Agonists of parathyroid hormone 1 and incretin receptors comprising substituted or modified diaminomethylene pyrimidine-2,4,6-triones

Abstract

Disclosed are compounds that are parathyroid hormone receptor 1 agonists, and methods useful for preventing or treating osteoporosis, fracture, osteomalacia, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia or tumoral calcinosis. Also disclosed are agonists of GLP-1R, GCGR, and / or GIPR, and methods of treating various therapeutic indications by administering an effective amount of an agonist of GLP-1R, GCGR, and / or GIPR.

Description

[0001] AGONISTS OF PARATHYROID HORMONE 1 AND INCRETIN RECEPTORS COMPRISING SUBSTITUTED OR MODIFIED DIAMINOMETHYLENE PYRIMIDINE-2, 4, 6-TRIONES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 63 / 706,308, filed October 11, 2024; and 63 / 637,989, filed April 24, 2024; each of which is incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005] Regulation of calcium concentration is important to normal function of the gastrointestinal tract, skeletal system, nervous system, muscular nervous system and cardiovascular system. Parathyroid hormone (PTH) synthesis and release is primarily controlled by serum calcium levels.

[0006] Osteoporosis is characterized by bone loss resulting in an increased incidence of fracture. This condition, which is most prevalent in the spine and hip, affects 1 in 3 postmenopausal women, a lesser but significant number of aging men, and is also caused by other conditions including hypogonadism and prolonged glucocorticoid use. Current therapies to treat osteoporosis, such as bisphosphonates, hormone replacement therapy, SERMs and calcitonin, serve to arrest further bone loss by inhibiting bone resorption. Although these treatments may slow or even prevent continued bone loss, new bone formation leading to increased bone mass and strength, does not occur. Consequently, there is still a need for a therapeutic agent capable of stimulating bone formation. Such a therapeutic agent would be beneficial both to patients who are at risk of developing osteoporosis or who present with established osteoporosis.

[0007] Parathyroid hormone (PTH) is a significant regulator of calcium homeostasis and acts, in part, by mobilizing calcium from the skeleton through increased bone resorption. Additionally, pulsatile administration of PTH can stimulate new bone formation, both in laboratory animals and in humans. Thus, there is evidence to suggest that targeting of the receptor for PTH with a small molecule agonist mimicking the actions of PTH, would be a suitable approach for generating an anabolic response in bone. PTH elicits its effects by binding and activating a class B, G protein-coupled receptor of the 7 transmembrane superfamily, designated PTH1R. PTH1R activates multiple signaling pathways, but predominantly the adenylyl cyclase / cyclic AMP and the phospholipase C / calcium mobilization pathways.

[0008] The glucagon- like peptide- 1 receptor (GLP-1R) is a receptor protein found on beta cells of the pancreas and on neurons of the brain. It is involved in the control of blood sugar level by enhancing insulin secretion. Glucagon-like peptide- 1 (GLP-1) is a hormone consisting of 30 amino acids. It is released by intestinal L cells when nutrients are consumed. GLP-1 has multiple effects, including enhancing insulin secretion from pancreatic beta cells in response to glucose, increasing insulin expression, preventing beta-cell apoptosis, promoting the formation of new beta cells, reducing glucagon secretion, slowing down stomach emptying, promoting satiety, and improving glucose disposal in peripheral tissues. GLP-1 R is also expressed in the brain, where it is involved in the control of appetite. Furthermore, mice that over express GLP-1 R display improved memory and learning.

[0009] Due to these diverse effects, there has been significant interest in developing long- lasting agonists of the GLP-1 receptor (GLP-1R) for the treatment of type 2 diabetes and other conditions. Indeed, glucagon-like peptide- 1 receptor agonists gained approval as drugs to treat diabetes and obesity starting in the 2000s.

[0010] The gastric inhibitory polypeptide receptor (GIPR), also known as the glucosedependent insulinotropic polypeptide receptor, is a protein that in humans is encoded by the GIPR gene. GIPR is a member of the class B family of G protein coupled receptors. GIPR is found on beta-cells in the pancreas where it serves as the receptor for the hormone Gastric inhibitory polypeptide (GIP). Gastric inhibitory polypeptide, also called glucose-dependent insulinotropic polypeptide, is a 42-amino acid polypeptide synthesized by K cells of the duodenum and small intestine. It was originally identified as an activity in gut extracts that inhibited gastric acid secretion and gastrin release, but subsequently was demonstrated to stimulate insulin release potently in the presence of elevated glucose. The insulinotropic effect on pancreatic islet beta-cells was then recognized to be the principal physiologic action of GIP. Together with glucagon-like peptide- 1, GIP is largely responsible for the secretion of insulin after eating. It is involved in several other facets of the anabolic response.

[0011] The glucagon receptor (GCGR) is a 62 kDa protein that is activated by glucagon and is a member of the class B G-protein coupled family of receptors, coupled to G alpha i, Gs and to a lesser extent G alpha q. Stimulation of the receptor results in the activation of adenylate cyclase and phospholipase C and in increased levels of the secondary messengers intracellular cAMP and calcium. Through interaction with GCGR, glucagon has been shown to increase hepatic glucose output in hypoglycemic state, reduce body weight and appetite, stimulate insulin secretion under certain circumstances and as a key hormone that modulates postprandial amino acid metabolism. In humans, the glucagon receptor is encoded by the GCGR gene.

[0012] Inactivating mutation of glucagon receptor in humans causes resistance to glucagon and is associated with pancreatic alpha cell hyperplasia, nesidioblastosis, hyperglucagonemia, and pancreatic neuroendocrine tumors, also known as Mahvash disease. Glucagon receptors are mainly expressed in liver and in kidney with lesser amounts found in heart, adipose tissue, spleen, thymus, adrenal glands, pancreas, cerebral cortex, and gastrointestinal tract.

[0013] Accordingly, there is a need in the art to provide small molecule therapeutics that treat or prevent hypoparathyroidism, osteoporosis and related conditions. In particular, there is a need for providing compounds that act as PTH1R agonists.

[0014] Additionally, there is a need in the art to provide small molecule modulators of the GLP-1R, GIPR, and / or GCGR in order to provide treatments for conditions such as diabetes, obesity or overweight, liver disease, and heart disease. More particularly, there is a need for GLP-1R / GIPR / GCGR triagonists to treat these conditions, which should be more efficacious compared to currently available GLP-1 agonists.

[0015] SUMMARY

[0016] The present disclosure provides in some embodiments a compound of Formula (I): or a pharmaceutically acceptable salt thereof; wherein:

[0017] R1is (C1-C6)alkyl;

[0018] R2is hydrogen or (C1-C6)alkyl optionally substituted with hydroxy or 4- to 7- membered heterocylcylalkyl;

[0019] R3is (C1-C6)alkyl optionally substituted with (C3-C8)cycloalkyl or phenyl;

[0020] R4aand R4bare independently hydrogen or (C1-C6)alkyl, wherein the (C1-C6)alkyl is optionally substituted with one or more instances of fluoro, hydroxy, or 4- to 7-membered heterocycloalkyl; or R4aand R4btaken together with the atoms to which they are attached form taken together with the nitrogen atom to which they are attached form a 4- to 7- membered heterocycloalkyl; and

[0021] R5aand R5bare independently hydrogen or (C1-C6)alkyl, wherein the (C1-C6)alkyl is optionally substituted with one or more instances of fluoro, hydroxy, or 4- to 7-membered heterocycloalkyl; or R5aand R5btaken together with the nitrogen atom to which they are attached form taken together with the atoms to which they are attached form a 4- to 7- membered heterocycloalkyl; wherein at least one of R4aand R4bR5aand R5bis hydrogen, and at least one of R4aand R4bR5aand R5bis not hydrogen; provided that the compound is not:

[0022] The present invention also provides a compound according to Formula (II) or a pharmaceutically acceptable salt thereof; wherein:

[0023] R1is (C1-C6)alkyl;

[0024] R2is hydrogen or (C1-C6)alkyl;

[0025] R3is (C1-C6)alkyl; and

[0026] R4a, R4b, R5aand R5bare each independently hydrogen or (C1-C6)alkyl; wherein at least one of R4aand R4bR5aand R5bis not hydrogen.

[0027] The present invention also provides a compound according to Formula (III): or a pharmaceutically acceptable salt thereof, wherein:

[0028] X is N or CH;

[0029] Y is hydrogen or NH2;

[0030] R6is:

[0031] R7is hydrogen, or

[0032] R6and R7taken together with the atom to which they are attached form:

[0033] R8is (C1-C6)alkyl;

[0034] R9and R10are independently (C1-C6)alkyl; and

[0035] R11is (C1-C6)alkyl; and

[0036] R12is (C1-C6)alkyl optionally substituted with heterocycloalkyl.

[0037] The present disclosure further provides a compound according to Formula (IV): or a pharmaceutically acceptable salt thereof, wherein:

[0038] R13and R14are each independently (C1-C6)alkyl; and

[0039] R15and R16are each independently (C1-C6)alkyl;

[0040] The present disclosure also provides a compound having the structure of Formula (V):

[0041] or a pharmaceutically acceptable salt thereof, wherein:

[0042] R17is:

[0043] R18is hydrogen; or

[0044] R17and R18taken together with the atom to which they are attached form:

[0045] R19is (C1-C6)alkyl;

[0046] R20is (C1-C6)alkyl or (C3-C8)cycloalkyl, each of which is optionally substituted with one or more fluoro, hydroxy, (C1-C6)alkoxy, and (C1-C6)thioalkoxy;

[0047] R21and R22are each independently (C1-C6)alkyl or R21and R22taken together with the atom to which they are attached form (C3-C8)cycloalkyl; and

[0048] R23and R24are each independently (C1-C6)alkyl.

[0049] The present invention also provides a compound of Formula (VI): or a pharmaceutically acceptable salt thereof, wherein:

[0050] R25and R26are independently (C1-C6) alkyl; and

[0051] R11is (C1-C6)alkyl optionally substituted with one or more fluorine atoms.

[0052] The present disclosure also provides a compound of Formula (VII): (VII), or a pharmaceutically acceptable salt thereof, wherein:

[0053] R28and R29are independently (C1-C6) alkyl; and

[0054] R30is (C1-C6)alkyl optionally substituted with (C3-C8)cycloalkyl.

[0055] Other aspects of the disclosure provide a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0056] In still other aspects, provided herein is a method for treating or preventing osteoporosis, fracture, osteomalacia, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia or tumoral calcinosis, comprising administering to a subject in need thereof a therapeutically amount of a compound of compound of the present invention, or a pharmaceutically acceptable salt thereof.

[0057] In still other aspects, provided herein is a method of: a) reducing of HbAlc; b) treating or preventing type 2 diabetes, hyperglycemia, impaired glucose tolerance, or non-insulin dependent diabetes, and / or obesity; c) reducing body weight and / or food intake, and / or inducing satiety; and / or d) treating or preventing Alzheimer’s disease, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and / or cardiovascular diseases; comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0058] 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 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present 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.

[0059] Other features, objects, and advantages of the invention will be apparent from the detailed description, and from the claims.

[0060] BRIEF DESCRIPTION OF THE FIGURES

[0061] Figure 1 tabulates exemplary compounds of the invention, and their characterization data and PTH1R activity.

[0062] DETAILED DESCRIPTION

[0063] Definitions

[0064] For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and as understood by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

[0065] In order for the present invention to be more readily understood, certain terms and phrases are defined below and throughout the specification.

[0066] The articles “a” and “an” are used herein to refer to one or to 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.

[0067] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0068] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0069] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0070] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0071] Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present invention may also be optically active. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

[0072] “Geometric isomer" means isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a cycloalkyl ring, or to a bridged bicyclic system. 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 or as “atropisomers.” 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 of the invention may be prepared as individual isomers by either isomer-specific synthesis or resolved from a mixture of isomers. 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.

[0073] If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically- active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

[0074] Percent purity by mole fraction is the ratio of the moles of the enantiomer (or diastereomer) or over the moles of the enantiomer (or diastereomer) plus the moles of its optical isomer. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least about 60%, about 70%, about 80%, about 90%, about 99% or about 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 about 60%, about 70%, about 80%, about 90%, about 99% or about 99.9% by mole fraction pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least about 60%, about 70%, about 80%, about 90%, about 99% or about 99.9% by mole fraction pure.

[0075] When a disclosed compound is named or depicted by 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 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 invention embraces all of these forms.

[0076] Structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds produced by the replacement of a hydrogen with deuterium or tritium, or of a carbon with a13C- or deenriched carbon are within the scope of this invention.

[0077] The term “prodrug” as used herein encompasses compounds that, under physiological conditions, are converted into therapeutically active agents. A common method for making a prodrug is to include selected moieties that are hydrolyzed under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. The phrase “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ or portion of the body, to another organ or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, not injurious to the patient, and substantially non-pyrogenic. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non- toxic compatible substances employed in pharmaceutical formulations. In certain embodiments, pharmaceutical compositions of the present invention are non-pyrogenic, i.e., do not induce significant temperature elevations when administered to a patient.

[0078] The term “pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the compound(s). These salts can be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting a purified compound(s) in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts, and the like. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19.)

[0079] In other cases, the compounds useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts” in these instances refers to the relatively non-toxic inorganic and organic base addition salts of a compound(s). These salts can likewise be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting the purified compound(s) in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).

[0080] The term “pharmaceutically acceptable cocrystals” refers to solid coformers that do not form formal ionic interactions with the small molecule.

[0081] A “therapeutically effective amount” (or “effective amount”) of a compound with respect to use in treatment, refers to an amount of the compound in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0082] The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0083] The term “patient” or “subject” refers to a mammal in need of a particular treatment. In certain embodiments, a patient is a primate, canine, feline, or equine. In certain embodiments, a patient is a human.

[0084] An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyl defined below. A straight aliphatic chain is limited to unbranched carbon chain moieties. As used herein, the term “aliphatic group” refers to a straight chain, branched-chain, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as an alkyl group, an alkenyl group, or an alkynyl group.

[0085] “Alkyl” refers to a fully saturated cyclic or acyclic, branched or unbranched carbon chain moiety having the number of carbon atoms specified, or up to 30 carbon atoms if no specification is made. For example, alkyl of 1 to 8 carbon atoms refers to moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, and those moieties which are positional isomers of these moieties. Alkyl of 10 to 30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl and tetracosyl. In certain embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30for straight chains, C3-C30for branched chains), and more preferably 20 or fewer. Alkyl groups may be substituted or unsubstituted.

[0086] As used herein, the term “heteroalkyl” refers to an alkyl moiety as hereinbefore defined which contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms.

[0087] As used herein, the term “haloalkyl” refers to an alkyl group as hereinbefore defined substituted with at least one halogen.

[0088] As used herein, the term “haloalkoxy” refers to an alkoxy group as defined herein substituted with at least one halogen atom. For example, haloalkoxy groups include fluoroalkoxy groups, such as trifluoromethoxy, difluoromethoxy, and the like.

[0089] As used herein, the term “hydroxyalkyl” refers to an alkyl group as hereinbefore defined substituted with at least one hydroxyl.

[0090] As used herein, the term “alkylene” refers to an alkyl group having the specified number of carbons, for example from 2 to 12 carbon atoms, that contains two points of attachment to the rest of the compound on its longest carbon chain. Non-limiting examples of alkylene groups include methylene -(CH2)-, ethylene -(CH2CH2)-, n-propylene - (CH2CH2CH2)-, isopropylene -(CH2CH(CH3))-, and the like. Alkylene groups can be cyclic or acyclic, branched or unbranched carbon chain moiety, and may be optionally substituted with one or more substituents.

[0091] "Cycloalkyl" means mono- or bicyclic or bridged or spirocyclic, or polycyclic saturated carbocyclic rings, each having from 3 to 12 carbon atoms. Preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3-6 carbons in the ring structure. Cycloalkyl groups may be substituted or unsubstituted.

[0092] “1,2-Disubstituted cyclohexyl” as used herein refers to a cyclohexane ring that contains points of attachment at the 1 and 2 positions of the cyclohexane ring to the rest of the compound. Similarly, “1,2-disubstituted cyclopentyl” as used herein refers to a cyclopentane ring that contains points of attachment at the 1 and 2 positions of the cyclopentane ring to the rest of the compound. 1 ,2-Disubstituted cyclohexyl and 1 ,2- disubstituted cyclopentyl can also be referred to as 1 ,2-cyclohexylene and 1 ,2- cyclopentylene, respectively.

[0093] As used herein, the term “halocyclo alkyl” refers to a cycloalkyl group as hereinbefore defined substituted with at least one halogen.

[0094] "Cycloheteroalkyl" refers to a cycloalkyl moiety as hereinbefore defined which contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms. Preferred cycloheteroalkyls have from 4-8 carbon atoms and heteroatoms in their ring structure, and more preferably have 4-6 carbons and heteroatoms in the ring structure. Cycloheteroalkyl groups may be substituted or unsubstituted.

[0095] Unless the number of carbons is otherwise specified, “lower alkyl,” as used herein, means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In certain embodiments, a substituent designated herein as alkyl is a lower alkyl.

[0096] “Alkenyl” refers to any cyclic or acyclic, branched or unbranched unsaturated carbon chain moiety having the number of carbon atoms specified, or up to 26 carbon atoms if no limitation on the number of carbon atoms is specified; and having one or more double bonds in the moiety. Alkenyl of 6 to 26 carbon atoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, and tetracosenyl, in their various isomeric forms, where the unsaturated bond(s) can be located anywhere in the moiety and can have either the (Z) or the (E) configuration about the double bond(s).

[0097] “Alkynyl” refers to hydrocarbyl moieties of the scope of alkenyl, but having one or more triple bonds in the moiety.

[0098] The term “aryl” as used herein includes 3- to 12-membered substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon (i.e., carbocyclic aryl) or where one or more atoms are heteroatoms (i.e., heteroaryl). Preferably, aryl groups include 5- to 12-membered rings, more preferably 6- to 10-membered rings The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Carboycyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. Heteroaryl groups include substituted or unsubstituted aromatic 3- to 12-membered ring structures, more preferably 5- to 12- membered rings, more preferably 5- to 10-membered rings, whose ring structures include one to four heteroatoms. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, indazole, quinoline, benzofuran, and the like. Aryl and heteroaryl can be monocyclic, bicyclic, or polycyclic.

[0099] The term “halo”, “halide”, or “halogen” as used herein means halogen and includes, for example, and without being limited thereto, fluoro, chloro, bromo, iodo and the like, in both radioactive and non-radioactive forms. In a preferred embodiment, halo is selected from the group consisting of fluoro, chloro and bromo. The terms “heterocyclyl” or “heterocyclic group” or “heterocycloalkyl” refer to 3- to 12-membered ring structures, more preferably 5- to 12- membered rings, more preferably 5- to 10-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can be monocyclic, bicyclic, spirocyclic, or polycyclic. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. Heterocycloalkyl groups can be fully saturated or partially saturated. Heterocycloalkyl groups include, for example, bicyclic ring systems having either or both constituent rings saturated (e.g., 2,3-dihydroindole, 4, 5,6,7- tetrahydro-benzofuran, decahydroquinoline, and the like) or partially saturated (e.g., octahydroquinoline and the like).

[0100] The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, and the like.

[0101] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. In preferred embodiments, the substituents on substituted alkyls are selected from Ci-6 alkyl, C3-6 cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In more preferred embodiments, the substituents on substituted alkyls are selected from fluoro, carbonyl, cyano, or hydroxyl. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

[0102] As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

[0103] As used herein, “small molecules” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

[0104] In some embodiments, a “small molecule” refers to an organic, inorganic, or organometallic compound typically having a molecular weight of less than about 1000. In some embodiments, a small molecule is an organic compound, with a size on the order of 1 nm. In some embodiments, small molecule drugs of the invention encompass oligopeptides and other biomolecules having a molecular weight of less than about 1000.

[0105] An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition depends on the composition selected. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions described herein can include a single treatment or a series of treatments.

[0106] The terms “decrease,” “reduce,” “reduced”, “reduction”, “decrease,” and “inhibit” are all used herein generally to mean a decrease by a statistically significant amount relative to a reference. However, for avoidance of doubt, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level and can include, for example, a decrease by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, up to and including, for example, the complete absence of the given entity or parameter as compared to the reference level, or any decrease between 10-99% as compared to the absence of a given treatment.

[0107] The terms “increased”, “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a

[0108] 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0109] As used herein, the term “modulate” includes up-regulation and down-regulation, e.g., enhancing or inhibiting a response.

[0110] A “radiopharmaceutical agent,” as defined herein, refers to a pharmaceutical agent which contains at least one radiation-emitting radioisotope. Radiopharmaceutical agents are routinely used in nuclear medicine for the diagnosis and / or therapy of various diseases. The radiolabeled pharmaceutical agent, for example, a radiolabeled antibody, contains a radioisotope (RI) which serves as the radiation source. As contemplated herein, the term “radioisotope” includes metallic and non-metallic radioisotopes. The radioisotope is chosen based on the medical application of the radiolabeled pharmaceutical agents. When the radioisotope is a metallic radioisotope, a chelator is typically employed to bind the metallic radioisotope to the rest of the molecule. When the radioisotope is a non-metallic radioisotope, the non-metallic radioisotope is typically linked directly, or via a linker, to the rest of the molecule.

[0111] For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

[0112] Compounds of the Invention

[0113] The present disclosure provides in some embodiments a compound of Formula (I): or a pharmaceutically acceptable salt thereof; wherein:

[0114] R1is (C1-C6)alkyl;

[0115] R2is hydrogen or (C1-C6)alkyl optionally substituted with hydroxy or 4- to 7- membered heterocylcylalkyl;

[0116] R3is (C1-C6)alkyl optionally substituted with (C3-C8)cycloalkyl or phenyl;

[0117] R4aand R4bare independently hydrogen or (C1-C6)alkyl, wherein the (C1-C6)alkyl is optionally substituted with one or more instances of fluoro, hydroxy, or 4- to 7-membered heterocycloalkyl; or R4aand R4btaken together with the atoms to which they are attached form taken together with the nitrogen atom to which they are attached form a 4- to 7- membered heterocycloalkyl; and

[0118] R5aand R5bare independently hydrogen or (C1-C6)alkyl, wherein the (C1-C6)alkyl is optionally substituted with one or more instances of fluoro, hydroxy, or 4- to 7-membered heterocycloalkyl; or R5aand R5btaken together with the nitrogen atom to which they are attached form taken together with the atoms to which they are attached form a 4- to 7- membered heterocycloalkyl; wherein at least one of R4aand R4bR5aand R5bis hydrogen, and at least one of R4aand R4bR5aand R5bis not hydrogen; provided that the compound is not:

[0119] In some embodiments, the compound has the structure of Formula (la) or lb): (lb), or a pharmaceutically acceptable salt thereof.

[0120] In some embodiments, R1is methyl, In some embodiments, R2is hydrogen. In some embodiments, R2is methyl.

[0121] In some embodiments, R2is (C1-C6)alkyl substituted with hydroxy.

[0122] In some embodiments, R2is 2-hydroxy-2-methylpropyl.

[0123] In some embodiments, R2is methyl substituted with 4- to 7-membered heterocycloalkyl. In more particular embodiments, the 4- to 7-membered heterocycloalkyl is oxetanyl, tetrahydrofuranyl, or tetrahydropyranyl, and in even more particular embodiments, the 4- to 7-membered heterocyclo alkyl is oxetan-3-yl, tetrahydrofuran-3-yl, or tetrahydropyran-4-yl.

[0124] In some embodiments, wherein R3is methyl optionally substituted with (C3- C8)cycloalkyl or phenyl.

[0125] In some embodiments, wherein R3is (C1-C6)alkyl optionally substituted with phenyl.

[0126] In some embodiments, R3is (C1-C6)alkyl optionally substituted with cyclopropyl, cyclobutyl, or cyclopentyl.

[0127] In some embodiments, R3is n-butyl.

[0128] In some embodiments, at least two of R4a, R4b, R5aand R5bare hydrogen.

[0129] In some embodiments, three of R4a, R4b, R5aand R5bare hydrogen.

[0130] In some embodiments, at least two of R4aand R4btaken together with the nitrogen atom to which they are attached form azetidine.

[0131] In some embodiments, one of R4aand R4b, or R5aand R5bis (C1-C6)alkyl optionally substituted with fluoro, hydroxy, or 4- to 7-membered heterocycloalkyl. In some embodiments, the 4- to 7-membered heterocycloalkyl is tetrahydrofuran-2-yl, while in other embodiments, the 4- to 7-membered heterocycloalkyl is 3-oxetanyl.

