position. Encouragingly, fully saturated systems 46 and 47 had modest potency. These compounds finally led us to synthesize compound 2, the only compound to surpass the potency of 1. The potency of compound 2 over compound 46 (fully saturated) and 1 (fully unsaturated) could be due to the conjugated system or the conformation provided by the partially unsaturated ring. The smaller Compound 48 was later synthesized but was not as active as compound 2.

[0241] The success of adding saturation to the distal ring system led us to test other heteroatoms in these rings. The distal tetrahydropyran (49), difluoro (51), tetrahydrothiopyran (52), and tetrahydropyran-ol (54) had modest activity. On the other hand, propargyl (50) and alcohol 53 had potency comparable to compound 2. Compound 53 also has an increased CNS MPO score as well as two more chiral centers. This series of compounds shows that potency can be maintained with a diversity of saturated systems. ADME, Selectivity, and Cross resistance for lead compounds from 1 to 2. Compound ID 1 37 41 53 50 47 2 EC90(µM) (stddev) 0.46(0.05) 0.71(0.22) 1.26(0.08) 0.52(0.06) 0.46(0.04) 1.21(0.09) 0.38(0.02) EC50(µM) (stddev) 0.27(0.02) 0.29(0.01) 0.52(0.15) 0.33(0.05) 0.25(0.08) 0.94(0.19) 0.20(0.09) THP-1 / HepG2 CC50(µM) 7.94 / 8.48 7.94 / 7.4 7.17 / 6.88 20.00 / 1.91 13.82 / 5.32 8.02 / 3.67 3.14 / 2.24 CNS MPO 2.18 2.03 2.45 2.31 1.82 2.17 2.05 AlogD 4.0 4.7 3.4 3.6 4.1 4.3 4.5 MDCK-MDR1 B-A (10-6cm / s) 1.28 nd 2.49 nd nd nd 0.66 MDCK-MDR1 A-B(10-6cm / s) 0.45 nd 0.02 nd nd nd 0.02 Plasma Protein binding (CD-1 >99.99 >99.99 99.9 99.47 99.81 99.8 >99.99 mouse%) Brain Homogenate binding (CD-1 nd nd nd 99.08 nd 99.97 99.99 mouse%) 71 CORE / 3510075.0137 / 196268526.1Microsomal stability 35.82 mouse / human (mouse) 75.39 / 53.77 nd 93.1 165.41 60.33 (mouse) (mouse) (mouse) 49.8 / 35.29 (µL / min / mg)

[0242] The Table above shows the selectivity, cross resistance, and ADME of seven selected compounds. These compounds had an EC50below 1 µM and an EC90below 1.5 µM. Compound 1 had average selectivity in the THP-1 (7.94 µM) and HepG2 (8.48 µM) assays in relation to the other compounds tested in this series.1 had the best microsomal stability and permeability with a B-A / A-B ratio of 2.84. Unfortunately, like most other compounds of this series the plasma protein binding is very high. Compound 37 was an unremarkable compound in terms of potency, selectivity, CNS MPO and AlogD score, plasma protein binding, and microsomal stability. Compound 41 was slightly less potent but has the best CNS MPO (2.45) and AlogD (3.4) score due to the distal pyridine ring. Compound 53 had a lower PPB and BHB than any other compound. Compound 50 had slightly lower PPB and BHB than the average, but the highest microsomal stability of all compounds. Compound 2 is the most potent compound synthesized but has the lowest selectivity (3.14 µM in THP-1 and 2.24 µM in HepG2). PK data for compounds 1, 2, 50, and 63 Compound ID 1 50 63 2 EC90(ng / mL) (stddev) 212(21) 217(20) 98.7(40) 117(9.6) Brain Tmax(h) 8 1 8 1 Cmax(ng / g) (stddev) 3042(1320) 87(19) 1790(607) 339(76) Auclast(h*ng / mL) 21207 NA NA 1081 Plasma Tmax(h) 3 1 8 1 Cmax(ng / mL) (stddev) 325(50) 94.5(41) 406(104) 112(22) Auclast(h*ng / mL) 1951 NA NA 268 The dose was 10 mg / kg and the formulation was 75% PEG300:24% D5W (solution).

[0243] The Table above shows the PK data for Compounds 1, 2, 50 and 63. These PK experiments were run in solution at 10mg / kg in mice.1 has the best PK data with very good brain exposure that reaches 10X the EC90at 8 hours with 3042 ng / g. Plasma exposure is not as high (325ng / mL) but does cover the EC90.1 also has the best B / P ratio of all three compounds.2 does not have a high brain exposure, but still covers the EC90 by 2X (Cmax is 339ng / g at 1 hour). Compound 50 has much lower exposure and a higher EC90. As a result, 72 CORE / 3510075.0137 / 196268526.1the EC90is not covered by the brain exposure. Compound 50 also has the lowest B / P ratio of the three compounds. The PK of Compound 63 shows that the EC90 is well covered by brain exposure (1790ng / g at 8 hours). While compounds 2 and 50 start to clear after 1 hour and 1 levels out after 3 hours, the Compound 63 exposure increases steadily until 8 hours. This poses a risk of accumulation and may be a challenge to dose safely. Compound 2 has a more typical drug PK profile than Compound 63 where EC90 is covered in the brain and begins to clear after a few hours. Comparison between 100mg / kg suspension dose and 10mg / kg solution dose of compound 2. Compound 2 Brain Compound 2 Plasma Dose (mg / kg) 100 10 Dose (mg / kg) 100 10 Tmax(h) 8 1 Tmax(h) 1 1 Cmax(ng / g) (stddev) 15,400(5467) 339(76) Cmax(ng / mL) 1584(1176) 112(22) Auclast(h*ng / mL) 59666 1081 Auclast(h*ng / mL) 11044 268

[0244] For the 10 mg / kg dose of compound 2 in the table above the formulation was 75% PEG300:24% D5W (solution). For the 100 mg / kg dose of compound 2 in the table above the formulation was 0.2% MC / Tween (suspension).

[0245] In order to better understand the PK profile of compound 2, a higher dose (100 mg / kg) in suspension was administered. The Table above shows a comparison between the 10mg / kg solution dosing vs. the 100mg / kg suspension dosing. In this experiment, brain exposure was much higher due to a delayed absorption of the compound. At 8 hours, the concentration in the brain reached 15,400 ng / g which covers the EC90 by more than 80X. In a later PK study, time points were taken from 8-48 hours to determine the clearance and the risk for accumulation. The compound started to clear at around 20 hours. The suspension dosing must have better absorption compared to the solution dosing because the concentration for 100 mg / kg in the brain was 45X higher than the 10mg / kg dosing and the AUC is 55X larger in the 100mg / kg dosing than the 10 mg / kg dosing. We would expect both measurements to be approximately 10X higher in the 100mg / kg dosing. The plasma Cmax was closer to expected with 100 mg / kg dosing reaching 1,584 ng / mL and 10 mg / kg reaching 112 ng / mL (about 14X).

[0246] In this work, investigation into 1 led us to the discovery of a more potent compound, 2. Compound 2 integrates sp3carbons into the distal ring and creates opportunities for diversification at this position. Our SAR investigations included core changes, stereochemistry changes, and aniline changes. In addition, many heteroaromatics and aliphatic groups were tested at the distal position, but only the cyclohexyl analog showed promise (37). This analog led us to synthesize compound 2 which proved to be more potent than 1. Other sp3analogs were therefore synthesized and tested. Solution PK of compound 2 shows that the EC9073 CORE / 3510075.0137 / 196268526.1is covered by 2X and in by more than 80X in a high dose suspension formulation. PK and biological assays for our most potent compounds highlight the potential for improvement in permeability and free fraction in the brain. The discovery of compound 2 creates possibilities for diversity at the distal ring and the potential for improvement in these areas.

[0247] Scheme 1 describes the synthetic route to compound 2 and other leads. Synthesis begins with the HATU coupling of commercially available Fmoc-D-Pro-OH (1) and the directing group 8-Aminquinoine in almost quantitative yield. Intermediate 2 is then used in the Palladium(II) acetate catalyzed C-H activation to form intermediate 3. Although conversion and yield in this reaction is modest, the aminoquinoline directing group gives almost exclusively the cis conformation. After quantitative deprotection to form 4, the directing group removal can be performed using 18%HCl in H2O solution to give intermediate 5. Racemization is observed from 10-50% depending on conditions and the scale of the reaction. Trans isomers could be used to synthesize the trans compounds. Reductive amination using NaBH(OAc)3 and the Fmoc-protected aldehyde, 6 adds the carbon chain that will become the 7-membered ring. One-pot Fmoc removal using Silia-Bond® piperazine and subsequent intramolecular ring formation gives lactam 8 at a good yield of 75%. Ruthenium-catalyzed reduction of the lactam using TMDS as the hydride source and then subsequent isocyanate addition gives urea 9 at 80% yield.9 can then be used in all SAR described in this paper through a Sonogashira-like coupling with the desired alkyne with the aryl bromide in decent yield. References cited 1. Dubey, J. P., Toxoplasmosis of Animals and Humans.2010; Vol.3. 2. Yarovinsky, F., Innate immunity to Toxoplasma gondii infection. Nat Rev Immunol 2014, 14 (2), 109-21. 74 CORE / 3510075.0137 / 196268526.13. Mayoral, J.; Shamamian, P., Jr.; Weiss, L. M., In Vitro Characterization of Protein Effector Export in the Bradyzoite Stage of Toxoplasma gondii. mBio 2020, 11 (2). 4. Dunay, I. R.; Gajurel, K.; Dhakal, R.; Liesenfeld, O.; Montoya, J. G., Treatment of Toxoplasmosis: Historical Perspective, Animal Models, and Current Clinical Practice. Clin Microbiol Rev 2018, 31 (4). 5. Kato, N.; Comer, E.; Sakata-Kato, T.; Sharma, A.; Sharma, M.; Maetani, M.; Bastien, J.; Brancucci, N. M.; Bittker, J. A.; Corey, V.; Clarke, D.; Derbyshire, E. R.; Dornan, G. L.; Duffy, S.; Eckley, S.; Itoe, M. A.; Koolen, K. M.; Lewis, T. A.; Lui, P. S.; Lukens, A. K.; Lund, E.; March, S.; Meibalan, E.; Meier, B. C.; McPhail, J. A.; Mitasev, B.; Moss, E. L.; Sayes, M.; Van Gessel, Y.; Wawer, M. J.; Yoshinaga, T.; Zeeman, A. M.; Avery, V. M.; Bhatia, S. N.; Burke, J. E.; Catteruccia, F.; Clardy, J. C.; Clemons, P. A.; Dechering, K. J.; Duvall, J. R.; Foley, M. A.; Gusovsky, F.; Kocken, C. H.; Marti, M.; Morningstar, M. L.; Munoz, B.; Neafsey, D. E.; Sharma, A.; Winzeler, E. A.; Wirth, D. F.; Scherer, C. A.; Schreiber, S. L., Diversity-oriented synthesis yields novel multistage antimalarial inhibitors. Nature 2016, 538 (7625), 344- 349. 6. Radke, J. B.; Melillo, B.; Mittal, P.; Sharma, M.; Sharma, A.; Fu, Y.; Uddin, T.; Gonse, A.; Comer, E.; Schreiber, S. L.; Gupta, A. K.; Chatterjee, A. K.; Sibley, L. D., Bicyclic azetidines target acute and chronic stages of Toxoplasma gondii by inhibiting parasite phenylalanyl t-RNA synthetase. Nat Commun 2022, 13 (1), 459. 7. Vinayak, S.; Jumani, R. S.; Miller, P.; Hasan, M. M.; McLeod, B. I.; Tandel, J.; Stebbins, E. E.; Teixeira, J. E.; Borrel, J.; Gonse, A.; Zhang, M.; Yu, X.; Wernimont, A.; Walpole, C.; Eckley, S.; Love, M. S.; McNamara, C. W.; Sharma, M.; Sharma, A.; Scherer, C. A.; Kato, N.; Schreiber, S. L.; Melillo, B.; Striepen, B.; Huston, C. D.; Comer, E., Bicyclic azetidines kill the diarrheal pathogen Cryptosporidium in mice by inhibiting parasite phenylalanyl-tRNA synthetase. Sci Transl Med 2020, 12 (563). 8. Forte, B.; Ottilie, S.; Plater, A.; Campo, B.; Dechering, K. J.; Gamo, F. J.; Goldberg, D. E.; Istvan, E. S.; Lee, M.; Lukens, A. K.; McNamara, C. W.; Niles, J. C.; Okombo, J.; Pasaje, C. F. A.; Siegel, M. G.; Wirth, D.; Wyllie, S.; Fidock, D. A.; Baragana, B.; Winzeler, E. A.; Gilbert, I. H., Prioritization of Molecular Targets for Antimalarial Drug Discovery. ACS Infect Dis 2021, 7 (10), 2764-2776. 9. Sharma, M.; Malhotra, N.; Yogavel, M.; Harlos, K.; Melillo, B.; Comer, E.; Gonse, A.; Parvez, S.; Mitasev, B.; Fang, F. G.; Schreiber, S. L.; Sharma, A., Structural basis of malaria parasite phenylalanine tRNA-synthetase inhibition by bicyclic azetidines. Nat Commun 2021, 12 (1), 343. 10. Sharma, M.; Mutharasappan, N.; Manickam, Y.; Harlos, K.; Melillo, B.; Comer, E.; Tabassum, H.; Parvez, S.; Schreiber, S. L.; Sharma, A., Inhibition of Plasmodium falciparum phenylalanine tRNA synthetase provides opportunity for antimalarial drug development. Structure 2022, 30 (7), 962-972 e3. 11. Maetani, M.; Zoller, J.; Melillo, B.; Verho, O.; Kato, N.; Pu, J.; Comer, E.; Schreiber, S. L., Synthesis of a Bicyclic Azetidine with In Vivo Antimalarial Activity Enabled by Stereospecific, Directed C(sp(3))-H Arylation. J Am Chem Soc 2017, 139 (32), 11300- 11306. 12. Cravatt, B.; Njomen, E.; Hayward, R.; DeMeester, K.; Ogasawara, D.; Dix, M.; Nguyen, T.; Ashby, P.; Simon, G.; Schreiber, S.; Melillo, B., ChemRxiv 2023. 75 CORE / 3510075.0137 / 196268526.113. Lovering, F.; Bikker, J.; Humblet, C., Escape from flatland: increasing saturation as an approach to improving clinical success. J Med Chem 2009, 52 (21), 6752-6. EXAMPLE 3: Synthesis of Compounds disclosed in Example 1

[0248] Reagents and solvents were commercially obtained and used without further purification. Compounds 1, 3 and 18 were synthesized following published procedures. Anhydrous solvents were used, and reactions were performed under an atmosphere of argon. Crude products were purified by silica gel chromatography or C18reverse-phase columns.1H NMR spectra were obtained on a Bruker Ultrashield 400 MHz. Shifts are expressed in parts permillion (ppm) and coupling constants (J) in Hertz (Hz). LC-MS was obtained on a Waters Acquity Ultra Performance LC system. Scheme 1. Synthesis of Compounds 3, 4, 5

[0249] (9R,9aR)-9-(4-((2-fluorophenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (3). 1 (8.0 mg, 0.018 mmol), 1-ethynyl-2-fluorobenzene (11.0mg, 0.09 mmol), and XPhos Pd G3 (1.5 mg, 0.0018mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (10uL, 0.072mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The crude was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give product as white solid (2.1mg, 24%).1H NMR (400 MHz, MeOD-d4) δ 7.58-7.39 (m, 6H), 7.23-7.17 (m, 4H), 6.85 (d, J = 9.1 Hz, 1H), 3.78 (s, 3H), 3.71-3.43 (m, 4H), 3.31-3.24 (m, 2H), 2.75-2.70 (m, 1H), 2.57-2.48 (m, 2H), 2.34-2.29 (m, 2H), 2.12-2.01 (m, 3H). LC-MS m / z calcd. C30H31FN3O2+[M+H]+= 484.24, found = 484.46.

[0250] (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(o-tolylethynyl)phenyl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide(4) Compound 1 (7.0 mg, 0.016 mmol.), 1-ethynyl-2-methylbenzene (9.1 mg, 0.08 mmol), and XPhos Pd G3 (1.3 mg, 0.0016 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (9 uL, 0.063 mmol) was added. The reaction was stirred at 70oC for 24h. The reaction was cooled 76 CORE / 3510075.0137 / 196268526.1to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give product as white solid (7.0 mg, 91%).1H NMR (400 MHz, MeOD-d4) δ 7.59 – 7.37 (m, 5H), 7.30-7.19 (m, 5H), 6.85 (d, J = 8.8 Hz, 2H), 3.78 (s, 3H), 3.70-3.44 (m, 4H), 3.29-3.23 (m, 2H), 2.73-2.69 (d, 1H), 2.56- 2.47 (m, 5H), 2.35-2.28 (m, 2H), 2.11-1.98 (m, 3H). LC-MS m / z calcd. C31H34N3O2+[M+H]+= 480.27, found = 480.50.

[0251] (9R,9aR)-9-(4-((2-cyanophenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide(5) Compound 1 (7.0 mg, 0.016 mmol), 2-ethynylbenzonitrile (8.0 mg, 0.08 mmol.), and XPhos Pd G3 (1.3 mg, 0.0016 mmol.), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (11 uL, 0.08 mmol) was added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give product as white solid (3.2 mg.41%).1H NMR (400 MHz, MeOD d4) δ 7.81 (d, J = 7.8 Hz, 1H), 7.75 – 7.69 (m, 2H), 7.59 – 7.52 (m, 3H), 7.47 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 9.0 Hz, 2H), 6.85 (d, J = 9.0 Hz, 2H), 3.78 (s, 3H), 3.72 – 3.53 (m, 3H), 3.51-3.41(m, 1H), 3.32 – 3.22 (m, 2H), 2.73 (t, J = 8.5 Hz, 1H), 2.60 – 2.45 (m, 2H), 2.41 – 2.26 (m, 2H), 2.16 – 1.95 (m, 3H). LC-MS m / z calcd. C31H31N4O2+[M+H]+= 491.24, found = 491.47. Scheme 2. Synthesis of 6

[0252] (9R,9aR)-9-(4-((2-(hydroxymethyl)phenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (6) Compound 1 (12.0 mg, 0.027 mmol), 2-(2-ethynylphenyl)-5,5-dimethyl-1,3-dioxane (29 mg, 0.14 mmol), and XPhos Pd G3 (2.3 mg, 0.0027 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (15 uL, 0.11 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The acetal intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give a white solid. To the solution 77 CORE / 3510075.0137 / 196268526.1of acetal in THF (0.1M), HCl (35% aq, 23uL, 0.27 mmol) was added. The reaction was quenched with sat. NaHCO3 and extract the aqueous phase with DCM (10mLx4). The organic solution was dried over Na2SO4 and concentrated under vacuum. To the crude aldehyde in DCM:MeOH=9:1, NaBH4(5.3 mg, 0.14 mmol) was added in one portion. The reaction was stirred at room temperature for 30min. The solvent was removed under vacuum and the crude was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to obtain the product as white solid (3.0 mg, 22% over 3 steps).1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.51-7.49 (m, 3H), 7.45-7.40 (m, 3H), 7.32-7.40 (m, 4H), 6.81 (d, J = 9.0 Hz, 2H), 5.35 (t, J = 5.6 Hz, 1H), 4.74 (d, J = 5.6 Hz, 2H), 3.75 – 3.58 (m, 6H), 3.29-3.12 (m, 3H), 2.36-2.09 (m, 5H), 1.98-1.75 (m, 3H). LC-MS m / z calcd. C31H34N3O2+[M+H]+= 496.26, found = 496.42. Scheme 3. Synthesis of Compound 7

[0253] (9R,9aR)-9-(4-(((1R,2S)-2-hydroxycyclohexyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (7) Compound 1 (23.0 mg, 0.052 mmol), tert-butyl(((1S,2R)-2- ethynylcyclohexyl)oxy)dimethylsilane (50 mg, 0.21 mmol), and XPhos Pd G3 (4.4 mg, 0.0052 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (30 uL, 0.21 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 104 uL, 0.104 mmol) solution was added at 0oC, then the reaction was stirred at 40oC for 18h. Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give white solid(15 mg, 59% over two steps)1H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.31 (m, 6H), 6.81 (d, J = 9.0 Hz, 2H), 4.90 (d, J = 5.2 Hz, 1H), 3.70 (s, 3H), 3.70-3.59 (m, 2H), 3.48 – 3.42 (m, 1H), 3.29-3.09 (m, 3H), 2.47-2.10 (m, 78 CORE / 3510075.0137 / 196268526.16H), 1.97-1.78 (m, 5H), 1.67-1.60 (m, 2H), 1.46-1.37 (m, 1H), 1.33-1.20 (m, 4H). LC-MS m / z calcd. C30H38N3O3+[M+H]+= 488.29, found = 488.28. Scheme 4. Synthesis of Compounds 8, 11, 12, 13, 14

[0254] (9R,9aR)-9-(4-((3-hydroxyphenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (8) Compound 1 (30 mg, 0.068 mmol), tert-butyl(3-ethynylphenoxy)dimethylsilane (78 mg, 0.34 mmol), and XPhos Pd G3 (5.7 mg, 0.0068 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (38 uL, 0.272 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To solution of TBS protected intermediate in THF (0.1M), TBAF (1M in THF, 80 uL, 0.08 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give white solid(13mg, 39% over two steps)1H NMR (400 MHz, DMSO- d6) δ 9.71 (s, 1H), 7.99 (s, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 9.1 Hz, 2H), 7.22 (t, J = 7.9 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 6.90 (s, 1H), 6.83-6.79 (m, 3H), 3.70 (s, 3H), 3.66-3.62 (m, 2H), 3.42 (m, 1H), 3.30 – 3.25 (m, 1H), 3.23 – 3.16 (m, 1H), 3.16- 3.10 (m, 1H), 2.48-2.41(m, 1H), 2.38– 2.08 (m, 4H), 1.95-1.80 (m, 3H). LC-MS m / z calcd. C30H32N3O3+[M+H]+= 482.24, found = 482.45.