[0132] In some embodiments, one of R4aand R4bis 2,2,2-trifluoroethyl.

[0133] In some embodiments, one of R4aand R4bis 2-hydroxy-2-methylpropyl.

[0134] In some embodiments, the 4- to 7-membered heterocycloalkyl is tetrahydrofuran-2-yl.

[0135] In some embodiments, wherein one of R4aand R4bis methyl, ethyl, isopropyl, or n- butyl.

[0136] In some embodiments, one of R4aand R4bis methyl.

[0137] In some embodiments, one of R5aand R5bis methyl.

[0138] The present invention also provides a compound according to Formula (II): (II), or a pharmaceutically acceptable salt thereof; wherein:

[0139] R1is (C1-C6)alkyl;

[0140] R2is hydrogen or (C1-C6)alkyl;

[0141] R3is (C1-C6)alkyl; and

[0142] R4a, R4b, R5aand R5bare each independently hydrogen or (C1-C6)alkyl; wherein at least one of R4aand R4bR5aand R5bis not hydrogen.

[0143] In some embodiments, the compound has the structure of Formula (Ila) or (Uh) : or a pharmaceutically acceptable salt thereof.

[0144] In some embodiments, R1is methyl.

[0145] In some embodiments, R2is hydrogen. In other embodiments, R2is methyl.

[0146] In some embodiments, R3is n-butyl.

[0147] In some embodiments, R4ais methyl.

[0148] In some embodiments, R4b, R5aand R5bare each hydrogen.

[0149] The present invention also provides a compound having the following structure:

[0150] or a pharmaceutically acceptable salt thereof, wherein:

[0151] X is N or CH;

[0152] Y is hydrogen or NH2;

[0153] R6is:

[0154] R7is hydrogen, or

[0155] R6and R7taken together with the atom to which they are attached form:

[0156] R8is (C1-C6)alkyl;

[0157] R9and R10are independently (C1-C6)alkyl; and

[0158] R11is (C1-C6)alkyl; and

[0159] R12is (C1-C6)alkyl optionally substituted with heterocycloalkyl.

[0160] In some embodiments, the compound has the structure of Formula (Illa), (Illb), (IIIc) or (Illd):

[0161] or a pharmaceutically acceptable salt thereof.

[0162] In some embodiments, X is CH2. In other embodiments, X is N.

[0163] In some embodiments, Y is hydrogen. In other embodiments, Y is NH2.

[0164] In some embodiments, R8is hydrogen.

[0165] In some embodiments, R8is (C1-C6)alkyl.

[0166] In some embodiments, R8is n-butyl.

[0167] In some embodiments, R9and R10are each methyl.

[0168] In some embodiments, R11is methyl.

[0169] In some embodiments, R12is methyl. In other embodiments, R12is 3- (oxetanyl)methyl. In yet other embodiments, R12is 3-(tetrahydrofurayl)methyl

[0170] The present disclosure further provides a compound according to Formula (IV): or a pharmaceutically acceptable salt thereof, wherein:

[0171] R13and R14are each independently (C1-C6)alkyl; and

[0172] R15and R16are each independently (C1-C6)alkyl;

[0173] In some embodiments, the compound has the structure of Formula (IVa), (IVb), (IVc), or (IVd): or a pharmaceutically acceptable salt thereof.

[0174] In some embodiments, R14is n-butyl.

[0175] In some embodiments, R13is methyl.

[0176] In some embodiments, R15and R16are each methyl.

[0177] The present disclosure also provides a compound having the structure of Formula (V): or a pharmaceutically acceptable salt thereof, wherein:

[0178] R17is:

[0179] R18is hydrogen; or

[0180] R17and R18taken together with the atom to which they are attached form:

[0181] R19is (C1-C6)alkyl;

[0182] R20is (C1-C6)alkyl or (C3-C8)cycloalkyl, each of which is optionally substituted with one or more fluoro, hydroxy, (C1-C6)alkoxy, and (C1-C6)thioalkoxy;

[0183] R21and R22are each independently (C1-C6)alkyl or R21and R22taken together with the atom to which they are attached form (C3-C8)cycloalkyl; and

[0184] R23and R24are each independently (C1-C6)alkyl. In some embodiments, the compound has the structure of Formula (Va), (Vb), (Vc), (Vd), (Vf), (Vg), or (Vh): or a pharmaceutically acceptable salt thereof.

[0185] In some embodiments, R19is n-butyl.

[0186] In some embodiments, R20is methyl.

[0187] In some embodiments, R20is cyclopropyl. In some embodiments, R21and R22taken together with the atom to which they are attached form cyclopropyl.

[0188] In some embodiments, R23and R24are each methyl.

[0189] The present invention also provides a compound of Formula (VI): or a pharmaceutically acceptable salt thereof, wherein:

[0190] R25and R26are independently (C1-C6) alkyl; and

[0191] R27is (C1-C6)alkyl optionally substituted with one or more fluorine atoms.

[0192] In some embodiments, the compound has the structure of Formula (Via) or (VIb): or a pharmaceutically acceptable salt thereof.

[0193] In some embodiments, R25is methyl.

[0194] In some embodiments, R26is methyl.

[0195] In some embodiments, R27is 2,2,2-trifluoroethyl.

[0196] The present disclosure also provides a compound of Formula (VII): or a pharmaceutically acceptable salt thereof, wherein:

[0197] R28and R29are independently (C1-C6) alkyl; and R30is (C1-C6)alkyl optionally substituted with (C3-C8)cycloalkyl.

[0198] In some embodiments, the compound has the structure of Formula (Vila) or (Vllb): (Vllb).

[0199] In some embodiments, R28is methyl.

[0200] In some embodiments, R29is methyl.

[0201] In some embodiments, R30is methyl substituted with (C3-C8)cycloalkyl.

[0202] In some embodiments, R30is cyclopentylmethyl.

[0203] The present invention also provides a compound having the structure: or a pharmaceutically acceptable salt thereof.

[0204] Methods of Treatment

[0205] One aspect of the invention provides a method for treating or preventing osteoporosis, fracture, osteomalacia, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia or tumoral calcinosis, comprising administering to a subject in need thereof an effective amount of a compound according to the present invention, or a pharmaceutically acceptable salt thereof.

[0206] Another aspect of this invention is a method for preventing or treating a condition mediated by PTH which comprises administering to a mammal in need thereof an effective amount of a compound a compound according to the present invention, or a pharmaceutically acceptable salt thereof, either alone or in admixture with a pharmaceutically excipient. Another aspect of the invention includes compounds of the present invention, or a pharmaceutically acceptable salt thereof, for use in the treatment and prevention of diseases and conditions characterized by loss of bone mineral density, mass, or strength, as well as in conditions wherein PTH would have a beneficial pharmacological effect. The invention includes administering compounds of formula (I) or (II) for use as a PTH mimetic. Another aspect of the invention includes use of the compounds of the present invention in the manufacture of a medicament for use in the treatment of osteopenia and osteoporosis in men and women for reduction in the risk of fractures, both vertebral and nonvertebral.

[0207] Another aspect of the invention provides a method of: a) reducing of HbAlc; b) treating or preventing type 2 diabetes, hyperglycemia, impaired glucose tolerance, or non-insulin dependent diabetes, and / or obesity; c) reducing body weight and / or food intake, and / or inducing satiety; and / or d) treating or preventing Alzheimer’s disease, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and / or cardiovascular diseases; comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to the present invention.

[0208] In certain embodiments, the compound is administered orally to the subject.

[0209] In certain embodiments, the compound is administered parenterally to the subject.

[0210] In certain embodiments, the disease is prevented. In other embodiments, the disease is treated.

[0211] Pharmaceutical Compositions, Routes of Administration, and Dosing

[0212] In certain embodiments, the invention is directed to a pharmaceutical composition, comprising a compound of the invention, e.g. a compound according to the present invention, and a pharmaceutically acceptable carrier.

[0213] In certain embodiments, the invention is directed to a pharmaceutical composition, comprising a compound of any of the disclosed embodiments, and a pharmaceutically acceptable carrier.

[0214] In certain embodiments, the pharmaceutical composition comprises a plurality of compounds of the invention and a pharmaceutically acceptable carrier.

[0215] Pharmaceutical compositions of the invention can be prepared by combining one or more compounds of the invention with a pharmaceutically acceptable carrier and, optionally, one or more additional pharmaceutically active agents. As stated above, an “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound of the invention being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound of the invention and / or other therapeutic agent without necessitating undue experimentation. A maximum dose may be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day may be contemplated to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be determined by, for example, measurement of the patient’s peak or sustained plasma level of the drug. “Dose” and “dosage” are used interchangeably herein.

[0216] In certain embodiments, intravenous administration of a compound may typically be from 0.1 mg / kg / day to 20 mg / kg / day. In one embodiment, intravenous administration of a compound may typically be from 0.1 mg / kg / day to 2 mg / kg / day. In one embodiment, intravenous administration of a compound may typically be from 0.5 mg / kg / day to 5 mg / kg / day. In one embodiment, intravenous administration of a compound may typically be from 1 mg / kg / day to 20 mg / kg / day. In one embodiment, intravenous administration of a compound may typically be from 1 mg / kg / day to 10 mg / kg / day.

[0217] Generally, daily oral doses of a compound will be, for human subjects, from about 0.01 milligrams / kg per day to 1000 milligrams / kg per day. It is expected that oral doses in the range of 0.5 to 50 milligrams / kg, in one or more administrations per day, will yield therapeutic results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, it is expected that intravenous administration would be from one order to several orders of magnitude lower dose per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound. For any compound described herein the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for compounds which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

[0218] The formulations of the invention can be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0219] For use in therapy, an effective amount of the compound can be administered to a subject by any mode that delivers the compound to the desired surface. Administering a pharmaceutical composition may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal (e.g., topical to eye), inhalation, and topical.

[0220] For intravenous and other parenteral routes of administration, a compound of the invention can be formulated as a lyophilized preparation, as a lyophilized preparation of liposome-intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a salt complex. Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.

[0221] For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions or may be administered without any carriers.

[0222] Also specifically contemplated are oral dosage forms of the above component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts”, In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981); Newmark et al., J Appl Biochem 4:185-9 (1982). Other polymers that could be used are poly- 1,3 -dioxolane and poly-l,3,6-tioxocane. For pharmaceutical usage, as indicated above, polyethylene glycol moieties are suitable.

[0223] For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the compound of the invention (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.

[0224] To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.

[0225] A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

[0226] The therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

[0227] Colorants and flavoring agents may all be included. For example, the compound of the invention (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

[0228] One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0229] Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0230] Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

[0231] An anti-frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

[0232] Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

[0233] To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents which can be used and can include benzalkonium chloride and benzethonium chloride. Potential non-ionic detergents that could be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound of the invention or derivative either alone or as a mixture in different ratios.

[0234] Pharmaceutical preparations which can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0235] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0236] For topical administration, the compound may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration. For administration by inhalation, compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro tetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0237] Also contemplated herein is pulmonary delivery of the compounds disclosed herein (or salts thereof). The compound is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (al- antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146 (a- 1 -proteinase); Oswein et al., 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor; incorporated by reference). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569 (incorporated by reference), issued Sep. 19, 1995 to Wong et al.

[0238] Contemplated for use in the practice of this invention are mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

[0239] Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

[0240] All such devices require the use of formulations suitable for the dispensing of the compounds of the invention. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and / or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified compound of the invention may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.

[0241] Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise a compound of the invention (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active compound of the invention per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for inhibitor stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the compound of the invention caused by atomization of the solution in forming the aerosol.

[0242] Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compound of the invention (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

[0243] Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing a compound of the invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. The compound of the invention (or derivative) should advantageously be prepared in particulate form with an average particle size of less than 10 micrometers (pm), most preferably 0.5 to 5 pm, for most effective delivery to the deep lung.

[0244] Nasal delivery of a pharmaceutical composition of the present invention is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

[0245] Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.

[0246] The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0247] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0248] Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0249] The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described above, a compound may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0250] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0251] Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and / or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249:1527-33 (1990).

[0252] The compound of the invention and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt or cocrystal. When used in medicine the salts or cocrystals should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts or cocrystals may conveniently be used to prepare pharmaceutically acceptable salts or cocrystals thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

[0253] Suitable buffering agents include: acetic acid and a salt (1-2% w / v); citric acid and a salt (1-3% w / v); boric acid and a salt (0.5-2.5% w / v); and phosphoric acid and a salt (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v). Pharmaceutical compositions of the invention contain an effective amount of a compound as described herein and optionally therapeutic agents included in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

[0254] The therapeutic agent(s), including specifically but not limited to a compound of the invention, may be provided in particles. Particles as used herein means nanoparticles or microparticles (or in some instances larger particles) which can consist in whole or in part of the compound of the invention or the other therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the compound of the invention in a solution or in a semi-solid state. The particles may be of virtually any shape.

[0255] Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney H S et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, poly anhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly (butylmethacrylate), poly (isobutyl methacrylate), poly (hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly (octadecyl acrylate).

[0256] The therapeutic agent(s) may be contained in controlled release systems. The term “controlled release” is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term “sustained release” (also referred to as “extended release”) is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term “delayed release” is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.”

[0257] Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. “Long-term” release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

[0258] It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the compositions and methods described herein are readily apparent from the description of the invention contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

[0259] EXAMPLES

[0260] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0261] Abbreviations:

[0262] Intermediates

[0263] Intermediate 1. l-(cyclopentylmethyl)-3-((5S,7s,10S)-3-methyl-l-(oxetan-3-ylmethyl)-

[0264] 2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0265] 4-nitrophenyl (cyclopentylmethyl)carbamate

[0266] To a solution of cyclopentylmethanamine (0.51 g, 5.14 mmol) in DCM (10 mL) was added pyridine (814 mg, 10.3 mmol, 830 μL), DMAP (628 mg, 5.14 mmol) and (4-nitrophenyl) carbonochloridate (1.04 g, 5.14 mmol). The mixture was stirred at 20 °C for 0.5 h to give the title compound (1.36 g, crude) as a colorless liquid, which was used without further purification. l-(cyclopentylmethyl)-3-((5S,7s,10S)-3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)urea

[0267] To a solution of (4-nitrophenyl) N-(cyclopentylmethyl)carbamate (1.34 g, 5.07 mmol) in DCM (15 mL) was added TEA (1.03 g, 10.1 mmol, 1.41 mL) and (5S,7s,10S)-10-amino-3- methyl-l-(oxetan-3-ylmethyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (1.56 g, 5.07 mmol). The mixture was stirred at 20 °C for 4 h. After completion, the mixture was concentrated under reduced pressure. The residue was diluted with saturated aqueous NH4CI (100 mL) and HO Ac (5 mL) and extracted with ethyl acetate (100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate) to give the title compound (2 g, 91.19%) as a white solid. MS (ESI): mass calcd. for C23H36N4O4: 432.27, found: 433.2 [M+H]+. l-(cyclopentylmethyl)-3-((5S,7s,10S)-3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0268] A solution of malonic acid (106 mg, 1.02 mmol) in AcOH (4.4 mL) and AC2O (4.4 mL) was stirred for 0.5 h at 80 °C. The reaction was added l-(cyclopentylmethyl)-3-((5S,7s,10S)- 3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)urea (440 mg, 1.02 mmol) in dioxane (8.8 mL) and stirred at 80 °C for 1 h. After completion, 3 parallel reactions were worked-up together. The reaction was quenched with H2O (60 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound (2 g, crude) as a yellow solid. MS (ESI): mass calcd. for C26H36N4O6: 500.26, found: 501.2 [M+H]+.

[0269] Intermediate 2. l-(cyclobutylmethyl)-3-((5S,7s,10S)-3-methyl-l-(oxetan-3-ylmethyl)-2,4- dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0270] The title compound was prepared following analogous procedures to those described for Intermediate 1. MS (ESI): mass calcd. for C25H34N4O6: 486.25, found: 487.3 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ ppm 4.60-4.71 (m, 2H), 4.43 (td, J = 6.4, 2.4 Hz, 3H), 3.63- 3.79 (m, 5H), 3.57 (s, 1H), 2.83 (s, 3H), 2.07-2.32 (m, 6H), 1.63-1.96 (m, 9H), 1.38-1.54 (m, 3H), 1.14-1.33 (m, 2H).

[0271] Intermediate 3. l-(cyclopropylmethyl)-3-(3-methyl-2,4-dioxo-l-(((R)-tetrahydrofuran-3- yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0272] The title compound was prepared following analogous procedures to those described for Intermediate 1. MS (ESI): mass calcd. for C25H34N4O6: 486.25, found: 487.3 [M+H]+.

[0273] Intermediate 4. l-benzyl-3-(3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yI)pyrimidine-2,4,6(lH,3H,5H)-trione

[0274] The title compound was prepared following analogous procedures to those described for Intermediate 1. MS (ESI): mass calcd. for C27H32N4O6: 508.23, found: 509.4 [M+H]+.

[0275] Intermediate 5 and Intermediate 6 (5R,7r,10R)-10-hydroxy-3-methyl-l,3 diazadispiro[4.1.57.l5]tridecane-2, 4-dione and (5S,7s,10S)-10-hydroxy-3-methyl-l,3 diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0276] To a suspension of 3-methyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4, 10-trione (8.43 g, 35.7 mmol) in DCM (270 mL) and methanol (27.0 mL, 668 mmol) was added NaBlL (1.41 g, 35.7 mmol) at 0 °C. The reaction was stirred at room temperature for 2.3 h. The resulting mixture was quenched with saturated aqueous NH4CI (200 mL) at 0 °C and stirred vigorously at room temperature for 30 min. Water (100 mL) was added followed by 4:1 mixture of CHCh / IPA (250 mL). After the layers were separated, the aqueous layer was extracted with 4: 1 mixture of CHCh / IPA (150 mL x 3) and the combined organic layer was dried over Na2SO4and concentrated. To the residue was added MeOH (20 mL) and CHCh (15 mL) and warmed up by a heat gun to form a solution. To the solution was added n-heptanes (250 mL) and sonicated to precipitate white particles. The solid was collected by filtration with Buchner funnel, washed with n-heptanes, and concentrated to afford a racemic mixture of alcohol (6.17 g, 25.9 mmol, 73%) as a white solid. The product was then purified by chiral SFC (Column Lux i-Amylose-3, 21.2 x250 mm, 5 pm, isocratic 30% MeOH + 0.1% NH4OH / 70% CO2, 40 °C, 50 mg / mL) to afford Intermediate 5: (5R,7r,10R)-10-hydroxy-3-methyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione as the second peak to elute (2.12 g, 25%, 97.6% ee) as a white solid. MS (ESI): mass calcd. for C12H18N2O3: 238.13, found: 239.2 [M+H]+. Also obtained was Intermediate 6: (5S,7s,10S)-10-hydroxy-3-methyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione as the first peak to elute (2.19 g, 26%, 99.9% ee). MS (ESI): mass calcd. for C31H44N4O6S2: 238.13, found: 239.2 [M+H]+.

[0277] Intermediate 7. 5-(bis(ethylthio)methylene)-l-(cyclobutyhnethyl)-3-((5S,7s,10S)-3- methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)- trione

[0278] 5-(bis(ethylthio)methylene)-l-(cyclobutylmethyl)-3-((5S,7s,10S)-3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0279] To a mixture of (5R,7r,10R)-10-hydroxy-3-methyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (500 mg, 2.10 mmol), 5-(bis(ethylthio)methylene)- l-(cyclobutylmethyl)pyrimidine-2,4,6(lH,3H,5H)-trione (827 mg, 2.52 mmol), and PPh? (834 mg, 3.15 mmol) in THF (21.0 mL) was added DIAD (632 uL, 3.15 mmol) drop wise before stirring at room temperature for 40 min. The resulting mixture was quenched with H2O (20 mL), diluted with EtOAc (30 mL), and extracted with EtOAc (20 mL x3). The combined organic layer was dried over Na2SO4and concentrated in vacuo. The product was purified by reverse phase column chromatography (Cl 8, 10-80%, MeCN / water). Fractions containing the title product were collected and extracted with EtOAc (x3). The combined organic layers were washed with brine, dried ( Na2SO4), and concentrated in vacuo to afford the title product (572 mg, 1.04 mmol, 50%). MS (ESI): mass calcd. for C26H36N4O5S2: 548.21, found: 549.4 [M+H]+.

[0280] Intermediate 8. 5-(bis(ethyIthio)methyIene)-l-(cycIopentyImethyI)-3-((5S,7s,10S)-3- methyI-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yI)pyrimidine-2,4,6(lH,3H,5H)- trione was prepared following analogous procedures to those described for Intermediate 7.

[0281] Intermediate 9. 5-(bis(ethylthio)methylene)-l-(cyclopentylmethyl)-3-((5S,7s,10S)-3- methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0282]

[0283] To 3-(iodomethyl)oxetane (36.3 mg, 178 μmo)l in DMSO (1.48 mL) was added 5- (bis(ethylthio)methylene)-l-(cyclopentylmethyl)-3-((5S,7s,10S)-3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (50.0 mg, 88.9 μmo)l followed by cesium carbonate (70.9 mg, 213 μmo)l. The solution was stirred at rt for 2.5 h and then poured in EtOAc and water. The phases were separated, and the organic phase was washed twice with brine, dried over Na2SO4, filtered, and concentrated to afford the title compound (54 mg, 96%). MS (ESI): mass calcd. for C31H44N4O6S2: 632.27, found: 633.4 [M+H]+

[0284] Intermediate 10. 4-butylisoindoline-l, 3-dione

[0285] 4-butylisoindoline- 1,3-dione was prepared following analogous procedures to those described for 4-amino-7-butylisoindoline-l, 3-dione. MS (ESI): mass calcd. for C12H13NO2: 203.09, found:

[0286] As illustrated in Example 1 and Example 4, the methylamino-substituted compounds in this application usually exist as a pair of interconverting tautomers. The later compounds were not drawn out as pair of tautomers for convenience.

[0287] Example 1. (5E)-5-[amino-(tetrahydrofuran-2-ylmethylamino)methylene]-l-butyl-3-(2- methyl-l,3-dioxo- 2, 4-diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4,6- trione (1)

[0288]

[0289] 11, 14-Dioxa-2,4-diazatrispiro[4.1.2.410.27.l5]heptadecane-l, 3-dione To a mixture of 8,1 l-dioxadispiro[3.2.47.24]tridecan-2-one (15 g, 76.4 mmol) in MeOH (150 mL) and H2O (150 mL) was added TMSCN (15.17 g, 152.9 mmol) and (NEU^CCh (29.38 g, 305.7 mmol) in one portion at 25 °C under N2. After heating at 90 °C for 12 h, the reaction was concentrated under reduced pressure to remove MeOH. The precipitate was collected by filtration, washed with H2O (3 x 100 mL) and dried under vacuum to provide the title compound (13.65 g, 67.1%) as a white solid, which was used directly in the next step without further purification.

[0290] 2-Methyl-ll,14-dioxa- 2, 4-diazatrispiro[4.1.2.410.27.l5]heptadecane-l, 3-dione

[0291] To a mixture of l l,14-dioxa-2,4-diazatrispiro[4.1.2.410.27.l5]heptadecane-l, 3-dione (6 g, 22.5 mmol) in DMF (50 mL) was added Mel (3.52 g, 24.8 mmol) and K2CO3 (3.43 g, 24.8 mmol) in one portion at 0 °C under N2. The reaction was stirred at 25 °C for 12 h. After completion, the crude residue was poured into water (20 mL) and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layer was washed with brine (3 x 30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (petroleum ether : EtOAc = 20 : 1 to 0 : 1) to afford the title compound (5.99 g, 94.8%) as a white solid. ’H NMR (400 MHz, DMSO-d6) 5 ppm 8.65 (s, 1H), 3.83 (s, 4H), 2.79 (s, 3H), 2.27 (d, 7 = 12.8 Hz, 2H), 1.97 (d, 7 = 13.6 Hz, 2H), 1.75 (m, 1.71-1.79, 2H), 1.64 (t, J = 6.4 Hz, 2H), 1.43-1.48 (m, 4H).

[0292] 2-Methyl-2,4-diazadispiro[4.1.57.l5]tridecane-l,3,10-trione

[0293] To a mixture of 2-methyl-l l,14-dioxa-2,4-diazatrispiro[4.1.2.410.27.l5]heptadecane- 1, 3-dione (5.99 g, 21.4 mmol) in acetone (40 mL) and H2O (20 mL) was added TSOH.H2O (8.13 g, 42.7 mmol) in one portion at 25 °C under N2. After stirring for 12 h, the mixture was poured into water (50 mL). The aqueous phase was extracted with EtOAc (3 x 30 mL). The combined organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and concentrated. The crude residue was purified by silica gel column chromatography (petroleum ether : EtOAc = 10 : 1 to 0 : 1) to afford the title compound (3.19 g, 63.2%) as a white solid.

[0294] 10-Amino-2-methyl- 2, 4-diazadispiro[4.1.57.l5]tridecane-l, 3-dione

[0295] To a mixture of 2-methyl-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3, 10-trione (3.19 g, 13.5 mmol) in MeOH (30 mL) was added ammonia (7 M in MeOH, 19.29 mL) at 25 °C. After stirring for 1 h, 10% Pd / C (502.9 mg) was added in one portion. The reaction was stirred at 25 °C for 3 h under H2(15 Psi). Then it was filtered though a Celite pad and the filtrate was concentrated under reduced pressure to provide the title compound (3 g) as a white solid, which was used in the next step without further purification. l-Butyl-3-(3-methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)urea

[0296] To a mixture of 10-amino-2-methyl-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3-dione (3 g, 12.6 mmol) in DCM (30 mL) was added 1-isocyanatobutane (1.25 g, 12.6 mmol), followed by addition of TEA (1.28 g, 12.6 mmol) at 25 °C under N2. After stirring for 30 min, H2O (20 mL) was added and the reaction was extracted with DCM (3 x 15 mL). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (petroleum ether : EtOAc = 5 : 1 to 0 : 1) to provide the title compound (2.7 g, 63.5%) as a white solid. MS (ESI): mass calcd. for C17H28N4O3: 336.22, found: 337.2 [M+H]+. l-Butyl-3-(3-methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2, 4, 6- trione

[0297] To a mixture of l-butyl-3-(2-methyl-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl)urea (2.6 g, 7.7 mmol) and malonic acid (804.2 mg, 7.3 mmol) in AcOH (25 mL) was added AC2O (5.52 g, 54.1 mmol) in one portion at 25 °C under N2. After heating at 80 °C for 4 h, the reaction was poured into water (30 mL) and the aqueous phase was extracted with EtOAc (3 x 20 mL). The combined organic layer was washed with brine, dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (petroleum ether : EtOAc = 20 : 1 to 1 : 1) to provide the title compound (2.3 g, 73.6%) as a yellow oil. MS (ESI): mass calcd. for C20H28N4O5: 404.21, found: 405.2 [M+H]+. l-Butyl-5-(diaminomethylene)-3-(2-methyl-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan- 10-yl)hexahydropyrimidine-2, 4, 6-trione

[0298] To a mixture of l-butyl-3-(2-methyl-l,3-dioxo-2,4-diazadispiro[4.1.57.15]tridecan-10- yl)hexahydropyrimidine-2, 4, 6-trione (2.3 g, 5.7 mmol) and cyanamide (2.39 g, 56.9 mmol) in THF (25 mL) was added Ni(acac)2 (438.3 mg, 1.7 mmol) at 25 °C under N2. After heating at 80 °C for 12 h, the reaction was filtered through a Celite pad. The filtrate was concentrated under reduced pressure and the crude residue was purified by reverse phase HPLC to provide the title compound (0.45 g, 17.7%) as a white solid. MS (ESI): mass calcd. for C21H30N6O5: 446.23, found: 447.2 [M+H]+. l-butyl-5-(diaminomethylene)-3-((5S,7s,10S)-3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione l-Butyl-5-(diaminomethylene)-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (0.23 g, 0.52 mmol) was separated by chiral SFC (column: DAICEL CHIRALCEL OD (250mm*30mm,10 um); mobile phase: CO2-MeOH (0.1% NH3H2O); 40% B with isocratic elution) to provide the title compound (0.064 g, 27.8%, second-eluting peak) as a white solid. MS (ESI): mass calcd. for C21H30N6O5: 446.23, found: 447.2 [M+H]+.

[0299] (5E)-5-[amino-(tetrahydrofuran-2-ylmethylamino)methylene]-l-butyl-3-(2-methyl-l,3- dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (1)

[0300]

[0301] To a solution of l-butyl-5-(diaminomethylene)-3-((5S,7s,10S)-3-methyl-2,4-dioxo- l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (200 mg, 447.93 μmo)l in DMF (2 mL) was added Cs2CO3(437.83 mg, 1.34 mmol) and 2- (bromomethyl)tetrahydrofuran (88.71 mg, 537.52 μmo)l at 25°C. The mixture was stirred at 50 °C for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (1.89 mg, 1%) as a white solid. MS (ESI): mass calcd. for C26H38N6O6: 530.29, found: 531.4 [M+H]+. ' H NMR (400 MHz, DMSO-d6) 5 ppm 10.58-10.76 (m, 1H), 10.10 (s, 1H), 8.65 (s, 1H), 7.73 (s, 1H), 4.53-4.78 (m, 1H), 3.99 (qd, 7 = 4.0, 7.2 Hz, 1H), 3.63-3.83 (m, 4H), 3.37-3.44 (m, 1H), 3.17-3.25 (m, 1H), 2.80 (s, 3H), 2.15-2.32 (m, 4H), 1.94-2.04 (m, 3H), 1.81-1.91 (m, 3H), 1.33-1.56 (m, 6H), 1.20- 1.31 (m, 4H), 0.88 (t, J - 7.2 Hz, 3H). This compound is a mixture of rapidly interconverting tautomers.

[0302] Example 2 and Example 3. (5E)-5-[amino-[(2-hydroxy-2-methyl- propyl)amino]methylene] - 1 -butyl-3- (2- methyl- 1 ,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (2) and ((5E)-5- [amino- [(2-hydroxy-2-methyl-propyl)amino]methylene] - 1 -butyl-3- [4- (2-hydroxy-2- methyl-propyl)-2-methyl-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (3)

[0303] (5E)-5-[amino-[(2-hydroxy-2-methyl-propyl)amino]methylene]-l-butyl-3-(2-methyl-l,3- dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (2) and ((5E)-5-[amino-[(2-hydroxy-2-methyl-propyl)amino]methylene]-l-butyl-3-[4-(2- hydroxy-2-methyl-propyl)-2-methyl-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (3)

[0304] A solution of l-butyl-5-(diaminomethylene)-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (60 mg, 134.38 μmo)l, 2,2-dimethyloxirane (193.8 mg, 2.69 mmol) and K2CO3 (37.14 mg, 269 μmo)l in NMP (2 mL) was heated at 150 °C for 2 h in a sealed tube. After completion, the mixture was filtered over Celite. The residue was purified by prep-HPLC to yield 2 (6.98 mg, 9.6%) as a white solid. MS (ESI): mass calcd. for C25H38N6O6: 518.29, found: 519.3 [M+H]+. ' H NMR (400 MHz, DMSO-d6) 5 ppm 10.73 (t, J = 4.8 Hz, 1H), 10.06 (s, 1H), 8.65 (s, 1H), 7.65 (s, 1H), 4.72-4.90 (m, 1H), 4.64 (t, 7 = 11.2 Hz, 1H), 3.70-3.77 (m, 2H), 3.14 (d, 7 = 5.2 Hz, 2H), 2.80 (s, 3H), 2.15-2.40 (m, 5H), 1.94-2.05 (m, 2H), 1.88 (d, 7 = 12.8 Hz, 1H), 1.33-1.49 (m, 5H), 1.22-1.30 (m, 3H), 1.15 (s, 6H), 0.87 (t, J - 7.2 Hz, 3H). 3 was also obtained from prep-HPLC separation (3.71 mg, 4.5%) as a white solid. MS (ESI): mass calcd. for C29H46N6O7: 590.34, found: 591.4 [M+H]+. ’H NMR (400 MHz, DMSO-d6) 5 ppm 10.66-10.81 (m, 1H), 10.07 (s, 1H), 7.64 (s, 1H), 4.81 (s, 1H), 4.73 (s, 1H), 4.65 (s, 1H), 3.73 0. 7 = 7.2 Hz, 2H), 3.30 (s, 2H), 3.14 (d, J = 5.6 Hz, 2H), 2.85 (s, 3H), 2.69 (t, J = 6.4 Hz, 2H), 2.55 (d, J = 12.8 Hz, 1H), 2.23- 2.38 (m, 2H), 2.14-2.19 (m, 1H), 2.06 (d, J = 12.4 Hz, 1H), 1.93-2.01 (m, 1H), 1.38-1.49 (m, 3H), 1.34 (d, J = 11.2 Hz, 2H), 1.21-1.29 (m, 3H), 1.15 (s, 12H), 0.87 (t, 7 = 7.2 Hz, 3H).

[0305] Example 4. (5E)-5-[amino(methylamino)methylene]-l-butyl-3-[2-methyl-4-(oxetan-3- ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-

[0306] 2,4,6-trione (4)

[0307] 5- [bis(ethylsulfanyl)methylene] - 1 -butyl-3-(2-methyl- 1 ,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione

[0308] To a solution of l-butyl-3-(2-methyl-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl)hexahydropyrimidine-2, 4, 6-trione (360 mg, 890 μmo)l in DMSO (3.6 mL) was added TEA (25.86 mmol, 3.6 mL) and CS2(4.55 g, 59.72 mmol). The mixture was stirred at 20 °C for 1 h. Bromoethane (5.26 g, 48.23 mmol) was added in a single portion. The resulting mixture was stirred at 20 °C for 12 h. The reaction mixture was quenched with saturated aqueous NH4CI (30 mL), diluted with H2O (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2. petroleum ether : ethyl acetate = 1 : 1) to give the title compound (230 mg, 48%) as a yellow solid. MS (ESI): mass calcd. for C25H36N4O5S2: 536.21, found: 537.4 [M+H]+.

[0309] 5-[bis(ethylsulfanyl)methylene]-l-butyl-3-[2-methyl-4-(oxetan-3-ylmethyl)-l,3-dioxo-

[0310] 2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2,4,6-trione

[0311] To a solution of 5-[bis(ethylsulfanyl)methylene]-l-butyl-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (230 mg, 428.5 μmo)l in DMF (2.3 mL) was added Cs2CO3(139.63 mg, 428.5 μmo)l and 3-(iodomethyl)oxetane (127.28 mg, 642.8 μmo)l. The mixture was stirred at 30 °C for 18 h. The reaction was quenched with H2O (15 mL) and extracted with ethyl acetate (15 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, petroleum ether : ethyl acetate = 1 : 1) to give the title compound (200 mg, 77%) as a yellow solid. MS (ESI): mass calcd. for C29H42N4O6S2: 606.25, found: 607.5 [M+H]+.

[0312] (5Z)-l-butyl-5-[ethylsulfanyl(methylamino)methylene]-3-[2-methyl-4-(oxetan-3- yImethyI)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yI]hexahydropyrimidine- 2, 4, 6- trione

[0313] A mixture of 5-[bis(ethylsulfanyl)methylene]-l-butyl-3-[2-methyl-4-(oxetan-3- ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4,6- trione (160 mg, 263.7 μmo)l, methylamine hydrochloride (13.71 mg, 131.8 μmo,l HC1 salt), TEA (53.36 mg, 527 μmo)l in DMF (1.6 mL) was degassed and purged with N2 3 times. The mixture was stirred at 30 °C for 12 h under N2. The reaction mixture was quenched with saturated aqueous NH4CI (15 mL), diluted with H2O (20 mL) and extracted with ethyl acetate (15 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiC>2, petroleum ether : ethyl acetate = 0 : 1) to give the title compound (110 mg, 72%) as a white solid. MS (ESI): mass calcd. for C28H41N5O6S: 575.28, found: 576.5 [M+H]+.

[0314] (5E)-5-[amino(methylamino)methylene]-l-butyl-3-[2-methyl-4-(oxetan-3-ylmethyl)-l,3- dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione (4)

[0315] To a solution of (5Z)-l-butyl-5-[ethylsulfanyl(methylamino)methylene]-3-[2-methyl- 4-(oxetan-3-ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (65 mg, 113 μmo)l in THF (0.7 mL) was added NH3.H2O (338.7 μmo,l 52.2 μL, 25% w / w NH3in water). The mixture was degassed and purged with N2 3 times, stirred at 30 °C for 16 h under N2. The reaction mixture was quenched with H2O (0.5 mL). The residue was purified by prep-HPLC (column: Phenomenex luna C18 100 x 40 mm x 3 um; mobile phase: H2O (0.2% FA)-ACN; gradient 35-65% B) to the title compound (22 mg, 35%) as a white solid. MS (ESI): mass calcd. for C26H38N6O6: 530.29, found: 531.4 [M+H]+. ' H NMR (400 MHz, DMSO-d6) 5 ppm 10.38 (d, 7 = 4.8 Hz, 1H), 10.05 (s, 1H), 7.65 (s, 1H), 4.63 (dd, J = 6.4, 7.6 Hz, 3H), 4.42 (q, J = 6.0 Hz, 2H), 3.74 (t, J = 7.2 Hz, 2H), 3.63- 3.70 (m, 2H), 2.85 (d, 7 = 5.2 Hz, 3H), 2.82 (s, 3H), 2.45 (s, 1H), 2.28-2.42 (m, 2H), 2.25 (d, J = 12.8 Hz, 2H), 2.09-2.20 (m, 3H), 1.85 (d, 7 = 11.2 Hz, 1H), 1.33-1.50 (m, 5H), 1.25 (qd, 7 = 7.2, 14.8 Hz, 3H), 0.87 0. 7 = 7.2 Hz, 3H). (6)

[0316] Example 4 (50 mg) was separated by chiral SFC (ChiralPak IH, 250 x 30 mm, 10 um; mobile phase: CO2-MeOH (0.1% NH3H2O); 30% B with isocratic elution) to give 5 (19.22 mg) from the first eluting peak from chiral SFC separation as white solid. MS (ESI): mass calcd. for C26H38N6O6: 530.29, found: 5 was obtained from the second eluting peak from chiral SFC separation as white solid. MS (ESI): mass calcd. for C26H38N6O6: 530.29, found: 531.5 [M+H]+. ’H NMR (400 MHz, DMSO-

[0317] Example 7. (E)-5-(amino(methylamino)methylene)-l-butyl-3-(3-methyl-2,4-dioxo-l- ((tetrahydro-2H-pyran-4-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6( 1H,3H,5H) -trione (7)

[0318]

[0319] 3-methyl-l-((tetrahydro-2H-pyran-4-yl)methyl)-ll,14-dioxa-l,3- diazatrispiro[4.1.2.410.27.l5]heptadecane-2, 4-dione

[0320] To a solution of 2-methyl-ll,14-dioxa-2,4-diazatrispiro[4.1.2.410.27.l5]heptadecane-

[0321] 1,3-dione (3 g, 10.7 mmol) and 4-(iodomethyl)tetrahydropyran (4.84 g, 21.4 mmol) in DMF (15 mL) was added Cs2CO3(6.97 g, 21.4 mmol). The solution was stirred at 80 °C for 16 h. After completion, the suspension was filtered through silica gel and washed with ethyl acetate (90 mL x 3). The organic phase was washed with saturated aqueous NaHCO3(60 mL x 2), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography SiO2. petroleum ether : ethyl acetate = 30 : 1 to 4 : 1) to give the title compound (2.5 g, 62%) as a yellow solid. MS (ESI): mass calcd. for C20H30N2O5: 378.22, found: 379.2 [M+H]+.

[0322] 3-methyl-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecane-

[0323] 2,4,10-trione

[0324] To a solution of 2-methyl-4-(tetrahydropyran-4-ylmethyl)-ll,14-dioxa-2,4- diazatrispiro[4.1.2.410.27.l5]heptadecane-l, 3-dione (2.5 g, 6.6 mmol) in acetone (25 mL) and H2O (12.5 mL) was added TSOH.H2O (2.51 g, 13.2 mmol). The solution was stirred at 30 °C for 16 h. After completion, the mixture was adjusted to pH = 9 with saturated aqueous NaHCO3and extracted with DCM (80 mL x 3). The combined organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound (2.2 g, crude) as a yellow solid. MS (ESI): mass calcd. for C18H26N2O4: 334.19, found: 335.1 [M+H]+.

[0325] 10-amino-3-methyl-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0326] A solution of 2-methyl-4-(tetrahydropyran-4-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecane-l, 3, 10-trione (1.9 g, 5.68 mmol) in NH3 / McOH (7 M, 16.23 mL) was stirred at 20 °C for 1 h under N2. Raney-Ni (243.4 mg) was added and the reaction was stirred at 20 °C for 16 h under H2(15 psi). After completion, the mixture was filtered through a celite pad. The filtrate was concentrated under reduced pressure to give the title compound (1.9 g, crude) as a white solid. MS (ESI): mass calcd. for C18H29N3O3: 335.22, found: 336.5 [M+H]+l-butyl-3-(3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)urea

[0327] The title compound was synthesized using the same conditions for the urea synthesis in

[0328] Example 1. l-butyl-3-(3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0329] The title compound was synthesized using the same conditions for the barbiturate synthesis in Example 1.

[0330] 5-(bis(ethylthio)methylene)-l-butyl-3-(3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4- yl)methyl)-l,3-diazadispiro[4.1.57.15]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0331] The title compound was synthesized using the same conditions for the bis(ethylthio)methylene synthesis in Example 4.

[0332] (5Z) - 1 -butyl-5- [ethylsulfanyl(methylamino)methylene] -3- [2-methyl- 1 ,3 -dioxo-4- (tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2,4,6-trione

[0333] To a solution of 5-[bis(ethylsulfanyl)methylene]-l-butyl-3-[2-methyl-l,3-dioxo-4- (tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine- 2, 4, 6-trione (360 mg, 567.06 μmo)l in DMF (3 mL) was added a solution of methylamine hydrochloride (19.14 mg, 284 μmo)l and TEA (114.76 mg, 1.13 mmol) in DMF (3 mL) at 20 °C. The resulting mixture was stirred at 20 °C for 12 h. The reaction was quenched with H2O (20 mL) and extracted with ethyl acetate (25 mL x 2). The combined organic layer was washed with saturated aqueous NH4CI, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC SiO2. petroleum ether : ethyl acetate = 0 : 1) to give the title compound (140 mg, 40%) as a colorless oil. MS (ESI): mass calcd. for C30H45N5O6S: 603.31, found: 604.5 [M+H]+.

[0334] (E)-5-(amino(methylamino)methylene)-l-butyl-3-(3-methyl-2,4-dioxo-l-((tetrahydro- 2H-pyran-4-yI)methyI)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yI)pyrimidine- 2,4,6(lH,3H,5H)-trione (7)

[0335] To a solution of (5Z)-l-butyl-5-[ethylsulfanyl(methylamino)methylene]-3-[2-methyl- l,3-dioxo-4-(tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (25 mg, 41.4 μmo)l in THF (0.5 mL) was added NH3.H2O (1 mL). The mixture was stirred at 30 °C for 16 h. The reaction was concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (6.3 mg, 27%) as a white solid. MS (ESI): mass calcd. for C28H42N6O6: 558.32, found: 559.3 [M+H]+. ’H NMR (400 MHz, DMSO-d6) 5 ppm 10.39 (d, J = 4.8 Hz, 1H), 10.05 (s, 1H), 7.66 (s, 1H), 4.60-4.78 (m, 1H), 3.86 (d, 7 = 11.2 Hz, 2H), 3.75 0. 7 = 7.2 Hz, 2H), 3.18-3.30 (m, 4H), 2.86 (s, 3H), 2.85 (s, 3H), 2.44-2.48 (m, 1H), 2.33-2.42 (m, 1H), 2.25-2.31 (m, 2H), 2.13-2.21 (m, 3H), 2.00-2.10 (m, 1H), 1.82 (d, J = 11.2 Hz, 1H), 1.59 (d, J = 11.6 Hz, 2H), 1.36-1.50 (m, 5H), 1.17-1.31 (m, 5H), 0.88 (t, 7 = 7.2 Hz, 3H).

[0336] Example 8 and Example 9. (E)-5-(amino(methylamino)methylene)-l-butyl-3- ((5S,7s,10S)-3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (8) and (E)-5- (amino(methylamino)methylene)-l-butyl-3-((5R,7r,10R)-3-methyl-2,4-dioxo-l- ((tetrahydro-2H-pyran-4-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6( 1H,3H,5H) -trione (9)

[0337] Example 7 (115 mg) was separated by chiral SFC ( column: REGIS (S,S)WHELK- 01(250mmx25mm,10 um); mobile phase: CO2-IPA; 60% B with isocratic elution) to give 8 (36.38 mg) from the first eluting peak from chiral SFC separation as a white solid. MS (ESI): mass calcd. for C28H42N6O6: 558.32, found: 559.3 [M+H]+. ’H NMR (400 MHz, DMSO-d6) 5 ppm 10.38 (d, 7 = 4.4 Hz, 1H), 10.05 (s, 1H), 7.65 (s, 1H), 4.55-4.72 (m, 1H), 3.85 (d, 7 = 11.2 Hz, 2H), 3.74 (t, J = 7.2 Hz, 2H), 3.17-3.31 (m, 4H), 2.80-2.88 (m, 6H), 2.43-2.48 (m, 1H), 2.22-2.42 (m, 3H), 2.13-2.21 (m, 3H), 1.98-2.08 (m, 1H), 1.81 (d, 7 = 11.6 Hz, 1H), 1.58 (d, J = 12.4 Hz, 2H), 1.35-1.52 (m, 5H), 1.15-1.31 (m, 5H), 0.87 (t, J = 7.2 Hz, 3H). 9 (35.12 mg) was obtained from the second eluting peak from chiral SFC separation as a white solid. MS (ESI): mass calcd. for C28H42N6O6: 558.32, found: 559.3 [M+H]+. ’H NMR (400 MHz, DMSO- d6) 5 ppm 10.38 (d, 7 = 4.8 Hz, 1H), 10.05 (s, 1H), 7.65 (s, 1H), 4.58-4.71 (m, 1H), 3.85 (d, J = 10.8 Hz, 2H), 3.74 0. 7 = 7.2 Hz, 2H), 3.17-3.30 (m, 4H), 2.80-2.89 (m, 6H), 2.42-2.47 (m, 1H), 2.24-2.40 (m, 3H), 2.13-2.23 (m, 3H), 1.98-2.09 (m, 1H), 1.81 (d, 7 = 11.6 Hz, 1H), 1.58 (d, J = 12.0 Hz, 2H), 1.34-1.51 (m, 5H), 1.13-1.31 (m, 5H), 0.87 (t, J = 7.2 Hz, 3H).