[0255] (9R,9aR)-9-(4-((4-hydroxyphenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (11) Compound 1 (12 mg, 0.027 mmol), tert-butyl(4-ethynylphenoxy)dimethylsilane (31 mg, 0.14 mmol), and XPhos Pd G3 (2.3 mg, 0.0027 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (15uL, 0.18 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM 79 CORE / 3510075.0137 / 196268526.1to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 30uL, 0.03mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4and was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give white solid (7.0mg, 54% over two steps).1H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 7.99 (s, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.37 (m, 4H), 7.31 (d, J = 9.1 Hz, 2H), 6.80 (m, 4H), 3.70 (s, 3H), 3.64 (m, 2H), 3.30-3.11(m, 3H), 2.47 (m, 1H), 2.39 – 2.08 (m, 4H), 1.95-1.77 (m, 3H). LC-MS m / z calcd. C30H32N3O3+[M+H]+= 482.24, found = 482.34.

[0256] (9R,9aR)-9-(4-((2-hydroxyphenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (12) Compound 1 (360 mg, 0.81 mmol), tert-butyl(2-ethynylphenoxy)dimethylsilane (850 mg, 3.65 mmol), and XPhos Pd G3 (69 mg, 0.081 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (0.45 mL, 3.24 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 0.9mL, 0.9 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4and was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give white solid (175mg, 45%). Note: heat under 40oC, in case of cyclizing to benzofuran.1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.00 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.3 Hz, 3H), 7.31 (d, J = 9.1 Hz, 2H), 7.26 – 7.17 (m, 1H), 6.92 (d, J = 9.5 Hz, 1H), 6.87 – 6.78 (m, 3H), 3.70 (s, 3H), 3.68-3.59 (m, 2H), 3.47 – 3.40 (m, 1H), 3.31 – 3.17 (m, 2H), 3.13 (dt, J = 12.7, 4.2 Hz, 1H), 2.49-2.47 (m, 1H), 2.40 – 2.29 (m, 2H), 2.30 – 2.19 (m, 2H), 2.20 – 2.07 (m, 1H), 1.85 (m, 3H). LC-MS m / z calcd. C30H32N3O3+[M+H]+= 482.24, found = 482.38.

[0257] (9R,9aR)-9-(4-((2,3-dihydroxyphenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (13) Compound 1 (10 mg, 0.023 mmol), ((3-ethynyl-1,2-phenylene)bis(oxy))bis(tert- butyldimethylsilane) (49 mg, 0.14 mmol), and XPhos Pd G3 (2.3 mg, 0.023 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (15 uL, 0.11 80 CORE / 3510075.0137 / 196268526.1mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 60uL, 0.06 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4and was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give white solid (2.0 mg, 17% over two steps). Note: heat under 40oC, in case of cyclizing to benzofuran.1H NMR (400 MHz, MeOD-d4) δ 7.51 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.8 Hz, 2H), 6.92 – 6.75 (m, 4H), 6.69 (t, J = 7.9 Hz, 1H), 3.78 (s, 3H), 3.71 – 3.43 (m, 6H), 2.85-2.79 (m, 1H), 2.59 (t, J = 12.1 Hz, 2H), 2.46 – 2.26 (m, 2H), 2.16-1.96 (m, 3H). LC-MS m / z calcd. C30H32N3O4+[M+H]+= 498.24, found = 498.36

[0258] (9R,9aR)-9-(4-((4-fluoro-2-hydroxyphenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (14) Compound 1 (10 mg, 0.023 mmol), tert-butyl(2-ethynyl-5-fluorophenoxy)dimethylsilane (27 mg, 0.011 mmol), and XPhos Pd G3 (2.3 mg, 0.023 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (12 uL, 0.09 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To solution of TBS protected intermediate in THF (0.1M), TBAF (1M in THF, 30uL, 0.03 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give white solid (3.0 mg, 26% over two steps)1H NMR (400 MHz, MeOD- d4) δ 7.49 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 8.1 Hz, 3H), 7.21 (d, J = 9.1 Hz, 2H), 6.85 (d, J = 9.1 Hz, 2H), 6.65 – 6.53 (m, 2H), 3.78 (s, 3H), 3.70 – 3.64 (m, 1H), 3.61 – 3.53 (m, 2H), 3.50- 3.42 (m, 2H), 3.28 – 3.21 (m, 2H), 2.72 (t, J = 9.2 Hz, 1H), 2.61 – 2.45 (m, 2H), 2.37-2.25 (m, 2H), 2.14 – 1.95 (m, 3H). LC-MS m / z calcd. C30H31FN3O3+[M+H]+= 500.23, found = 500.42. Scheme 5. Synthesis of Compound 9 81 CORE / 3510075.0137 / 196268526.1

[0259] (9R,9aR)-9-(4-((2-fluoro-3-hydroxyphenyl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (9) Compound 1 (27.0 mg, 0.061 mmol), 1-ethynyl-2-fluoro-3-(methoxymethoxy)benzene (55.0 mg, 0.31 mmol), and XPhos Pd G3 (5.2 mg, 0.0061 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (34 uL, 0.24 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of MOM protected intermediate in DCM (0.1 M), HCl (4M in dioxane, 78uL, 0.31 mmol) was added. The reaction was stirred for 30 min and reaction progress was monitored by LC-MS until all starting material was consumed. The crude was diluted with DCM and quenched with sat. NaHCO3(aq). The aqueous phase was extracted with DCM 3 times and the combined organic phase was washed with water and brine. The organic solvent was removed under vacuum (heat under 40oC, in case of cyclizing to benzofuran) and crude was purified on flash chromatography (100% DCM to 5 % MeOH in DCM) to give white solid (12.0 mg, 40% over two steps).1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 9.1 Hz, 2H), 7.08 – 6.96 (m, 3H), 6.81 (d, J = 9.1 Hz, 2H), 3.71 (s, 3H), 3.64 (dd, J = 14.1, 6.4 Hz, 2H), 3.48 – 3.40 (m, 1H), 3.30-3.25 (m, 1H), 3.23 – 3.07 (m, 2H), 2.50-2.47 (m, 1H), 2.41 – 2.19 (m, 3H), 2.14 (t, J = 11.0 Hz, 1H), 1.85 (m, 3H).. LC-MS m / z calcd. C30H31FN3O3+[M+H]+= 500.23, found = 500.34. Scheme 6. Synthesis of Compound 10.82 CORE / 3510075.0137 / 196268526.1

[0260] (9R,9aR)-9-(4-ethynylphenyl)-N-(4-methoxyphenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (2) Compound 1 (132mg, 0.30 mmol), Ethynyl-trimethyl-silane (208 uL, 1.49mmol), and XPhos Pd G3 (25.1mg, 0.03 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (0.16mL, 1.19 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To solution of TMS protected intermediate in THF(0.1M), TBAF (1M in THF, 1.7mL, 1.70mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4and was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give yellow solid (117mg, 85% over two steps).

[0261] (9R,9aR)-9-(4-((5-hydroxypyridin-3-yl)ethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide(10) Compound 2 (18 mg, 0.046 mmol), 3-bromo-5-(methoxymethoxy)pyridine (20.0 mg, 0.092 mmol), and XPhos Pd G3 (7.8 mg, 0.0092 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) was added to solid reagents and then triethylamine (13 uL, 0.092mmol) was added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of MOM protected intermediate (1.0 equiv.) in DCM (0.1 M), HCl (4M in dioxane, 58uL, 0.23 mmol) was added. The reaction was stirred for 30 min and reaction progress was monitored by LC- MS until all starting material was consumed. The crude was diluted with DCM and quenched with sat. NaHCO3(aq). The aqueous phase was extracted with DCM 3 times and the combined organic phase is washed with water and brine. The organic solvent was removed under vacuum and crude was purified on flash chromatography (100% DCM to 5 % MeOH in DCM) to give white solid (10mg, 45%).1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J = 1.8 Hz, 1H), 8.15 (d, J = 2.8 Hz, 1H), 7.99 (s, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 9.1 Hz, 2H), 7.29 – 7.26 (m, 1H), 6.80 (d, J = 9.1 Hz, 2H), 3.70 (s, 3H), 3.63 (m, 2H), 3.30 – 3.24 (m, 2H), 3.24 – 3.17 (m, 1H), 3.12 (dt, J = 12.6, 4.2 Hz, 1H), 2.49-2.47 (m, 1H), 2.41 – 2.29 (m, 1H), 2.29 – 2.19 (m, 2H), 2.16-2.08 (m, 1H),1.96 – 1.74 (m, 3H). LC-MS m / z calcd. C29H31N4O3+[M+H]+= 483.24, found = 483.39. Scheme 7. Synthesis of Compounds 19, 20, 21, 22, 25, 26 83 CORE / 3510075.0137 / 196268526.1

[0262] (9R,9aR)-9-(4-bromophenyl)-N-(5-ethoxypyridin-2-yl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (4a) Compound 3 (50 mg, 0.162 mmol) was added to a seal tube along with Ru3CO12( 10.3 mg, 0.016 mmol) Dry toluene (0.1M) and TMDS (0.29mL, 1.62 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (85uL, 0.48 mmol) and 4-nitrophenyl (5- ethoxypyridin-2-yl)carbamate in DMF (100 mg, 0.33mmol, 0.1M) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until completion. The crude was concentrated under vacuum and purified on silica column with 10% MeOH in DCM to give brown solid (40 mg, 54%).

[0263] (9R,9aR)-9-(4-bromophenyl)-N-(6-morpholinopyridin-3-yl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (4b) Compound 3 (50 mg, 0.162 mmol) was added to a seal tube along with Ru3CO12( 10.3 mg, 0.016 mmol) Dry toluene (0.1M) and TMDS (0.29mL, 1.62 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (85uL, 0.48 mmol) and 4-nitrophenyl (6- morpholinopyridin-3-yl) carbamate in DMF (114 mg, 0.33 mmol, 0.1M) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until completion. The crude was concentrated under vacuum and purified on silica column with 15% MeOH in DCM to give yellow-brown solid (49 mg, 60%).

[0264] (9R,9aR)-9-(4-bromophenyl)-N-(5-ethoxypyrazin-2-yl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (4c) Compound 3 (50 mg, 0.162 mmol) was added to a seal tube along with Ru3CO12( 10.3 mg, 0.016 mmol) Dry toluene (0.1M) and TMDS (0.29mL, 1.62 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (85uL, 0.48 mmol) and 4-nitrophenyl (5- 84 CORE / 3510075.0137 / 196268526.1ethoxypyrazin-2-yl)carbamate in DMF (100mg, 0.33mmol) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until completion. The crude was concentrated under vacuum and purified on silica column with 10% MeOH in DCM to give brown solid (35mg, 47%).

[0265] (9R,9aR)-9-(4-bromophenyl)-N-(6-morpholinopyridin-3-yl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (4d) Compound 3 (50 mg, 0.162 mmol) was added to a seal tube along with Ru3CO12( 10.3 mg, 0.016 mmol) Dry toluene (0.1M) and TMDS (0.29mL, 1.62 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (85uL, 0.48 mmol) and 4-nitrophenyl (5- ethoxypyrazin-2-yl)carbamate in DMF (96mg, 0.33mmol) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until completion. The crude was concentrated under vacuum and purified on silica column with 10% MeOH in DCM to give brown solid (36mg, 50%).

[0266] (9R,9aR)-9-(4-bromophenyl)-N-(6-(2-fluoroethoxy)pyridin-3- yl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (4e) Compound 3 (50 mg, 0.162 mmol) was added to a seal tube along with Ru3CO12( 10.3 mg, 0.016 mmol) Dry toluene (0.1M) and TMDS (0.29mL, 1.62 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (85uL, 0.48 mmol) and 4- nitrophenyl (6-(2-fluoroethoxy)pyridin-3-yl)carbamate in DMF (106mg, 0.33mmol) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until completion. The crude was concentrated under vacuum and the crude was concentrated and purified on silica column with 5% MeOH in DCM to give brown solid (58mg, 75%).

[0267] (9R,9aR)-9-(4-bromophenyl)-N-(6-(cyclopropylmethoxy)pyridin-3- yl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (4f) Compound 3 (50 mg, 0.162 mmol) was added to a seal tube along with Ru3CO12( 10.3 mg, 0.016 mmol) Dry toluene (0.1M) and TMDS (0.29mL, 1.62 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (85uL, 0.48 mmol) and 4- nitrophenyl (6-(cyclopropylmethoxy)pyridin-3-yl)carbamate in DMF (109 mg, 0.33mmol) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until 85 CORE / 3510075.0137 / 196268526.1completion. The crude was concentrated under vacuum and purified on silica column with 5% MeOH in DCM to give brown solid (59mg, 70%).

[0268] (9R,9aR)-N-(6-ethoxypyridin-3-yl)-9-(4-((2- hydroxyphenyl)ethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)- carboxamide (19) Compound 4a (35.0mg. 0.076 mmol), tert-butyl(2- ethynylphenoxy)dimethylsilane (89 mg, 0.38 mmol), and XPhos Pd G3 (6.4 mg, 0.0076 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and then triethylamine (42uL, 0.30 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 80 uL, 0.08 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4and was purified on flash silica chromatography with 10 % MeOH in DCM to give white solid (12.0 mg.32% over two steps)1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.15 (s, 1H), 8.12 (s, 1H), 7.45 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 5.8 Hz, 3H), 7.22 (t, J = 7.8 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.82 (t, J = 7.5 Hz, 1H), 6.69 (d, J = 9.0 Hz, 1H), 4.25 (d, J = 6.9 Hz, 2H), 3.68-3.58 (m, 2H), 3.48-3.39 (m, 2h)3.23 – 3.06 (m, 2H), 2.69-2.62 (m, 1H), 2.41 – 2.21 (m, 4H), 2.20-2.10 (m, 1H), 1.96-1.79 (d, 3H), 1.29 (t, J = 7.1 Hz, 3H). LC-MS m / z calcd. C30H33N4O3+[M+H]+= 497.26, found = 497.47.

[0269] (9R,9aR)-9-(4-((2-hydroxyphenyl)ethynyl)phenyl)-N-(6- morpholinopyridin-3-yl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (20) Compound 4b (25.0 mg, 0.05 mmol), tert-butyl(2-ethynylphenoxy)dimethylsilane (58.0 mg, 0.25 mmol), and XPhos Pd G3 (4.2 mg, 0.005 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (28uL, 0.20mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 15 % MeOH in DCM. To the solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 0.06 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 15 % MeOH in DCM to give white solid 86 CORE / 3510075.0137 / 196268526.1(16.0 mg.60 % over two stes)1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.14 (d, J = 2.7 Hz, 1H), 8.04 (s, 1H), 7.62 (dd, J = 9.0, 2.7 Hz, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.38 (d, J = 8.1 Hz, 3H), 7.27 – 7.18 (m, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 6.76 (d, J = 9.1 Hz, 1H), 3.70 (s, 4H), 3.66 – 3.57 (m, 2H), 3.48 – 3.40 (m, 1H), 3.34 – 3.32 (m, 4H), 3.27 (s, 1H), 3.24-3.09 (m, 2H), 2.50 – 2.44 (m, 1H), 2.41 – 2.20 (m, 3H), 2.15 (t, J = 13.0 Hz, 1H), 1.98 – 1.74 (m, 3H).LC-MS m / z calcd. C32H36N5O3+[M+H]+= 538.28, found = 538.37.

[0270] (9R,9aR)-N-(5-ethoxypyrazin-2-yl)-9-(4-((2- hydroxyphenyl)ethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)- carboxamide (21) Compound 4c (20 mg, 0.043 mmol), tert-butyl(2- ethynylphenoxy)dimethylsilane (51 mg, 0.22 mmol), and XPhos Pd G3 (3.7 mg, 0.0043 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (24 uL, 0.17 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 60uL, 0.06 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 10 % MeOH in DCM to give white solid (9.7 mg. 45 % over two steps).1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.79 (s, 1H), 8.52 (s, 1H), 8.00 (s, 1H), 7.50-7.35 (m, 5H), 7.22 (t, J = 8.7 Hz, 1H), 6.92 (d, J = 9.3 Hz, 1H), 6.83 (t, J = 6.9 Hz, 1H), 4.29 (q, J = 7.0 Hz, 2H), 3.75-3.59 (m, 2H), 3.48 – 3.45 (m, 1H), 3.35 – 3.29 (m, 1H), 3.23 – 3.06 (m, 2H), 2.50 – 2.44 (m, 1H), 2.38 – 2.07 (m, 4H), 1.94-1.71 (m, 3H), 1.33 (t, J = 7.0 Hz. , 3H).. LC-MS m / z calcd. C29H32N5O3+[M+H]+= 498.25, found = 498.35.

[0271] (9R,9aR)-9-(4-((2-hydroxyphenyl)ethynyl)phenyl)-N-(5- methoxypyrazin-2-yl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (22) Compound 4d (20.0 mg, 0.045 mmol), tert-butyl(2-ethynylphenoxy)dimethylsilane (52 mg, 0.22 mmol), and XPhos Pd G3 (3.8 mg, 0.0045 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) was added to solid reagents and then triethylamine (25 uL, 0.18 mmol) was added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF (0.1M), TBAF (1M in THF, 50uL, 0.05 mmol) solution was 87 CORE / 3510075.0137 / 196268526.1added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 15 % MeOH in DCM to give white solid (11.0 mg.51 % over two steps).1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 8.81 (s, 1H), 8.55 (s, 1H), 8.03 (s, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.42 – 7.33 (m, 3H), 7.22 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 3.87 (s, 3H), 3.78 – 3.57 (m, 2H), 3.45-3.37 (m, 1H), 3.31-3.25 (m, 1H), 3.20 (t, J = 8.4 Hz, 1H), 3.11 (d, J = 12.4 Hz, 1H), 2.59-2.55 (m, 1H), 2.43 – 2.19 (m, 3H), 2.14 (t, J = 11.6 Hz, 1H), 1.94-1.74 (m, 3H).. LC-MS m / z calcd. C28H30N5O3+[M+H]+= 484.23, found = 484.37.

[0272] (9R,9aR)-N-(6-(2-fluoroethoxy)pyridin-3-yl)-9-(4-((2- hydroxyphenyl)ethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)- carboxamide (25) Compound 4e (11.5mg, 0.024 mmol), tert-butyl(2- ethynylphenoxy)dimethylsilane (28 mg, 0.12 mmol), and XPhos Pd G3 (2.0 mg, 0.0024 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (13 uL, 0.1 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF (0.1M), TBAF (1M in THF, 30 uL, 0.03 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 10 % MeOH in DCM to give white solid (4.6 mg.37% over two steps).1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 8.19 (s, 1H), 8.15 (s, 1H), 7.76 (d, J = 9.6 Hz, 1H), 7.46 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 7.5 Hz, 3H), 7.22 (t, J = 7.8 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.82 (t, J = 7.4 Hz, 1H), 6.77 (d, J = 9.1 Hz, 1H), 4.78 (d, J = 4.6 Hz, 1H), 4.66 (d, J = 4.4 Hz, 1H), 4.48 (d, J = 4.1 Hz, 1H), 4.41 (d, J = 4.4 Hz, 1H), 3.71-3.56 (m, 3H), 3.31-3.26 (m, 1H), 3.24 – 3.08 (m, 2H), 2.44 – 2.05 (m, 5H), 1.96-1.76(m, 3H). LC-MS m / z calcd. C30H32FN4O3+[M+H]+= 515.25, found = 515.40.