[0338] Example 10. (E)-5-(amino(methylamino)methylene)-l-butyl-3-((5S,7r,10S)-3-methyl- 2,4-dioxo-l-(((S)-tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6(lH,3H,5H)-trione (10)

[0339] 10

[0340] Synthetic scheme:

[0341] l-butyl-5-(l,3-dithian-2-ylidene)-3-(3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0342] To a solution of l-butyl-3-(2-methyl-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl)hexahydropyrimidine-2, 4, 6-trione (5.0 g, 12.3 mmol) in DMSO (50.0 mL) was added TEA (6.2 g, 61.8 mmol) and Cs2(2.8 g, 37 mmol) at 25 °C for 1 h. 1,3-dibromopropane (7.4 g, 37 mmol) was added at 0 °C. The mixture was stirred at 25 °C for 2 h. After completion, the reaction was triturated with ice water (100 mL) at 25 °C for 30 min. The reaction was filtered. The filter cake was washed with H2O (100 mL) and dried under reduced pressure. The crude product was triturated with MTBE (100 mL) at 25 °C for 30 min to give the title compound (20 g, 77%) as a white solid. MS (ESI): mass calcd. for C24H32N4O5S2: 520.18, found: 521.3 [M+H]+. l-butyl-5-(l,3-dithian-2-ylidene)-3-((5S,7s,10S)-3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yI)pyrimidine-2,4,6(lH,3H,5H)-trione l-butyl-5-(l,3-dithian-2-ylidene)-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (20 g) was separated by prep-SFC (column: ChiralPak IH, 250x50mm, 10 um; mobile phase: CCL-MeOH; 60% B with isocratic elution) to give the title compound (12.9 g) from the second eluting peak as a white solid. MS (ESI): mass calcd. for C24H32N4O5S2: 520.18, found: 521.2 [M+H]+. l-butyl-5-(l,3-dithian-2-ylidene)-3-((5S,7r,10S)-3-methyl-2,4-dioxo-l-(((S)- tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2,4,6(lH,3H,5H)-trione

[0343] To a mixture of l-butyl-5-(l,3-dithian-2-ylidene)-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (0.2 g, 384 μmo)l and (3R)-3-(iodomethyl)tetrahydrofuran (162.9 mg, 768.3 μmo)l in DMF (2 mL) was added NaH (30.73 mg, 768.3 μmo,l 60% purity) at 0 °C. The mixture was stirred at 20 °C for 6 h. After completion, the mixture was poured into NH4CI (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by prep-HPLC to the title compound (40 mg, 14.8%) as a white solid. MS (ESI): mass calcd. for C29H40N4O6S2: 604.24, found: 605.2 [M+H]+. l-butyl-5-(diaminomethylene)-3-((5S,7r,10S)-3-methyl-2,4-dioxo-l-(((S)- tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2,4,6(lH,3H,5H)-trione and (5Z,5'Z)-5,5'-(((disulfanediylbis(propane-3,l- diyl))bis(sulfanediyl))bis(aminomethaneylylidene))bis(l-butyl-3-((5S,7r,10S)-3-methyl- 2,4-dioxo-l-(((S)-tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6( 1H,3H,5H) -trione)

[0344]

[0345] To a mixture of l-butyl-5-(l,3-dithian-2-ylidene)-3-[2-methyl-l,3-dioxo-4-[[(3S)- tetrahydrofuran-3-yl]methyl]-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine- 2, 4, 6-trione (40 mg, 66.14 μmo)l in THF (0.5 mL) was added NH4OH (25.47 μL, 661.4 μmo,l 25% NH3in water). The mixture was stirred at 30 °C for 12 h. After completion, the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give 1- butyl-5-(diaminomethylene)-3-((5S,7r,10S)-3-methyl-2,4-dioxo-l-(((S)-tetrahydrofuran-3- yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (2 mg, 5%) as a white solid. MS (ESI): mass calcd. for C26H38N6O6: 530.29, found: 531.3 [M+H]+. (((disulfanediylbis(propane-3,l-diyl))bis(sulfanediyl))bis(aminomethaneylylidene))bis(l- butyl-3-((5S,7r,10S)-3-methyl-2,4-dioxo-l-(((S)-tetrahydrofuran-3-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione) (30 mg, 35.8%) was also obtained from prep-HPLC purification as a white solid. MS (ESI): mass calcd. for C58H84N10O12S4: 1240.52, found: 1241.5 [M+H]+.

[0346] (E)-5-(amino(methylamino)methylene)-l-butyl-3-((5S,7r,10S)-3-methyl-2,4-dioxo-l- (((S)-tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2,4,6(lH,3H,5H)-trione (10)

[0347]

[0348] To a solution of (5Z)-5-[amino-[3-[3-[(Z)-amino-[l-butyl-3-[2-methyl-l,3-dioxo-4- [[(3S)-tetrahydrofuran-3-yl]methyl]-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]-2,4,6-trioxo- hexahydropyrimidin-5-ylidene]methyl]sulfanylpropyldisulfanyl]propylsulfanyl]methylene]- l-butyl-3-[2-methyl-l,3-dioxo-4-[[(3S)-tetrahydrofuran-3-yl]methyl]-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione (30 mg, 24.16 μmo)l in DMF (0.3 mL) was added a solution of TEA (12.22 mg, 120.8 μmo)l and methylamine hydrochloride (8.16 mg, 120.81 μmo)l in DMF (0.3 mL). The mixture was stirred at 40 °C for 12 h. After completion, the mixture was poured into water (5 mL) and extracted with ethyl acetate (5 mL x 3). The combined organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (4.79 mg, 36.4%) as a white solid. MS (ESI): mass calcd. for C27H40N6O6: 544.30, found: 545.3 [M+H]+. ’H NMR (400 MHz, DMSO-d6) 5 ppm 10.38 (d, J = 4.8 Hz, 1H), 10.04 (s, 1H), 7.66 (s, 1H), 4.56-4.75 (m, 1H), 3.71-3.82 (m, 3H), 3.61-3.69 (m, 2H), 3.49 (dd, 7 = 4.8, 8.4 Hz, 1H), 3.35-3.36 (m, 1H), 2.82-2.87 (m, 6H), 2.68-2.76 (m, 1H), 2.14-2.49 (m, 8H), 1.93-2.01 (m, 1H), 1.79 (d, 7= 10.4 Hz, 1H), 1.60-1.67 (m, 1H), 1.36-1.49 (m, 5H), 1.21-1.29 (m, 3H), 0.87 (t, 7 = 7.2 Hz, 3H).

[0349] Example 11 was prepared in a similar fashion to Example 10 using (3S)-3- (iodomethyl)tetrahydrofuran instead of (3R)-3-(iodomethyl)tetrahydrofuran.

[0350] Example 12. (5E)-5-[amino(methylamino)methylene]-l-benzyl-3-[2-methyl-l,3-dioxo-4- (tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (12)

[0351] l-benzyl-3-(3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.15]tridecan-10-yl)urea

[0352]

[0353] The title compound was prepared in a similar fashion to the urea synthesis in Example 7 using benzyl isocyanate instead of butyl isocyanate. l-benzyl-3-(3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.15]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0354] The title compound was prepared in a similar fashion to the barbiturate synthesis in Example 7. l-benzyl-5-[bis(ethylsulfanyl)methylene]-3-[2-methyl-l,3-dioxo-4-(tetrahydropyran-4- ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione

[0355] To a solution of l-benzyl-3-[2-methyl-l,3-dioxo-4-(tetrahydropyran-4-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione (280 mg, 521.8 μmo)l in DMSO (2.8 mL) was added TEA (2.04 g, 20.12 mmol, 2.80 mL) and CS2 (3.54 g, 46.45 mmol, 2.80 mL). The mixture was stirred at 20 °C for 1 h. Bromoethane (4.09 g, 37.52 mmol) was added. The resulting mixture was stirred at 20 °C for 12 h. The reaction was quenched with H2O (20 mL) and extracted with ethyl acetate (30 mL x 2). The combined organic layer was washed with saturated aqueous NH4CI, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether : ethyl acetate = 99 : 1 to 77 : 23) to give the title compound (170 mg, 47%) as a yellow solid. MS (ESI): mass calcd. for C34H44N4O6S2: 668.27, found: 669.2 [M+H]+.

[0356] (5Z)-l-benzyl-5-[ethylsulfanyl(methylamino)methylene]-3-[2-methyl-l,3-dioxo-4-

[0357] (tetrahydropyran-4-yImethyI)-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2,4,6-trione

[0358] To a solution of l-benzyl-5-[bis(ethylsulfanyl)methylene]-3-[2-methyl-l,3-dioxo-4- (tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine- 2,4,6-trione (116 mg, 173.4 μmo)l in DMF (1.1 mL) was added a solution of methylamine hydrochloride (5.85 mg, 86.71 μmo)l and TEA (35.1 mg, 346.86 μmo)l in DMF (1.1 mL) at 20 °C. The resulting mixture was stirred at 20 °C for 3 h. The reaction was quenched with H2O (20 mL) and extracted with ethyl acetate (25 mL x 2). The combined organic layer was washed with saturated aqueous NH4CI, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, petroleum ether : ethyl acetate = 1 : 1) to give the title compound (75 mg, 65%) as a white solid. MS (ESI): mass calcd. for C33H43N5O6S: 637.29, found: 638.3 [M+H]+.

[0359] (5E)-5- [amino(methylamino)methylene] - 1 -benzyl-3- [2-methyl- 1 ,3-dioxo-4- (tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (12)

[0360]

[0361] To a solution To a solution of (5Z)-l-benzyl-5- [ethylsulfanyl(methylamino)methylene]-3-[2-methyl-l,3-dioxo-4-(tetrahydropyran-4- ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione (65 mg, 101.9 μmo)l in THF (0.5 mL) was added NH4OH (2.81 mmol, 433.3 μL, 25% w / w NH3in water). The mixture was stirred at 20 °C for 2 h. The reaction was concentrated under reduced pressure. The crude product was purified by prep-HPLC to give the title compound (51.3 mg, 80%) as a white solid. MS (ESI): mass calcd. for C31H40N6O6: 592.30, found: 593.3

[0362] Example 13 and Example 14. (E)-5-(amino(methylamino)methylene)-l-benzyl-3- ((5S,7s,10S)-3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (13) and (E)-5- (amino(methylamino)methylene)-l-benzyl-3-((5R,7r,10R)-3-methyl-2,4-dioxo-l- ((tetrahydro-2H-pyran-4-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6(lH,3H,5H) -trione (14)

[0363]

[0364] Example 12 (45 mg) was purified by chiral SFC (column: DAICEL CHIRALPAK IC (250mmx30mm,10 um); mobile phase: CO2-EtOH (0.1% NH3.H2O); 50% B with isocratic elution) to yield 13 (19.4 mg) from the first eluting peak from chiral SFC separation as a white solid. MS (ESI): mass calcd. for

[0365] Example 15 and Example 16. (E)-5-(amino(methylamino)methylene)-l-

[0366] (cyclobutylmethyl) -3- ((5R,7r, 10R)-3 -methyl-2,4-dioxo- l-((tetrahydro-2H-pyran-4- yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0367] (15) and (E)-5-(amino(methylamino)methylene)-l-(cyclobutylmethyl)-3-((5S,7s,10S)-3- methyl-2,4-dioxo- 1 -((tetrahydro-2H-pyran-4-yl)methyl) -1,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (16)

[0368] Synthetic scheme:

[0369]

[0370] 3-methyl-l-((tetrahydro-2H-pyran-4-yl)methyl)-ll,14-dioxa-l,3- diazatrispiro[4.1.2.410.27.l5]heptadecane-2, 4-dione

[0371] To a solution of 2-methyl-l l,14-dioxa-2,4-diazatrispiro[4.1.2.410.27.l5]heptadecane- 1, 3-dione (3 g, 10.7 mmol) and 4-(iodomethyl)tetrahydropyran (4.84 g, 21.4 mmol) in DMF (15 mL) was added Cs2CO3(6.97 g, 21.4 mmol). The solution was stirred at 80 °C for 16 h. After completion, the suspension was filtered through silica gel and washed with ethyl acetate (90 mL x 3). The organic phase was washed with saturated aqueous NaHCO3(60 mL x 2), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiCL, petroleum ether : ethyl acetate = 30 : 1 to 4 : 1) to give the title compound (2.5 g, 62%) as a yellow solid. MS (ESI): mass calcd. for C20H30N2O5: 378.22, found: 379.2 [M+H]+.

[0372] 3-methyl-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecane- 2,4,10-trione

[0373] To a solution of 2-methyl-4-(tetrahydropyran-4-ylmethyl)-ll,14-dioxa-2,4- diazatrispiro[4.1.2.410.27.l5]heptadecane-l, 3-dione (2.5 g, 6.6 mmol) in acetone (25 mL) and H2O (12.5 mL) was added TsOH.ILO (2.51 g, 13.2 mmol). The solution was stirred at 30 °C for 16 h. After completion, the mixture was adjusted to pH = 9 with saturated aqueous NaHCO3and extracted with DCM (80 mL x 3). The combined organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the title compound (2.2 g, crude) as a yellow solid. MS (ESI): mass calcd. for C18H26N2O4: 334.19, found: 335.1 [M+H]+.

[0374] 10-amino-3-methyl-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0375] A solution of 2-methyl-4-(tetrahydropyran-4-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecane-l, 3, 10-trione (1.9 g, 5.68 mmol) in NH3 / McOH (7 M, 16.23 mL) was stirred at 20 °C for 1 h under N2. Raney-Ni (243.4 mg) was added and the reaction was stirred at 20 °C for 16 h under H2(15 psi). After completion, the mixture was filtered through a celite pad. The filtrate was concentrated under reduced pressure to give the title compound (1.9 g, crude) as a white solid. MS (ESI): mass calcd. for C18H29N3O3: 335.22, found: 336.5 [M+H]+l-(3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)urea

[0376] To a solution of 10-amino-2-methyl-4-(tetrahydropyran-4-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecane-l, 3-dione (1.14 g, 3.4 mmol) in H2O (12 mL) was added potassium cyanate (275.7 mg, 3.4 mmol). The reaction was stirred at 50 °C for 16 h. After completion, the reaction was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (60 mL), MeOH (6 mL) and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, Ethyl acetate : MeOH = 100 : 0 to 30 : 1) to give the title compound (1.2 g, 93%) as a white solid. MS (ESI): mass calcd. for To a solution of [2-methyl-l,3-dioxo-4-(tetrahydropyran-4-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl]urea (1.2 g, 3.17 mmol) in HOAc (12 mL) was added acetic anhydride (2.27 g, 22.19 mmol) and malonic acid (659.9 mg, 6.34 mmol). The solution was stirred at 80 °C for 2 h. After completion, the reaction was poured into water (100 mL) and extracted with ethyl acetate (60 mL x 2). The combined organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2. petroleum ether : ethyl acetate = 20 : 1 to 1 : 1) to give the title compound (830 mg, 59%) as a white solid. MS (ESI): mass calcd. for

[0377] To a solution of l-[2-methyl-l,3-dioxo-4-(tetrahydropyran-4-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione (560 mg, 1.25 mmol) in DMSO (10 mL) was added TEA (507.7 mg, 5.02 mmol) and CS2(286.5 mg, 3.76 mmol). The reaction was stirred at 40 °C for 2 h. 1,3-dibromopropane (506.4 mg, 2.51 mmol) was added and the reaction was stirred at 20 °C for 1 h. After completion, the reaction was partitioned between saturated aqueous NH4CI (10 mL) and ethyl acetate (20 mL). The organic phase was separated, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether : ethyl acetate = 41 : 59) to give the title compound (204 mg, 29%) as a yellow solid. MS (ESI): mass calcd. for

[0378]

[0379] To a solution of 5-(l,3-dithian-2-ylidene)-l-[2-methyl-l,3-dioxo-4-(tetrahydropyran- 4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione (170 mg, 302 μmo)l in DMF (6 mL) was added K2CO3(208.8 mg, 1.51 mmol) and iodomethylcyclobutane (118.5 mg, 604 μmo)l. The reaction was stirred at 20 °C for 16 h. After completion, the reaction was partitioned between saturated aqueous NH4CI (5 mL) and ethyl acetate (8 mL). The organic phase was separated, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether : ethyl acetate = 1 : 1) to give the title compound (90 mg, 47%) as a yellow solid. MS (ESI): mass calcd. for C31H42N4O6S2: 630.25, found: 631.3 [M+H]+. l-(cyclobutylmethyl)-5-(diaminomethylene)-3-(3-methyl-2,4-dioxo-l-((tetrahydro-2H- pyran-4-yI)methyI)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yI)pyrimidine- 2,4,6(lH,3H,5H)-trione and (5Z,5'E)-5,5'-(((disulfanediylbis(propane-3,l- diyI))bis(suIfanediyI))bis(aminomethaneyIyIidene))bis(l-(cycIobutyImethyI)-3-(3-methyI- 2,4-dioxo-l-((tetrahydro-2H-pyran-4-yI)methyI)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yI)pyrimidine-2,4,6(lH,3H,5H)-trione)

[0380] To a solution of l-(cyclobutylmethyl)-5-(l,3-dithian-2-ylidene)-3-[2-methyl-l,3- dioxo-4-(tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (170 mg, 269.49 μmo)l in THF (8.5 mL) was added NH4OH (1.13 g, 8.08 mmol, 1.25 mL, 25% w / w NH3in water). The mixture solution was stirred at 30 °C for 16 h. After the completion, the reaction was concentrated under reduced pressure. The residue was purified by prep-HPLC to give 1- (cyclobutylmethyl) -5- (diaminomethylene)-3-(3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (40 mg, 27%) as a white solid. MS (ESI): mass calcd. for C28H40N6O6: 556.30, found: 557.3 [M+H]+. (5Z,5'E)- 5 ,5’-(((disulfanediylbis(propane-3 , 1 -diyl))bis(sulfanediyl))bis(aminomethaneylylidene))bis( 1 - (cyclobutylmethyl)-3-(3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione) was also obtained from prep-HPLC (45 mg, 12.9%) as a white solid. MS (ESI): mass calcd. for C62H88N10O12S4: 1292.55, found: 1293.8 [M+H]+.

[0381] (E)-5-(amino(methylamino)methylene)-l-(cyclobutylmethyl)-3-(3-methyl-2,4-dioxo-l- ((tetrahydro-2H-pyran-4-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6( 1H,3H,5H) -trione

[0382] To a solution of (5Z)-5-[amino-[3-[3-[(E)-amino-[l-(cyclobutylmethyl)-3-[2-methyl-

[0383] 1.3-dioxo-4-(tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]-2,4,6- trioxo-hexahydropyrimidin-5 - ylidene]methyl]sulfanylpropyldisulfanyl]propylsulfanyl]methylene]-l-(cyclobutylmethyl)-3- [2-methyl-l,3-dioxo-4-(tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (45 mg, 34.8 μmo)l in DMF (5 mL) was added TEA (17.6 mg, 173.92 μmo)l and methylamine hydrochloride (11.74 mg, 173.9 μmo)l. The mixture solution was stirred at 30 °C for 16 h. After completion, the mixture was concentrated under reduced pressure to give the title compound (90 mg, crude) as a white solid. MS (ESI): mass calcd. for C29H42N6O6: 570.32, found: 571.3 [M+H]+.

[0384] (E)-5-(amino(methylamino)methylene)-l-(cyclobutylmethyl)-3-((5S,7s,10S)-3-methyl-

[0385] 2.4-dioxo-l-((tetrahydro-2H-pyran-4-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6(lH,3H,5H)-trione (15) and (E)-5-(amino(methylamino)methylene)- l-(cyclobutylmethyl)-3-((5R,7r,10R)-3-methyl-2,4-dioxo-l-((tetrahydro-2H-pyran-4- yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (16)

[0386]

[0387] (5E)-5-[amino(methylamino)methylene]-l-(cyclobutylmethyl)-3-[2-methyl-l,3- dioxo-4-(tetrahydropyran-4-ylmethyl)-2,4-diazadispiro[4.1.57.15]tridecan- 10- yl]hexahydropyrimidine-2, 4, 6-trione (90 mg) was separated by chiral SFC (condition: column: DAICEL CHIRALCEL OX (250mmx30mm,10 um); mobile phase: CO2-MeOH (0.1% NH3H2O); 50% B with isocratic elution) to give 15 (12 mg) from the first eluting peak as a white solid. MS (ESI): mass calcd. for

[0388] Example 17. (5E)-5-[amino(methylamino)methylene]-l-benzyl-3-[2-methyl-l,3-dioxo-4- [[(3S)-tetrahydrofuran-3-yl]methyl]-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (17)

[0389] l-benzyl-3-(3-methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)urea

[0390] The title compound was prepared in a similar fashion to the urea synthesis in Example 1 using benzyl isocyanate instead of butyl isocyanate. l-benzyl-3-(3-methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-

[0391] 2,4,6(lH,3H,5H)-trione

[0392] The title compound was prepared in a similar fashion to the barbiturate synthesis in

[0393] Example 1. l-benzyl-5-[bis(ethylsulfanyl)methylene]-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yI)hexahydropyrimidine-2, 4, 6-trione

[0394] To a solution of l-benzyl-3-(2-methyl-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan- 10-yl)hexahydropyrimidine-2, 4, 6-trione (1 g, 2.28 mmol) in DMSO (10 mL) was added TEA (7.27 g, 71.85 mmol) and CS2 (12.63 g, 165.88 mmol). After addition, the mixture was stirred at 25 °C for 1 h. Bromoethane (14.6 g, 134 mmol) was added. The resulting mixture was stirred at 25 °C for 12 h. The reaction was quenched with saturated aqueous NH4CI (30 mL), diluted with H2O (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2. petroleum ether : ethyl acetate = 1 : 0 to 1 : 1) to give the title compound (810 mg, 62%) as a yellow solid. MS (ESI): mass calcd. for C28H34N4O5SX: 570.20, found: 571.2 [M+H]+. (5Z)-l-benzyl-5-[ethylsulfanyl(methylamino)methylene]-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione

[0395] A mixture of l-benzyl-5-[bis(ethylsulfanyl)methylene]-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (700 mg, 1.23 mmol), methylamine hydrochloride (41.4 mg, 613.3 μmo)l, TEA (248.22 mg, 2.45 mmol) in DMF (7 mL) was degassed and purged with N2 3 times. The mixture was stirred at 25 °C for 5 h under N2. The reaction mixture was quenched with saturated aqueous NH4CI (30 mL), diluted with H2O (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether : ethyl acetate = 1 : 0 to 1 : 2) to give the title compound (307 mg, 46%) as a yellow solid. MS (ESI): mass calcd.

[0396] To a solution of (5Z)-l-benzyl-5-[ethylsulfanyl(methylamino)methylene]-3-(2-methyl- l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (420 mg, 778.29 μmo)l in THF (4.2 mL) was added NH4OH (1.56 mmol, 239.79 μL, 25% w / w NH3in water). The mixture was degassed and purged with N2 3 times and stirred at 30 °C for 1 h under N2. The mixture was concentrated under reduced pressure to give the title compound (410 mg, 89%) as a yellow solid, which was used in the next step without further purification. MS (ESI): mass calcd. for C25H30N6O5: 494.23, found: 495.3 [M+H]+.

[0397] (5E)-5-[amino(methylamino)methylene]-l-benzyl-3-[2-methyl-l,3-dioxo-4-[[(3S)- tetrahydrofuran-3-yl]methyl]-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (17)

[0398] To a solution of (5E)-5-[amino(methylamino)methylene]-l-benzyl-3-(2-methyl-l,3- dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (410 mg, 696.4 μmo)l in DMF (4.1 mL) was added Cs2CO3(453.8 mg, 1.39 mmol) and (3R)-3- (iodomethyl)tetrahydrofuran (147.66 mg, 696 μmo)l. The mixture was stirred at 40 °C for 16 h. The reaction was cooled to 20 °C and a second portion of (3R)-3- (iodomethyl)tetrahydrofuran (147.66 mg, 696 μmo)l was added. The reaction was heated at 50 °C for 5 h. The reaction was quenched with H2O (50 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (27.28 mg, 6%) as a white solid.

[0399] Example 18 and Example 19. (E)-5-(amino(methylamino)methylene)-l-benzyl-3- ((5R,7s,10R)-3-methyI-2,4-dioxo-l-(((S)-tetrahydrofuran-3-yI)methyI)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yI)pyrimidine-2,4,6(lH,3H,5H)-trione (18) & (E)-5- (amino(methylamino)methylene)-l-benzyl-3-((5S,7r,10S)-3-methyl-2,4-dioxo-l-(((S)- tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-

[0400] 2,4,6(lH,3H,5H)-trione (19)

[0401] Example 17 (27.28 mg) was separated by chiral SFC (DAICEL CHIRALPAK IK (250 mm x 25 mm, 10 um); mobile phase

[0402] Example 20 was prepared in a similar fashion to the urea synthesis in Example 17 using (3S)- 3-(iodomethyl)tetrahydrofuran instead of (3R)-3-(iodomethyl)tetrahydrofuran for the final step.

[0403] Example 21 and Example 22 were prepared from the chiral SFC separation of Example 20 using conditions similar to those reported in Example 18 and Example 19. Example 23. (E)-5-(amino(methylamino)methylene)-l-(cyclopentylmethyl)-3- ((5S,7r,10S)-3-methyl-2,4-dioxo-l-(((S)-tetrahydrofuran-3-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (23)

[0404] Synthetic scheme:

[0405]

[0406] 4-nitrophenyl (cyclopentylmethyl)carbamate

[0407] To a solution of cyclopentylmethanamine (4 g, 40.33 mmol) in DCM (80 mL) was added DMAP (4.93 g, 40.33 mmol) and pyridine (80.67 mmol, 6.51 mL) and (4-nitrophenyl) chloroformate (8.13 g, 40.33 mmol). The mixture was stirred at 25 °C for 10 min. After completion, the title compound (10.66 g, crude) was used in the next step without further purification. MS (ESI): mass calcd. forC13H16N2O4: 264.11, found: 265.0 [M+H]+. l-(cyclopentylmethyl)-3-(3-methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)urea

[0408] To a solution of 10-amino-2-methyl-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3-dione (9.57 g, 40.34 mmol) in DCM (200 mL) was added TEA (8.16 g, 80.67 mmol) and 4- nitrophenyl (cyclopentylmethyl)carbamate (10.66 g, 40.34 mmol). The mixture was stirred at

[0409] 25 °C for 16 h. After completion, the mixture was poured into water (200 mL) and extracted with DCM (200 mL x 3). The combined organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether : ethyl acetate = 50 : 1 to 0 : 1) to give the title compound (9.6 g, 66%) as a white solid. MS (ESI): mass calcd. for

[0410] To a solution of l-(cyclopentylmethyl)-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)urea (9.6 g, 26.48 mmol) in AcOH (100 mL) was added malonic acid (5.51 g, 52.97 mmol) and AC2O (18.93 g, 185.4 mmol). The mixture was stirred at 80 °C for 3 h. After completion, the mixture was poured into water (200 mL) and extracted with ethyl acetate (150 mL x 3). The combined organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether : ethyl acetate = 80 : 1 to 0 : 1) to give the title compound (6.3 g, 55.3%) as a white solid. MS (ESI): mass calcd. for C22H30N4O5: 430.22, found: 431.2 [M+H]+.

[0411] 5-(bis(ethylthio)methylene)-l-(cyclopentylmethyl)-3-(3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0412]

[0413] To a solution of l-(cyclopentylmethyl)-3-(3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (6.3 g, 14.63 mmol) in DMSO (63 mL) was added TEA (45.91 g, 453.66 mmol) and CS2 (80.22 g, 1053.7 mmol). After addition, the mixture was stirred at 25 °C for 1 h. Bromoethane (75.93 g, 696.9 mmol) was added. The resulting mixture was stirred at 25 °C for 2 h. The mixture was poured into water (200 mL) and extracted with ethyl acetate (150 mL x 3). The combined organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (4.4 g, 63.5%) as a yellow solid. MS (ESI): mass calcd. for

[0414] 5-(bis(ethylthio)methylene)-l-(cyclopentylmethyl)-3-(3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (4.4 g) was separated by chiral SFC (column: DAICEL CHIRALPAK IG (250mmx30mm,10 um); mobile phase: CO2-EtOH; 50% B with isocratic elution) to give 5-(bis(ethylthio)methylene)-l- (cyclopentylmethyl)-3-((5S,7s,10S)-3-methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan- 10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (1.1 g) from the first eluting peak as a yellow solid. MS (ESI): mass calcd. for C27H38N4O5S2: 562.23, found: 563.3 [M+H]+. 5- (bis(ethylthio)methylene)-l-(cyclopentylmethyl)-3-((5R,7r,10R)-3-methyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (1.1 g) was obtained from the second eluting peak as a yellow solid. MS (ESI): mass calcd. for

[0415] To a solution of 5-(bis(ethylthio)methylene)-l-(cyclopentylmethyl)-3-((5S,7s,10S)-3- methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)- trione (0.47 g, 835.2 μmo)l in DMF (5 mL) was added TEA (169.03 mg, 1.67 mmol) and methylamine hydrochloride (28.2 mg, 417.6 μmo)l in DMF (5 mL). The mixture was stirred at 20 °C for 1 h. After completion, the mixture was poured into water (15 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with brine, dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, petroleum ether : ethyl acetate = 0 : 1) to give the title compound as a white solid. MS (ESI): mass calcd. for

[0416] of (5Z)-l-(cyclopentylmethyl)-5-

[0417] [ethylsulfanyl(methylamino)methylene]-3-(2-methyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydro pyrimidine-2, 4, 6-trione (0.12 g, 225.7 μmo)l and (3R)-3-(iodomethyl)tetrahydrofuran (47.86 mg, 225.7 μmo)l in DMF (5 mL) was added NaH (18.05 mg, 451.4 μmo,l 60% purity) at 0 °C. The mixture was stirred at 20 °C for 12 h. After completion, the mixture was poured into saturated aqueous NH4CI (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with brine, dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, petroleum ether : ethyl acetate = 1 : 1) to give the title compound (32 mg, 13%) as a white solid. MS (ESI): mass calcd. for C31H45N5O6S: 615.31, found: 616.4 [M+H]+.

[0418] (E)-5-(amino(methylamino)methylene)-l-(cyclopentylmethyl)-3-((5S,7r,10S)-3-methyl-

[0419] 2,4-dioxo-l-(((S)-tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6( 1H,3H,5H) -trione (23)

[0420] To a mixture of (Z)-l-(cyclopentylmethyl)-5-((ethylthio)(methylamino)methylene)-3- ((5S,7r,10S)-3-methyl-2,4-dioxo-l-(((S)-tetrahydrofuran-3-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (30 mg, 48.7 μmo)l in THF (0.5 mL) was added NH3.H2O (1 mL). The mixture was stirred at 40 °C for 1 h. After completion, the mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (10 mg, 35.6%) as a white solid. MS (ESI): mass calcd. for C29H42N6O6: 570.32, found: 571.3 [M+H]+. ’H NMR (400 MHz, DMSO-d6) 5 ppm 9.54 (s, 2H), 7.31 (s, 2H), 4.56-4.74 (m, 1H), 3.75-3.82 (m, 1H), 3.61-3.72 (m, 4H), 3.49 (dd, 7 = 3.6. 8.8 Hz, 1H), 3.33-3.38 (m, 2H), 2.84 (s, 3H), 2.68-2.75 (m, 1H), 2.33-2.47 (m, 2H), 2.14-2.31 (m, 6H), 1.92-2.01 (m, 1H), 1.79 (d, 7 = 11.2 Hz, 1H), 1.51-1.67 (m, 5H), 1.35-1.49 (m, 5H), 1.18-1.29 (m, 3H).

[0421] Example 24 was prepared in a similar fashion to Example 23 using (3S)-3- (iodomethyl)tetrahydrofuran instead of (3R)-3-(iodomethyl)tetrahydrofuran.

[0422] Example 25. (5E)-5-[amino(methylamino)methylene]-l-(cyclopentylmethyl)-3-[2- methyI-4-(oxetan-3-yImethyI)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yI]hexahydropyrimidine-2, 4, 6-trione (25)

[0423] Synthetic scheme:

[0424]

[0425] 5-[bis(ethylsulfanyl)methylene]-l-(cyclopentylmethyl)-3-[2-methyl-4-(oxetan-3- yImethyI)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yI]hexahydropyrimidine- 2, 4, 6- trione

[0426] To a solution of 5-[bis(ethylsulfanyl)methylene]-l-(cyclopentylmethyl)-3-(2-methyl- l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (330 mg, 586.4 μmo)l in DMSO (3.3 mL) was added Cs2CO3(191.06 mg, 586.4 μmo)l and 3- (iodomethyl)oxetane (174.17 mg, 879.6 μmo)l. The mixture was stirred at 40 °C for 16 h. The residue was poured into water (10 mL) and extracted with ethyl acetate (20 mL x 2). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2petroleum ether : ethyl acetate = 10 : 1 to 1 : 1) to afford the title compound (195 mg, 53%) as a yellow solid. MS (ESI): mass calcd. for C31H44N4O6S2: 632.27, found: 633.3 [M+H]+. (5Z)-l-(cycIopentyImethyI)-5-[ethyIsuIfanyI(methyIamino)methyIene]-3-[2-methyI-4-

[0427] (oxetan-3-yImethyI)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2,4,6-trione

[0428] To a solution of 5-[bis(ethylsulfanyl)methylene]-l-(cyclopentylmethyl)-3-[2-methyl- 4-(oxetan-3-ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (190 mg, 300.2 μmo)l in DMF (2 mL) was added the solution of TEA (60.76 mg, 600.5 μmo)l and methylamine hydrochloride (10.14 mg, 150.1 μmo)l in DMF (2 mL). The mixture was stirred at 20 °C for 3 h. The residue was poured into water (10 mL) and extracted with ethyl acetate (20 mL x 2). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2. petroleum ether : ethyl acetate = 0 : 1) to give the title compound (91 mg, 50%) as a white solid. MS (ESI): mass calcd. for C30H43N5O6S: 601.29, found: 602.4 [M+H]+.

[0429] (5E)-5-[amino(methylamino)methylene]-l-(cyclopentylmethyl)-3-[2-methyl-4-(oxetan-3- yImethyI)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yI]hexahydropyrimidine- To a solution of (5Z)-l-(cyclopentylmethyl)-5-

[0430] [ethylsulfanyl(methylamino)methylene]-3-[2-methyl-4-(oxetan-3-ylmethyl)-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione (20 mg, 33.2 μmo)l in THF (0.2 mL) was added NH4OH (33.2 μmo,l 5.12 μL, 25% w / w NH3in water). The mixture was stirred at 20 °C for 1 h. This reaction mixture was blown dry with N2 to remove THF. The residue was purified by prep-HPLC to give the title compound (5.66 mg, 30%) as a white solid. MS (ESI): mass calcd. for C28H40N6O6: 556.30, found: 557.5 [M+H]+. ' H NMR (400 MHz, DMSO-d6) 5 ppm 10.39 (d, J = 4.4 Hz, 1H), 10.06 (s, 1H), 7.66 (s, 1H), 4.63 (dd, J = 6.4, 7.6 Hz, 3H), 4.42 (q, 7 = 6.0 Hz, 2H), 3.65-3.72 (m, 4H), 2.79-2.88 (m, 6H), 2.11-2.39 (m, 8H), 1.81-1.89 (m, 1H), 1.34-1.61 (m, 10H), 1.16-1.27 (m, 3H).

[0431] Example 26 and Example 27. (E)-5-(amino(methylamino)methylene)-l- (cyclopentylmethyl)-3-((5R,7r,10R)-3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (26) and (E)-5- (amino(methylamino)methylene)-l-(cyclopentylmethyl)-3-((5S,7s,10S)-3-methyl-l- (oxetan-3-ylmethyl)-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-

[0432] 2,4,6(lH,3H,5H)-trione (27)

[0433] Example 25 (50 mg) was separated by chiral SFC (column: DAICEL CHIRALCEL OD(250mmx30mm,10 um); mobile phase: NMR (400 MHz, DMSO-d6) 5 ppm 10.39 (d, 7 = 4.8 Hz, 1H), 10.06 (s, 1H), 7.66 (s, 1H), 4.63 (dd, 7 = 6.0, 7.6 Hz, 3H), 4.42 (q, 7 = 6.2 Hz, 2H), 3.65-3.72 (m, 4H), 2.79-2.90 (m, 6H), 2.11- 2.41 (m, 8H), 1.85 (d, 7 = 12.8 Hz, 1H), 1.34-1.64 (m, 10H), 1.17-1.28 (m, 3H).

[0434] Example 28. (5E) -5- [amino(ethylamino)methylene] - 1- (cyclopentylmethyl)-3- [2-methyl-

[0435] 4-(oxetan-3-ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (28)

[0436]

[0437] (5E)-5-[amino(ethylsulfanyl)methylene]-l-(cyclopentylmethyl)-3-[2-methyl-4-(oxetan-3- ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-

[0438] 2, 4, 6- trione

[0439] To a solution of 5-[bis(ethylsulfanyl)methylene]-l-(cyclopentylmethyl)-3-[2-methyl- 4-(oxetan-3-ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (90 mg, 142.2 μmo)l in THF (0.9 mL) was added NH4OH (0.9 mL, 25% w / w NH3in water). The mixture was stirred at 30 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give the title compound (80 mg, 95.7%) as a yellow solid. MS (ESI): mass calcd. for C29H41N5O6S: 587.28, found: 588.3 [M+H]+. (5E)-5-[amino(ethylamino)methylene]-l-(cyclopentylmethyl)-3-[2-methyl-4-(oxetan-3- yImethyI)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yI]hexahydropyrimidine- 2,4,6-trione (28)

[0440]

[0441] A solution of (5Z)-5-[amino(ethylsulfanyl)methylene]-l-(cyclopentylmethyl)-3-[2- methyl-4-(oxetan-3-ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (80 mg, 136.12 μmo)l in ethanamine (0.8 mL) was stirred at 30 °C for 1 h. This reaction was concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (50 mg, 63%) as a white solid. MS (ESI): mass calcd. for C29H42N6O6: 570.32, found: 571.4 [M+H]+. ’H NMR (400 MHz, DMSO-d6) 5 ppm 10.51 (t, 7 = 4.8 Hz, 1H), 10.07 (s, 1H), 7.72 (s, 1H), 4.63 (dd, 7 = 6.4, 7.6 Hz, 3H), 4.38- 4.46 (m, 2H), 3.65-3.72 (m, 4H), 3.20-3.32 (m, 3H), 2.82 (s, 3H), 2.12-2.40 (m, 7H), 1.85 (d, J = 12.4 Hz, 1H), 1.33-1.64 (m, 10H), 1.14-1.27 (m, 6H).

[0442] (5E)-5-[amino-(2,2,2-trifluoroethylamino)methylene]-l-(cyclopentylmethyl)-3-[2- methyI-4-(oxetan-3-yImethyI)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (29)

[0443] (5E)-5-[amino-(2,2,2-trifluoroethylamino)methylene]-l-(cyclopentylmethyl)-3-[2- methyI-4-(oxetan-3-yImethyI)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (29)

[0444] To a solution of (5E)-5-[amino(ethylsulfanyl)methylene]-l-(cyclopentylmethyl)-3-[2- methyl-4-(oxetan-3-ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10- yl]hexahydropyrimidine-2, 4, 6-trione (92 mg, 156.53 μmo)l in THF (1 mL) was added 2,2,2- trifluoroethanamine (1.25 g, 12.62 mmol). The mixture was stirred at 30 °C for 1 h and concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (33 mg, 33.1%) as a white solid. MS (ESI): mass calcd. for C29H39F3N6O6: 624.29, found: 625.5[M+H]+’H NMR (400 MHz, DMSO-d6) 5 ppm 11.11 (t, J = 6.4 Hz, 1H), 10.48 (s, 1H), 8.28 (s, 1H), 4.61-4.65 (m, 3H), 4.42 (q, J = 6.0 Hz, 2H), 4.27-4.38 (m, 2H), 3.63-3.77 (m, 4H), 3.26-3.33 (m, 1H), 2.82 (s, 3H), 2.11-2.38 (m, 7H), 1.85 (d, J = 11.2 Hz, 1H), 1.36- 1.62 (m, 10H), 1.15-1.29 (m, 3H). Example 30 was prepared using a similar synthetic scheme to the one reported for Example 28. Dimethylamine was used instead of ethylamine.

[0445] Example 31 was prepared using a similar synthetic scheme to the one reported for Example 28. Azetidine was used instead of ethylamine.

[0446] Example 32. (E)-5-(amino(methylamino)methylene)-l-(cyclopentylmethyl)-3-(l-((3- fluorooxetan-3-yl)methyl)-3-methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6(lH,3H,5H)-trione (32)

[0447] Synthetic scheme:

[0448] - I l l -

[0449]

[0450] 5-(bis(ethylthio)methylene)-l-(cyclopentylmethyl)-3-(l-((3-fluorooxetan-3-yl)methyl)-3- methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)- trione.

[0451] The title compound was prepared in a similar fashion to the corresponding hydantoin alkylation in Example 25. 3-fluoro-3-(iodomethyl)oxetane was used in place of 3-(iodomethyl)oxetane.

[0452] (E)-5-(amino(methylamino)methylene)-l-(cyclopentylmethyl)-3-(l-((3-fluorooxetan-3- yl)methyl)-3-methyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2,4,6(lH,3H,5H)-trione (32)

[0453]

[0454] 5-(Bis(ethylthio)methylene)-l-(cyclopentylmethyl)-3-((5S',7s',10S')-3-methyl-l-((3- fluorooxetan-3-yl)methyl)-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2,4,61 ( 1H, 3 , 5H / )-tr ione (231 mg, 355 μmo)l was dissolved in methylamine (2 M in THF) (178 μL, 355 μmo)l and stirred for 30 min. The reaction was concentrated and then redissolved in THF (1.5 mL) and ammonium hydroxide (28%, 13.8 μL, 355 μmo)l was added. The solution was stirred at 45 °C for 18 h, diluted with EtOAc and washed with water (2x). The organic phase was dried over MgSO4, filtered and concentrated. The crude was purified by prep-HPLC to provide the title compound (72.6 mg, 36%) as a colorless solid. MS (ESI): mass calcd. for 10H), 1.20 (dd, 7 = 13.1, 8.7 Hz, 3H).

[0455] 33

[0456] Synthetic scheme:

[0457] 3-methyl-l-(oxetan-3-ylmethyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4, 10-trione

[0458] To a solution of 2-methyl-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3, 10-trione (50 g, 211.63 mmol) in DMF (500 mL) was added 3-(iodomethyl)oxetane (46.09 g, 232.8 mmol) and Cs2CO3(137.9 g, 423.3 mmol). The mixture was stirred at 40 °C for 12 h under N2. The mixture was poured into water (2 L) and extracted with ethyl acetate (1 L x 2). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether : ethyl acetate = 5 : 1 to 0 : 1) to give the title compound (44 g, 68%) as a white solid. MS (ESI): mass calcd. for C16H22N2O4: 306.16, found: 307.1 [M+H]+. 10-amino-3-methyl-l-(oxetan-3-ylmethyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0459] To a solution of 2-methyl-4-(oxetan-3-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecane- 1,3, 10-trione (22 g, 71.81 mmol) in NH3 / McOH (200 mL) was added Raney-Ni (6.15 g, 71.8 mmol) under N2. The suspension was degassed and purged with H23 times. The mixture was stirred under H2(15 psi) at 25 °C for 12 h. The three parallel reactions were combined and worked-up together. The mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure to give the title compound (65 g, crude) as a green oil, which was used in the next step without further purification. MS (ESI): mass calcd. for C16H25N3O3: 307.19, found: 308.1 [M+H]+. l-(cyclobutylmethyl)-3-(3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)urea

[0460] The title compound was prepared in a similar fashion to the urea synthesis in Example 25. 4-nitrophenyl (cyclobutylmethyl)carbamate was used in place of 4-nitrophenyl (cyclopentylmethyl)carbamate. l-(cyclobutylmethyl)-3-(3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.15]tridecan-10-yI)pyrimidine-2,4,6(lH,3H,5H)-trione

[0461] The title compound was prepared in a similar fashion to the barbiturate synthesis in

[0462] Example 25.

[0463] 5-(bis(ethylthio)methylene)-l-(cyclobutylmethyl)-3-(3-methyl-l-(oxetan-3-ylmethyl)-2,4- dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yI)pyrimidine-2,4,6(lH,3H,5H)-trione

[0464] The title compound was prepared in a similar fashion to the bis(ethylthio)methylene synthesis in Example 25.

[0465] (Z)-l-(cyclobutylmethyl)-5-((ethylthio)(methylamino)methylene)-3-(3-methyl-l-(oxetan-3- ylmethyl)-2,4-dioxo-l,3-diazadispiro[4.1.57.ls]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0466] To a solution of 5-[bis(ethylsulfanyl)methylene]-l-(cyclobutylmethyl)-3-[2-methyl-4- (oxetan-3-ylmethyl)-l ,3-dioxo-2,4-diazadispiro[4.1.57. l5]tridecan- 10- yl]hexahydropyrimidine-2, 4, 6-trione (20 mg, 32.3 μmo)l in DMF (1 mL) was added methylamine hydrochloride (2.18 mg, 32.3 μmo)l and TEA (16.35 mg, 161.6 μmo)l in DMF (0.5 mL). The mixture was stirred at 20 °C for 10 min. The reaction was used directly in the next step without further purification as a solution in DMF. MS (ESI): mass calcd. for

[0467] 33 NH4OH (649.15 μmo,l 0.1 mL, 25% w / w NH3in water) was added to a solution of (5Z)- 1 -(cyclobutylmethyl)-5 - [ethylsulfanyl(methylamino)methylene] -3 - [2-methyl-4-(oxetan- 3-ylmethyl)-l,3-dioxo-2,4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2,4,6- trione (19 mg, 32.33 μmo)l in DMF (1.5 mL). The mixture was stirred at 20 °C for 6 h. The residue was poured into water (10 mL) and extracted with ethyl acetate (10 mL). The organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (6 mg, 16%) as a white solid. MS (ESI): mass calcd. for

[0468] Example 34 and Example 35. (E)-5-(amino(methylamino)methylene)-l- (cyclobutylmethyl)-3-((5R,7r,10R)-3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione and (E)-5- (amino(methylamino)methylene)-l-(cyclobutylmethyl)-3-((5S,7s,10S)-3-methyl-l- (oxetan-3-ylmethyl)-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2,4,6(lH,3H,5H)-trione

[0469] Example 33 (35 mg) was separated by chiral SFC (column: DAICEL CHIRALCEL OD (250mmx30mm,10um); mobile phase: CO2-EtOH (0.1% NH3H2O); 30% B with isocratic elution) to give 34 (12.8 mg) from the first eluting peak as a white solid. MS (ESI): mass calcd. for (d, J = 7.2 Hz, 2H), 2.78-2.90 (m, 6H), 2.51-2.62 (m, 2H), 2.45 (s, 1H), 2.20-2.41 (m, 3H), 2.09-2.19 (m, 3H), 1.82-1.93 (m, 3H), 1.62-1.80 (m, 4H), 1.32-1.47 (m, 3H), 1.18-1.29 (m, 1H).

[0470] Example 36. l-(cyclopropylmethyl)-3-[2-methyl-4-(oxetan-3-yhnethyl)-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl]urea (36) methylcyanamide

[0471] To a solution of cyanogen bromide (5 g, 47.20 mmol, 3.47 mL) in ACN (75 mL) was added methylamine hydrochloride (3.19 g, 47.2 mmol) and K2CO3(6.52 g, 47.2 mmol). The mixture was stirred at 20 °C for 3 h under N2. After completion, the reaction mixture was filtered and concentrated under reduced pressure to give the title compound (2.28 g, 86%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) 5 ppm 6.50 (br, 1H) 2.65 (d, 7 = 4.8 Hz, 3H). l-(cyclopropylmethyl)-3-(3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yI)pyrimidine-2,4,6(lH,3H,5H)-trione

[0472] The title compound was prepared in a similar fashion to the corresponding barbiturate intermediate in Example 33. 4-nitrophenyl (cyclopropylmethyl)carbamate was used in place of 4-nitrophenyl (cyclobutylmethyl)carbamate. l-(cyclopropylmethyl)-3-[2-methyl-4-(oxetan-3-ylmethyl)-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl]urea (36)

[0473] To a solution of l-(cyclopropylmethyl)-3-[2-methyl-4-(oxetan-3-ylmethyl)-l,3-dioxo- 2, 4-diazadispiro[4.1.57.l5]tridecan-10-yl]hexahydropyrimidine-2, 4, 6-trione (0.28 g, 592.6 μmo)l in THF (3 mL) was added methylcyanamide (332.22 mg, 5.93 mmol) and Ni(acac)2 (45.67 mg, 177.8 μmo)l. The mixture was stirred at 80 °C for 12 h. After completion, the reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (47 mg, 15%) as white solid. MS (ESI): mass calcd. for Example 37 and Example 38. (E)-5-(amino(methylamino)methylene)-l- (cyclopropylmethyl)-3-((5R,7r,10R)-3-methyl-l-(oxetan-3-ylmethyl)-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (37) & (E)-5- (amino(methylamino)methylene)-l-(cyclopropylmethyl)-3-((5S,7s,10S)-3-methyl-l- (oxetan-3-ylmethyl)-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2,4,6(lH,3H,5H)-trione (38)

[0474] Example 36 (40 mg) was separated by chiral SFC (column: DAICEL CHIRALPAK AD(250mmx30mm,10 um); (mobile phase: CO2-EtOH (0.1% NH3H2O); 35% B with isocratic elution) to give 37 (11.54 mg) from the first eluting peak as a white solid. MS (ESI): mass calcd. for

[0475] Example 39. 5-(bis(methylamino)methylene)-l-butyl-3-(l,3-dimethyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (39)

[0476] 10-amino-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0477] The title compound was prepared in a similar sequence to 10-amino-3-methyl-l- (oxetan-3-ylmethyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione in Example 33. lodomethane was used instead of 3-(iodomethyl)oxetane for the alkylation of the hydantoin. l-butyl-3-(l,3-dimethyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)urea The title compound was prepared in a similar fashion to the urea synthesis in Example 25. Butyl isocyanate was used in place of 4-nitrophenyl (cyclopentylmethyl)carbamate. l-butyl-3-(l,3-dimethyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine- 2,4,6(lH,3H,5H)-trione

[0478] The title compound was prepared using the same conditions for the barbiturate synthesis reported in Example 25.

[0479] 5-(bis(ethylthio)methylene)-l-butyl-3-(l,3-dimethyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione

[0480] The title compound was prepared in a similar fashion to the bis(ethylthio)methylene synthesis in Example 25.