[0273] (9R,9aR)-N-(6-(cyclopropylmethoxy)pyridin-3-yl)-9-(4-((2- hydroxyphenyl)ethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)- carboxamide (26) Compound 4f (20.0 mg, 0.041 mmol), tert-butyl(2- ethynylphenoxy)dimethylsilane (48 mg, 0.2 mmol), and XPhos Pd G3 (3.5 mg, 0.004 mmol), 88 CORE / 3510075.0137 / 196268526.1were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (23 uL, 0.17 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF (0.1M), TBAF (1M in THF, 50uL, 0.05 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4and was purified on flash silica chromatography with 15 % MeOH in DCM to give white solid (6.7 mg.30 % over two steps).1H NMR (400 MHz, DMSO-d6).1H NMR (400 MHz, MeOD-d6) δ 8.03 (d, J = 2.7 Hz, 1H), 7.67 (dd, J = 8.9, 2.7 Hz, 1H), 7.51 (d, J = 8.3 Hz, 2H), 7.44 – 7.36 (m, 3H), 7.21 (ddd, J = 8.8, 7.3, 1.7 Hz, 1H), 6.92 – 6.82 (m, 2H), 6.75 (d, J = 8.8 Hz, 1H), 4.06 (d, J = 7.1 Hz, 2H), 3.73 – 3.43 (m, 4H), 3.31 – 3.21 (m, 2H), 2.73 (t, J = 9.3 Hz, 1H), 2.65 – 2.55 (m, 1H), 2.56 – 2.46 (m, 1H), 2.40 – 2.24 (m, 2H), 2.15 – 1.95 (m, 3H), 1.36 – 1.22 (m, 1H), 0.66 – 0.56 (m, 2H), 0.35 (dt, J = 6.2, 4.5 Hz, 2H).. LC-MS m / z calcd. C32H35N4O3+[M+H]+= 523.24, found = 523.54. Scheme 14. Synthesis of Compound 27

[0274] (9R,9aR)-9-(4-bromophenyl)-N-(5-methoxypyrimidin-2-yl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (4g) Compound 3 (30 mg, 0.097 mmol) was added to a seal tube along with Ru3CO12( 6.2 mg, 0.0097 mmol) Dry toluene (0.1M) and TMDS (0.17 mL, 0.97 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (35 uL, 0.2 mmol) and (5-methoxypyrimidin- 2-yl)carbamic chloride in THF (0.1M, , 36 mg, 0.2 mmol) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until completion and the crude was concentrated and purified on silica column with 5% MeOH in DCM and further purified with C18column: H2O:MeCN= 60:40. (8.0 mg, 18%). 89 CORE / 3510075.0137 / 196268526.1

[0275] (9R,9aR)-9-(4-((2-hydroxyphenyl)ethynyl)phenyl)-N-(5- methoxypyrimidin-2-yl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (27) Compound 4g (8.0 mg, 0.018 mmol), 1-ethynyl-2-(methoxymethoxy)benzene (15 mg, 0.09 mmol), and XPhos Pd G3 (1.5 mg, 0.0018 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) was added to solid reagents and then triethylamine (10 uL, 0.072 mmol) was added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of the MOM protected intermediate in DCM (0.1 M), HCl (4M in dioxane, 23 uL, 0.09 mmol) was added. The reaction was stirred for 30 min and reaction progress was monitored by LC-MS until all starting material was consumed. The crude was diluted with DCM and quenched with sat. NaHCO3(aq). The aqueous phase was extracted with DCM 3 times and the combined organic phase was washed with water and brine. The organic solvent was removed under vacuum (heat under 40oC, in case of cyclizing to benzofuran) and crude was purified on flash chromatography (100% DCM to 5 % MeOH in DCM) (1.5 mg, 17% over two steps).1H NMR (400 MHz, DMSO-d6)1H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.32 (s, 2H), 7.43 (d, J = 8.2 Hz, 2H), 7.38-7.30 (m, 3H), 7.20 (t, J = 7.9 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.78 (s, 1H), 3.85 (s, 4H), 3.62 – 3.51 (m, 4H), 3.23 – 3.11 (m, 3H), 2.63-2.56 (m, 1H), 2.42 – 2.13 (m, 5H), 1.93-1.71 (m, 3H). LC-MS m / z calcd. C29H29N5O3Na+[M+Na]+= 506.22, found = 506.2. Scheme 8. Synthesis of Compound 24

[0276] (9R,9aR)-9-(4-bromophenyl)-N-(2-fluoro-4-methoxyphenyl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (4h) Compound 3 (50 mg, 0.162 mmol) was added to a seal tube along with Ru3CO12 ( 10.3 mg, 0.016 mmol) Dry toluene (0.1M) and TMDS (0.29mL, 1.62 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (85uL, 0.48 mmol) and 4-nitrophenyl (2- fluoro-4-methoxyphenyl)carbamate in DMF (101 mg, 0.33 mmol, 0.1M) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until completion. The crude 90 CORE / 3510075.0137 / 196268526.1was concentrated under vacuum and purified on silica column with 5% MeOH in DCM to give off-white solid (56 mg, 75%).

[0277] (9R,9aR)-N-(2-fluoro-4-methoxyphenyl)-9-(4- (phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (24) Compound 4h (8.0 mg, 0.017 mmol), ethynylbenzene (8.8 mg, 0.08 mmol), and XPhos Pd G3 (1.5 mg, 0.0017 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (10 uL, 0.07 mmol) was added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM to give white solid (4.0mg, 48%).1H NMR (400 MHz, DMSO-d6) δ 7.77 (s, 1H), 7.58 – 7.51 (m, 2H), 7.51 – 7.45 (m, 2H), 7.45 – 7.40 (m, 3H), 7.40 – 7.35 (m, 2H), 7.19 (t, J = 9.0 Hz, 1H), 6.80 (dd, J = 12.2, 2.8 Hz, 1H), 6.69 (ddd, J = 8.8, 2.8, 1.1 Hz, 1H), 3.74 (s, 3H), 3.67 – 3.52 (m, 2H), 3.46 – 3.38 (m, 2H), 3.25-3.16 (m, 1H), 3.16 – 3.05 (m, 1H), 2.54-2.52 (m, 1H), 2.40 – 2.06 (m, 5H), 1.95-1.74 (m, 3H).. LC-MS m / z calcd. C30H31FN3O2+[M+H]+= 484.24, found = 484.44. Scheme 9. Synthesis of Compound 15

[0278] (9H-fluoren-9-yl)methyl (2R,3R)-3-(6-chloropyridin-3-yl)-2-(quinolin- 8-ylcarbamoyl)pyrrolidine-1-carboxylate (6) Compound 5 (9.6 g, 20.7 mmol), Pd(OAc)2 (465 mg, 2.07 mmol), dibenzyl phosphate (1.15 g, 41.4 mmol), AgOAc (6.91 g, 41.4 mmol), and 2- chloro-5-iodopyridine (14.9 g, 62.1 mmol) werepurged with argon and then dissolved in dry toluene (96 mL, 0.22 M). The reaction was heated to reflux for 24 h under argon and then filtered through Celite. The crude was further purified by flash column chromatography (silica, 1:2 Hexane / EthylAcetate), affording product as brown foam solid (8.2g, 69%). 91 CORE / 3510075.0137 / 196268526.1

[0279] (2R,3R)-3-(6-chloropyridin-3-yl)-N-(quinolin-8-yl)pyrrolidine-2- carboxamide (7) Compound 6 (2.34 g, 4.07 mmol) was dissolved in dry THF (15 mL, 0.3 M). Pyrrolidine (1.67 mL, 20.3 mmol)was then added dropwise, and the reaction was stirred for 1 h at room temperature. The reaction mixture was thenconcentrated and purified by column chromatography (1:1 Hexane / EtOAc, then DCM to 9:1 DCM / MeOH)to afford the title product as a yellow solid (98% yield, 1.4g).

[0280] (2R,3R)-3-(6-chloropyridin-3-yl)pyrrolidine-2-carboxylic acid, HCl (8) Compound 7 (1.4 g, 3.2 mmol) was dissolved in 27 mL of sat. HCl(aq) and heated in a sealed vessel 90 °C for 17h (note: too concentrated acid or heating too long / hot willcause isomerization to trans compound). The reactionwas cooled to 0oC. Aqueous NaOH was added slowly to adjust pH to 12, and the aqueous phase was washed with EtOAc to remove starting material and 8-aminoquinoline. The mixture was acidified withaqueous HCl to pH 2, and water was removed. To the solid residue, 7:3 CHCl3:iPrOH was added to redissolve the crude product and the solids were removed by filtration. Solvent was removed under vacuum to give product as yellow solids.

[0281] (2R,3R)-1-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-3- (6-chloropyridin-3-yl)pyrrolidine-2-carboxylic acid (9) Compound 8 (1.40 g, 5.33 mmol) was dissolved in DCM solution (60mL, 0.1M), and NaBH(OAc)3 (3.39 g, 16 mmol) was addedat 0 °C while stirring. (9H-fluoren-9-yl)methyl (2-oxoethyl)carbamate (3.94 g, 13.3 mmol) was added in three portions at 0 °C. Thereaction mixture was warmed to room temperature and stirred for 1h. The reaction was then quenched with NH4Cl andextracted with DCM. The product was purified by columnchromatography (DCM: MeOH=7:3) to give white product(1.65 g, 61%).

[0282] (9R,9aR)-9-(6-chloropyridin-3-yl)octahydro-1H-pyrrolo[1,2- a][1,4]diazepin-1-one (10) Compound 9 (1.65 g, 3.26 mmol) was dissolved in dry DMF (30 mL, 0.11M). SiliaBond Piperazine (9.69 g, 9.78 mmol) was added and heated to 50 °C for 2 h. The reactionwas then cooled to 0 °C, and HATU (1.86 g, 4.89 mmol) and DIPEA (1.14 mL, 6.52 mmol) were added. The reaction was thenwarmed to room temperature and stirred for 30 min. The reaction crude was filtered and rinsed with 20% MeOH in DCM, and the liquids were concentrated under vacuum below 40oC. The crude was purified on C18 column (H2O: MeCN= 80:20) to give the product as a white solid (0.65 g, 75% yield).

[0283] (9R,9aR)-9-(6-chloropyridin-3-yl)-N-(4-methoxyphenyl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (11a) Compound 10 (170 mg, 0.64 92 CORE / 3510075.0137 / 196268526.1mmol) was added to a seal tube along with Ru3CO12(41 mg, 0.064 mmol). Dry toluene (5mL, 0.1M) and TMDS (1.1mL, 6.4 mmol) was added stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry THF (0.1 M) and 1-isocyanato-4-methoxybenzene (83 uL, 0.64 mmol) was added at 0oC dropwisely. The mixture is stirred at 0oC for 30min before quenched with MeOH and then crude was purified on column with 5% MeOH in DCM to give white solid (150mg, 58%).

[0284] (9R,9aR)-9-(6-((2-hydroxyphenyl)ethynyl)pyridin-3-yl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (15) Compound 11a (70 mg, 0.175 mmol), tert-butyl(2-ethynylphenoxy)dimethylsilane (203 mg, 0.87 mmol), and XPhos Pd G3 (30 mg, 0.035 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (1.7 mL, 0.1M) and triethylamine (97uL, 0.7 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 0.21mL, 0.21 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 10 % MeOH in DCM to give white solid (32 mg.38% over two steps).1H NMR (400 MHz, DMSO- d6) δ 10.17 (s, 1H), 8.53 (s, 1H), 8.02 (s, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.31 (d, J = 9.1 Hz, 2H), 7.26 (t, J = 7.9 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.83 (dd, J = 18.9, 8.1 Hz, 3H), 3.71 (s, 3H), 3.69-3.61 (m, 2H), 3.46 (s, 1H), 3.30 – 3.18 (m, 2H), 3.18-3.10 (m, 1H), 2.54-2.51 (m, 1H), 2.42 – 2.08 (m, 4H), 1.96-1.74 (m, 3H).. LC-MS m / z calcd. C29H31N4O3+[M+H]+= 483.24, found = 483.31. Scheme 10. Synthesis of Compound 23

[0285] (9R,9aR)-9-(6-chloropyridin-3-yl)-N-(6-ethoxypyridin-3-yl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (11b) Compound 10 (30 mg, 0.11 mmol) was added to a seal tube along with Ru3CO12 ( 7.2 mg, 0.011 mmol) Dry toluene (1.6 93 CORE / 3510075.0137 / 196268526.1mL, 0.1M) and TMDS (0.2mL, 1.13 mmol) was added and the reaction was stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry acetonitrile (0.1 M) and DIPEA (40 uL, 0.23 mmol) and 4- nitrophenyl (5-ethoxypyridin-2-yl)carbamate in DMF (68.5 mg, 0.23 mmol) was added at 0oC dropwisely. The mixture is stirred at room temperature for 2 hours until completion. The crude was concentrated under vacuum and purified on silica column with 5% MeOH in DCM to give the product as brown solid (22 mg, 47%).

[0286] (9R,9aR)-N-(6-ethoxypyridin-3-yl)-9-(6-((2- hydroxyphenyl)ethynyl)pyridin-3-yl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)- carboxamide (23) Compound 11b (8.5 mg, 0.02 mmol), 1-ethynyl-2- (methoxymethoxy)benzene (17 mg, 0.1 mmol), and XPhos Pd G3 (1.7 mg, 0.002 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (11 uL, 0.08 mmol) was added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of the MOM protected intermediate in DCM (0.1 M), HCl (4M in dioxane, 25 uL, 0.1 mmol) was added. The reaction was stirred for 30 min and reaction progress was monitored by LC-MS until all starting material was consumed. The crude was diluted with DCM and quenched with sat. NaHCO3(aq). The aqueous phase was extracted with DCM 3 times and the combined organic phase was washed with water and brine. The organic solvent was removed under vacuum (heat under 40oC, in case of cyclizing to benzofuran) and crude was purified on flash chromatography (100% DCM to 10 % MeOH in DCM) (3.0 mg, 30% over two steps).1H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.54 (d, J = 2.4 Hz, 1H), 8.18 (s, 1H), 8.13 (d, J = 2.7 Hz, 1H), 7.82 (dd, J = 8.2, 2.2 Hz, 1H), 7.72 (dd, J = 8.9, 2.8 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.43 (dd, J = 7.7, 1.7 Hz, 1H), 7.31 – 7.22 (m, 1H), 6.98 – 6.92 (m, 1H), 6.85 (td, J = 7.5, 1.1 Hz, 1H), 6.70 (d, J = 8.8 Hz, 1H), 4.24 (q, J = 7.1 Hz, 2H), 3.71-3.59 (m, 2H), 3.52-3.43 (m, 1H), 3.31 – 3.27 (m, 1H), 3.22 (t, J = 8.5 Hz, 1H), 3.17-3.09 (m, 1H), 2.41 – 2.08 (m, 5H), 1.98-1.78 (m, 3H), 1.30 (t, J = 7.1 Hz, 3H).. LC-MS m / z calcd. C29H32N5O3+[M+H]+= 498.61, found = 498.42. Scheme 11. Synthesis of Compound 16 94 CORE / 3510075.0137 / 196268526.1

[0287] (9H-fluoren-9-yl)methyl (2R,3R)-3-(6-chloro-5-fluoropyridin-3-yl)-2- (quinolin-8-ylcarbamoyl)pyrrolidine-1-carboxylate (12) Compound 5 (2.0 g, 4.31 mmol), Pd(OAc)2 (97 mg, 0.43 mmol), dibenzyl phosphate (240 mg, 0.86 mmol), AgOAc (1.44 g, 8.63 mmol), and 2-chloro-3-fluoro-5-iodopyridine (3.33 g, 12.9 mmol) were purged with argon and then dissolved in dry toluene (20 mL, 0.22 M). The reaction was heated to reflux for 24 h under argon and then filtered through Celite. The crude was further purified by flash column chromatography (silica, 1:2 Hexane / EthylAcetate), affording product as brown foam solid (1.3g, 50%).

[0288] (2R,3R)-3-(6-chloro-5-fluoropyridin-3-yl)-N-(quinolin-8- yl)pyrrolidine-2-carboxamide (13) Compound 12 (256 mg, 0.43 mmol) was dissolved in dry THF (3 mL, 0.14 M). Pyrrolidine (0.18 mL, 2.2 mmol)was then added dropwise, and the reaction was stirred for 1 h at room temperature. The reaction mixture was thenconcentrated and purified by column chromatography (1:1 Hexane / EtOAc, then DCM to 9:1 DCM / MeOH)to afford the title product as a yellow solid (0.14g, 95%)

[0289] (2R,3R)-3-(6-chloro-5-fluoropyridin-3-yl)pyrrolidine-2-carboxylic acid (14) Compound 13 (110mg, 0.3 mmol) was dissolved in 2.5mL of sat. HCl(aq) and heated in a sealed vessel 90 °C for 17h (note: too concentrated acid or heating too long / hot willcause isomerization to trans compound). The reactionwas cooled to 0oC. Aqueous NaOH was added slowly to adjust pH to 12, and the aqueous phase was washed with EtOAc to remove starting material and 8-aminoquinoline. The mixture was acidified withaqueous HCl to pH 2, and water was removed. To the solid residue, 7:3CHCl3:iPrOH was added to redissolve the crude product and the solids were removed by filtration. Solvent was removed under vacuum to give product as yellow solids.

[0290] (2R,3R)-1-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-3- (6-chloro-5-fluoropyridin-3-yl)pyrrolidine-2-carboxylic acid (15) Compound 14 (84 mg, 0.3 95 CORE / 3510075.0137 / 196268526.1mmol) was dissolved in DCM solution (3mL, 0.1M), and NaBH(OAc)3(189 mg, 0.9 mmol) was addedat 0 °C while stirring. (9H-fluoren-9-yl)methyl (2-oxoethyl)carbamate (189 mg, 0.9 mmol) was added in three portions at 0 °C. Thereaction mixture was warmed to room temperature and stirred for 1h. The reaction was then quenched with NH4Cl andextracted with DCM. The product was purified by column chromatography (DCM: MeOH=7:3) to give white product(88 mg, 57%).

[0291] (9R,9aR)-9-(6-chloro-5-fluoropyridin-3-yl)octahydro-1H-pyrrolo[1,2- a][1,4]diazepin-1-one (16) Compound 15 (88 mg, 0.17 mmol) was dissolved in dry DMF (1.5mL, 0.11M). SiliaBond Piperazine (500 mg, 0.5mol) was added and heated to 50 °C for 2 h. The reactionwas then cooled to 0 °C, and HATU (96 mg, 0.25 mmol) and DIPEA (59 uL, 0.34 mmol) were added. The reaction was then warmed to room temperature and stirred for 30 min. The reaction crude was filtered and rinsed with 20% MeOH in DCM, and the liquids were concentrated under vacuum below 40oC. The crude was purified on C18 column (H2O: MeCN= 70:30) to give the product as a white solid (23 mg, 48%).

[0292] (9R,9aR)-9-(6-chloro-5-fluoropyridin-3-yl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (17) Compound 16 (23 mg, 0.081 mmol) was added to a seal tube along with Ru3CO12(5.2 mg, 0.008 mmol). Dry toluene (0.5mL, 0.15M) and TMDS (0.14mL, 0.81 mmol) was added stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry THF (0.1 M) and 1-isocyanato-4-methoxybenzene (10.5 uL, 0.081 mmol) was added at 0oC. The mixture is stirred at 0oC for 30min before quenched with MeOH and then crude was purified on column with 5% MeOH in DCM to give white solid (14 mg, 40%).

[0293] (9R,9aR)-9-(5-fluoro-6-((2-hydroxyphenyl)ethynyl)pyridin-3-yl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (16) Compound 17 (8.0 mg, 0.019 mmol), tert-butyl(2-ethynylphenoxy)dimethylsilane (22 mg, 0.095 mmol), and XPhos Pd G3 (1.6 mg, 0.0019 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (11uL, 0.076 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF(0.1M), TBAF (1M in THF, 25 uL, 0.025 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the 96 CORE / 3510075.0137 / 196268526.1organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4 and was purified on flash silica chromatography with 10 % MeOH in DCM to give white solid (2.6 mg.27% over two steps).1H NMR (400 MHz, DMSO- d6) δ 8.42 (s, 1H), 8.02 (s, 1H), 7.76 (d, J = 11.0 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.31 (d, J = 9.1 Hz, 2H), 7.29-7.23 (m, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.81 (d, J = 9.0 Hz, 2H), 3.70 (s, 3H), 3.68 – 3.60 (m, 1H), 3.56-3.47 (m, 2H), 3.24 (dd, J = 18.9, 9.2 Hz, 3H), 3.13 (d, J = 13.5 Hz, 2H), 2.58-2.55 (m, 1H) 2.43 – 2.35 (m, 1H), 2.31-2.22 (m, 2H), 2.21 – 2.07 (m, 1H), 1.99 – 1.74 (m, 3H). LC-MS m / z calcd. C29H30FN4O3+[M+H]+= 501.23, found = 501.42. Scheme 12. Synthesis of Compound 17

[0294] (2R,3S)-3-(5-bromopyridin-2-yl)-1-pivaloyl-N-(quinolin-8- yl)pyrrolidine-2-carboxamide (19) Compound 18 ( 1 equiv.), Pd(OAc)2 (0.1equiv), dibenzyl phosphate (0.2 equiv), AgOAc (2 equiv), and 5-bromo-2-iodopyridine (4 equiv) were purged with argon and then dissolved in dry toluene (0.2 M). The reaction was heated to reflux for 48 h under argon and then filtered through Celite for purification by column chromatography (1:2 Hexane / EthylAcetate), affording the title product as brown foam solid.

[0295] (2R,3S)-3-(5-bromopyridin-2-yl)pyrrolidine-2-carboxylic acid (20) Compound 19 (1.0 equiv.) was dissolved in sat. HCl(aq, 150 equiv.) and heated in a sealed vessel 50 °C for 18h (note: too concentrated acid or heating too long / hot willcause isomerization to trans compound). The reactionwas cooled to 0oC. Aqueous NaOH was added slowly to adjust pH to 12, and the aqueous phase was washed with EtOAc to remove starting material and 8-aminoquinoline. The mixture was acidified with aqueous HCl to pH 2, and water was removed. To the solid residue, 7:3CHCl3:iPrOH was added to redissolve the crude product and the solids were removed by filtration. Solvent was removed under vacuum to give product as brown solids. 97 CORE / 3510075.0137 / 196268526.1

[0296] (2R,3S)-1-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-3- (5-bromopyridin-2-yl)pyrrolidine-2-carboxylic acid (21) Compound 20 (1 equiv.) was dissolved in dry DCM (0.1M), and NaBH(OAc)3 (3 equiv) was addedat 0 °C while stirring. Aldehyde (3 equiv.) was dissolved in dry DCM(0.1 M) and added dropwise to compound 5 at 0 °C. Thereaction mixture was warmed to room temperature and stirred for 1h. The reaction was then quenched with NH4Cl andextracted with DCM. The combined organic solution was dried over Na2SO4. Then the organic solvent was removed under vacuum and the product was purified by column chromatography (DCM: MeOH=7:3) as an off-white solid.