[0481] 5-(bis(methylamino)methylene)-l-butyl-3-(l,3-dimethyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (39)

[0482] A mixture of 5-[bis(ethylsulfanyl)methylene]-l-butyl-3-(2,4-dimethyl-l,3-dioxo-2,4- diazadispiro[4.1.57.l5]tridecan-10-yl)hexahydropyrimidine-2, 4, 6-trione (60 mg, 109 μmo)l, TEA (44.1 mg, 435.8 μmo,l 60.7 μL) and methylamine hydrochloride (45.31 mg, 435.8 μmo)l in DMF (0.6 mL) was degassed and purged with N23 times. The mixture was stirred at 20 °C for 12 h under N2. The reaction was quenched with saturated aqueous NH4CI (5.0 mL), diluted with H2O (5 mL), and extracted with ethyl acetate (5 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2. petroleum ether : ethyl acetate = I : 1) to afford the title compound (43 mg, 81%) as a white solid. MS (ESI): mass calcd. for C24H36N6O5: 488.27, found: 489.4 [M+H]+. ’H NMR (400 MHz, DMSO-d6) 5 ppm 10.71 (s, 1H), 4.63 (s, 1H), 3.72 0. 7 = 7.2 Hz, 2H), 3.04 (s, 5H), 2.94 (s, 3H), 2.83 (s, 3H), 2.03-2.45 (m, 7H), 1.81 (d, 7 = 11.6 Hz, 1H), 1.11-1.57 (m, 10H), 0.87 (t, 7 = 7.2 Hz, 3H).

[0483] Example 40 and Example 41 were obtained from the chiral SFC separation of Example 39 using similar conditions to those reported in Example 37 and Example 38.

[0484] Example 42. (5S,7s,10S)-10-(5-amino-l-(cyclopentylmethyl)-2,4-dioxo-l,4- dihydropyrido[2,3-d]pyrimidin-3(2H)-yl)-l,3-dimethyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (42)

[0485] Synthetic scheme:

[0486]

[0487] (5S, 7s, 10S)-10-amino-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione and

[0488] (5R,7r,10R)-10-amino-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0489] 10-amino-2,4-dimethyl-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3-dione (8.5 g) was separated by chiral SFC (column: DAICEL CHIRALPAK AD (250mmx50mm,10 um); mobile phase: CO2-EtOH (0.1% NH3H2O); 40% B with isocratic elution) to give (5S,7s,10S)-10- amino-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (3.8 g) from the first eluting peak as a white solid. MS (ESI): mass calcd. for C13H21N3O2: 251.16, found: 252.0 [M+H]+. (5R,7r,10R)-10-amino-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (3.8 g) was obtained from the second eluting peak as a white solid. MS (ESI): mass calcd. for C13H21N3O2: 251.16, found: 251.9 [M+H]+.

[0490] 2-fluoro-4-iodo-pyridine-3-carbonyl chloride

[0491] To a solution of 2-fluoro-4-iodo-pyridine-3-carboxylic acid (235 mg, 880.2 μmo)l in DCM (2.4 mL) was added DMF (6.43 mg, 88 μmo)l and then added oxalyl dichloride (139.64 mg, 1.10 mmol) drop wise over 10 min. The mixture was stirred at 20 °C for 40 min. The mixture was concentrated under reduced pressure to give the title compound (250 mg, crude) as a yellow oil, which was used in the next step directly.

[0492] N-((5S,7s,10S)-l,3-dimethyl-2,4-dioxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)-2- fluoro-4-iodonicotinamide

[0493] To a solution of (5S,7s,10S)-10-amino-l,3-dimethyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (200 mg, 795.79 μmo)l in DCM (2 mL) was added TEA (120.8 mg, 1.19 mmol). A solution of 2-fluoro-4-iodo-pyridine-3-carbonyl chloride (249.87 mg, 875.36 μmo)l in DCM (2 mL) was added dropwise at 0 °C. The mixture was stirred at 20 °C for 1 h. The reaction mixture was quenched with H2O (10 mL) and extracted with DCM (5 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, ethyl acetate) to give the title compound (280 mg, 70.3%) as a yellow solid. MS (ESI): mass calcd. for C19H22FIN4O3: 500.07, found: 501.1 [M+H]+.

[0494] N-((5S,7s,10S)-l,3-dimethyl-2,4-dioxo-l,3-diazadispiro[4.1.57.ls]tridecan-10-yl)-4- iodonicotinamide

[0495]

[0496] To a solution of N-((5S,7s,10S)-l,3-dimethyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)-2-fluoro-4-iodonicotinamide (140 mg, 279.8 μmo)l in DMSO (1.4 mL) was added cyclopentylmethanamine (83.26 mg, 839.5 μmo)l. The reaction was stirred at 60 °C for 12 h. The reaction was quenched with H2O (30 mL) and extracted with ethyl acetate (15 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep- TLC (SiC>2, ethyl acetate : petroleum ether = 2 : 1) to give the title compound (160 mg, 98.7%) as a yellow solid. MS (ESI): mass calcd. for C25H34IN5O3: 579.17, found: 580.2 [M+H]+.

[0497] (5S,7s,10S)-10-(l-(cyclopentylmethyl)-5-iodo-2,4-dioxo-l,4-dihydropyrido[2,3- d]pyrimidin-3(2H)-yl)-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0498] To a solution of N-((5S,7s,10S)-l,3-dimethyl-2,4-dioxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)-4-iodonicotinamide (200 mg, 345.1 μmo)l in THF (2 mL) was added DIPEA (133.82 mg, 1.04 mmol) and triphosgene (102.4 mg, 345.1 μmo)l at 0 °C. The mixture was stirred at 20 °C for 10 min under N2. DIPEA (133.82 mg, 1.04 mmol) was added to the above mixture. The mixture was stirred at 20 °C for 2 h under N2. The reaction was quenched with H2O (20 mL) and extracted with ethyl acetate (15 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, ethyl acetate : petroleum ether = 2 : Ito give the title compound (200 mg, 96%) as white solid. MS (ESI): mass calcd. for C26H32IN5O4: 605.15, found: 606.1 [M+H]+. (5S,7s,10S)-10-(l-(cyclopentylmethyl)-5-((diphenylmethylene)amino)-2,4-dioxo-l,4- dihydropyrido[2,3-d]pyrimidin-3(2H)-yl)-l,3-dimethyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0499] A mixture of (5S,7s,10S)-10-(l-(cyclopentylmethyl)-5-iodo-2,4-dioxo-l,4- dihydropyrido[2,3-d]pyrimidin-3(2H)-yl)-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane- 2, 4-dione (50 mg, 82.6 μmo)l, diphenylmethanimine (29.93 mg, 165.2 μmo)l, BINAP (10.28 mg, 16.5 μmo)l, t-BuONa (23.81 mg, 247.7 μmo)l and Pd2(dba)3 (7.56 mg, 8.3 μmo)l in toluene (1.25 mL) was degassed and purged with N2 3 times. The mixture was stirred at 80 °C for 3 h under N2. The residue was poured into water (20 mL) and extracted with ethyl acetate (20 mL). The organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiCL, petroleum ether : ethyl acetate = 2 : 1) to give the title compound (25 mg, crude) as a yellow solid, which was used in the next step without further purification. MS (ESI): mass calcd. for C39H42N6O4: 658.33, found: 659.3 [M+H]+.

[0500] (5S,7s,10S)-10-(5-amino-l-(cyclopentylmethyl)-2,4-dioxo-l,4-dihydropyrido[2,3- d]pyrimidin-3(2H)-yl)-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (42)

[0501] To a solution of (5S,7s,10S)-10-(l-(cyclopentylmethyl)-5- ((diphenylmethylene)amino)-2,4-dioxo-l,4-dihydropyrido[2,3-d]pyrimidin-3(2H)-yl)-l,3- dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (25 mg, 38 μmo)l in THF (0.5 mL) was added aqueous HC1 (5 M, 136.6 μL). The mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (6.47 mg, 31%) as yellow solid. MS (ESI): mass calcd. for

[0502] Example 43. 10-(4-amino-l,3-dioxoisoindolin-2-yl)-3-methyl-l-(oxetan-3-ylmethyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (42)

[0503] 10-hydroxy-3-methyl-l-(oxetan-3-yhnethyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2,4-

[0504] To a solution of 2-methyl-4-(oxetan-3-ylmethyl)-2,4-diazadispiro[4.1.57.l5]tridecane- 1,3, 10-trione (1 g, 3.26 mmol) in EtOH (10 mL) was added NaBH4(37.05 mg, 979.3 μmol). The mixture was stirred at 20 °C for 1 h under N2. The reaction was quenched with saturated aqueous NH4CI (10 mL) and extracted with ethyl acetate (10 mL). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, ethyl acetate) to give the title compound (750 mg, 74.5%) as a white solid. MS (ESI): mass calcd. for C16H24N2O4: 308.17, found: 309.1 [M+H]+.

[0505] 10-(4-amino-l,3-dioxoisoindolin-2-yl)-3-methyl-l-(oxetan-3-ylmethyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (42)

[0506] To a solution of 10-hydroxy-2-methyl-4-(oxetan-3-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecane-l, 3-dione (150 mg, 486.43 μmo)l in toluene (2 mL) was added 4-aminoisoindoline- 1,3-dione (157.74 mg, 973 μmo)l and 2-(tributyl-phosphanylidene) acetonitrile (410.9 mg, 1.7 mmol). The mixture was stirred at 100 °C for 16 h. After completion, the reaction was concentrated under reduced pressure. The residue was diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL x 2). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (4.17 mg, 14%) as a yellow solid. MS (ESI): mass calcd. for

[0507] Example 44 and Example 45. (5S,7s,10S)-10-(4-amino-l,3-dioxoisoindolin-2-yl)-3- methyI-l-(oxetan-3-yImethyI)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (44) and (5R,7r,10R)-10-(4-amino-l,3-dioxoisoindoIin-2-yI)-3-methyI-l-(oxetan-3-yImethyI)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (45)

[0508] Example 43 (150 mg) was separated by chiral SFC (column: DAICEL CHIRALCEL OD (250mmx30mm,10 um); mobile phase: elution) to give 44 (21 mg) from the first eluting peak as a yellow solid. MS (ESI): mass calcd. for

[0509] 10-(4-amino-7-butyl-l,3-dioxoisoindolin-2-yl)-3-methyl-l-(oxetan-3-ylmethyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (46)

[0510] Synthetic scheme:

[0511]

[0512] 4-amino-7 -bromoisoindoline- 1 ,3-dione

[0513] To a solution of 4- aminoisoindoline- 1,3-dione (1 g, 6.17 mmol) in MeOH (75 mL) was added NBS (1.15 g, 6.48 mmol). The mixture was stirred at 25 °C for 4 h. The reaction was filtered and washed with MeOH (10 mL x 2). The filter cake was dried under vacuum to give the title compound (1 g, 60.7%) as a yellow solid. MS (ESI): mass calcd. for

[0514] 4-amino-7 -butylisoindoline- 1 ,3-dione

[0515] To a solution of 4-amino-7-bromo-isoindoline-l, 3-dione (1 g, 4.15 mmol) and potassium butyl(trifluoro)borate (748.5 mg, 4.56 mmol) in toluene (15 mL) and H2O (5 mL) was added Cs2CO3(4.06 g, 12.45 mmol) and [2-(2-aminophenyl)phenyl]-chloro- palladium;bis(l-adamantyl)-butyl-phosphane (277.4 mg, 414.9 μmo)l. The mixture was stirred at 85 °C for 16 h under N2. The reaction was filtered to remove the insoluble precipitate. The filtrate was diluted with H2O (20 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiCL, petroleum ether : ethyl acetate = 1 : 0 to 2 : 1) to give the title compound (210 mg, 19.4%) as a yellow solid. MS (ESI): mass calcd. for

[0516] 10-(4-amino-7-butyl-l,3-dioxoisoindolin-2-yl)-3-methyl-l-(oxetan-3-ylmethyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (46)

[0517] To a solution of 10-hydroxy-2-methyl-4-(oxetan-3-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecane-l, 3-dione (150.71 mg, 488.73 μmo)l and 4-amino-7-butyl- isoindoline- 1,3 -dione (128 mg, 586.48 μmo)l in THF (2 mL) was added PPh3(192.28 mg, 733.1 μmo)l and DIAD (148.24 mg, 733.1 μmo)l. The mixture was stirred at 25 °C for 1 h. The reaction mixture was quenched with H2O (10 mL) and extracted with ethyl acetate (10 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiCL, petroleum ether : ethyl acetate = 1 : 0 to 1 : 1) to give the crude product (165 mg) as a yellow solid which was further purified by prep-HPLC to give the title compound (26 mg, 10%) as a yellow solid. MS (ESI): mass calcd. for

[0518] Example 47 and Example 48. (5S,7s,10S)-10-(4-amino-7-butyl-l,3-dioxoisoindolin-2-yl)- 3-methyl-l-(oxetan-3-ylmethyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (47) and (5R,7r,10R)-10-(4-amino-7-butyI-l,3-dioxoisoindoIin-2-yI)-3-methyI-l-(oxetan-3- yImethyI)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (48)

[0519] Example 46 (30 mg) was separated by chiral SFC (column: DAICEL CHIRALCEL OJ (250mmx30mm,10 um); mobile phase: CO2-EtOH; 55% B with isocratic elution) to give 47 (9.38 mg) from the first eluting peak as a white solid. MS (ESI): mass calcd. for C

[0520] Example 49. 10-(l,3-dioxoisoindolin-2-yl)-3-methyl-l-(oxetan-3-ylmethyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (49)

[0521] Synthetic scheme:

[0522] 10-(l,3-dioxoisoindolin-2-yl)-3-methyl-l-(oxetan-3-ylmethyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (49)

[0523] To a solution of 10-amino-2-methyl-4-(oxetan-3-ylmethyl)-2,4- diazadispiro[4.1.57.l5]tridecane-l, 3-dione (200 mg, 650.65 μmo)l in toluene (2 mL) was added TEA (164.6 mg, 1.63 mmol) and isobenzofuran- 1,3-dione (125.3 mg, 845.8 μmo)l. The mixture solution was stirred at 100 °C for 16 h. After completion, the reaction was diluted with H2O (10 mL) extracted with ethyl acetate (10 mL x 2). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC to give the title compound (90 mg, 30.9%) as a yellow solid. MS (ESI): mass calcd. for

[0524] Example 50 and Example 51. (5S,7s,10S)-10-(l,3-dioxoisoindolin-2-yl)-3-methyl-l- (oxetan-3-yImethyI)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (50) and (5R,7r,10R)- 10-(l,3-dioxoisoindoIin-2-yI)-3-methyI-l-(oxetan-3-yImethyI)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (51)

[0525] Example 49 (90 mg) was separated by chiral SFC (column: REGIS (s,s) WHELK-01 (250mmx30mm, 5 um); mobile phase: CO2-EtOH; 50% B with isocratic elution) to give 50 (22.99 mg) from the first eluting peak as a yellow solid. MS (ESI): mass calcd. for 1.22-1.39 (m, 1H). 51 (24.01 mg) was obtained from the second eluting peak as a yellow solid. MS (ESI): mass calcd. for

[0526] Example 52. 10-(4-amino-7-butyl-l,3-dioxoisoindolin-2-yl)-l,3-dimethyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (52)

[0527] Synthetic scheme:

[0528]

[0529] 10-hydroxy-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0530] The title compound was prepared in a similar fashion to the alcohol in Example 43. l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4, 10-trione was used instead of 3-methyl- l-(oxetan-3-ylmethyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4, 10-trione.

[0531] 10-(4-amino-7-butyl-l,3-dioxoisoindolin-2-yl)-l,3-dimethyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (52)

[0532] To a solution of 4-amino-7-butyl-isoindoline-l, 3-dione (100.0 mg, 458.1 μmo)l in toluene (2 mL) was added 10-hydroxy-2,4-dimethyl-2,4-diazadispiro[4.1.57.l5]tridecane-l,3- dione (173.4 mg, 687.2 μmo)l and 2-(tributyl-phosphanylidene)acetonitrile (387 mg, 1.6 mmol). The mixture was stirred at 100 °C for 16 h. The reaction was concentrated under reduced pressure. The residue was diluted with H2O (5 mL) and extracted with DCM (10 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiCL, petroleum ether : ethyl acctatc= l : I ) to give the title compound (88.0 mg, 40.3%) as a yellow solid. MS (ESI): mass calcd. for

[0533] Example 53 and Example 54. (5S,7s,10S)-10-(4-amino-7-butyl-l,3-dioxoisoindolin-2-yl)- l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (53) and (5R,7r,10R)-10-(4- amino-7-butyl-l,3-dioxoisoindolin-2-yl)-l,3-dimethyl-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (54)

[0534] Example 52 (100 mg) was separated by chiral SFC (column: DAICEL CHIRALPAK IC (250mmx30mm,10 um); mobile phase: CO2-IPA (0.1% NH3H2O); 50% B with isocratic elution) to give 53 (30.4 mg) from the first eluting peak as a yellow solid. MS (ESI): mass calcd. for 1H), 2.39-2.46 (m, 1H), 2.27-2.34 (m, 2H), 2.21-2.27 (m, 1H), 2.15-2.21 (m, 1H), 2.09-2.14 (m, 2H), 2.01-2.09 (m, 1H), 1.81-1.92 (m, 1H), 1.49-1.59 (m, 3H), 1.40-1.49 (m, 2H), 1.23- 1.35 (m, 3H), 0.88 (d, J = 7.2 Hz, 3H).

[0535] Example 55. 4-amino-7-butyl-2-((ls,4s)-4-((5,5-dimethyl-2,4-dioxoimidazolidin-l- yl)methyl)cyclohexyl)isoindoline-l, 3-dione (55)

[0536] (lr,4r)-ethyl 4-hydroxycyclohexanecarboxylate

[0537] To a suspension of (lr,4r)-4-hydroxycyclohexane-l-carboxylic acid (2 g, 13.5 mmol) in EtOH (9.7 mL) was added H2SO4(52.9 μL, 942 μmo)l. The mixture was stirred at 70 °C for 2.5 h. After cooling to rt, the resulting mixture was basified with 1 M aqueous Na2CO3solution and concentrated to remove most of the solvent. The aqueous mixture was diluted with water and extracted with EtOAc (x3). The combined organic layer was washed with water (xl), dried over Na2SO4, filtered, and concentrated to provide the crude title compound (2.27 g, 98%) as a yellow oil. ' H NMR (400 MHz, CDCh) 5 4.11 (q, J = 7.1 Hz, 2H), 3.53 - 3.65 (m, 1H), 2.15 - 2.30 (m, 1H), 1.91 - 2.10 (m, 4H), 1.64 (br s, 1H), 1.41 - 1.59 (m, 2H), 1.18 - 1.36 (m, 5H).

[0538] (lr,4r)-ethyl 4-(benzyloxy)cyclohexanecarboxylate

[0539] A mixture of (lr,4r)-ethyl 4-hydroxycyclohexanecarboxylate (2.27 g, 13.2 mmol), N,N- diisopropylethylamine (5.8 mL, 33 mmol), and benzyl bromide (1.7 mL, 13.8 mmol) was stirred at 150 °C for 2.5 h. The resulting mixture was poured into 1 M aqueous HC1 (46 mL) at 0 °C and vigorously stirred for 5 min. The quenched mixture was extracted with EtOAc (x3). The combined organic layer was washed with brine (xl), dried over Na2SO4, filtered and concentrated. The crude residue was purified by flash column chromatography (silica, 5-10% EtOAc / heptanes to provide the title compound (1.82 g, 53%). ' H NMR (400 MHz, CDCl3) 5 7.30 - 7.38 (m, 4H), 7.23 - 7.30 (m, 1H), 4.56 (s, 2H), 4.11 (q, J = 7.1 Hz, 2H), 3.34 (tt, J = 10.3, 4.1 Hz, 1H), 2.27 (tt, J = 11.6, 3.7 Hz, 1H), 2.08 - 2.19 (m, 2H), 1.97 - 2.07 (m, 2H), 1.47 (ddd, 7 = 24.7, 13.2, 3.1 Hz, 2H), 1.28 - 1.39 (m, 2H), 1.21 - 1.27 (m, 3H).

[0540] ((lr,4r)-4-(benzyloxy)cyclohexyl)methanol

[0541] To a solution of (lr,4r)-ethyl 4-(benzyloxy)cyclohexanecarboxylate (1.82 g, 6.94 mmol) in THF (17.3 mL) was added lithium aluminum hydride (624 mg, 15.6 mmol) at 0 °C. The mixture was stirred at 0 °C for 5 min then at 60 °C for 5 h. The resulting mixture was cooled to rt and quenched by a slow addition of sodium sulfate decahydrate. The mixture was filtered through a pad of Celite by aid of EtOAc and concentrated to provide the crude title compound (1.50 g, 98%) as a white solid which was used as such in the next step. ’H NMR (400 MHz, CDCh) 5 7.30 - 7.39 (m, 4H), 7.26 (s, 1H), 4.57 (s, 2H), 3.45 (t, J = 6.0 Hz, 2H), 3.31 (tt, 7 = 10.9, 4.2 Hz, 1H), 2.09 - 2.19 (m, 2H), 1.80 - 1.91 (m, 2H), 1.41 - 1.59 (m, 1H), 1.23 - 1.40 (m, 3H), 0.98 (qd, 7 = 13.3, 3.4 Hz, 2H). l-(((lr,4r)-4-(benzyloxy)cyclohexyl)methyl)-5,5-dimethyl-3-((2- (trimethylsilyl)ethoxy)methyl)imidazolidine-2, 4-dione

[0542] To a solution of ((lr,4r)-4-(benzyloxy)cyclohexyl)methanol (1.20 g, 5.45 mmol) and triethylamine (835 μL, 5.99 mmol) in MeCN (27.2 mL) was added methanesulfonyl chloride (465 μL, 5.99 mmol) at 0 °C dropwise. The reaction was stirred at 0 °C for 15 min then was quenched with a saturated aqueous NaHCO3solution and a small amount of water and extracted with DCM (x4). The combined organic layer was dried over Na2SO4, filtered, and concentrated to give the crude mesylate as a colorless oil. To a solution of the crude mesylate and 5, 5-dimethyl-3-((2-(trimethylsilyl)ethoxy)methyl)imidazolidine-2, 4-dione (2.11 g, 8.17 mmol) in MeCN (27.2 mL) was added potassium iodide (913 mg, 5.45 mmol) followed by cesium carbonate (5.43 g, 16.3 mmol). The reaction was stirred at 90 °C for 15.5 h. After cooled to rt, the resulting mixture was quenched with water and extracted with EtOAc (x3). The combined organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was purified by flash column chromatography (silica, 1-50% EtOAc / heptane) to afford the title compound (2.27 g, 90%) as a colorless oil. MS (ESI): mass calcd. for C25H40N2O4Si: 460.28 found: 483.5 [M+Na]+. l-(((lr,4r)-4-hydroxycyclohexyl)methyl)-5,5-dimethyl-3-((2- (trimethyIsiIyI)ethoxy)methyI)imidazoIidine-2, 4-dione

[0543] To a solution of l-(((lr,4r)-4-(benzyloxy)cyclohexyl)methyl)-5,5-dimethyl-3-((2- (trimethylsilyl)ethoxy)methyl)imidazolidine-2, 4-dione (2.55 g, 5.54 mmol) in THF (111 mL) was added 10% palladium on carbon (895 mg, 8.41 mmol) under N2. The reaction flask was submitted to 3 cycles of vacuum / hydrogen purges and stirred under hydrogen for 23 h. The mixture was filtered through a pad of Celite by aid of MeOH and concentrated. The crude residue was purified by flash column chromatography (silica, 1-80% EtOAc / heptane) to afford the title compound (1.47 g, 72%) as a yellow oil. ’H NMR (400 MHz, CDCl35 4.92 (s, 2H), 3.50 - 3.66 (m, 3H), 3.11 (d. 7 = 7.5 Hz, 2H), 1.94 - 2.04 (m, 2H), 1.65 - 1.86 (m, 3H), 1.41 (s, 6H), 1.15 - 1.32 (m, 2H), 0.98 - 1.14 (m, 2H), 0.89 - 0.98 (m, 2H), -0.05 - 0.04 (m, 9H). 4-amino-7-butyl-2-((ls,4s)-4-((5,5-dimethyl-2,4-dioxo-3-((2- (trimethylsilyl)ethoxy)methyl)imidazolidin-l-yl)methyl)cydohexyl)isoindoline-l, 3-dione

[0544] , r)-4-hydroxycyclohexyl)methyl)-5 ,5-dimethyl-3-((2- (trimethylsilyl)ethoxy)methyl)imidazolidine-2, 4-dione (93 mg, 251 μmol) and 4-amino-7- butylisoindoline- 1,3-dione (65.7 mg, 301 μmo)l in THF (5 mL) were added PPh ? (98.7 mg, 376 μ mol) and DIAD (75.6 μL, 376 μmo)l. The reaction mixture was stirred at rt for 1 h and was quenched with water and extracted with Et2O (x4). The combined organic layer was dried over Na2SO4, filtered, and concentrated. The crude was purified by flash column chromatography (silica, 5-50% EtOAc / heptane) to afford the title compound (73.4 mg, 51%) as yellow solid). MS (ESI): mass calcd. for C30H46N4O5Si 570.32 found: 593.4 [M+Na]+.