[0297] (9S,9aR)-9-(5-bromopyridin-2-yl)octahydro-1H-pyrrolo[1,2- a][1,4]diazepin-1-one (22) Compound 21 (1.0 equiv.) was dissolved in dry DMF (0.1M). SiliaBond Piperazine (3 equiv) was added and heated to 50 °C for 2 h. The reaction was then cooled to 0 °C, and HATU (1.5 equiv) and DIPEA (2.0 equiv.) were added. The reaction was then warmed to room temperature and stirred for 20 min. The reaction crude was filtered and rinsed with 20% MeOH in DCM, and the liquids were concentrated under vacuum below 40oC. The crude was purified on C18 column (H2O: MeCN= 70:30) to give the product as white solid.

[0298] (9S,9aR)-9-(5-bromopyridin-2-yl)-N-(4-methoxyphenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (23) Compound 22 (33 mg, 0.11 mmol) was added to a seal tube along with Ru3CO12 ( 6.8mg, 0.01 mmol). Dry toluene (0.1M) and TMDS (0.19 mL, 1.1 mmol) was added stirred at 80oC for 3 hours. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry THF (0.1 M) and 1-isocyanato-4-methoxybenzene (14 uL, 0.11 mmol) was added at 0oC dropwisely. The mixture is stirred at 0oC for 30min before quenched with MeOH and then crude was further purified on column with 10% MeOH in DCM give the title product as a white solid (32 mg, 65%).

[0299] (9S,9aR)-9-(5-((2-hydroxyphenyl)ethynyl)pyridin-2-yl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (17) Compound 23 (14.2 mg, 0.032 mmol), 1-ethynyl-2-(methoxymethoxy)benzene (20.7 mg, 0.13 mmol), and XPhos Pd G3 (2.7 mg, 0.0032 mmol), were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) and triethylamine (22 uL, 0.16 mmol) were added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of the MOM protected intermediate (1.0 equiv.) in 98 CORE / 3510075.0137 / 196268526.1DCM (0.1 M), HCl (4M in dioxane, 40 uL, 0.16 mmol) was added. The reaction was stirred for 30 min and reaction progress was monitored by LC-MS until all starting material was consumed. The crude was diluted with DCM and quenched with sat. NaHCO3(aq). The aqueous phase was extracted with DCM 3 times and the combined organic phase was washed with water and brine. The organic solvent was removed under vacuum (heat under 40oC, in case of cyclizing to benzofuran) and crude was purified on flash chromatography (100% DCM to 5 % MeOH in DCM).1H NMR (400 MHz, DMSO-d6) to give a white solid (8.0 mg, 52% over two steps)1H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 8.63 (d, J = 3.0 Hz, 1H), 8.00 (s, 1H), 7.88 (dd, J = 8.2, 2.3 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.42 (dd, J = 7.7, 1.8 Hz, 1H), 7.31 (d, J = 9.1 Hz, 3H), 6.94 (d, J = 8.2 Hz, 1H), 6.89 – 6.77 (m, 3H), 3.70 (s, 3H), 3.68 – 3.56 (m, 3H), 3.32 – 3.25 (m, 1H), 3.19 (t, J = 7.8 Hz, 1H), 3.15 – 3.05 (m, 1H), 2.68-2.61 (m, 1H), 2.41 (q, J = 8.6 Hz, 1H), 2.28 (dd, J = 14.0, 10.3 Hz, 1H), 2.23 – 2.07 (m, 3H), 1.92 – 1.74 (m, 2H). LC-MS m / z calcd. C29H31N4O3+[M+H]+= 483.24, found = 483.37 Scheme 13. Synthesis of Compound 18

[0300] (2R,3S)-3-(6-chloropyridazin-3-yl)-1-pivaloyl-N-(quinolin-8- yl)pyrrolidine-2-carboxamide (24) Compound 18 (2.22 g, 6.82 mmol), Pd(OAc)2(153 mg, 0.682 mmol), dibenzyl phosphate (380 mg, 1.36mmol ), AgOAc (2.28g, 13.6 mmol), and 3- chloro-6-iodopyridazine (6.56 g, 27.3 mmol) were purged with argon and then dissolved in dry 1,4-dioxane (30 mL, 0.2M). The reaction was heated to 100oC for 24 h under argon and then concentrated onto Celite for purification by flash silica chromatography (1:2 Hexane / EthylAcetate) and C18 column(H2O:MeCN=50:50), affording title product as light yellow solid (200mg, 5%).

[0301] (2R,3S)-3-(6-chloropyridazin-3-yl)pyrrolidine-2-carboxylic acid (25) Compound 24 (200 mg, 0.46 mmol) was dissolved in 3.8 mL of sat. HCl(aq) and heated in a 99 CORE / 3510075.0137 / 196268526.1sealed vessel 50 °C for 18h (note: too concentrated acid or heating too long / hot willcause isomerization to trans compound). The reactionwas cooled to 0oC. Aqueous NaOH was added slowly to adjust pH to 12, and the aqueous phase was washed with EtOAc to remove starting material and 8-aminoquinoline. The mixture was acidified with aqueous HCl to pH 2, and water was removed. To the solid residue, 7:3CHCl3:iPrOH was added to redissolve the crude product and the solids were removed by filtration. Solvent was removed under vacuum to give product as yellow solids.

[0302] (2R,3S)-1-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-3- (6-chloropyridazin-3-yl)pyrrolidine-2-carboxylic acid (26) Compound 25 (77 mg, 0.342 mmol) was dissolved in dry DCM (4 mL), and NaBH(OAc)3(217 mg, 1.0 mmol) was addedat 0 °C while stirring. Aldehyde (295 mg, 1.0 mmol) was added at 0 °C in three portions. The reaction mixture was warmed to room temperature and stirred for 1h. The solvent was removed under vacuum and crude was purified by C18 column (H2O: MeCN=70:30) as white solid (90 mg, 53%).

[0303] (9S,9aR)-9-(6-chloropyridazin-3-yl)octahydro-1H-pyrrolo[1,2- a][1,4]diazepin-1-one (27) Compound 26 (90 mg, 0.18 mmol) was dissolved in dry DMF (6 mL). SiliaBond Piperazine (534 mg, 0.54 mmol) was added and heated to 50 °C for 2 h. The reaction was then cooled to 0 °C, and HATU (103 mg, 0.27 mmol) and DIPEA (62 uL, 0.36 mmol) were added. The reaction was then warmed to room temperature and stirred for 30 min. The reaction crude was filtered and rinsed with 20% MeOH in DCM, and the liquids were concentrated under vacuum below 40oC. The crude was purified on silica column with 15% MeOH in DCM to give the title product as a white solid (20 mg, 42%).

[0304] (9S,9aR)-9-(6-chloropyridazin-3-yl)-N-(4-methoxyphenyl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (28) Compound 27 (20.0 mg, 0.075 mmol) was added to a seal tube along with Ru3CO12 (4.8mg, 0.0075 mmol). Dry toluene (0.1M) and TMDS (27uL, 0.15mmol) were added and stirred at 70oC for 2 hours under dark. Reaction was cooled to room temperature and concentrated under vacuum. Crude residue was dissolved in dry THF (0.1 M) and 1-isocyanato-4-methoxybenzene (9.7 uL, 0.075 mmol) was added at 0oC. The mixture is stirred at 0oC for 30min before quenched with MeOH and then crude was further purified on column with 10% MeOH in DCM to give the product as white solid(10.0 mg, 45%).

[0305] (9S,9aR)-9-(6-((2-hydroxyphenyl)ethynyl)pyridazin-3-yl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (18) 100 CORE / 3510075.0137 / 196268526.1Compound 28 (11.0 mg, 0.027 mmol.), tert-butyl(2-ethynylphenoxy)dimethylsilane (32 mg, 0.14mmol), and XPhos Pd G3 (2.3mg, 0.0027 mmol) were added to a dry vial and flushed with Argon. Dry acetonitrile (0.1M) was added to solid reagents and then triethylamine (15uL, 0.11 mmol) was added. The reaction was stirred at 70oC for 24h. The reaction was cooled to room temperature and concentrated under vacuum. The intermediate was purified on flash silica chromatography with 100% DCM to 10 % MeOH in DCM. To the solution of TBS protected intermediate in THF (0.1M), TBAF (1M in THF, 35uL, 0.035 mmol) solution was added at 0oC, then the reaction was stirred at 0oC for 30min. Quench the reaction with aq. NH4Cl solution, Dilute the organic solution with DCM and wash the organic phase with water and brine. The organic solution was dried over Na2SO4and was purified on flash silica chromatography with 10 % MeOH in DCM to give white solid (5.0 mg.38% over two steps).1H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.38-7.26 (m, 3H), 6.97 (d, J = 8.3 Hz, 1H), 6.88 (d, J = 14.7 Hz, 1H), 6.79 (d, J = 9.0 Hz, 2H), 3.98 (d, J = 14.4 Hz, 1H), 3.69 (s, 3H), 3.62 – 3.53 (m, 2H), 3.18 – 2.96 (m, 3H), 2.78-2.64 (m, 3H), 2.41 – 2.25 (m, 2H), 2.03-1.80 (m, 3H). LC-MS m / z calcd. C28H30N5O3+[M+H]+= 484.23, found = 484.37. References (1) Petersen, E.; Kijlstra, A.; Stanford, M. Epidemiology of ocular toxoplasmosis. Ocul Immunol Inflamm 2012, 20 (2), 68-75. DOI: 10.3109 / 09273948.2012.661115 From NLM Medline. (2) Glasner, P. D.; Silveira, C.; Kruszon-Moran, D.; Martins, M. C.; Burnier, M.; Silveira, S.; Camargo, M. E.; Nussenblatt, R. B.; Kaslow, R. A.; Belfort, R. An unusually high prevalence of ocular toxoplasmosis in southern Brazil. Am. J. Opthalmol. 1992, 114, 136-144. (3) Grigg, M. E.; Dubey, J. P.; Nussenblatt, R. B. Ocular toxoplasmosis: lessons from Brazil. Am J Ophthalmol 2015, 159 (6), 999-1001. DOI: 10.1016 / j.ajo.2015.04.005. (4) de-la-Torre, A.; Sauer, A.; Pfaff, A. W.; Bourcier, T.; Brunet, J.; Speeg-Schatz, C.; Ballonzoli, L.; Villard, O.; Ajzenberg, D.; Sundar, N.; et al. Severe South American ocular toxoplasmosis is associated with decreased Ifn-gamma / Il-17a and increased Il- 6 / Il-13 intraocular levels. PLoS Negl Trop Dis 2013, 7 (11), e2541. DOI: 10.1371 / journal.pntd.0002541. (5) Pfaff, A. W.; de-la-Torre, A.; Rochet, E.; Brunet, J.; Sabou, M.; Sauer, A.; Bourcier, T.; Gomez-Marin, J. E.; Candolfi, E. New clinical and experimental insights into Old World and neotropical ocular toxoplasmosis. Int J Parasitol 2013. DOI: S0020- 7519(13)00255-5 [pii] 10.1016 / j.ijpara.2013.09.007. 101 CORE / 3510075.0137 / 196268526.1(6) Dunay, I. R.; Gajurel, K.; Dhakal, R.; Liesenfeld, O.; Montoya, J. G. Treatment of Toxoplasmosis: Historical Perspective, Animal Models, and Current Clinical Practice. Clin Microbiol Rev 2018, 31 (4). DOI: 10.1128 / CMR.00057-17. (7) Commodaro, A. G.; Belfort, R. N.; Rizzo, L. V.; Muccioli, C.; Silveira, C.; Burnier, M. N., Jr.; Belfort, R., Jr. Ocular toxoplasmosis: an update and review of the literature. Mem Inst Oswaldo Cruz 2009, 104 (2), 345-350. DOI: 10.1590 / s0074-02762009000200030 From NLM Medline. (8) Gill, J.; Sharma, A. Exploration of aminoacyl-tRNA synthetases from eukaryotic parasites for drug development. J Biol Chem 2023, 299 (3), 102860. DOI: 10.1016 / j.jbc.2022.102860 From NLM Medline. (9) Kato, N.; Comer, E.; Sakata-Kato, T.; Sharma, A.; Sharma, M.; Maetani, M.; Bastien, J.; Brancucci, N. M.; Bittker, J. A.; Corey, V.; et al. Diversity-oriented synthesis yields novel multistage antimalarial inhibitors. Nature 2016, 538 (7625), 344-349. DOI: 10.1038 / nature19804. (10) Vinayak, S.; Jumani, R. S.; Miller, P.; Hasan, M. M.; McLeod, B. I.; Tandel, J.; Stebbins, E. E.; Teixeira, J. E.; Borrel, J.; Gonse, A.; et al. Bicyclic azetidines kill the diarrheal pathogen Cryptosporidium in mice by inhibiting parasite phenylalanyl-tRNA synthetase. Sci Transl Med 2020, 12 (563). DOI: 10.1126 / scitranslmed.aba8412. (11) Radke, J. B.; Melillo, B.; Mittal, P.; Sharma, M.; Sharma, A.; Fu, Y.; Uddin, T.; Gonse, A.; Comer, E.; Schreiber, S. L.; et al. Bicyclic azetidines target acute and chronic stages of Toxoplasma gondii by inhibiting parasite phenylalanyl t-RNA synthetase. Nat Commun 2022, 13 (1), 459. DOI: 10.1038 / s41467-022-28108-y. (12) Sharma, M.; Mutharasappan, N.; Manickam, Y.; Harlos, K.; Melillo, B.; Comer, E.; Tabassum, H.; Parvez, S.; Schreiber, S. L.; Sharma, A. Inhibition of Plasmodium falciparum phenylalanine tRNA synthetase provides opportunity for antimalarial drug development. Structure 2022, 30 (7), 962-972 e963. DOI: 10.1016 / j.str.2022.03.017. (13) Sharma, M.; Malhotra, N.; Yogavel, M.; Harlos, K.; Melillo, B.; Comer, E.; Gonse, A.; Parvez, S.; Mitasev, B.; Fang, F. G.; et al. Structural basis of malaria parasite phenylalanine tRNA-synthetase inhibition by bicyclic azetidines. Nat Commun 2021, 12 (1), 343. DOI: 10.1038 / s41467-020-20478-5. (14) Ence, C. C.; Uddin, T.; Borrel, J.; Mittal, P.; Xie, H.; Zoller, J.; Sharma, A.; Comer, E.; Schreiber, S. L.; Melillo, B.; et al. Bicyclic pyrrolidine inhibitors of Toxoplasma gondii phenylalanine t-RNA synthetase with antiparasitic potency in vitro and brain exposure. bioRxiv 2024. DOI: 10.1101 / 2024.02.28.582607 From NLM PubMed-not-MEDLINE. (15) McFadden, D. C.; Tomavo, S.; Berry, E. A.; Boothroyd, J. C. Characterization of cytochrome b from Toxoplasma gondii and Qodomain mutations as a mechanism of atovaquone-resistance. Mol Biochem Parasitol 2000, 108 (1), 1-12. (16) Reynolds, M. G.; Oh, J.; Roos, D. S. In vitro generation of novel pyrimethamine resistance mutations in the Toxoplasma gondii dihydrofolate ˆreductase. Antimicrob. Agents Chemother.2001, 45 (4), 1271-1277. (17) Roos, D. S. Primary structure of the dyhydrofolate reductase-thymidylate synthase gene from Toxoplasma gondii. Journal of Biological Chemistry 1993, 268, 6269. 102 CORE / 3510075.0137 / 196268526.1(18) Uddin, T.; Xia, J.; Fu, Y.; McNamara, C. W.; Chatterjee, A. K.; Sibley, L. D. High Throughput Repurposing Screen Reveals Compounds with Activity Against Toxoplasma gondii Bradyzoites. bioRxiv 2024. DOI: 10.1101 / 2024.07.01.601569 From NLM PubMed-not-MEDLINE. (19) Xia, J.; Fu, Y.; Huang, W.; Uddin, T.; Sibley, L. D. Constitutive upregulation of transcription factors underlies permissive bradyzoite differentiation in a natural isolate of Toxoplasma gondii. mBio 2024, 15 (9), e0064124. DOI: 10.1128 / mbio.00641-24 From NLM Medline. (20) Jeffers, V.; Tampaki, Z.; Kim, K.; Sullivan, W. J., Jr. A latent ability to persist: differentiation in Toxoplasma gondii. Cell Mol Life Sci 2018, 75 (13), 2355-2373. DOI: 10.1007 / s00018-018-2808-x. (21) Tu, V.; Tomita, T.; Sugi, T.; Mayoral, J.; Han, B.; Yakubu, R. R.; Williams, T.; Horta, A.; Ma, Y.; Weiss, L. M. The Toxoplasma gondii Cyst Wall Interactome. mBio 2020, 11 (1). DOI: 10.1128 / mBio.02699-19. (22) Djurkovic-Djakovic, O.; Milenkovic, V.; Nikolic, A.; Bobic, B.; Grujic, J. Efficacy of atovaquone combined with clindamycin against murine infection with a cystogenic (Me49) strain of Toxoplasma gondii. J Antimicrob Chemother 2002, 50 (6), 981-987. (23) Dunay, I. R.; Heimesaat, M. M.; Bushrab, F. N.; Muller, R. H.; Stocker, H.; Arasteh, K.; Kurowski, M.; Fitzner, R.; Borner, K.; Liesenfeld, O. Atovaquone maintenance therapy prevents reactivation of toxopasmic encepahalitis in the murine model of reactivated toxoplasmosis. Antimicrob. Agents Chem.2004, 48, 4848-4854. (24) Wang, Q.; Sibley, L. D. Assays for Monitoring Toxoplasma gondii Infectivity in the Laboratory Mouse. Methods Mol Biol 2020, 2071, 99-116. DOI: 10.1007 / 978-1-4939- 9857-9_5. (25) Tobin, C. M.; Knoll, L. J. A patatin-like protein protects Toxoplasma gondii from degradation in a nitric oxide-dependent manner. Infect Immun 2012, 80 (1), 55-61. DOI: 10.1128 / IAI.05543-11. (26) Vizcarra, E. A.; Goerner, A. L.; Ulu, A.; Hong, D. D.; Bergersen, K. V.; Talavera, M. A.; Le Roch, K.; Wilson, E. H.; White, M. W. An ex vivo model of Toxoplasma recrudescence reveals developmental plasticity of the bradyzoite stage. mBio 2023, 14 (5), e0183623. DOI: 10.1128 / mbio.01836-23. EXAMPLE 4: Synthesis of bicyclic pyrrolidines (2R,3R)-3-(4-bromophenyl)-N-(quinolin-8-yl)pyrrolidine-2-carboxamide (330-017).103 CORE / 3510075.0137 / 196268526.1

[0306] To a solution of (9H-fluoren-9-yl)methyl (2R,3R)-3-(4-bromophenyl)- 2-(quinolin-8-ylcarbamoyl)pyrrolidine-1-carboxylate (6.05 g, 9.78 mmol, 1 equiv) in THF (49 mL) was added pyrrolidine (8.14 mL, 97.7 mmol, 10 equiv). The resulting mixture was stirred at rt for 2 h, then concentrated in vacuo. The resulting orange oil was purified by column chromatography (0:100 → 100:0 EtOAc / hexane then 0:100 → 5:95 MeOH / CH2Cl2) to afford 330-017 (3.34 g, 86%) as an orange solid.

[0307] [^]23D= –147 (c = 1.0, THF)

[0308] 1H NMR (400 MHz, CDCl3, 300 K) δ 10.93 (s, 1H), 8.84 (dd, J = 4.3, 1.6 Hz, 1H), 8.44 (dd, J = 7.0, 1.9 Hz, 1H), 8.12 (dd, J = 8.3, 1.6 Hz, 1H), 7.48 – 7.38 (m, 3H), 7.16 (q, J = 8.6 Hz, 4H), 4.23 (d, J = 8.7 Hz, 1H), 3.72 (q, J = 7.8 Hz, 1H), 3.57 (ddd, J = 9.6, 7.4, 4.1 Hz, 1H), 3.26 (dt, J = 9.6, 7.8 Hz, 1H), 2.36 – 2.23 (m, 1H), 2.13 (dq, J = 12.6, 7.6 Hz, 1H).

[0309] 13C NMR (101 MHz, CDCl3, 300K) δ 171.1, 148.5, 140.0, 139.0, 136.3, 134.0, 131.2, 130.1, 128.1, 127.3, 121.7, 121.5, 120.6, 116.6, 67.1, 48.3, 46.3, 32.8.