[0545] 4-amino-7-butyl-2-((ls,4s)-4-((5,5-dimethyl-2,4-dioxoimidazolidin-l- yl)methyl)cyclohexyl)isoindoline-l, 3-dione (55) mino-7-butyl-2-((l.s,4.s)-4-((5,5-dimethyl-2,4-dioxo-3-((2- (trimethylsilyl)ethoxy)methyl)imidazolidin- 1 -yl)methyl)cyclohexyl)isoindoline- 1 ,3 -dione (143 mg, 251 μmo)l in DCM (5 mL) was added TFA (576 μL, 7.52 mmol). The mixture was stirred at rt for 5 h and was basified with a 1 M aqueous NaOH (10 mL) until pH = 12-13. The resulting solution was diluted with EtOAc, layers were separated, and the aqueous layer was extracted with EtOAc (x3). The combined organic layer was dried over Na2SO4, filtered, and concentrated. The reaction was purified by prep-HPLC to provide the title compound (34.2 mg, 84%) as a yellow fluorescent solid. MS (ESI): mass calcd. for C24H32N4O4: 440.24, found: 441.4 [M+H]+. ' H NMR (400 MHz, DMSO) 5 10.80 (s, 1H), 7.24 (d, j = 8.5 Hz, 1H), 6.89 (d,

[0546] J = 8.5 Hz, 1H), 6.31 (s, 2H), 3.98 - 3.83 (m, 1H), 3.36 - 3.33 (m, 2H), 2.89 - 2.78 (m, 2H), 2.42 - 2.28 (m, 2H), 2.02 (brs, 1H), 1.76 (brs, 1H), 1.72 (brs, 1H), 1.60 - 1.37 (m, 6H), 1.38 - 1.21 (m, 8H), 0.89 (t, J = 7.3 Hz, 3H). Example 56 was synthesized following the same sequence of steps of Example 55 using isoindoline- 1,3 -dione instead of 4-amino-7-butylisoindoline-l, 3-dione in the Mitsunobu reaction.

[0547] Example 57. 7-amino-4-butyl-2-((ls,4s)-4-((5,5-dimethyl-2,4-dioxoimidazolidin-l- yl)methyl)cyclohexyl)-lH-pyrrolo[3,4-c]pyridine-l,3(2H)-dione (57)

[0548] Ethyl 2-butyloxazole-5-carboxylate

[0549] In a sealed tube a mixture of ethyl 2-bromooxazole-5-carboxylate (800 mg, 3.64 mmol), A-butylboronic acid (1.16 g, 10.9 mmol), and K3PO4 (2.36 g, 10.9 mmol) in toluene (16.2 mb) and water (2.0 mL) was degassed with N2 bubbling for 10 min before addition of Pd(dppf)Cl2- DCM (299 mg, 364 μmo)l. The mixture was stirred at 120 °C for 3 h then after cooling to rt, the resulting mixture was filtered through a pad of Celite by aid of EtOAc. The filtrate was diluted with water (10 mL), and the layers were separated. The aqueous layer was extracted with EtOAc (10 mL x3). The combined organic layer was dried over Na2SO4, filtered, and concentrated. The reaction was purified by reverse phase chromatography (Cl 8, 5-95% MeCN / 10 mM AmB buffer). Fractions containing the desired product were combined and extracted with DCM (x3). The combined organic layer was dried over Na2SO4, filtered, and concentrated to provide the title compound (157.2 mg, 22%) as a dark brown oil. MS (ESI): mass calcd. for C10H15NO3: 197.11, found: 198.3 [M+H]+.

[0550] 2-butyloxazole-5-carboxylic acid

[0551] To a solution of ethyl 2-butyloxazole-5-carboxylate (157 mg, 797 μmo)l in THF (2.5 mL) and water (613 p L) was added LiOH (76.3 mg, 3.19 mmol). The mixture was stirred at rt for 3 h. The resulting mixture was acidified with 1 M aqueous HC1 until pH 1~2 and extracted with DCM (x4). The combined organic layer was dried over Na2SO4, filtered, and concentrated to provide the crude title compound (121.7 mg, 90%) as a brown solid. MS (ESI): mass calcd. for

[0552] Tert-butyl (2-butyloxazol-5-yl)carbamate

[0553]

[0554] To a solution of 2-butyloxazole-5-carboxylic acid (113 mg, 668 μmo)l and triethylamine (130 μL, 935 μmo)l in t-BuOH (2.23 mL) was added DPPA (163 μL, 735 μmo)l. The mixture was stirred at 88 °C for 6.5 h then was cooled to rt. The resulting mixture was quenched with saturated aqueous NaHCO3solution and was extracted with EtOAc (x3). The combined organic layer was dried over Na2SO4, filtered, and concentrated. The crude residue was purified by flash column chromatography (silica, 5-50% EtOAc / heptane) to afford the title compound (112 mg, 70%) as a white solid. MS (ESI): mass calcd. for C12H20N2O3: 240.15, found: 241.3 [M+H]+.

[0555] A mixture of tert-butyl (2-butyloxazol-5-yl)carbamate (103 mg, 427 μmo)l and 1H- pyrrole-2,5-dione (207 mg, 2.13 mmol) in toluene (4.3 mL) was stirred at 110 °C for 8 h. The resulting mixture was concentrated with EtOAc to give the crude title compound as a yellow solid which was directly used for the next reaction without further purification. MS (ESI): mass calcd. for A solution of tert-butyl (4-butyl-l,3-dioxo-2,3,3α,4,7,7a-hexahydro-l / f-4,7- epoxypyrrolo[3,4-c]pyridin-7-yl)carbamate (144 mg, 427 μmo)l in a solution of TFA in HFIP (5% w / w) (4.6 mL, 2.99 mmol) was stirred at rt for 5 h then was concentrated with z'-PrOH. The reaction was purified by prep-HPLC to provide the title compound (51.5 mg, 55%) as a yellow fluorescent solid. MS (ESI): mass calcd. for

[0556] 7-amino-4-butyl-2-((ls,4s)-4-((5,5-dimethyl-2,4-dioxo-3-((2-

[0557] (trimethylsilyl)ethoxy)methyl)imidazolidin- 1 -yl)methyl)cyclohexyl) - 1 H- py r rolo[3,4- c]pyridine-l,3(2H)-dione

[0558] To a solution of l-(((lr,4r)-4-hydroxycyclohexyl)methyl)-5,5-dimethyl-3-((2- (trimethylsilyl)ethoxy)methyl)imidazolidine-2, 4-dione (86.5 μL, 214 μmo)l, 7-amino-4-butyl- l / f-pyrrolo[3,4-c]pyridine-l,3(2H)-dione (51.5 mg, 235 μmo)l, and PPh3(84.1 mg, 321 μmo)l in THF (4.2 mL) was added DIAD (64.4 μL, 321 μmo)l. The mixture was stirred at rt for 2 h. The resulting mixture was quenched with water and extracted with EtOAc (x4). The combined organic layer was dried over Na2SO4, filtered, and concentrated. The crude residue was purified by flash column chromatography (silica, 5-80% EtOAc / heptane) to afford the title compound (58.1 mg, 48%) as a yellow fluorescent solid. MS (ESI): mass calcd. for

[0559] 7-amino-4-butyl-2-((ls,4s)-4-((5,5-dimethyl-2,4-dioxoimidazolidin-l- yI)methyI)cycIohexyI)-lH-pyrroIo[3,4-c]pyridine-l,3(2H)-dione (57)

[0560] mino-4-butyl-2-((l.v,4.v)-4-((5,5-dimethyl-2,4-dioxo-3-((2- (trimethylsilyl)ethoxy)methyl)imidazolidin-l-yl)methyl)cyclohexyl)-177-pyrrolo[3,4- c]pyridine-l,3(2H)-dione (55.7 mg, 97.4 μmo)l in DCM (1.0 mL) was added a solution of TFA (224 μL, 2.92 mmol) in DCM (1.0 mL) and the mixture was stirred at rt for 7 h. The reaction was basified with NaOH (1 N), then extracted with EtOAc (3x). The organics were combined, dried over Na2SO4, filtered, and evaporated. The reaction was purified by prep-HPLC to provide the title compound (29.8 mg, 69%) as a yellow solid. MS (ESI): mass calcd. for C23H31N5O4: 441.24, found: 442.3 [M+H]+. ’H NMR (400 MHz, DMSO) 5 10.79 (s, 1H), 8.32

[0561] (s, 1H), 6.44 (s, 2H), 3.91 (tt, J = 11.7, 3.3 Hz, 1H), 3.32 - 3.35 (m, 2H), 2.89 - 2.99 (m, 2H), 2.26 - 2.39 (m, 2H), 1.95 - 2.05 (m, 1H), 1.67 - 1.79 (m, 2H), 1.39 - 1.63 (m, 6H), 1.24 - 1.36 (m, 8H), 0.87 (t, J = 7.4 Hz, 3H).

[0562] Example 58. 10-(3-amino-7-butyl-4,6-dioxo-2H-pyrazolo[3,4-d]pyrimidin-5-yl)-2- methyl-4-(2, 2, 2-trifluoroethyl)-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3-dione (58)

[0563] Synthetic scheme:

[0564] 3-methyl-l-(2,2,2-trifluoroethyl)-ll,14-dioxa-l,3- diazatrispiro[4.1.2.410.27.l5]heptadecane-2, 4-dione

[0565] To a solution of 2-methyl-l l,14-dioxa-2,4-diazatrispiro[4.1.2.410.27.l5]heptadecane- 1, 3-dione (40 g, 142.7 mmol) in DMF (400 mL) was added 2,2,2-trifluoroethyl trifluoromethanesulfonate (66.24 g, 285.4 mmol) and Cs2CO3(69.74 g, 214 mmol). The mixture was heated to 25 °C for 12 h. After completion, the mixture was poured into water

[0566] (500 mL) and extracted with ethyl acetate (500 mL x 3). The combined organic phase was washed with brine, dried with anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (SiCL, petroleum ether : ethyl acetate = 10 : 1 to 0 : 1) to afford the title compound (36.8 g, 71%) as a white solid. ' H NMR (400 MHz, DMSO-d6) 5 ppm 4.24-4.34 (m, 2H), 3.84 (s, 4H), 2.88 (s, 3H), 2.26 (s, 4H), 1.88 (s, 2H), 1.62-1.69 (m, 2H), 1.45-1.51 (m, 4H).

[0567] 3-methyl-l-(2, 2, 2-trifluoroethyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4, 10-trione

[0568] To a solution of 2-methyl-4-(2,2,2-trifluoroethyl)-l l,14-dioxa-2,4- diazatrispiro[4.1.2.410.27.l5]heptadecane-l, 3-dione (36.8 g, 101.56 mmol) in acetone (400 mL) and H2O (200 mL) was added TsOH.H2O (38.64 g, 203.1 mmol). The mixture was stirred at 25 °C for 12 h. The mixture was poured into water (500 mL), and then extracted with ethyl acetate (500 mL x2). The organic layer was dried, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (SiO2, petroleum ether : ethyl acetate = 10 : 1 to 0 : 1) to give the title compound (29 g, 90%) as a white solid.1H NMR (400

[0569] To a mixture of 6-chloro- 1 H-pyri midinc-2, 4-dione (5 g, 34.1 mmol) and 1 -iodobutane (6.91 g, 37.5 mmol) in DMSO (25 mL) was added K2CO3 (2.36 g, 17.1 mmol) in one portion at 25 °C under N2. After stirring at 25 °C for 12 h, the combined mixture was poured into water (30 mL) and EtOAc (30 mL). The aqueous phase was extracted with EtOAc (3 x 20 mL). The combined organic layer was washed with brine (3 x 20 mL), dried over anhydrous Na2SO4, filtered and concentrated under redued pressure. The crude residue was triturated with petroleum ether (20 mL) and stirred for 5 min. The precipitate was collected by filtration, washed with petroleum ether (3 x 20 mL) and dried under vacuum to provide the title compound (4.5 g, 22.2 mmol, 65.1%) as a white solid, which was used directly in the next step without further purification. MS (ESI): mass calcd. for C8H11CIN2O2: 202.05, found: 203.2 [M+H]+.

[0570] 10-hydroxy-2-methyl-4-(2,2,2-trifluoroethyl)-2,4-diazadispiro[4.1.57.l5]tridecane-l,3- dione

[0571] To a solution of 2-methyl-4-(2,2,2-trifluoroethyl)-2,4-diazadispiro[4.L57.l5]tridecane- 1,3, 10-trione (1 g, 3.14 mmol) in EtOH (10 mL) was added NaBH4(35.66 mg, 942.53 μmo)l. The mixture was stirred at 20 °C for 1 h. The reaction mixture was quenched with saturated aqueous NH4CI (5 mL), and concentrated under reduced pressure. The residue was diluted with H2O (15 mL) and extracted with ethyl acetate (25 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2. petroleum ether : ethyl acetate = 19 : 1 to 2 : 1) to afford the title compound (780 mg, 69%) as a colorless oil. MS (ESI): mass calcd. for

[0572] A mixture of l-butyl-6-chloro-pyrimidine-2, 4-dione (230 mg, 1.14 mmol), 10- hydroxy-2-methyl-4-(2,2,2-trifluoroethyl)-2,4-diazadispiro[4.1.57. l5]tridecane-l ,3-dione

[0573] (545.34 mg, 1.7 mmol), DIAD (344.27 mg, 1.7 mmol), PPh3(446.55 mg, 1.7 mmol) in toluene (5 mL) was degassed and purged with N23 times. The mixture was stirred at 100 °C for 16 h under N2. The reaction was quenched with H2O (20 mL) and extracted with ethyl acetate (25 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiCL, petroleum ether : ethyl acetate = 1 : 1) to give the title compound (270 mg, 44%) as a white solid. MS (ESI): mass calcd. for

[0574] To a solution of 10-(3-butyl-4-chloro-2,6-dioxo-pyrimidin-l-yl)-2-methyl-4-(2,2,2- trifluoroethyl)-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3-dione (270 mg, 534.73 μmo)l in EtOH (3 mL) was added NH2NH2.H2O (1.6 mmol, 97.27 μL, 80% in EtOH). The mixture was stirred at 80 °C for 1.5 h under N2. The reaction was quenched with H2O (10 mL) and extracted with DCM (25 mL x 2). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound (240 mg, 89.7%) as a yellow solid. MS (ESI): mass calcd. for

[0575] 10-[7-butyl-3-[(4-methoxyphenyl)methylamino]-4,6-dioxo-2H-pyrazolo[3,4-d]pyrimidin- 5-yI]-2-methyI-4-(2, 2, 2-trifluoroethyI)-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3-dione

[0576] To a solution of 10-(3-butyl-4-hydrazino-2,6-dioxo-pyrimidin-l-yl)-2-methyl-4-(2,2,2- trifluoroethyl)-2,4-diazadispiro[4.1.57.l5]tridecane-l, 3-dione (150 mg, 300 μmo)l in DMA (1.5 mL) was added l-(isothiocyanatomethyl)-4-methoxy-benzene (53.72 mg, 300 μmo)l. The mixture was stirred at 80 °C for 4 h. Then the mixture was stirred at 120 °C for 12 h. The reaction was quenched with H2O (15 mL) and extracted with ethyl acetate (25 mL x 2). The combined organic layer was washed with H2O, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2. petroleum ether : ethyl acetate = 1 : 1) to give the title compound (100 mg, 47.4%) as a yellow solid. MS (ESI): mass calcd. for

[0577]

[0578] A solution of 10-[7-butyl-3-[(4-methoxyphenyl)methylamino]-4,6-dioxo-2H- pyrazolo[3,4-d]pyrimidin-5-yl]-2-methyl-4-(2,2,2-trifluoroethyl)-2,4- diazadispiro[4.1.57.l5]tridecane-l, 3-dione (23 mg, 35.62 μmo)l in TFA (1 mF) was stirred at 20 °C for 4 h. The reaction was concentrated under reduced pressure. The crude product was purified by prep-HPLC to give the title compound (5.82 mg, 31.1%) as a white solid. MS (ESI): mass calcd. for

[0579] Example 59 and Example 60 were obtained from the chiral SFC separation of Example 58 using similar conditions to those used in Example 26 and Example 27.

[0580] Example 61, Example 62, Example 63, and Example 64. (E')-5-aminomethylene-l-butyl- 3-{(ls,4s)-4-[(5,5-dimethyI-2,4-dioxo-l-imidazoIidinyI)methyI]-4-methyIcycIohexyI}- 2,4,6( 1H,3H,5H )-pyrimidinetrione (61), (Z)-5-(aminomethylene)-l-butyl-3-((ls,4s)-4- ((5,5-dimethyI-2,4-dioxoimidazoIidin-l-yI)methyI)-4-methyIcycIohexyI)pyrimidine- 2,4,6(lH,3H,5H)-trione (62), (E')-5-aminomethylene-l-butyl-3-{(lr,4r)-4-[(5,5-dimethyl- 2,4-dioxo-l-imidazoIidinyI)methyI]-4-methyIcycIohexyI}-2,4,6(177,377,5H)- pyrimidinetrione (63), and (Z)-5-aminomethylene-l-butyl-3-{(lr,4r)-4-[(5,5-dimethyl- 2,4-dioxo-l-imidazoIidinyI)methyI]-4-methyIcycIohexyI}-2,4,6(177,377,5H)- pyrimidinetrione (64).

[0581]

[0582] 8-Methyl-l,4-dioxaspiro[4.5]decane-8-carbaldehyde

[0583] To a solution of DMSO (9.69 g, 124 mmol) in DCM (80 mL) was added oxalyl dichloride (10.49 g, 82.7 mmol) dropwise at -70 °C. After 1 h, (8-methyl-l,4- dioxaspiro[4.5]decan-8-yl)methanol (7.7 g, 41.3 mmol) in DCM (30 mL) was added and reaction was stirred at -70 °C for another 30 min, before TEA (25.1 g, 248.1 mmol) was added. After 2 h at the same temperature, the reaction was quenched with saturated NH4CI (100 mL) and extracted with DCM (3 x 100 mL). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to provide the title compound (8.61 g, crude) as a yellow oil, which was used in the next step directly without further purification.

[0584] Ethyl 2-methyl-2-(((8-methyl-l,4-dioxaspiro[4.5]decan-8-yl)methyl)amino)propanoate

[0585] To a solution of crude 8-methyl-l,4-dioxaspiro[4.5]decane-8-carbaldehyde (8.61 g, 46.7 mmol) and ethyl 2-amino-2-methyl-propanoate hydrochloride (9.4 g, 56.1 mmol) in DCM (100 mL) was added AcOH (2.81 g, 46.7 mmol). After stirring at 20 °C for 30 min, NaBH(OAc)3 (19.81 g, 93.5 mmol) was added. The reaction was stirred at 20 °C for 12 h, poured into ice-water (100 mL), and adjusted to pH = 8 using Na2CO3. The mixture was extracted with DCM (2 x 200 mL). The organic layer was dried, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (petroleum ether : EtOAc = 10 : 1 to 0 : 1) to provide the title compound (6 g, 42.9%) as a white solid. MS (ESI): mass calcd. for C16H29NO4: 299.21, found: 300.2 [M+H]+.

[0586] 5, 5-Dimethyl-l-((l-methyl-4-oxocyclohexyl)methyl)imidazolidine-2, 4-dione

[0587] To a solution of ethyl 2-methyl-2-(((8-methyl-l,4-dioxaspiro[4.5]decan-8- yl)methyl)amino)propanoate (6 g, 20 mmol) in AcOH (25 mL) was added potassium isocyanate (8.13 g, 3.95 mL). After heating at 100 °C for 12 h, the reaction was poured into ice-water (100 mL), adjusted to pH = 8 using aqueous Na2CO3, and extracted with EtOAc (2 x 200 mL). The organic layer was dried, filtered and concentrated under reduced pressure and the crude residue was purified by silica gel column chromatography (petroleum ether : EtOAc = 20 : 1 to 0 : 1) to provide the title compound (5 g, 98.9%) as a yellow oil. MS (ESI): mass calcd. for C13H20N2O3: 252.15, found: 253.2 [M+H]+.

[0588] 5,5-Dimethyl-l-((l-methyl-4-oxocyclohexyl)methyl)-3-((2-(trimethylsilyl)ethoxy)methyl) imidazolidine-2, 4-dione

[0589] To a solution of 5,5-dimethyl-l-((l-methyl-4-oxocyclohexyl)methyl) imidazolidine- 2, 4-dione (5 g, 19.8 mmol) in DCM (60 mL) was added SEM-C1 (6.61 g, 39.6 mmol) and DIPEA (12.81 g, 99.1 mmol). After stirring at 20 °C for 12 h, the reaction was quenched with saturated NH4CI (60 mL) and extracted with DCM (3 x 60 mL). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (petroleum ether : EtOAc = 10 : 1 to 0 : 1) to provide the title compound (5.5 g, 72.6%) as a yellow oil. MS (ESI): mass calcd. for

[0590] To a solution of 5,5-dimethyl-l-((l-methyl-4-oxocyclohexyl)methyl)-3-((2- (trimethylsilyl)ethoxy)methyl)imidazolidine-2, 4-dione (5.5 g, 14.4 mmol) in MeOH (100 mL) was added NH3 / McOH (7 M, 20.5 mL) and Raney-Ni (2.22 g, 25.9 mmol) under N2. The suspension was degassed under vacuum and purged with H2several times and stirred under H2(15 psi) at 20 °C for 1 h. After filtering through a Celite pad, the filtrate was concentrated under reduced pressure to provide the title compound (5.5 g) as a yellow oil, which was used in the next step directly without further purification. MS (ESI): mass calcd. for 383.26, found: 384.3 [M+H]+. l-butyl-3-(4-((5,5-dimethyl-2,4-dioxo-3-((2-(trimethylsilyl)ethoxy)methyl)imidazolidin- l-yl)methyl)-4-methylcyclohexyl)urea

[0591] To l-((4-amino-l-methylcyclohexyl)methyl)-5,5-dimethyl-3-((2-

[0592] (trimethylsilyl)ethoxy)methyl)imidazolidine-2, 4-dione (250 mg, 652 p mol) in DCM (6.5 mL) was added a solution of butyl isocyanate (82.4 μL, 717 μmo)l in DCM (6.5 mL) and the solution was stirred for 10 min. 1 M aqueous HC1 solution was added, and the mixture was extracted with DCM (3x). The combined organic phases was dried over Na2SO4, filtered, and concentrated. The crude was purified by flash column chromatography (silica, 0-10% MeOH / DCM) to provide the title compound (167 mg, 53%). MS (ESI): mass calcd. for l-butyl-3-(4-((5,5-dimethyl-2,4-dioxo-3-((2-

[0593] (trimethylsilyl)ethoxy)methyl)imidazolidin- 1 -yl)methyl)-4-methylcyclohexyl)urea (167 mg, 346 μmo)l in DCM (3.5 mL) was added malonyl chloride (69.4 μL, 692 μmo)l and the mixture was stirred at rt for 1 h. Water was added and the mixture was extracted with DCM (3x). The combined organic phases was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude was purified by flash column chromatography (silica, 20-50% EtOAc / heptane) to provide the title compound (50 mg, 26%). MS (ESI): mass calcd. for

[0594] To l-butyl-3-(4-((5,5-dimethyl-2,4-dioxo-3-((2-

[0595] (trimethylsilyl)ethoxy)methyl)imidazolidin-l-yl)methyl)-4-methylcyclohexyl)pyrimidine- 2,4,6(1H,3H,5H)-trione (100 mg, 182 μmo)l in EtOH (1.8 mL) was added 1,3, 5 -triazine (16.2 mg, 200 μmo)l. The tube was sealed and heated to 80 °C for 30 min. The solvent was removed to get the crude compound which was used as such in the next step. MS (ESI): mass calcd. for