[0310] HRMS (ESI+) m / z Calculated for C20H19BrN3O [M+H]+396.0706; Found 395.85. (2R,3R)-3-(4-bromophenyl)-2-carboxypyrrolidin-1-ium chloride (330-022)

[0311] To a precooled (0 ºC) solution of 330-017 (2.51 g, 6.33 mmol, 1 equiv) in dimethylformamide (31.6 mL) was added protionwise Boc2O (5.56 g, 25.5 mmol, 4 equiv). The resulting mixture was allowed to warm to rt and stirred for 1 h. The mixture was cooled to 0 ºC, DMAP (154.7 mg, 1.26 mmol, 0.2 equiv) and Boc2O (20.98 g, 96.1 mmol, 15 equiv) were added then the resulting mixture was heated to 60 ºC and stirred for 3 h (the initially colorless mixture turned black). The mixture was cooled to rt and concentrated in vacuo to afford a dark brown oil. The crude oil was reconstituted in THF (63.3 mL) and water (21.1 mL), and the mixture was cooled to 0 ºC. LiOH (1.06 g, 44.3 mmol, 7 equiv) and H2O2(30% w / w in water, 9 mL, 88.6 mmol, 14 equiv) were added next. The resulting mixture was heated to 50 ºC and stirred for 5.5 h. The reaction was deemed incomplete by LC-MS analysis. The mixture was therefore cooled to 0 ºC; LiOH (0.56 g, 22.1 mmol, 3.5 equiv) and H2O2(30% 104 CORE / 3510075.0137 / 196268526.1w / w in water, 4.5 mL, 44.3 mmol, 7 equiv) were added, and the resulting mixture was heated to 50 ºC and stirred for 15.5 h. The mixture was cooled to 0 ºC, quenched with aq. Na2S2O3 (1.5 M, 50 mL) and concentrated in vacuo. The resulting mixture was washed with CH2Cl2 (3 × 50 mL). Combined organic layers were extracted with aq. NaOH (2 M, 5 × 40 mL). All aqueous layers were combined and acidified (pH = 2) by addition of aq. HCl (2 M) then extracted with chloroform / isopropanol (7:3, 3 × 50 mL). Organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuo to afford an orange solid. To the crude solid was added CH2Cl2(126 mL), the mixture was cooled to 0 ºC and HCl (4.0 M solution in dioxane, 31.5 mL, 126 mmol, 20 equiv) was slowly added. The mixture was allowed to warm to rt and stirred for 15 h. The resulting suspension was sparged with N2to remove excess HCl then concentrated in vacuo. The resulting orange solid was triturated with hexane then CH2Cl2(75 mL) to afford 330-022 (1.18 g, 61%) as an orange solid.

[0312] [^]23D= –72.6 (c = 0.1, DMSO / MeOH, 1:1)

[0313] 1H NMR (400 MHz, DMSO, 330 K) δ 7.52 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 4.54 (d, J = 9.0 Hz, 1H), 3.90 (q, J = 8.4 Hz, 1H), 3.57 (ddd, J = 12.1, 8.4, 4.2 Hz, 1H), 3.31 (dt, J = 11.3, 8.4 Hz, 1H), 2.48 – 2.39 (m, 1H), 2.17 (dq, J = 13.0, 8.5 Hz, 1H).

[0314] 13C NMR (101 MHz, DMSO, 330 K) δ 168.7, 137.8, 131.6, 131.0, 121.1, 63.4, 45.2, 45.0, 30.5.

[0315] HRMS (ESI+) m / z Calculated for C11H13BrNO2[M+H]+270.0130; Found 270.10. (9H-fluoren-9-yl)methyl (3-oxopropyl)carbamate (330-045)

[0316] To a precooled (–78 ºC) solution of oxalyl chloride (693 ^L, 8.23 mmol, 1.25 equiv) in CH2Cl2 (65.9 mL) was added dimethyl sulfoxide (1.16 mL, 16.4 mmol, 2.5 equiv). The resulting mixture was stirred at –78 ºC for 10 min. A solution of (9H-fluoren-9- yl)methyl (3-hydroxypropyl)carbamate1(1.96 g, 6.59 mmol, 1 eq) in CH2Cl2(65.9 mL + 6 mL rinse) was then added slowly via syringe and the resulting mixture was stirred at –78 ºC for 1 h. Triethylamine (4.57 mL, 32.9 mmol, 5 equiv) was added, the cold bath was removed and the reaction was stirred allowing to warm to rt for 30 min. The resulting mixture was concentrated in vacuo and co-evaporated with CH2Cl2(3 × 15 mL) to afford an orange oil. The crude material obtained was used without further purification in the 2-3 hours following isolation. 105 CORE / 3510075.0137 / 196268526.1(2R,3R)-1-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-3-(4- bromophenyl)pyrrolidine-2-carboxylic acid (330-046)

[0317] To a precooled (0 ºC) solution of 330-022 (779 mg, 2.54 mmol, 1 equiv) and sodium triacetoxyborohydride (1.6 g, 7.54 mmol, 3 equiv) in CH2Cl2(12.7 mL) was added a solution of crude aldehyde 330-045 (1.95 g, 6.59 mmol, 2.5 equiv) in CH2Cl2 (12.7 mL + 2 × 3 mL rinse). The resulting mixture was allowed to gradually warm to rt and was stirred for 16.5 h. The reaction was quenched with sat. aq. NH4Cl (30 mL). The mixture was extracted with CH2Cl2 (3 × 20 mL). Combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to afford an orange oil. The crude oil was purified by column chromatography (0:100 → 80:20 EtOAc / hexane then 0:100 → 20:80 MeOH / CH2Cl2) to afford 330-046 (869 mg, 63%) as white foamy solid.

[0318] 1H NMR (300 MHz, pyridine-d5, 300 K) δ 8.1 (d, J = 6.3 Hz, 1H), 7.9 (d, J = 7.5 Hz, 2H), 7.8 (dd, J = 7.6, 2.9 Hz, 2H), 7.4 (s, 4H), 7.4 (td, J = 7.7, 7.1, 3.3 Hz, 2H), 7.3 – 7.3 (m, 1H), 4.6 (d, J = 7.3 Hz, 2H), 4.4 (t, J = 7.2 Hz, 1H), 3.8 – 3.7 (m, 2H), 3.7 – 3.5 (m, 1H), 3.3 (td, J = 8.4, 3.0 Hz, 1H), 3.2 – 3.1 (m, 1H), 2.6 – 2.3 (m, 2H), 2.3 – 2.0 (m, 2H), 2.0 – 1.8 (m, 3H).

[0319] HRMS (ESI+) m / z Calculated for C29H30BrN2O4[M+H]+549.1383; Found 549.25. (2R,3R)-3-(4-bromophenyl)-1-((R)-3-((tert-butoxycarbonyl)amino)-2- hydroxypropyl)pyrrolidine-2-carboxylic acid (330-233)

[0320] To a precooled (0 °C) solution of 330-022 (49.9 mg, 163 ^mol, 1 equiv) in CH2Cl2(815 ^L) was added NaBH(OAc)3(207 mg, 978 ^mol, 6 equiv). To the 106 CORE / 3510075.0137 / 196268526.1mixtures was added a solution of crude tert-butyl (R)-(2-hydroxy-3-oxopropyl)carbamate3(154 mg, 815 ^mol, 5 equiv) in CH2Cl2 (815 ^L) and the mixture was allowed to gradually warm to rt and was stirred for 16h. The reaction was deemed incomplete by LC-MS analysis (presence of the imine). The mixture was cooled to 0 ºC, NaBH(OAc)3(100 mg, 489 ^mol, 3 equiv) was added and the mixture was allowed to gradually warm to rt and was stirred for 3h. The reaction was quenched sat. aq. NH4Cl (3 mL) and extracted with CH2Cl2(3 x 4 mL). Combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (0:100 → 100:0 EtOAc / hexane then 0:100 → 10:90 MeOH / CH2Cl2) to afford 330-233 (48.1 mg, 67%) as an orange foam.

[0321] HRMS (ESI+) m / z Calculated for C19H28BrN2O5[M+H]+443.1176; Found 443.30. (2R,3R)-3-(4-bromophenyl)-1-((S)-3-((tert-butoxycarbonyl)amino)-2- hydroxypropyl)pyrrolidine-2-carboxylic acid (330-243)

[0322] To a precooled (0 °C) solution of 330-022 (50 mg, 163 ^mol, 1 equiv) in CH2Cl2(815 ^L) was added NaBH(OAc)3(241 mg, 1.14 mmol, 7 equiv). To the mixtures was added a solution of crude tert-butyl (S)-(2-hydroxy-3-oxopropyl)carbamate3(198 mg, 1.05 mmol, 6 equiv) in CH2Cl2(815 ^L) and the mixture was allowed to gradually warm to rt and was stirred for 16h. The reaction was quenched sat. aq. NH4Cl (3 mL) and extracted with CH2Cl2 (3 x 4 mL). Combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (0:100 → 100:0 EtOAc / hexane then 0:100 → 10:90 MeOH / CH2Cl2) to afford 330-243 (40 mg, 55%) as an orange foam.

[0323] HRMS (ESI+) m / z Calculated for C19H28BrN2O5[M+H]+443.1176; Found 443.30. 107 CORE / 3510075.0137 / 196268526.1(9R,9aR)-9-(4-bromophenyl)octahydro-1H-pyrrolo[1,2-a][1,4]diazepin-1-one (330-028)

[0324] To a solution of 330-047 (1.034 g, 1.87 mmol, 1 equiv) in dimethylformamide (187 mL) was added Silia-Bond® piperazine (6.7 g, 0.84 mmol / g, 5.62 mmol, 3 equiv). The resulting suspension was heated to 50 ºC and stirred for 1.5 h. The mixture was cooled to 0 ºC, following which DIPEA (1.61 mL, 9.35 mmol, 5 equiv) and HATU (1.07 g, 2.81 mmol, 1.5 equiv) were added. The resulting yellow suspension was stirred at rt for 1.5 h then concentrated in vacuo. Silica and CH2Cl2were added and the mixture was concentrated in vacuo. The resulting yellow solid was transferred to a fritted funnel and washed with hexane. The hexane washings were discarded. The remaining solid was washed with a mixture of CH2Cl2 / MeOH (9:1). These washings were concentrated in vacuo, EtOAc was added to the residue and the mixture was washed sat. aq. NaHCO3 (with 5 × 20 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to afford 330-028 as an orange oil (507 mg, 94.5% w / w [tetramethylurea, DMF: see NMR], 83% yield).

[0325] [^]22D= 26.4 (c = 0.2, CH2Cl2)

[0326] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.36 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.9 Hz, 2H), 5.64 (s, 1H), 3.78 – 3.62 (m, 2H), 3.41 (d, J = 7.0 Hz, 1H), 3.33 (td, J = 8.8, 3.6 Hz, 1H), 3.22 – 3.10 (m, 1H), 2.87 – 2.73 (m, 1H), 2.60 – 2.43 (m, 2H), 2.39 – 2.19 (m, 1H), 2.10 – 1.94 (m, 1H), 1.92 – 1.76 (m, 2H).

[0327] HRMS (ESI+) m / z Calculated for C14H18BrN2O [M+H]+309.0597; Found 309.15. General procedure: reduction of lactam 330-028 and formation of urea derivatives.

[0328] A sealed vial containing 330-028 (1 equiv) and Ru3CO12 (0.1 equiv) was evacuated and backfilled with N2 (3x). Toluene (1.6 M) was added and the vial was vortexed for 30 s. Tetramethyldisiloxane (15 equiv) was finally added, and the reactor was sealed, heated to 90 ºC and stirred for 24 h. The initially orange mixture quickly turns dark red upon heating. Diverse ureas were synthesized from this product either by addition of a commercially available isocyanate (Procedure A) or by formation of the corresponding trifluoroacetate salt and reaction with freshly prepared isocyanates (Procedure B). 108 CORE / 3510075.0137 / 196268526.1

[0329] Procedure A: The reaction was allowed to cool to rt then was concentrated in vacuo to afford a dark red oil. THF (0.25 M) was added, to this solution was added isocyanate portion wise, between each portion the reaction was stirred 15 min at rt and presence of the starting material was monitored by LC-MS. When total conversion of the starting material was observed, the reaction was quenched with MeOH. The resulting mixture was concentrated in vacuo, reconstituted in CH2Cl2 and purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) to afford the desired product.

[0330] Procedure B: The reaction was allowed to cool to rt then was concentrated in vacuo to afford a dark red oil. The crude product was purified by reverse phase chromatography (0:100 → 20:80 CH3CN (+ 0.1% v / v TFA) / H2O (+ 0.1% v / v TFA)). To the TFA salt obtained was added THF (0.2 M) and triethylamine. To this solution was added freshly prepared isocyanate portion wise, between each portion the reaction was stirred 15 min at rt and presence of the starting material was monitored by LC-MS. When total conversion of the starting material was observed, AcOEt (1 mL) and sat. aq. NaHCO3(2 mL) were added and the mixture was extracted with AcOEt (3 x 2 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The crude mixture was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) to afford the desired product. (9R,9aR)-9-(4-bromophenyl)-N-(4-methoxyphenyl)hexahydro-1H-pyrrolo[1,2- a][1,4]diazepine-2(3H)-carboxamide (330-033)

[0331] Prepared according to General Procedure A from 330-028 (144.1 mg, 465 ^mol) using 4-Methoxyphenyl isocyanate (0.9 equiv) to afford 330-033 as a brown oil (124.2 mg, 60%).

[0332] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.39 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.2 Hz, 4H), 6.82 (d, J = 8.4 Hz, 2H), 6.06 (s, 1H), 3.77 (s, 3H), 3.70 (d, J = 14.4 Hz, 1H), 3.50 (t, J = 6.4 Hz, 2H), 3.36 (q, J = 7.6 Hz, 1H), 3.30 – 3.13 (m, 2H), 2.60 (t, J = 8.9 Hz, 1H), 2.39 (h, J = 9.9, 9.4 Hz, 2H), 2.33 – 2.22 (m, 2H), 2.03 – 1.85 (m, 3H). 109 CORE / 3510075.0137 / 196268526.1

[0333] 13C NMR (101 MHz, CDCl3, 300 K) δ 155.8, 155.6, 132.1, 131.1 (2C), 130.6 (2C), 122.2 (2C), 120.2, 114.1 (2C), 69.7, 55.5, 55.2, 53.3, 46.8, 46.2, 32.2, 27.6.

[0334] HRMS (ESI+) m / z Calculated for C22H27BrN3O2 [M+H]+444.1281; Found 444.22. (9R,9aR)-9-(4-bromophenyl)-N-phenylhexahydro-1H-pyrrolo[1,2-a][1,4]diazepine- 2(3H)-carboxamide (330-067)

[0335] Prepared according to General Procedure A from 330-028 (40.4 mg, 130 ^mol) using phenyl isocyanate (0.85 equiv) to afford 330-067 as a brown oil (31 mg, 58%).

[0336] 1H NMR (400 MHz, MeOD, 300 K) δ 7.4 (d, J = 7.3 Hz, 2H), 7.3 (t, J = 6.6 Hz, 4H), 7.2 (t, J = 7.6 Hz, 2H), 7.0 (t, J = 7.3 Hz, 1H), 3.7 – 3.5 (m, 2H), 3.5 – 3.4 (m, 2H), 3.3 – 3.2 (m, 2H), 2.7 (t, J = 9.3 Hz, 1H), 2.5 – 2.4 (m, 2H), 2.3 (dt, J = 14.4, 7.8 Hz, 2H), 2.1 – 1.9 (m, 3H).

[0337] 13C NMR (101 MHz, MeOD, 300 K) δ 158.3, 143.5, 140.8, 132.2 (2C), 131.9 (2C), 129.5 (2C), 124.2, 122.7 (2C), 121.2, 70.6, 56.4, 54.6, 49.9 (see HSQC), 47.9, 47.0, 33.0, 28.5.

[0338] HRMS (ESI+) m / z Calculated for C21H25BrN3O [M+H]+414.1176; Found 414.10. (9R,9aR)-9-(4-bromophenyl)-N-(4-fluorophenyl)hexahydro-1H-pyrrolo[1,2- a][1,4]diazepine-2(3H)-carboxamide (330-068)

[0339] Prepared according to General Procedure A from 330-028 (40 mg, 129 ^mol) using 4-Fluorophenyl isocyanate (0.7 equiv) to afford 330-068 as a brown oil (27 mg, 49%). 110 CORE / 3510075.0137 / 196268526.1

[0340] 1H NMR (400 MHz, MeOD, 300 K) δ 7.4 (d, J = 7.4 Hz, 2H), 7.4 – 7.2 (m, 4H), 7.0 (t, J = 8.5 Hz, 2H), 3.7 – 3.5 (m, 2H), 3.5 – 3.4 (m, 2H), 3.3 – 3.1 (m, 2H), 2.7 (t, J = 9.2 Hz, 1H), 2.6 – 2.4 (m, 2H), 2.3 (dt, J = 13.7, 7.5 Hz, 2H), 2.1 – 1.8 (m, 3H).

[0341] 13C NMR (101 MHz, MeOD) δ 160.4 (d, J = 240.6 Hz), 158.3, 143.5, 137.0 (d, J = 2.8 Hz), 132.2 (2C), 131.9 (2C), 124.8 (d, J = 7.8 Hz, 2C), 121.2, 115.9 (d, J = 22.7 Hz, 2C), 70.6, 56.5, 54.6, 49.9, 47.9, 47.0, 33.0, 28.5.

[0342] HRMS (ESI+) m / z Calculated for C21H24BrFN3[M+H]+432.1081; Found 432.39. (9R,9aR)-9-(4-bromophenyl)-N-(6-methoxypyridin-3-yl)hexahydro-1H-pyrrolo[1,2- a][1,4]diazepine-2(3H)-carboxamide (330-080)

[0343] Prepared according to General Procedure B from 330-028 (51.7 mg, 167 ^mol) and 330-079 (1.2 equiv) to afford 330-080 as an orange oil (14.4 mg, 32.3 ^mol, 32%).

[0344] 1H NMR (300 MHz, CDCl3, 300 K) δ 7.92 (d, J = 2.3 Hz, 1H), 7.75 (dd, J = 8.9, 2.8 Hz, 1H), 7.44 – 7.34 (m, 2H), 7.25 – 7.16 (m, 2H), 6.67 (d, J = 8.9 Hz, 1H), 6.14 (s, 1H), 3.88 (s, 3H), 3.67 (d, J = 14.1 Hz, 1H), 3.50 (t, J = 5.7 Hz, 2H), 3.39 – 3.28 (m, 1H), 3.28 – 3.13 (m, 2H), 2.57 (t, J = 8.8 Hz, 1H), 2.46 – 2.31 (m, 2H), 2.31 – 2.18 (m, 2H), 2.02 – 1.86 (m, 3H).

[0345] HRMS (ESI+) m / z Calculated for C21H26BrN4O2[M+H]+445.1234; Found 445.36. 111 CORE / 3510075.0137 / 196268526.1(9R,9aR)-9-(4-bromophenyl)-N-(5-methoxypyridin-2-yl)hexahydro-1H-pyrrolo[1,2- a][1,4]diazepine-2(3H)-carboxamide (330-088)

[0347] Prepared according to General Procedure A from 330-028 (24.2 mg, 78.2 ^mol) and freshly prepared isocyanate 330-189 (1.5 equiv) to afford 330-188 (19.2 mg, 52%) as an orange foam.

[0348] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.39 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 9.2 Hz, 4H), 6.95 (d, J = 8.6 Hz, 2H), 6.08 (s, 1H), 3.79 – 3.62 (m, 2H), 3.59 – 3.43 (m, 2H), 3.42 – 3.31 (m, 1H), 3.29 – 3.15 (m, 2H), 2.69 – 2.50 (m, 1H), 2.48 – 2.15 (m, 4H), 2.02 – 1.89 (m, 3H), 0.74 (d, J = 4.5 Hz, 4H).

[0349] HRMS (ESI+) m / z Calculated for C24H29BrN3O2[M+H]+470.1438; Found 470.21. 112 CORE / 3510075.0137 / 196268526.1(4S,9R,9aR)-9-(4-bromophenyl)-4-hydroxy-N-(4-methoxyphenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-212)

[0350] A sealed vial containing 330-028 (14.6 mg, 44.8 ^mol, 1 equiv) and Ru3CO12(2.9 mg, 4.5 ^mol, 0.1 equiv) was evacuated and backfilled with N2(3x). Toluene (22.4 ^L) was added and the vial was vortexed for 30 s. Tetramethyldisiloxane (118 ^L, 672 ^mol, 15 equiv) was finally added, and the reactor was sealed, heated to 90 ºC and stirred for 24 h. The initially orange mixture quickly turns dark red upon heating. The mixture was cooled to rt, AcOEt (0.5 mL) and HCl (4 M solution in dioxane, 44.7 ^L, 179 ^mol, 4 equiv) were added. The mixture was stirred at rt for 30 min. The precipitate obtained was triturated with AcOEt and dried in vacuo. The orange solid obtained was taken up in CH2Cl2 (1.79 mL) and the solution was cooled to 0 °C then DIPEA (23.3 ^L, 134 ^mol, 3quiv) was added. Finally, 4- methoxyphenyl isocyanate (3.5 ^L, 26.8 ^mol, 0.6 equiv) was added portion-wise, between each portion the reaction was stirred 15 min at rt and presence of the starting material was monitored by LC-MS. The reaction mixture was quenched by the addition of MeOH (1 mL) and the mixture was concentrated in vacuo. The residue was purified by column chromatography (0:100 → 100:0 EtOAc / hexane then 0:100 → 10:90 MeOH / CH2Cl2) to afford 330-212 (4.8 mg, 23%).

[0351] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.40 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 9.0 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 6.80 (d, J = 8.9 Hz, 2H), 4.26 – 4.16 (m, 1H), 3.96 – 3.80 (m, 2H), 3.76 (s, 3H), 3.37 (td, J = 8.7, 6.2 Hz, 1H), 3.31 (d, J = 4.7 Hz, 1H), 3.29 – 3.21 (m, 2H), 2.71 (ddd, J = 11.7, 8.2, 3.7 Hz, 1H), 2.54 (q, J = 9.0 Hz, 1H), 2.33 (dd, J = 13.1, 9.3 Hz, 1H), 2.30 – 2.20 (m, 1H), 2.08 (d, J = 14.6 Hz, 1H), 1.99 – 1.85 (m, 1H).

[0352] HRMS (ESI+) m / z Calculated for C22H27BrN3O3 [M+H]+460.1230; Found 460.26. 113 CORE / 3510075.0137 / 196268526.1(4R,9R,9aR)-9-(4-bromophenyl)-4-hydroxy-N-(4-methoxyphenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-215)

[0353] A sealed vial containing 330-028 (16.2 mg, 49.8 ^mol, 1 equiv) and Ru3CO12(3.2 mg, 5 ^mol, 0.1 equiv) was evacuated and backfilled with N2(3x). Toluene (24.9 ^L) was added and the vial was vortexed for 30 s. Tetramethyldisiloxane (131 ^L, 747 ^mol, 15 equiv) was finally added, and the reactor was sealed, heated to 90 ºC and stirred for 24 h. The initially orange mixture quickly turns dark red upon heating. The mixture was cooled to rt, AcOEt (0.5 mL) and HCl (4 M solution in dioxane, 49.7 ^L, 199 ^mol, 4 equiv) were added. The mixture was stirred at rt for 30 min. The precipitate obtained was triturated with AcOEt and dried in vacuo. The orange solid obtained was taken up in CH2Cl2 (2 mL) and the solution was cooled to 0 °C then DIPEA (25.8 ^L, 149 ^mol, 3 equiv) was added. Finally, 4- methoxyphenyl isocyanate (3.3 ^L, 24.9 ^mol, 0.5 equiv) was added portion-wise, between each portion the reaction was stirred 15 min at rt and presence of the starting material was monitored by LC-MS. The reaction mixture was quenched by the addition of MeOH (1 mL) and the mixture was concentrated in vacuo. The residue was purified by column chromatography (0:100 → 100:0 EtOAc / hexane then 0:100 → 10:90 MeOH / CH2Cl2) to afford 330-215 (7.3 mg, 32%).

[0354] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.42 (d, J = 8.4 Hz, 2H), 7.15 (dd, J = 16.9, 8.7 Hz, 4H), 6.80 (d, J = 8.9 Hz, 2H), 6.56 (s, 1H), 4.01 (s, 1H), 3.76 (s, 3H), 3.63 – 3.44 (m, 3H), 3.24 (ddd, J = 9.0, 6.9, 2.1 Hz, 1H), 3.19 (dd, J = 13.0, 3.8 Hz, 1H), 3.11 (dd, J = 13.4, 3.3 Hz, 1H), 2.92 – 2.82 (m, 1H), 2.75 – 2.64 (m, 2H), 2.58 (dd, J = 12.9, 1.7 Hz, 1H), 2.23 – 2.08 (m, 2H).

[0355] HRMS (ESI+) m / z Calculated for C22H27BrN3O3[M+H]+460.1230; Found 460.28. 114 CORE / 3510075.0137 / 196268526.1General Procedure C: Isocyanate formation starting from carboxylic acid derivate using DPPA (Curtius rearrangement)

[0356] To a solution of carboxylic acid (1 equiv) in toluene (0.2 M) was added DPPA (1.2 equiv) then Et3N (1.2 equiv) was added dropwise. The resulting mixture was stirred at rt for 30 min then heated to 80 ºC and stirred for 2 h. The reaction was allowed to cool to rt and the product was used as a crude solution (~ 0.2 M in toluene) without further purification. 5-isocyanato-2-methoxypyridine (330-079)

[0357] Prepared according to General Procedure C from 2-Methoxy-5- pyridinecarboxylic acid (40.9 mg, 267 ^mol). 2-isocyanato-5-methoxypyridine (330-087)

[0358] Prepared according to General Procedure C from 5-Methoxy-2- pyridinecarboxylic acid (50.8 mg, 332 ^mol). 1-cyclopropoxy-4-isocyanatobenzene (330-189)

[0359] A sealed vial containing triphosgene (19 mg, 64 ^mol, 0.55 equiv) was evacuated and backfilled with N2(3x) and toluene (468 ^L) was added. To the mixture was added a solution of 4-cyclopropoxyaniline2(17 mg, 117 ^mol, 1 equiv) in dioxane (84 ^L) 115 CORE / 3510075.0137 / 196268526.1under N2and the mixture was stirred at 110 °C for 2h. The reaction mixture was concentrated in vacuo and used crude without further purification. General Procedure D: Palladium coupling between bromo-phenyl derivate and aromatic acetynyl derivate

[0360] A sealed vial containing the aryl bromide intermediate (1 equiv) was evacuated and backfilled with N2 (× 3) then were added CH3CN previously sparged with argon for 40 min, NEt3 and alkyne, followed by XPhos-Pd-G3. The vial was sealed, heated to 70 ºC and stirred. The reaction was allowed to cool to rt then sat. aq. NaHCO3(1 mL) was added and the mixture was extracted with CH2Cl2(2 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. General Procedure E: Palladium coupling between bromo-phenyl derivate and aromatic acetynyl derivate (optimized conditon)

[0361] A sealed vial containing the aryl bromide intermediate (1 equiv) was evacuated and backfilled with N2 (× 3) then were added CH3CN (0.1 M) previously sparged with argon for 40 min, NEt3 (4 equiv) and alkyne (5 equiv), followed by XPhos-Pd-G3 (0.1 equiv). The vial was sealed, heated to 70 ºC and stirred for 1h30. The reaction was allowed to cool to rt then sat. aq. NaHCO3 (1 mL) was added and the mixture was extracted with CH2Cl2 (2 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2- a][1,4]diazepine-2(3H)-carboxamide (330-032)

[0362] Prepared according to General Procedure D from 330-033 (21.3 mg, 47.9 ^mol), using Et3N (25 ^L, 178 ^mol, 3.7 equiv), phenylacetylene (24.6 ^L, 223 ^mol, 4.6 equiv), XPhos-Pd-G3 (9.9 mg, 11.6 ^mol, 0.24 equiv) and CH3CN (230 ^L). The reaction was stirred for 4h. The crude product was purified by normal phase column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by reverse phase chromatography (0:100 → 50:50 CH3CN / H2O + 0.1% TFA) to afford 330-032 as a clear colorless oil (8.3 mg, 37%). 116 CORE / 3510075.0137 / 196268526.1

[0363] 1H NMR (400 MHz, CDCl3, 300K) δ 7.45 (d, J = 7.7 Hz, 2H), 7.38 (d, J = 7.4 Hz, 2H), 7.32 – 7.21 (m, 5H), 7.18 (d, J = 7.8 Hz, 2H), 6.74 (dd, J = 8.9, 2.0 Hz, 2H), 5.98 (s, 1H), 3.69 (s, 3H), 3.61 (d, J = 14.2 Hz, 1H), 3.44 (t, J = 6.3 Hz, 2H), 3.40 – 3.28 (m, 1H), 3.25 – 3.09 (m, 2H), 2.65 – 2.51 (m, 1H), 2.45 – 2.28 (m, 2H), 2.28 – 2.12 (m, 2H), 2.06 – 1.76 (m, 3H).

[0364] 13C NMR (101 MHz, CDCl3) δ 15f5.9, 155.7, 132.3, 131.7 (2C), 131.5(2C), 129.0 (2C), 128.5 (2C), 128.3, 123.5, 122.4 (2C), 121.4, 114.2 (2C), 89.5, 89.3, 69.8, 55.7 (2C), 55.5, 53.5, 48.5, 47.4, 46.2, 32.1, 27.8.

[0365] HRMS (ESI+) m / z Calculated for C30H32N3O2 [M+H]+466.2489; Found 466.02. (9R,9R)-N-(4-methoxyphenyl)-9-(4-(pyridin-2-ylethynyl)phenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-036)

[0366] Prepared according to General Procedure D from 330-033 (31.4 mg, 70.4 ^mol), using Et3N (39.1 ^L, 281 ^mol, 4 equiv), 2-ethynylpyridine (35.4 ^L, 353 ^mol, 5 equiv), XPhos-Pd-G3 (12.1 mg, 14.2 ^mol, 0.2 equiv) and CH3CN (352 ^L). The reaction was stirred for 22h. The crude product was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by preparative HPLC to afford 330-036 (5 mg, 15%) as a yellow oil.

[0367] 1H NMR (300 MHz, CDCl3, 300 K) δ 8.65 – 8.58 (m, 1H), 7.68 (td, J = 7.7, 1.8 Hz, 1H), 7.52 (d, J = 8.2 Hz, 3H), 7.35 (d, J = 8.3 Hz, 2H), 7.25 – 7.19 (m, 3H), 6.87 – 6.74 (m, 2H), 6.05 (s, 1H), 3.77 (s, 3H), 3.69 (d, J = 14.3 Hz, 1H), 3.51 (t, J = 6.5 Hz, 2H), 3.48 – 3.35 (m, 1H), 3.32 – 3.16 (m, 2H), 2.72 – 2.54 (m, 1H), 2.51 – 2.17 (m, 4H), 2.11 – 1.87 (m, 3H).

[0368] HRMS (ESI+) m / z Calculated for C29H31N4O2 [M+H]+467.2442; Found 477.04. 117 CORE / 3510075.0137 / 196268526.1(9R,9R)-N-(4-methoxyphenyl)-9-(4-(pyrimidin-2-ylethynyl)phenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-037)

[0369] Prepared according to General Procedure D from 330-033 (31 mg, 69.7 ^mol), using Et3N (38.7 ^L, 278 ^mol, 4 equiv), 2-ethynylpyrimidine (37 mg, 355 ^mol, 5.1 equiv), XPhos-Pd-G3 (17.8 mg, 21 ^mol, 0.3 equiv) and CH3CN (348 ^L). The reaction was stirred for 2h30. The crude product was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by preparative HPLC to afford 330-038 (4.3 mg, 13%) as a yellow oil.

[0370] 1H NMR (400 MHz, CDCl3, 300 K) δ 8.75 (d, J = 4.9 Hz, 2H), 7.59 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H), 7.25 – 7.20 (m, 3H), 6.81 (d, J = 8.5 Hz, 2H), 6.05 (s, 1H), 3.77 (s, 3H), 3.71 (d, J = 14.8 Hz, 1H), 3.56 – 3.47 (m, 2H), 3.47 – 3.37 (m, 1H), 3.30 – 3.18 (m, 2H), 2.71 – 2.58 (m, 1H), 2.50 – 2.20 (m, 4H), 2.09 – 1.88 (m, 3H).

[0371] HRMS (ESI+) m / z Calculated for C28H30N5O2 [M+H]+468.2394; Found 468.08. (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(pyridin-3-ylethynyl)phenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-039)

[0372] Prepared according to General Procedure D from 330-033 (30.2 mg, 67.7 ^mol), using Et3N (37.6 ^L, 270 ^mol, 4 equiv), 3-ethynylpyridine (37.4 mg, 362 ^mol, 5.3 equiv), XPhos-Pd-G3 (17.4 mg, 20.5 ^mol, 0.3 equiv) and CH3CN (230 ^L). The reaction was stirred for 2h30. The crude product was purified by column chromatography (0:100 → 118 CORE / 3510075.0137 / 196268526.15:95 MeOH / CH2Cl2) then by preparative HPLC to afford 330-039 as a yellow oil (10.4 mg, 33%).

[0373] 1H NMR (400 MHz, CDCl3, 300K) δ 8.76 (s, 1H), 8.54 (d, J = 4.7 Hz, 1H), 7.80 (d, J = 8.0, 2.0 Hz, 1H), 7.46 (d, J = 7.5 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 7.29 (d, J = 5.9 Hz, 1H), 7.23 (d, J = 8.1 Hz, 2H), 6.96 – 6.73 (m, 2H), 6.10 (s, 1H), 3.77 (s, 3H), 3.72 (d, J = 14.6 Hz, 1H), 3.51 (t, J = 6.4 Hz, 2H), 3.43 (q, J = 7.7 Hz, 1H), 3.31 – 3.19 (m, 2H), 2.64 (t, J = 8.9 Hz, 1H), 2.52 – 2.35 (m, 2H), 2.35 – 2.21 (m, 2H), 2.13 – 1.89 (m, 3H)

[0374] 13C NMR (101 MHz, CDCl3, 300K) δ 155.9, 155.8, 152.4, 148.6, 138.5, 132.3, 131.6 (2C), 129.1 (2C), 123.2, 122.4 (2C), 120.8, 120.6, 114.2 (2C), 92.9, 85.9, 70.0, 55.7 (2C), 55.4, 53.5, 48.3, 47.4, 46.3, 32.2, 27.8.

[0375] HRMS (ESI+) m / z Calculated for C29H31N4O2[M+H]+467.2442; Found 467.50. (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(pyridazin-3-ylethynyl)phenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-040)

[0376] Prepared according to General Procedure D from 330-033 (12.7 mg, 28.8 ^mol), using Et3N (15.9 ^L, 270 ^mol, 4 equiv), 3-ethynylpyridazine (15 mg, 144 ^mol, 5 equiv), XPhos-Pd-G3 (2.4 mg, 2.9 ^mol, 0.1 equiv) and CH3CN (143 ^L). The reaction was stirred for 6h. The crude product was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by preparative HPLC to afford 330-x040 as a yellow oil (0.7 mg, 7%).

[0377] 1H NMR (300 MHz, CDCl3, 300 K) δ 9.13 (dd, J = 5.0, 1.7 Hz, 1H), 7.64 (dd, J = 8.5, 1.7 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.47 (dd, J = 8.5, 5.0 Hz, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 9.0 Hz, 2H), 6.86 – 6.76 (m, 2H), 6.05 (s, 1H), 3.77 (s, 3H), 3.71 (d, J = 3.8 Hz, 1H), 3.52 (t, J = 6.3 Hz, 2H), 3.46 – 3.37 (m, 1H), 3.31 – 3.18 (m, 2H), 2.70 – 2.57 (m, 1H), 2.50 – 2.14 (m, 4H), 2.11 – 1.91 (m, 3H).

[0378] HRMS (ESI+) m / z Calculated for C28H30N5O2 [M+H]+468.2394; Found 468.57. 119 CORE / 3510075.0137 / 196268526.1(9R,9aR)-N-phenyl-9-(4-(phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2- a][1,4]diazepine-2(3H)-carboxamide (330-069)

[0379] Prepared according to General Procedure D from 330-067 (20.8 mg, 50.1 ^mol), using Et3N (27.8 ^L, 200 ^mol, 4 equiv), phenylacetylene (27.4 ^L, 250 ^mol, 5 equiv), XPhos-Pd-G3 (4.2 mg, 5.0 ^mol, 0.1 equiv) and CH3CN (250 ^L). The reaction was stirred for 2h. The crude product was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by preparative HPLC to afford 330-069 as a yellow oil (6.3 mg, 29%).

[0380] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.58 – 7.50 (m, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.41 – 7.30 (m, 7H), 7.30 – 7.23 (m, 2H), 7.01 (t, J = 7.3 Hz, 1H), 6.18 (s, 1H), 3.69 (d, J = 14.3 Hz, 1H), 3.60 – 3.40 (m, 3H), 3.35 – 3.17 (m, 2H), 2.68 (s, 1H), 2.56 – 2.38 (m, 2H), 2.38 – 2.25 (m, 2H), 2.15 – 1.90 (m, 3H).

[0381] HRMS (ESI+) m / z Calculated for C29H30N3O [M+H]+436.2383; Found 436.15. (9R,9aR)-N-(4-fluorophenyl)-9-(4-(phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2- a][1,4]diazepine-2(3H)-carboxamide (330-070)

[0382] Prepared according to General Procedure D from 330-068 (19.9 mg, 46.0 ^mol), using Et3N (25.6 ^L, 184 ^mol, 4 equiv), phenylacetylene (25.1 ^L, 230 ^mol, 5 equiv), XPhos-Pd-G3 (4.0 mg, 54.7 ^mol, 0.1 equiv) and CH3CN (230 ^L). The reaction was stirred for 3h. The crude product was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by preparative HPLC to afford 330-070 as a yellow oil (6.6 mg, 30%). 120 CORE / 3510075.0137 / 196268526.1

[0383] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.57 – 7.49 (m, 2H), 7.49 – 7.43 (m, 2H), 7.39 – 7.31 (m, 5H), 7.31 – 7.27 (m, 2H), 7.01 – 6.91 (m, 2H), 6.13 (s, 1H), 3.66 (d, J = 14.2 Hz, 1H), 3.60 – 3.39 (m, 3H), 3.25 (d, J = 15.2 Hz, 2H), 2.68 (s, 1H), 2.45 (s, 2H), 2.38 – 2.25 (m, 2H), 2.15 – 1.89 (m, 3H).

[0384] HRMS (ESI+) m / z Calculated for C29H29FN3O [M+H]+454.2289; Found 454.12. (9R,9aR)-N-(6-methoxypyridin-3-yl)-9-(4-(phenylethynyl)phenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-083)

[0385] Prepared according to General Procedure D from 330-080 (15.8 mg, 35.5 ^mol), using Et3N (19.8 ^L, 142 ^mol, 4 equiv), phenylacetylene (19.3 ^L, 177 ^mol, 5 equiv), XPhos-Pd-G3 (3.4 mg, 4.0 ^mol, 0.1 equiv) and CH3CN (177 ^L). The reaction was stirred for 4h. The crude product was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by preparative HPLC to afford 330-083 as a yellow oil (7.3 mg, 44%).

[0386] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.93 (d, J = 2.7 Hz, 1H), 7.77 (dd, J = 8.9, 2.8 Hz, 1H), 7.57 – 7.49 (m, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.40 – 7.30 (m, 5H), 6.69 (d, J = 8.8 Hz, 1H), 6.04 (s, 1H), 3.89 (s, 3H), 3.74 – 3.39 (m, 4H), 3.36 – 3.19 (m, 2H), 2.78 – 2.59 (m, 1H), 2.57 – 2.39 (m, 2H), 2.39 – 2.25 (m, 2H), 2.14 – 1.89 (m, 3H).

[0387] HRMS (ESI+) m / z Calculated for C29H31N4O2 [M+H]+467.2442; Found 467.33. 121 CORE / 3510075.0137 / 196268526.1(9R,9aR)-N-(5-methoxypyridin-2-yl)-9-(4-(phenylethynyl)phenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-095)

[0388] Prepared according to General Procedure E from 330-088 (6.6 mg, 14.8 ^mol) using phenylacetylene (6.3 ^L, 57 ^mol). The crude product was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by reverse phase chromatography (0:100 → 50:50 CH3CN (+ 0.1% v / v TFA) / H2O (+ 0.1% v / v TFA)) to afford 330-095 as a colorless oil (2.2 mg, 32%).

[0389] 1H NMR (300 MHz, CDCl3, 300 K) δ 7.96 (d, J = 9.1 Hz, 1H), 7.87 (d, J = 3.0 Hz, 1H), 7.57 – 7.49 (m, 2H), 7.49 – 7.42 (m, 2H), 7.39 – 7.30 (m, 5H), 7.23 (d, J = 3.1 Hz, 1H), 6.92 (d, J = 3.6 Hz, 1H), 3.85 – 3.67 (m, 4H), 3.66 – 3.53 (m, 1H), 3.53 – 3.36 (m, 2H), 3.32 – 3.14 (m, 2H), 2.61 (t, J = 8.8 Hz, 1H), 2.49 – 2.17 (m, 4H), 2.10 – 1.89 (m, 3H).

[0390] HRMS (ESI+) m / z Calculated for C29H31N4O2 [M+H]+467.2442; Found 467.37. (9R,9aR)-N-(4-cyclopropoxyphenyl)-9-(4-(phenylethynyl)phenyl)hexahydro-1H- pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-191)

[0391] Prepared according to General Procedure E from 330-188 (19.2 mg, 40.8 ^mol) using phenylacetylene (22.2 ^L, 203 ^mol). The crude product was purified by reverse phase chromatography (0:100 → 50:50 CH3CN (+ 0.1% v / v TFA) / H2O (+ 0.1% v / v TFA)) to afford 330-191 as am orange oil (10.8 mg, 54%). 122 CORE / 3510075.0137 / 196268526.1

[0392] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.56 – 7.49 (m, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.39 – 7.29 (m, 5H), 7.25 – 7.19 (m, 2H), 6.99 – 6.91 (m, 2H), 6.06 (s, 1H), 3.75 – 3.61 (m, 2H), 3.59 – 3.48 (m, 2H), 3.47 – 3.36 (m, 1H), 3.33 – 3.15 (m, 2H), 2.65 (s, 1H), 2.53 – 2.36 (m, 2H), 2.36 – 2.21 (m, 2H), 2.11 – 1.89 (m, 3H), 0.74 (d, J = 4.5 Hz, 4H).

[0393] HRMS (ESI+) m / z Calculated for C32H34N3O2 [M+H]+492.2646; Found 492.80. (4S,9R,9aR)-4-hydroxy-N-(4-methoxyphenyl)-9-(4-(phenylethynyl)phenyl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-246)

[0394] Prepared according to General Procedure E from 330-212 (70.7 mg, 153 ^mol) using phenylacetylene (89.9 ^L, 765 ^mol). The crude product was purified by reverse phase chromatography (0:100 → 50:50 CH3CN (+ 0.1% v / v TFA) / H2O (+ 0.1% v / v TFA)) to afford 330-246 as an orange oil (28.3 mg, 38%).

[0395] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.56 – 7.50 (m, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.39 – 7.31 (m, 3H), 7.23 (dd, J = 11.3, 8.4 Hz, 4H), 6.85 – 6.78 (m, 2H), 4.16 (d, J = 10.8 Hz, 1H), 3.93 – 3.79 (m, 1H), 3.76 (s, 3H), 3.41 (d, J = 8.4 Hz, 1H), 3.34 – 3.20 (m, 4H), 2.72 (d, J = 9.3 Hz, 1H), 2.55 (q, J = 9.0 Hz, 1H), 2.38 – 2.22 (m, 2H), 2.10 (t, J = 12.8 Hz, 1H), 2.00 (s, 1H).

[0396] HRMS (ESI+) m / z Calculated for C30H32N3O3[M+H]+482.2438; Found 482.39. 123 CORE / 3510075.0137 / 196268526.1(4R,9R,9aR)-4-hydroxy-N-(4-methoxyphenyl)-9-(4-(phenylethynyl)phenyl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide (330-216)

[0397] Prepared according to General Procedure E from 330-215 (7.3 mg, 15.8 ^mol) using phenylacetylene (8.7 ^L, 79 ^mol). The crude product was purified by reverse phase chromatography (0:100 → 50:50 CH3CN (+ 0.1% v / v TFA) / H2O (+ 0.1% v / v TFA)) to afford 330-216 as an orange oil (2.4 mg, 32%).

[0398] 1H NMR (400 MHz, CDCl3, 300 K) δ 7.57 – 7.50 (m, 2H), 7.48 (d, J = 8.1 Hz, 2H), 7.38 – 7.32 (m, 3H), 7.29 – 7.23 (m, 2H), 7.18 (d, J = 9.0 Hz, 2H), 6.81 (d, J = 9.0 Hz, 2H), 6.48 (s, 1H), 4.03 (d, J = 4.0 Hz, 1H), 3.76 (s, 3H), 3.62 (dd, J = 14.8, 3.7 Hz, 1H), 3.57 (d, J = 8.3 Hz, 1H), 3.51 (dd, J = 14.8, 3.3 Hz, 1H), 3.32 – 3.26 (m, 1H), 3.22 (dd, J = 13.0, 3.8 Hz, 1H), 3.14 (dd, J = 13.3, 3.2 Hz, 1H), 2.92 (t, J = 9.8 Hz, 1H), 2.79 – 2.67 (m, 2H), 2.60 (d, J = 13.0 Hz, 1H), 2.26 – 2.18 (m, 2H).

[0399] HRMS (ESI+) m / z Calculated for C30H32N3O3[M+H]+482.2438; Found 482.83. (9R,9aR)-9-([1,1'-biphenyl]-4-yl)-N-(4-methoxyphenyl)hexahydro-1H-pyrrolo[1,2- a][1,4]diazepine-2(3H)-carboxamide (330-117)

[0400] A sealed vial containing 330-033 (18 mg, 40.5 ^mol, 1 equiv) and phenylboronic acid (7.4 mg, 60.7 ^mol, 1.5 equiv) was evacuated and backfilled with N2 (× 3) then were added THF (540 ^L) previously sparged with argon for 1h and a mixture of aq. K3PO4 / THF (1:2, 270 ^L), followed by XPhos-Pd-G3 (5.1 mg, 6.07 ^mol, 0.15 equiv). The 124 CORE / 3510075.0137 / 196268526.1vial was sealed, heated to 50 ºC and stirred for 3h. The reaction was allowed to cool to rt then pH = 7 buffer (10 mL) was added and the mixture was extracted with AcOEt (3 x 7 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (0:100 → 5:95 MeOH / CH2Cl2) then by reverse phase chromatography (0:100 → 50:50 CH3CN (+ 0.1% v / v TFA) / H2O (+ 0.1% v / v TFA)) to afford 330-117 as a colorless oil (5.5 mg, 31%).

[0401] 1H NMR (300 MHz, CDCl3, 300 K) δ 7.62 – 7.55 (m, 2H), 7.55 – 7.49 (m, 2H), 7.47 – 7.37 (m, 4H), 7.37 – 7.29 (m, 1H), 7.25 – 7.18 (m, 2H), 6.85 – 6.77 (m, 2H), 6.04 (s, 1H), 3.76 (s, 3H), 3.68 (d, J = 13.9 Hz, 1H), 3.61 – 3.42 (m, 3H), 3.34 – 3.20 (m, 2H), 2.68 (t, J = 9.1 Hz, 1H), 2.57 – 2.39 (m, 2H), 2.37 – 2.22 (m, 2H), 2.18 – 1.87 (m, 3H).

[0402] HRMS (ESI+) m / z Calculated for C28H32N3O2[M+H]+442.2489; Found 442.40. References cited:1Kong Thoo Lin, P.; Kuksa, V. A.; Maguire, N. M. Synthesis 1998, 6, 859-866.2WO 2017 / 153952, p.1573Tetrahedron Letters, 2015 (56), 6659–6663. EXAMPLE 4A: Synthesis of Compounds disclosed in Example 2

[0403] The following compounds can be made similarly to the compounds prepared in Examples 3 and 4.

[0404] Compound 55: (9R,9aR)-9-([1,1'-biphenyl]-4-yl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) δ 7.67 (d, J = 7.9 Hz, 2H), 7.60 (d, J =8.3 Hz, 2H), 7.50 – 7.42 (m, 5H), 7.39 (d, J =9.3 Hz, 2H), 7.36 (d, J =7.3 Hz, 1H), 6.81 – 6.77 (m, 2H), 3.83 (d, J =14.9 Hz, 1H), 3.74 (s, 4H), 3.48 – 3.35 (m, 2H), 3.25 (t, J =7.9 Hz, 1H), 3.18 (dd, J =12.8, 4.5 Hz, 1H), 2.62 – 2.56 (m, 1H), 2.44 – 2.37 (m, 2H), 2.35 – 2.20 (m, 2H), 2.01 – 1.90 (m, 3H). LC-MS m / z calcd. C28H32N3O2+[M+H]+= 442.2495, found = 422.3695.

[0405] Compound 56: (9R,9aR)-9-([1,1'-biphenyl]-4-yl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) δ 7.67 (d, J =7.9 Hz, 2H), 7.60 (d, J =8.3 Hz, 2H), 7.50 – 7.42 (m, 5H), 7.39 (d, J =9.3 Hz, 2H), 7.36 (d, J =7.3 Hz, 1H), 6.81 – 6.77 (m, 2H), 3.83 (d, J =14.9 Hz, 1H), 3.74 (s, 4H), 3.48 – 3.35 (m, 2H), 3.25 (t, J =7.9 Hz, 1H), 3.18 (dd, J =12.8, 4.5 Hz, 1H), 2.62 – 2.56 (m, 1H), 2.44 – 2.37 (m, 2H), 2.35 – 2.20 (m, 2H), 2.01 – 1.90 (m, 3H). LC-MS m / z calcd. C28H32N3O2+[M+H]+= 442.2495, found = 422.3695. 125 CORE / 3510075.0137 / 196268526.1

[0406] Compound 57: (9R,9aR)-9-(4-phenethylphenyl)-N-(p-tolyl)hexahydro- 1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) δ 7.45 (s, 1H), 7.38 (d, J = 8.5 Hz, 2H), 7.31 – 7.20 (m, 6H), 7.16 (dd, J = 11.2, 7.5 Hz, 3H), 7.01 (d, J = 8.2 Hz, 2H), 3.76 (d, J = 14.8 Hz, 2H), 3.42 – 3.33 (m, 2H), 3.26 – 3.13 (m, 2H), 2.54 (d, J = 5.8 Hz, 2H), 2.42 – 2.28 (m, 3H), 2.23 (d, J = 8.5 Hz, 5H), 2.01 – 1.86 (m, 5H). LC-MS m / z calcd. C30H36N3O+ [M+H]+= 454.42858, found = 454.4629.

[0407] Compound 58: (9R,9aR)-9-(4-(phenylethynyl)phenyl)octahydro-1H- pyrrolo[1,2-a][1,4]diazepin-1-on:1H NMR (400 MHz, Acetone) δ 7.58 – 7.49 (m, 2H), 7.47 – 7.34 (m, 7H), 6.53 (s, 1H), 3.70 – 3.60 (m, 2H), 3.38 (d, J = 7.0 Hz, 1H), 3.33 (td, J = 8.9, 3.9 Hz, 1H), 3.24 – 3.16 (m, 1H), 2.96 – 2.89 (m, 1H), 2.58 – 2.48 (m, 2H), 2.34 – 2.24 (m, 1H), 2.03 – 1.95 (m, 1H), 1.88 – 1.73 (m, 2H). LC-MS m / z calcd.C22H23N2O+[M+H]+= 331.1810, found = 331.2686.

[0408] Compound 59: (9R,9aS)-9-(4-(phenylethynyl)phenyl)octahydro-1H- pyrrolo[1,2-a][1,4]diazepin-1-one:1H NMR (400 MHz, Acetone) δ 7.57 – 7.52 (m, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.45 – 7.38 (m, 3H), 7.35 (d, J = 8.4 Hz, 2H), 6.88 (s, 1H), 4.26 – 4.16 (m, 1H), 3.37 – 3.20 (m, 5H), 2.83 – 2.76 (m, 1H), 2.58 (dd, J = 11.6, 3.9 Hz, 1H), 2.30 – 2.20 (m, 1H), 1.93 – 1.86 (m, 1H), 1.81 – 1.66 (m, 2H). LC-MS m / z calcd. C22H23N2O+[M+H]+= 331.1810, found = 331.2686.

[0409] Compound 60: (9R,9aS)-N-(4-methoxyphenyl)-9-(4- (phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, CD3CN) 67.59 – 7.54 (m, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.46 – 7.39 (m, 3H), 7.37 (d, J = 7.5 Hz, 2H), 7.22 (d, J = 9.0 Hz, 2H), 6.82 (d, J = 9.5 Hz, 2H), 6.71 (s, 1H), 3.96 (d, J = 13.9 Hz, 1H), 3.73 (s, 3H), 3.58 (t, J = 6.6 Hz, 2H), 3.20 – 3.10 (m, 2H), 3.04 – 2.94 (m, 2H), 2.74 – 2.65 (m, 1H), 2.49 (t, J = 9.9 Hz, 1H), 2.40 – 2.30 (m, 2H), 1.91 – 1.71 (m, 2H). LC-MS m / z calcd. C30H32N3O2+[M+H]+= 466.2495, found = 466.3749.

[0410] Compound 28: (9R,9aR)-N-phenyl-9-(4-(pyridin-4- ylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 68.62 (d, J = 6.8 Hz, 2H), 7.59 (s, 1H), 7.55 – 7.46 (m, 8H), 7.21 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 7.5 Hz, 1H), 3.82 (d, J = 14.5 Hz, 1H), 3.75 (dd, J = 9.5, 3.9 Hz, 1H), 3.49 – 3.38 (m, 2H), 3.26 (t, J = 8.9 Hz, 1H), 3.19 (dt, J = 12.6, 4.3 Hz, 1H), 2.60 (t, J = 8.3 Hz, 1H), 2.43 – 2.31 (m, 3H), 2.26 – 2.20 (m, 1H), 1.95 (ddd, J = 13.1, 7.7, 4.7 Hz, 3H). LC-MS m / z calcd. C28H29N4O2+[M+H]+=437.2341, found = 437.3722.

[0411] Compound 29: (9R,9aR)-N-phenyl-9-(4-(pyridin-3- ylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR 126 CORE / 3510075.0137 / 196268526.1(400 MHz, CDCl3) 68.77 (s, 1H), 8.55 (dd, J = 4.9, 1.7 Hz, 1H), 7.82 (dt, J = 7.9, 1.9 Hz, 1H), 7.53 – 7.47 (m, 2H), 7.47 – 7.39 (m, 2H), 7.39 – 7.33 (m, 2H), 7.32 – 7.28 (m, 3H), 7.04 (t, J = 8.5 Hz, 1H), 6.34 (s, 1H), 3.75 (d, J = 13.9 Hz, 1H), 3.69 – 3.48 (m, 3H), 3.43 – 3.24 (m, 2H), 2.70 – 2.48 (m, 2H), 2.47 – 2.28 (m, 2H), 2.28 – 2.10 (m, 2H), 2.13 – 1.95 (m, 2H). LC- MS m / z calcd. C28H29N4O2+[M+H]+=437.2341, found = 437.3730.

[0412] Compound 30: (9R,9aR)-N-phenyl-9-(4-(pyrimidin-5- ylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 69.13 (s, 1H), 8.93 (s, 2H), 7.60 (s, 1H), 7.56 – 7.48 (m, 6H), 7.21 (t, J = 8.2 Hz, 2H), 6.93 (t, J = 7.4 Hz, 1H), 3.82 (d, J = 12.9 Hz, 1H), 3.79 – 3.72 (m, 1H), 3.49 – 3.39 (m, 2H), 3.26 (d, J = 8.7 Hz, 1H), 3.22 – 3.17 (m, 1H), 2.60 (t, 1H), 2.42 – 2.32 (m, 3H), 2.25 – 2.20 (m, 1H), 1.99 – 1.93 (m, 3H). LC-MS m / z calcd. C27H28N5O+[M+H]+=468.2400, found = 438.4229.

[0413] Compound 31: (9R,9aR)-N-phenyl-9-(4-(pyridazin-3- ylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 69.19 (dd, J = 5.0, 1.7 Hz, 1H), 7.85 (dd, J = 8.5, 1.7 Hz, 1H), 7.72 (dd, J = 8.5, 5.0 Hz, 1H), 7.61 (d, J = 8.4 Hz, 3H), 7.52 (d, J = 8.1 Hz, 3H), 7.20 (t, J = 8.4 Hz, 2H), 6.93 (t, J = 7.5 Hz, 1H), 3.84 (d, J = 14.3 Hz, 1H), 3.75 (dd, J = 13.5, 6.8 Hz, 1H), 3.52 – 3.45 (m, 1H), 3.45 – 3.37 (m, 1H), 3.26 (t, J = 8.4 Hz, 1H), 3.19 (td, J = 13.0, 3.6 Hz, 1H), 2.60 (t, J = 8.0 Hz, 1H), 2.44 – 2.32 (m, 3H), 2.28 – 2.20 (m, 1H), 2.01 – 1.91 (m, 3H). LC-MS m / z calcd. C27H28N5O+[M+H]+=468.2400, found = 438.3992.

[0414] Compound 64: (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(pyridin-2- ylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, DMSO) 68.62 (d, J = 4.9 Hz, 1H), 8.00 (s, 1H), 7.86 (t, J = 7.7 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.43 (d, J = 7.6 Hz, 3H), 7.31 (d, J = 8.3 Hz, 2H), 6.80 (d, J = 8.3 Hz, 2H), 3.70 (d, J = 1.5 Hz, 5H), 3.24 – 3.07 (m, 4H), 2.39 (s, 4H), 2.31 – 2.09 (m, 2H), 1.87 (s, 3H). LC-MS m / z calcd. C29H30N4O2+[M+H]+= 467.2369, found = 467.4611.

[0415] Compound 32: (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(pyridin-4- ylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, CDCl3) 68.62 (d, J = 5.2 Hz, 2H), 7.50 (d, J = 8.2 Hz, 2H), 7.46 – 7.32 (m, 4H), 7.26 (d, J = 8.1 Hz, 2H), 6.84 (d, 2H), 6.20 (s, 1H), 3.79 (s, 4H), 3.55 (t, J = 6.3 Hz, 2H), 3.52 – 3.43 (m, 1H), 3.37 – 3.20 (m, 2H), 2.81 – 2.63 (m, 1H), 2.59 – 2.40 (m, 2H), 2.40 – 2.25 (m, 2H), 2.13 – 1.95 (m, 3H). LC-MS m / z calcd. C29H30N4O2+[M+H]+= 467.2447, found = 467.4249. 127 CORE / 3510075.0137 / 196268526.1

[0416] Compound 33: (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(pyrazin-2- ylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, MeOD) 68.81 (s, 1H), 8.62 (dd, J = 2.6, 1.6 Hz, 1H), 8.56 (d, J = 2.6 Hz, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 9.1 Hz, 2H), 3.76 (s, 3H), 3.71 – 3.60 (m, 2H), 3.58 – 3.53 (m, 1H), 3.44 (dt, J = 13.6, 5.6 Hz, 1H), 3.29 – 3.21 (m, 2H), 2.70 (t, J = 9.8 Hz, 1H), 2.53 – 2.45 (m, 2H), 2.35 – 2.26 (m, 2H), 2.08 – 1.97 (m, 3H). LC-MS m / z calcd. C28H29N5O2+[M+H]+= 468.2400, found = 468.4341.

[0417] Compound 34: (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(pyridazin-3- ylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 69.19 (d, J = 5.0 Hz, 1H), 7.85 (dd, J = 8.5, 1.7 Hz, 1H), 7.72 (dd, J = 8.5, 5.0 Hz, 1H), 7.61 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.45 (s, 1H), 7.39 (d, J = 9.1 Hz, 2H), 6.79 (d, J = 9.1 Hz, 2H), 3.83 (d, J = 13.1 Hz, 1H), 3.74 (s, 3H), 3.48 (d, J = 5.5 Hz, 1H), 3.39 (d, J = 13.2 Hz, 1H), 3.29 – 3.17 (m, 2H), 2.63 – 2.59 (m, 1H), 2.45 – 2.33 (m, 3H), 2.24 – 2.20 (m, 1H), 2.00 – 1.88 (m, 4H). LC-MS m / z calcd. C28H29N5O2+[M+H]+= 468.2400, found = 468.4335.

[0418] Compound 35: (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(3-methylbut-1- yn-1-yl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.43 (s, 1H), 7.38 (d, J = 9.1 Hz, 2H), 7.36 – 7.27 (m, 4H), 6.79 (d, J = 8.7 Hz, 2H), 3.77 (d, J = 12.9 Hz, 1H), 3.74 (s, 3H), 3.42 – 3.32 (m, 2H), 3.22 (d, J = 8.9 Hz, 1H), 3.19 – 3.14 (m, 1H), 2.81 – 2.75 (m, 1H), 2.56 (d, J = 8.9 Hz, 1H), 2.42 – 2.31 (m, 2H), 2.32 – 2.15 (m, 3H), 1.98 – 1.87 (m, 3H), 1.24 (d, J = 6.8 Hz, 6H). LC-MS m / z calcd. C27H34N3O2+[M+H]+= 432.2651, found = 432.4846.

[0419] Compound 36: (9R,9aR)-9-(4-(cyclopropylethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.45 (s, 1H), 7.38 (d, J = 9.3 Hz, 2H), 7.35 – 7.24 (m, 4H), 6.79 (d, J = 8.8 Hz, 2H), 3.79 – 3.68 (m,5H), 3.40-3.33 (m, 2H, 3.21 (t, J = 8.5 Hz, 1H), 3.16 (dt, J = 12.6, 4.3 Hz, 1H), 2.55 (t, J = 8.7 Hz, 1H), 2.42 – 2.3 (m, 2H, 2.28 – 2.15 (m, 2H), 1.97-1.86 (m, 3H), 1.54 – 1.44 (m, 1H), 0.92 – 0.86 (m, 2H), 0.76 – 0.65 (m, 2H). LC-MS m / z calcd. C27H32N3O2+[M+H]+= 430.2495, found = 430.4569.

[0420] Compound 37: (9R,9aR)-9-(4-(cyclohexylethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.45 (s, 1H), 7.38 (d, J = 9.1 Hz, 2H), 7.35 – 7.29 (m, 4H), 6.79 (d, J = 9.1 Hz, 2H), 3.80 – 3.69 (m, 5H), 3.41 – 3.34 (m, 2H), 3.22 (d, J = 8.0 Hz, 1H), 3.15 (dt, J = 128 CORE / 3510075.0137 / 196268526.112.6, 3.8 Hz, 1H), 2.66 – 2.59 (m, 1H), 2.55 (t, J = 8.2 Hz, 1H), 2.40 – 2.34 (m, 1H), 2.31 – 2.26 (m, 1H), 2.24 – 2.17 (m, 1H), 1.97 – 1.84 (m, 5H), 1.79 – 1.72 (m, 2H), 1.58 – 1.47 (m, 3H), 1.43 – 1.35 (m, 3H). LC-MS m / z calcd. C30H38N3O2+[M+H]+= 472.2964, found = 472.5300.

[0421] Compound 38: (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(m- tolylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.49 – 7.42 (m, 5H), 7.40 – 7.33 (m, 4H), 7.30 (d, J = 6.9 Hz, 1H), 7.22 (d, J = 7.4 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 3.81 (d, 1H), 3.74 (s, 4H), 3.47 – 3.34 (m, 2H), 3.25 (t, J = 8.6 Hz, 1H), 3.18 (dt, J = 12.6, 4.2 Hz, 1H), 2.58 (t, J = 8.8 Hz, 1H), 2.44 – 2.38 (m, 1H), 2.35 (s, 4H), 2.34 – 2.28 (m, 1H), 2.28 – 2.18 (m, 1H), 1.98 – 1.89 (m, 3H). LC-MS m / z calcd. C31H34N3O2+[M+H]+= 480.2651, found = 480.4952.

[0422] Compound 39: (9R,9aR)-N-(4-methoxyphenyl)-9-(4-(3-phenylprop-1- yn-1-yl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.45 (d, J = 7.8 Hz, 3H), 7.43 – 7.31 (m, 8H), 7.26 (t, J = 6.8 Hz, 1H), 6.79 (d, J = 9.3 Hz, 2H), 3.88 (s, 2H), 3.81 – 3.70 (m, 5H), 3.41 (s, 2H), 3.29 – 3.15 (m, 2H), 2.68 – 2.53 (m, 1H), 2.48 – 2.21 (m, 4H), 2.02 – 1.88 (m, 3H). LC-MS m / z calcd. C31H34N3O2+[M+H]+= 480.2651, found = 480.4558.

[0423] Compound 40: (9R,9aR)-N-phenyl-9-(4- (phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.63 – 7.53 (m, 3H), 7.53 – 7.38 (m, 8H), 7.21 (t, J = 7.5 Hz, 2H), 6.92 (tt, J = 7.5, 1.1 Hz, 1H), 3.82 (d, J = 14.5 Hz, 1H), 3.79 – 3.70 (m, 1H), 3.49 – 3.38 (m, 2H), 3.31 – 3.14 (m, 2H), 2.64 – 2.57 (m, 1H), 2.47 – 2.28 (m, 3H), 2.27 – 2.20 (m, 1H), 2.02 – 1.84 (m, 3H). LC-MS m / z calcd. C29H30N3O+[M+H]+= 436.2389, found = 436.4008.

[0424] Compound 41: (9R,9aR)-9-(4-(pyridin-3-ylethynyl)phenyl)-N-(p- tolyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)- carboxamide:1H NMR (400 MHz, Acetone) 68.74 (s, 1H), 8.57 (dd, J = 4.9, 1.7 Hz, 1H), 7.92 (dt, J = 7.9, 1.7 Hz, 1H), 7.55 – 7.40 (m, 6H), 7.38 (d, J = 9.0 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 3.82 (d, J = 14.3 Hz, 1H), 3.73 (dd, J = 13.9, 6.0 Hz, 1H), 3.48 – 3.36 (m, 2H), 3.25 (t, J = 8.8 Hz, 1H), 3.18 (dt, J = 13.1, 4.0 Hz, 1H), 2.62 – 2.57 (m, 1H), 2.43 – 2.31 (m, 3H), 2.26 – 2.20 (m, 4H), 1.99 – 1.90 (m, 3H). LC-MS m / z calcd. C29H31N4O+[M+H]+= 451.2498, found = 451.3491.

[0425] Compound 42: (9R,9aR)-N-(2,4-difluorophenyl)-9-(4- (phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.81 – 7.71 (m, 1H), 7.58 – 7.52 (m, 2H), 7.49 (d, J = 8.4 Hz, 129 CORE / 3510075.0137 / 196268526.12H), 7.46 – 7.37 (m, 5H), 7.23 (s, 1H), 7.01 (t, J = 10.8 Hz, 1H), 6.91 (t, J = 9.0 Hz, 1H), 3.82 – 3.68 (m, 2H), 3.51 – 3.40 (m, 2H), 3.30 – 3.13 (m, 2H), 2.62 (t, J = 8.9 Hz, 1H), 2.49 – 2.37 (m, 2H), 2.35 – 2.21 (m, 2H), 2.01 – 1.91 (m, 3H). LC-MS m / z calcd. C29H28F2N3O+[M+H]+= 472.2200, found = 472.3851.

[0426] Compound 43: (9R,9aR)-N-benzyl-9-(4- (phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1HNMR (400 MHz, Acetone) 67.58 – 7.53 (m, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.44 – 7.37 (m, 5H), 7.30 – 7.24 (m, 4H), 7.22 – 7.16 (m, 1H), 4.34 (d, J = 5.9 Hz, 2H), 3.75 (d, J = 14.3 Hz, 1H), 3.71 – 3.59 (m, 1H), 3.44 – 3.35 (m, 1H), 3.32 – 3.20 (m, 2H), 3.15 (dt, J = 12.8, 4.0 Hz, 1H), 2.52 (t, J = 8.7 Hz, 1H), 2.41 – 2.25 (m, 3H), 2.19 (t, J = 11.3 Hz, 1H), 2.10 (s, 2H), 1.97 – 1.84 (m, 2H). LC-MS m / z calcd. C30H32N3O+[M+H]+= 450.2545, found = 450.4077.

[0427] Compound 44: (9R,9aR)-N-cyclohexyl-9-(4- (phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.56 – 7.53 (m, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.44 – 7.39 (m, 5H), 5.16 (d, J = 7.8 Hz, 1H), 3.74 (d, J = 14.6 Hz, 1H), 3.58 – 3.51 (m, 2H), 3.42 – 3.36 (m, 1H), 3.25 – 3.18 (m, 2H), 3.13 (dt, J = 12.4, 4.1 Hz, 1H), 2.49 (t, J = 8.4 Hz, 1H), 2.39 – 2.34 (m, 1H), 2.24 – 2.13 (m, 2H), 1.93 – 1.82 (m, 5H), 1.71 – 1.66 (m, 2H), 1.61 – 1.55 (m, 1H), 1.32 – 1.26 (m, 3H), 1.18 – 1.10 (m, 3H). LC-MS m / z calcd. C29H36N3O+[M+H]+= 442.2858, found = 442.4058.

[0428] Compound 45: (9R,9aR)-N-cyclopropyl-9-(4- (phenylethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.58 – 7.51 (m, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.44 – 7.37 (m, 5H), 5.69 (s, 1H), 3.70 (d, J = 14.3 Hz, 1H), 3.57 – 3.47 (m, 1H), 3.43 – 3.35 (m, 1H), 3.24 – 3.10 (m, 3H), 2.60 – 2.52 (m, 1H), 2.48 (t, J = 9.8 Hz, 1H), 2.40 – 2.13 (m, 4H), 1.95 – 1.81 (m, 3H), 0.59 – 0.49 (m, 2H), 0.39 – 0.32 (m, 2H). LC-MS m / z calcd. C26H30N3O+[M+H]+= 400.2389, found = 400.4387.

[0429] Compound 46: (9R,9aR)-9-(4-(cyclohexylethynyl)phenyl)-N-(p- tolyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.48 (s, 1H), 7.41 – 7.26 (m, 6H), 7.01 (d, J = 8.3 Hz, 2H), 3.81 – 3.68 (m, 2H), 3.44 – 3.34 (m, 2H), 3.25 – 3.13 (m, 2H), 2.64 – 2.53 (m, 2H), 2.42 – 2.27 (m, 3H), 2.25 – 2.18 (m, 4H), 1.97 – 1.85 (m, 5H), 1.79 – 1.72 (m, 2H), 1.59 – 1.48 (m, 3H), 1.42 – 1.34 (m, 3H). LC-MS m / z calcd. C30H38N3O+[M+H]+= 456.3015, found = 456.8526. 130 CORE / 3510075.0137 / 196268526.1

[0430] Compound 47: (9R,9aR)-9-(4-(cyclopentylethynyl)phenyl)-N-(4- methoxyphenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.54 (s, 1H), 7.38 – 7.32 (m, 4H), 7.28 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 3.78 – 3.67 (m, 5H), 3.45 – 3.33 (m, 3H), 2.89 – 2.78 (m, 1H), 2.61 – 2.52 (m, 1H), 2.40 – 2.15 (m, 4H), 2.03 – 1.86 (m, 6Y415H), 1.81 – 1.71 (m, 2H), 1.71 – 1.56 (m, 4H). LC-MS m / z calcd. C29H36N3O2+[M+H]+= 458.2808, found = 458.4819.

[0431] Compound 48: (9R,9aR)-9-(4-(cyclopent-1-en-1-ylethynyl)phenyl)-N- (p-tolyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.49 (s, 1H), 7.42 – 7.31 (m, 6H), 7.01 (d, J =8.1 Hz, 2H), 6.10 (ddd, J =4.7, 2.7, 1.9 Hz, 1H), 3.79 (d, J =15.3 Hz, 1H), 3.76 – 3.66 (m, 1H), 3.44 – 3.35 (m, 2H), 3.23 (td, J =8.5, 1.9 Hz, 1H), 3.17 (dt, J =12.6, 4.2 Hz, 1H), 2.60 – 2.54 (m, 1H), 2.54 – 2.45 (m, 4H), 2.43 – 2.33 (m, 2H), 2.33 – 2.29 (m, 1H), 2.24 (s, 3H), 2.23 – 2.17 (m, 1H), 1.99 – 1.89 (m, 5H). LC-MS m / z calcd. C29H34N3O2+[M+H]+= 440.2702, found = 440.4143.

[0432] Compound 49: (9R,9aR)-N-(4-methoxyphenyl)-9-(4-((tetrahydro-2H- pyran-4-yl)ethynyl)phenyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.48 (s, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.35 – 7.31 (m, 3H), 6.79 (d, J = 9.2 Hz, 2H), 3.91 – 3.80 (m, 2H), 3.80 – 3.67 (m, 5H), 3.49 (t, J = 10.7 Hz, 1H), 3.45 – 3.30 (m, 3H), 3.24 – 3.14 (m, 2H), 2.90 – 2.81 (m, 1H), 2.58 – 2.51 (m, 1H), 2.41 – 2.27 (m, 3H), 2.24 – 2.16 (m, 1H), 2.03 – 1.79 (m, 5H), 1.72 – 1.54 (m, 2H). LC-MS m / z calcd. C29H36N3O3+[M+H]+= 474.2757, found = 474.4851.

[0433] Compound 50: (9R,9aR)-9-(4-((1-hydroxycyclohexyl)ethynyl)phenyl)- N-(p-tolyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.49 (s, 1H), 7.44 – 7.30 (m, 6H), 7.03 (d, J = 8.2 Hz, 2H), 4.42 (s, 1H), 3.83 – 3.70 (m, 2H), 3.47 – 3.37 (m, 2H), 3.27 – 3.16 (m, 2H), 2.58 (t, J = 8.7 Hz, 1H), 2.45 – 2.31 (m, 3H), 2.27 – 2.22 (m, 4H), 1.99 – 1.89 (m, 5H), 1.77 – 1.53 (m, 7H), 1.37 – 1.28 (m, 1H). LC-MS m / z calcd. C30H38N3O2+[M+H]+= 472.2964, found = 472.4214.

[0434] Compound 51: (9R,9aR)-9-(4-((4,4- difluorocyclohexyl)ethynyl)phenyl)-N-(p-tolyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine- 2(3H)-carboxamide:1H NMR (400 MHz, Acetone) 67.48 (s, 1H), 7.39 – 7.32 (m, 6H), 7.01 (d, J = 8.6 Hz, 2H), 3.78 (d, J = 13.1 Hz, 1H), 3.76 – 3.68 (m, 1H), 3.43 – 3.34 (m, 2H), 3.22 (td, J = 8.6, 2.0 Hz, 1H), 3.18 – 3.13 (m, 1H), 2.54 (t, J =9.2 Hz, 1H), 2.42 – 2.33 (m, 2H), 2.32 – 2.26 (m, 2H), 2.24 (s, 3H), 2.22 – 2.12 (m, 3H), 2.01 – 1.90 (m, 7H), 1.84 – 1.77 (m, 2H). LC- MS m / z calcd. C30H36F2N3O+[M+H]+= 492.2826, found = 492.4428. 131 CORE / 3510075.0137 / 196268526.1

[0435] Compound 52: (9R,9aR)-9-(4-((tetrahydro-2H-thiopyran-4- yl)ethynyl)phenyl)-N-(p-tolyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)- carboxamide:1H NMR (400 MHz, Acetone) 67.47 (s, 1H), 7.40 – 7.31 (m, 6H), 7.01 (d, I = 8.7 Hz, 2H), 3.82 – 3.69 (m, 2H), 3.45 – 3.34 (m, 2H), 3.27 – 3.12 (m, 2H), 2.63 – 2.54 (m, 3H), 2.43 – 2.33 (m, 2H), 2.33 – 2.24 (m, 2H), 2.24 (s, 3H), 2.23 – 2.20 (m, 1H), 2.20 – 2.11 (m, 2H), 2.02 – 1.84 (m, 7H). LC-MS m / z calcd. C29H36N3OS+[M+H]+= 474.2579, found = 474.3765.

[0436] Compound 53: (9R,9aR)-9-(4-(((1S,2S)-2- hydroxycyclopentyl)ethynyl)phenyl)-N-(p-tolyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine- 2(3H)-carboxamide: H NMR (400 MHz, Acetone) 67.48 (s, 1H), 7.41 – 7.34 (m, 4H), 7.31 (d, I = 8.0 Hz, 2H), 7.03 (d, I = 8.3 Hz, 2H), 4.28 – 4.23 (m, 1H), 4.12 (d, I = 4.2 Hz, 1H), 3.82 – 3.70 (m, 2H), 3.44 – 3.36 (m, 2H), 3.26 – 3.16 (m, 2H), 2.81 – 2.76 (m, 1H), 2.60 – 2.55 (m, 1H), 2.42 – 2.31 (m, 3H), 2.26 – 2.20 (m, 5H), 2.01 – 1.91 (m, 4H), 1.81 – 1.70 (m, 3H), 1.65 – 1.59 (m, 1H). LC-MS m / z calcd. C29H36N3O2+[M+H]+= 458.2808, found = 458.3733.

[0437] Compound 54: (9R,9aR)-9-(4-((4-hydroxytetrahydro-2H-pyran-4- yl)ethynyl)phenyl)-N-(p-tolyl)hexahydro-1H-pyrrolo[1,2-a][1,4]diazepine-2(3H)- carboxamide:1H NMR (400 MHz, Acetone) 67.48 (s, 1H), 7.42 – 7.33 (m, 6H), 7.01 (d, I = 8.4 Hz, 2H), 4.72 (s, 1H), 3.90 – 3.82 (m, 2H), 3.81 – 3.76 (m, 1H), 3.75 – 3.69 (m, 1H), 3.65 (ddd, I = 11.6, 8.8, 3.0 Hz, 2H), 3.45 – 3.35 (m, 2H), 3.23 (t, I = 9.3 Hz, 1H), 3.17 (dt, I = 12.8, 4.9 Hz, 1H), 2.57 (t, I = 9.6 Hz, 1H), 2.43 – 2.28 (m, 3H), 2.24 (s, 3H), 2.23 – 2.18 (m, 1H), 1.98 – 1.89 (m, 5H), 1.80 (ddd, I = 12.9, 8.9, 3.9 Hz, 2H). LC-MS m / z calcd. C29H36N3O3+[M+H]+= 474.2757, found = 474.3402. 132 CORE / 3510075.0137 / 196268526.1

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:R1 is alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, or alkaryl; L1is a bond, alkylene, alkenylene, or alkynylene; R2 is aryl or substituted aryl; and R3 is cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, or alkaryl.

2. The compound of claim 1, wherein R1is propyl, phenyl, substituted phenyl, cyclohexenyl, cyclohexyl, substituted cyclohexyl, benzyl, cyclopropyl, pyridinyl, substituted pyridinyl, pyrimidinyl, pyridazinyl, or pyrazinyl.

3. The compound of claim 1, wherein R1 is phenyl, substituted phenyl, cyclohexenyl, cyclohexyl, substituted cyclohexyl, or substituted pyridyl.

4. The compound of claim 1, wherein R1 is substituted phenyl.

5. The compound of claim 4, wherein R1is substituted phenyl and the phenyl is substituted with halo, hydroxyl, cyano, alkylene-OH, or alkyl.

6. The compound of claim 4, wherein R1is substituted phenyl and the phenyl is substituted with fluoro, chloro, hydroxyl, cyano, C1-C3alkylene-OH, or C1-C3alkyl.

7. The compound of claim 4, wherein R1 is substituted phenyl and the phenyl is substituted with fluoro or hydroxyl. 133 CORE / 3510075.0137 / 196268526.

18. The compound of any one of claims 1 to 7, wherein L1is C2-C4alkylene, C2-C4alkenylene, or C2-C4 alkynylene.

9. The compound of any one of claims 1 to 7, wherein L1is ethylene, ethenylene, or ethynylene.

10. The compound of any one of claims 1 to 7, wherein L1is ethynylene.

11. The compound of any one of claims 1 to 10, wherein R2 is phenyl, pyridinyl, substituted pyridinyl, or pyridazinyl.

12. The compound of any one of claims 1 to 10, wherein R2 is phenyl.

13. The compound of any one of claims 1 to 12, wherein R3is phenyl, substituted phenyl, benzyl, cyclohexyl, cyclopropyl, substituted pyridinyl, substituted pyrizinyl, or substituted pyrimidinyl.

14. The compound of any one of claims 1 to 12, wherein R3 is phenyl, substituted phenyl, substituted pyridinyl, substituted pyrizinyl, or substituted pyrimidinyl.

15. The compound of any one of claims 1 to 12, wherein R3is substituted phenyl or substituted pyridinyl.

16. The compound of any one of claims 1 to 12, wherein R3is substituted phenyl.

17. The compound of claim 1, wherein the compound has a structure corresponding to Formula 2, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:wherein 134 CORE / 3510075.0137 / 196268526.1R1is C1-C6alkyl, C3-C6cycloalkyl, substituted cycloalkyl, 5- or 6-membered ring aryl, substituted 5- or 6-membered ring aryl, or alkaryl; L1 is a bond, C2-C4 alkylene, C2-C4 alkenylene, or C2-C4 alkynylene; E2is N or C(H); E3 is N or C(H); E4 is N or C(R32); E5is N or C(H); E6is N or C(R32); R21 and R22 are independently hydrogen or halo; R31is hydrogen, alkyl, alkoxy, substituted alkoxy, halo, or morpholino; R32is hydrogen or halo.

18. The compound of claim 17, wherein R1 is phenyl or substituted phenyl.

19. The compound of claim 17, wherein R1 is substituted phenyl.

20. The compound of any one of claims 17 to 19, wherein L1is ethylene, ethenylene, or ethynylene.

21. The compound of any one of claims 17 to 19, wherein L1is ethynylene.

22. The compound of any one of claims 17 to 21, wherein E2 is N.

23. The compound of any one of claims 17 to 21, wherein E2is C(H).

24. The compound of any one of claims 17 to 23, wherein E3 is C(H).

25. The compound of any one of claims 17 to 24, wherein E4 is C(R32).

26. The compound of claim 25, wherein R32is hydrogen.

27. The compound of claim 25, wherein R32 is fluoro. 135 CORE / 3510075.0137 / 196268526.

128. The compound of any one of claims 17 to 27, wherein E5is C(H).

29. The compound of any one of claims 17 to 27, wherein E5 is N.

30. The compound of any one of claims 17 to 29, wherein E6 is N.

31. The compound of any one of claims 17 to 29, wherein E6is C(R32).

32. The compound of claim 31, wherein R32 is fluoro.

33. The compound of any one of claims 17 to 32, wherein R21is hydrogen.

34. The compound of any one of claims 17 to 33, wherein R22 is hydrogen.

35. The compound of any one of claims 17 to 33, wherein R22 is fluoro.

36. The compound of any one of claims 17 to 35, wherein R31is C1-C3alkoxy.

37. The compound of any one of claims 17 to 35, wherein R31 is methoxy.

38. The compound of claim 1, wherein the compound has a structure corresponding to Formula 3, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:wherein R13, R14, and R15are independently hydrogen, alkyl, hydroxyl, halo, or cyano; E1 is N or C(H); 136 CORE / 3510075.0137 / 196268526.1E2is N or C(H); E3 is N or C(H); E4 is N or C(R32); E5is N or C(H); E6 is N or C(R32); R21 and R22 are independently hydrogen or halo; R31is hydrogen, alkyl, alkoxy, substituted alkoxy, halo, or morpholino; R32is hydrogen or halo.

39. The compound of claim 38, wherein E1is N.

40. The compound of claim 38, wherein E1 is C(H).

41. The compound of any one of claims 38 to 40, wherein E2is N.

42. The compound of any one of claims 38 to 40, wherein E2 is C(H).

43. The compound of any one of claims 38 to 42, wherein E3 is C(H).

44. The compound of any one of claims 38 to 43, wherein E4is C(R32).

45. The compound of claim 44, wherein R32 is hydrogen.

46. The compound of claim 44, wherein R32is fluoro.

47. The compound of any one of claims 38 to 46, wherein E5 is C(H).

48. The compound of any one of claims 38 to 46, wherein E5 is N.

49. The compound of any one of claims 38 to 48, wherein E6is N.

50. The compound of any one of claims 38 to 48, wherein E6 is C(R32). 137 CORE / 3510075.0137 / 196268526.

151. The compound of claim 50, wherein R32is fluoro.

52. The compound of any one of claims 38 to 51, wherein R21 is hydrogen.

53. The compound of any one of claims 38 to 52, wherein R22 is hydrogen.

54. The compound of any one of claims 38 to 52, wherein R22is fluoro.

55. The compound of any one of claims 38 to 54, wherein R31 is C1-C3 alkoxy.

56. The compound of any one of claims 38 to 54, wherein R31is methoxy.

57. A method of treatment of a Toxoplasma gondii infection or infection caused by a parasite closely related to Toxoplasma gondii in a subject in need thereof, the method comprising administering a therapeutic amount of a compound of any one of claims 1 to 56 to the subject.

58. The method of claim 57, wherein the subject is human.

59. The method of claim 57 or 58, wherein the subject is a mouse, rat cat, dog, chicken, turkey, rabbit, non-human primate, lizard, gecko, cow, calf, sheep, goat, lamb, horse, foal, pig, or piglet.

60. The method of any one of claims 57 to 59, wherein the compound is coadministered with one or more antibiotics selected from pyrimethamine, sulfamethoxazole, sulfadiazine, or clindamycin.

61. The method of any one of claims 57 to 60, wherein the infection is caused by a parasite closely related to Toxoplasma gondii selected from Sarcocystis neurona or Neospora caninum.

62. The method of any one of claims 57 to 60, wherein the infection is caused by Toxoplasma gondii. 138 CORE / 3510075.0137 / 196268526.1

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