[0596] To 5-(aminomethylene)-l-butyl-3-(4-((5,5-dimethyl-2,4-dioxo-3-((2-

[0597] (trimethylsilyl)ethoxy)methyl)imidazolidin-l-yl)methyl)-4-methylcyclohexyl)pyrimidine- 2,4,6(lH,3H,5H)-trione (105 mg, 182 μmo)l in DCM (1.8 mL) was added trifluoroacetic acid (417 μL, 5.45 mmol) and the solution was stirred for 30 min. A saturated aqueous sodium bicarbonate solution was added, and the bi-phasic mixture was stirred for 10 min and then extracted with DCM (3x). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The residue was dissolved in MeOH and a few drops of 28% ammonium hydroxide were added. The solution was filtered with a syringe filter, and concentrated. The racemate was subjected to chiral separation (Phenomenex, z-Amylose-3, isocratic 4% MeOH / (0.1% formic acid in MeCN)). The first peak to elute was collected to provide 61 (3.5 mg, 4%) as a white solid. MS (ESI): mass calcd. for C22H33N5O5: 447.25 found: 448.5 [M+H]+. ’H NMR (400 MHz, DMSO) 5 10.78 (br s, 1H), 9.51 - 9.78 (m, 1H), 9.26 (br s, 1H), 8.08 (dd, J = 16.6, 8.7 Hz, 1H), 4.64 (br s, 1H), 3.69 - 378 (m, 2H), 3.24 (s, 2H), 2.58 (d, J = 13.3 Hz, 1H), 1.64 - 1.78 (m, 2H), 1.40 - 1.50 (m, 2H), 1.30 (s, 6H), 1.11 - 1.36 (m, 7H), 0.83 - 0.91 (m, 6H). 62 (5.0 mg, 6%) was obtained from the second eluting peak. MS (ESI): mass calcd. for C22H33N5O5: 447.25 found: 448.5 [M+H]+. ’H NMR (400 MHz, DMSO) 5 10.80 (br s, 1H), 9.70 - 9.55 (m, 1H), 9.26 (br s, 1H), 8.09 (dd, 7 = 14.9, 6.9 Hz, 1H), 4.64 (br s, 1H), 3.78 - 3.68 (m, 2H), 3.24 (s, 2H), 2.59 (d, 7 = 12.2 Hz, 1H), 1.70 (s, 1H), 1.49 - 1.42 (m, 3H), 1.30 (s, 6H), 1.35 - 1.16 (m, 7H), 0.92 - 0.80 (m, 6H). 63 (4.6 mg, 6%) was obtained from the third eluting peak. MS (ESI): mass calcd. for C22H33N5O5: 447.25 found: 448.5 [M+H]+.:H NMR (400 MHz, DMSO) 5 10.82 (br s, 1H), 9.60 (br s, 1H), 9.27 (br s, 1H), 8.09 (dd, J = 15.2, 6.5 Hz, 1H), 4.55 (br s, 1H), 3.75 - 3.70 (m, 2H), 2.95 (s, 2H), 1.47 - 1.20 (m, 17H), 0.98 (d, J = 2.9 Hz, 3H), 0.87 (td, J - 7.2, 3.5 Hz, 4H). 64 (4.0 mg, 5%) was obtained from the fourth eluting peak. MS (ESI): mass calcd. for C22H33N5O5: 447.25 found: 448.5 [M+H]+. ’H NMR (400 MHz, DMSO) 5 10.82 (br s, 1H), 9.64 (br s, 1H), 9.28 (br s, 1H), 8.17 - 7.98 (m, 1H), 4.53 (br s, 1H), 3.77 - 3.67 (m, 2H), 2.95 (s, 2H), 1.49 - 1.16 (m, 17H), 0.98 (d, J = 3.2 Hz, 3H), 0.90 - 0.76 (m, 4H). The absolute stereochemistry for these four compounds is arbitrarily assigned.

[0598] Example 65 and Example 66. (E)-5-(amino(methylamino)methylene)-l-butyl-3- ((6R,8r,llR)-3-methyl-2,4-dioxo-l,3-diazadispiro[5.1.58.16] tetradecan-11- yl)pyrimidine-2,4,6(lH,3H,5H)-trione (65) and (E)-5-(amino(methylamino)methylene)-l- butyl-3-((6R,8r,llR)-l,3-dimethyl-2,4-dioxo-l,3-diazadispiro[5.1.58.16]tetradecan-ll- yl)pyrimidine-2,4,6(lH,3H,5H)-trione (66)

[0599] o[3.2.47.24]tridecan-2-ylidene)-2-methylpropane-2-sulfinamide

[0600] To a solution of 8,1 l-dioxadispiro[3.2.47.24]tridecan-2-one (5.00 g, 24.2 mmol) in THF (150 mL) under N2 was added titanium(IV) ethoxide (15.2 mL, 72.6 mmol). The resulting solution was stirred at rt for 5 min and (R)-(+)-2-methyl-2-propanesulfinamide (4.54 g, 36.3 mmol) was added and stirred at rt for 17 h. The reaction was then poured into saturated aqueous NaHCO3solution with rapid stirring then filtered through Celite and the filtered cake washed with EtOAc. Phases were separated and the aqueous phase was extracted with EtOAc (x2) and the combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated. The crude was purified by flash column chromatography (silica, 10-100% EtOAc / heptane) to provide the title compound (5.65 g, 78%) as a white solid. MS (ESI): mass calcd. for

[0601] To a -78 °C solution of lithium diisopropylamide (8.1 mL, 16.1 mmol) in THF (27.6 mL) was added ethyl acetate (1.5 mL, 15.4 mmol) dropwise. After stirring for 20 min, a solution of chlorotitanium triisopropoxide (7.7 mL, 30.7 mmol) in THF (9.2 mL) was added dropwise. After 1 h, (R)-A-(8, 11 -dioxadispiro[3.2.47.24]tridecan-2-ylidene)-2-methylpropane- 2-sulfinamide (2.3 g, 7.68 mmol) in THF (10 mL) was added dropwise. The reaction was stirred at -78 °C for 2.5 h, and then quenched by the addition of 10 mL ammonium chloride solution and warmed to rt. The mixture was filtered through Celite and the cake was washed with EtOAc. Phases were separated and the organic phase was dried over Na2SO4, filtered and concentrated. The crude was purified by flash column chromatography (silica, 0-100% EtOAc / heptanes) to afford the title compound (1.77 g, 59%) as an oil. MS (ESI): mass calcd. for To ethyl (R)-2-(2-((tert-butylsulfinyl)amino)-8,l l-dioxadispiro[3.2.47.24]tridecan-2- yl)acetate (1.70 g, 4.39 mmol) in dioxane (10.0 mL) was added hydrochloric acid 4 N in dioxane (10 mL) and the mixture was stirred at rt for 4 h. The mixture was concentrated to dryness to give the crude title compound which was carried forward as is for next step. ' H

[0602] 13-methyl-l,4-dioxa-ll,13-diazatrispiro[4.2.1.510.l8.25]octadecane-12, 14-dione

[0603] To ethyl 2-(2-amino-8,l l-dioxadispiro[3.2.47.24]tridecan-2-yl)acetate (500 mg, 1.76 mmol) and IV-methyl-lH-imidazole-l -carboxamide (265 mg, 2.12 mmol) dissolved in THF (12.5 mL) was added triethylamine (738 μL, 5.29 mmol) and the solution was stirred at 60 °C for 1 h and the uncyclized urea adduct was observed majorly. Cesium carbonate (1.76 g, 5.29 mmol) was added along with MeCN (12.5 mL) and the mixture stirred at 60 °C for 17 h. The reaction mixture was cooled to rt. DCM was added and washed with saturated aqueous NaHCO3solution and brine, dried over Na2SO4, filtered, and concentrated to provide the crude title compound (400 mg, 77%) which was used such as for the next step. MS (ESI): mass calcd. for

[0604] To 13 -methyl- 1 ,4-dioxa- 11,13 -diazatrispiro [4.2.1.5 .18.25] octadecane- 12,14-dione (400 mg, 1.36 mmol) in THF (8 mL) was added HC1 1 N (1.2 mL, 1.17 mmol) and the reaction was stirred for 3 h at 50 °C. The mixture was cooled down to rt and DCM was added and washed with water, brine, dried over Na2SO4, filtered, and concentrated to provide the title compound (291 mg, 99 %) as a light-yellow solid which was carried forward to next step. MS (ESI): mass calcd. for C13H18N2O3: 250.13, found: 251.4 [M+H]+. ll-amino-3-methyl-l,3-diazadispiro[5.1.58.l6]tetradecane-2, 4-dione

[0605] In a flask was dissolved 3-methyl-l,3-diazadispiro[5.1.58.l6]tetradecane-2, 4, 11-trione (291 mg, 1.16 mmol) in ammonia 7 M in MeOH (2.5 mL, 17.5 mmol) and the solution was stirred at rt for 4 h. MeOH (6.0 mL) and 10% palladium on carbon (248 mg, 116 μmo)l were added. The suspension was purged with hydrogen and the mixture was stirred under hydrogen balloon at rt for 17 h. The reaction was filtered and concentrated to afford the title compound (259 mg, 89%) which was carried forward as crude material. MS (ESI): mass calcd. for

[0606] To l l-amino-3-methyl-l,3-diazadispiro[5.1.58.l6]tetradecane-2, 4-dione (259 mg, 1.03 mmol) in DCM (4.4 mL) was added butyl isocyanate (134 μL, 1.19 mmol) dropwise. The reaction was stirred at rt for 1 h and concentrated to silica gel. The crude was purified by flash column chromatography (silica, 0-10% MeOH / DCM) to provide the title compound (160 mg, 44%) as a white solid. MS (ESI): mass calcd. for

[0607] In a vial were dissolved l-butyl-3-(3-methyl-2,4-dioxo-l,3- diazadispiro[5.1.58.l6]tetradecan-ll-yl)urea (160 mg, 457 μmo)l and malonic acid (57.6 mg, 548 μmo)l in AcOH (3.0 mL) to which acetic anhydride (95.9 μL, 1 mmol) was added. The solution was stirred at 90 °C for 4 h, quenched with water (2 mL) and continued to stir at 90 °C for 30 min. EtOAc and additional water were added and the phases were separated. The organic phase was dried over Na2SO4, filtered, and concentrated to afford the title compound which was used as such in the next step. MS (ESI): mass calcd. for

[0608] To 1 -butyl- 3- (3 -methyl-2 ,4-dioxo- 1 , 3 -diazadispiro [5.1.58.16] tetradecan- 11- yl)pyrimidine- (180 mg, 430 μmo)l in THF (17.4 mL) was added cyanamide (110 mg, 2.58 mmol) and nickel(II) acetylacetonate (22.1 mg, 86 μmo)l and the mixture was stirred at 80 °C for 18 h. The reaction was concentrated under reduced pressure and the crude was purified by reverse phase chromatography (C18, 0-100% MeCN / 10 mM AmB buffer) to provide a white solid (66.0 mg, 33 %) as a racemate. The racemic mixture was subjected to chiral separation (Phenomenex, z-Amylose, 70% Hexane, 30% Ethanol / methanol +0.1% NH4OH as a modifier), and the first peak to elute gave the title compound (17.0 mg, 9%) as a white solid. MS (ESI): mass calcd. for The stereochemistry for this compound is arbitrarily assigned.

[0609] The title compound was the second peak to elute in the chiral SFC separation from the l-butyl-3-(3-methyl-2,4-dioxo-l,3-diazadispiro[5.1.58.l6]tetradecan-l l-yl)pyrimidine- and was isolated as a white solid (17.7 mg, 9%). MS (ESI): mass calcd. for The stereochemistry for this compound is arbitrarily assigned.

[0610] (E)-5-(amino(methylamino)methylene)-l-butyl-3-((6R,8r,llR)-3-methyl-2,4-dioxo-l,3- diazadispiro[5.1.58.16]tetradecan-ll-yI)pyrimidine-2,4,6(lH,3H,5H)-trione (65) and (E)- 5-(amino(methyIamino)methyIene)-l-butyI-3-((6R,8r,llR)-l,3-dimethyI-2,4-dioxo-l,3- diazadispiro[5.1.58.16]tetradecan-ll-yI)pyrimidine-2,4,6(lH,3H,5H)-trione (66)

[0611] To μmo)l dissolved in DMF (0.5 mL) was added cesium carbonate (7.22 mg, 21.7 μmo)l followed by a solution of iodomethane diluted in DMF (10% v / v, 8.2 pF, 13 μmo)l. The reaction was stirred at room temperature for 4 h and quenched with a few drops of water. The products were directly purified by HPLC to afford 65 (0.3 mg, 6%) as an off-white solid. MS (ESI): mass calcd. for

[0612] Example 67 and Example 68 were prepared in a similar fashion to last step in the scheme for Example 65 and Example 66 using Example 69 and Example 70. (E)-5-(l-aminoethylidene)-l-butyl-3-((5R,7r,10R)-l,3- dimethyl-2-oxo-l,3-diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)- trione (69) and (E)-5-(l-aminoethylidene)-l-butyl-3-((5S,7s,10S)-l,3-dimethyl-2-oxo-l,3- diazadispiro[4.1.57.l5]tridecan-10-yl)pyrimidine-2,4,6(lH,3H,5H)-trione (70) l,3-dimethyl-ll,14-dioxa-l,3-diazatrispiro[4.1.2.410.27.l5]heptadecan-2-one

[0613] To a solution of sodium borohydride (450 mg, 11.4 mmol) in THF (3.5 mL) at 0 °C was added boron trifluoride diethyl etherate (1.5 mL, 12.1 mmol) dropwise. The mixture was stirred at 0 °C for 15 min then a solution of l,3-dimethyl-l l,14-dioxa-l,3- diazatrispiro[4.1.2.410'27.l5]heptadecane-2, 4-dione (0.500 g, 1.70 mmol) in THF (2 mL) was added. The mixture was slowly warmed up to rt and stirred for 17 h. MeOH (5 mL) was slowly added at 0 °C and the solution was stirred at rt for 1 h. Solvent was removed and the residue was added water and extracted with EtOAC (x2). The combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The crude was purified by flash column chromatography (silica, 5-100% of a solution mix of 20% MeOH / EtOAc in DCM) to provide the title compound (367 mg, 77%). ’H NMR (400 MHz, DMSO) 5 3.83 (s, 4H), 3.34 (s, 2H), 2.66 (s, 3H), 2.61 (s, 3H), 2.16 - 2.22 (m, 2H), 1.77 - 1.83 (m, 2H), 1.42 - 1.63 (m, 8H). l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 10-dione

[0614] To 1 ,3-dimethyl- 11 , 14-dioxa- 1 ,3-diazatrispiro[4.1.2.410.27. l5]heptadecan-2-one (0.500 g, 1.78 mmol) in acetone (4 mL) and water (2 mL) was added p-toluenesulfonic acid monohydrate (172 mg, 892 μmo)l and the mixture was heated at 50 °C for 17 h. The reaction was cooled down to rt and diluted with EtOAc, washed with 50% aqueous K2CO3solution and brine, dried over MgSCO4filtered, and concentrated to give the crude title compound (421 mg, 100 %) as a colorless solid which was used as such in the next step.

[0615] 10-amino-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecan-2-one To a solution of l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecane-2, 10-dione (300 mg, 1.27 mmol) in MeOH (750 μL) was added ammonia 7 M in MeOH (3.7 mL, 25.9 mmol), followed by 10% palladium on carbon (135 mg, 1.27 mmol). The reaction was submitted to 3 cycles of vacuum / hydrogen purges and stirred under hydrogen for 72 h. The reaction was purged with nitrogen then filtered through Celite and concentrated to give the title compound which was used as such in the next step. MS (ESI): mass calcd. for

[0616] To 10-amino-l,3-dimethyl-l,3-diazadispiro[4.1.57.l5]tridecan-2-one (300 mg, 1.26 mmol) in DCM (2.3 mL) was added triethylamine (705 μL, 5.06 mmol) at rt and the reaction was stirred for 5 min when butyl isocyanate (726 μL, 6.32 mmol) was added and the reaction was stirred for 17 h. The mixture was concentrated onto silica gel and the crude was purified by flash column chromatography (silica, 0-20% MeOH / DCM) to provide the title compound (250 mg, 59 %). MS (ESI): mass calcd. for

[0617] To a solution l-butyl-3-(l,3-dimethyl-2-oxo-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)urea (125 mg, 372 μmo)l in AcOH (1.1 mL) was added malonic acid (78.1 mg, 743 μmo)l. The reaction was heated to 60 °C and acetic anhydride (281 μL, 2.97 mmol) was added. The reaction was sealed and stirred at 90 °C for 1 h. The mixture was cooled down to rt and neutralized with saturated aqueous NaHCO3solution, extracted with DCM (3x). The combined organic phase was dried over Na2SO4, filtered, and concentrated to give the crude title compound which was used as such in the next step. MS (ESI): mass calcd. for C21H32N4O4: 404.24, found: 405.4 [M+H]+.

[0618]

[0619] To l-butyl-3-(l,3-dimethyl-2-oxo-l,3-diazadispiro[4.1.57.l5]tridecan-10- yl)pyrimidine-2,4,6(1H,3H,5H)-trione (40 mg, 98.9 μmo)l in THF (594 μL) was added cyanamide (42.0 mg, 989 μmo)l and nickel(II) acetylacetonate (7.6 mg, 29.7 μmo)l and the mixture was stirred at 85 °C for 17 h. The reaction was concentrated, and the crude was purified by reverse phase chromatography (C18, 0-100% MeCN / 10 mM AmF buffer) to provide the title compound as an impure mixture of desired product and byproduct. MS (ESI): mass calcd. for

[0620] Crude 3-butyl-5-diaminomethylene- 1 -(1 ,3-dimethyl-2-oxo- 1 ,3-diaza- 10- dispiro[4.1.5.1]tridecyl)-2,4,6(1H,3H,5H)-pyrimidinetrione was submitted for chiral SFC separation (Cellulose tris(3,5-dichlorophenylcarbamate, 35% ( IPA + 0.1% NH4OH ) / 65% supercritical CO2). The first peak to elute was collected to provide 1-{(5R,7R,1OR)-1,3- dimethyl-2-oxo- 1 ,3-diaza- 10-dispiro[4.1.5.1 Jtridecyl } -3-butyl-5-diaminomethylene- 2,4,6(1H,3H,5H)-pyrimidinetrione as a white solid. Mass calcd. for C22H34N6O4: 446.26, found: 447.3 [M+H]+. The second peak to elute was collected to provide 1-{(55,7S,105)-1,3- dimethyl-2-oxo- 1 ,3-diaza- 10-dispiro[4.1.5.1 Jtridecyl } -3-butyl-5-diaminomethylene- 2,4,6(1H,3H,5H)-pyrimidinetrione. Mass calcd. for [M+H]+. 69 was obtained from the third eluting peak from chiral SFC separation as a white solid. Mass calcd. for

[0621] Example 71 and Example 72 were prepared following analogous procedures to those described for Example 10, with chiral SFC separation moved to the end of the synthetic sequence. Stereochemistry was assigned arbitrarily.

[0622] Example 73 and Example 74 were prepared following analogous procedures to those described for Example 10, with chiral SFC separation moved to the end of the synthetic sequence. Stereochemistry was assigned arbitrarily.

[0623] Example 75 and Example 76 were obtained after chiral SFC separation of Example 28. Stereochemistry was assigned arbitrarily.

[0624] Example 77 and Example 78 were obtained after chiral SFC separation of Example 29. Stereochemistry was assigned arbitrarily.

[0625] Example 79 was prepared following analogous procedures to those described for Example 28.

[0626] Example 80 was prepared following analogous procedures to those described for Example 28. Example 81 was prepared following analogous procedures to those described for Example 28.

[0627] Example 82 was prepared following analogous procedures to those described for Example 28.

[0628] Example 83 and Example 84 were prepared following analogous procedures to those described for Example 36 followed by chiral SFC separation. Stereochemistry was assigned arbitrarily.

[0629] Example 85 and 86 and were prepared following analogous procedures to those described for Example 36 followed by chiral SFC separation. Stereochemistry was assigned arbitrarily.

[0630] Example 87 and Example 88. (5S,7s,10S)-10-(4-amino-7-butyl-l,3-dioxoisoindolin-2-yl)-

[0631] 3-methyl-l-(((R)-tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2,4- dione (87) and (5R,7r,10R)-10-(4-amino-7-butyl-l,3-dioxoisoindolin-2-yl)-3-methyl-l-

[0632] (((R)-tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2, 4-dione (88)

[0633] To a solution of 4-amino-7-butylisoindoline- 1,3-dione (86.4 mg, 321 μmo)l, (R)-10- hydroxy-3-methyl-l-((tetrahydrofuran-3-yl)methyl)-l,3-diazadispiro[4.1.57.l5]tridecane-2,4- dione (134 mg, 417 μmo)l, and PPh3(127 mg, 481 μmo)l in THF (6.41 mL) was added DIAD (95 μL, 481 μmo)l drop wise at room temperature and the reaction was stirred for 3 h. The resulting mixture was quenched with H2O and extracted with EtOAc (x3). The combined organic layer wwa dried over Na2SO4, filtered, and concentrated. The crude residue was purified by reverse -phase column chromatography (C18, 10-100% MeCN / 10 mM AmB buffer) to afford a diastereomeric mixture of the title compound. The material was subjected to chiral separation by SFC-MS (Lux Cellulose-3, 5 pm, 10 x 250 mm; 60% MeOH + 0.1% NH4OH, 40% supercritical CO2). The first peak to elute was collected to provided the title compound (Example 87) (11.4 mg, 7%) as a yellow solid. MS (ESI): mass calcd. for

[0634] The second peak to elute from the chiral separation above was collected to provide the title compound (Example 88) (11.0 mg, 7%) as a yellow solid. MS (ESI): mass calcd. for

[0635] Example 89. (5S,7s,10S)-10-(7-amino-4-butyl-l,3-dioxo-l,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-yl)-3-methyl-l-(((R)-tetrahydrofuran-3-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione

[0636] To a solution of 7-amino-4-butyl-lH-pyrrolo[3,4-c]pyridine-l,3(2H)-dione (13.2 mg, 60.2 μmo)l, (5R,7r,10R)-10-hydroxy-3-methyl-l-(((R)-tetrahydrofuran-3-yl)methyl)-l,3- diazadispiro[4.1.57.l5]tridecane-2, 4-dione (67.9 mg, 211 μmo)l, and PPh3(55.8 mg, 211 μmo)l in THF (1.20 mL) was added DIAD (41.5 μL, 211 μmo)l dropwise and the reaction was stirred at room temperature for 2 h. The resulting mixture was quenched with H2O and extracted with EtOAc (x4). The combined organic layer was dried over Na2SO4and concentrated in vacuo. The product was purified by reverse-phase column chromatography (Cl 8 column, 5-60% MeCN in 10 mM AmB pH 10.0 buffered solution) to afford title compound (14.4 mg, 46%) as a yellow solid. MS (ESI): mass calcd. for

[0637] Example 90 was prepared following an analogous procedure to that described for Example 89 but using the diastereomer (5S,7s,10S)-10-hydroxy-3-methyl-l-(((R)-tetrahydrofuran-3- yl)methyl)-l ,3-diazadispiro[4.1.57.15] tridecane-2, 4-dione.

[0638] Example 91 and Example 92 were prepared using Intermediate 10 following analogous procedures to those described for Example 43 followed by chiral SFC separation. Stereochemistry was assigned arbitrarily.

[0639] Example 93 and Example 94 were prepared using Intermediate 12 following analogous procedures to those described for Example 43 followed by chiral SFC separation. Stereochemistry was assigned arbitrarily. Example 95 and Example 96 were prepared using Intermediate 12 following analogous procedures to those described for Example 43 followed by chiral SFC separation. Stereochemistry was assigned arbitrarily.

[0640] PTH1R Assay

[0641] Assays were performed using Expi293F Inducible cells (Invitrogen) stably expressing hPTHIR via a pcZeo TetO DNA plasmid. Cell lines were maintained in suspension in Expi293 Expression Medium (ThermoFisher Scientific) supplemented with 10 pg / mL Blasticidin and 10 μg / mL Zeocin and incubated at 37°C, 8% CO2, with shaking. To induce receptor expression, hPTHIR cells were incubated in induction medium (Expi293 Expression Medium with 4 pg / mL Doxycycline (Millipore Sigma), 5 mM sodium butyrate (Millipore Sigma) and 100 ng / mL Pertussis toxin (Millipore Sigma)) for 24 hours at 32°C, 5% CO2, with shaking. Assay-ready aliquots were prepared by harvesting cells 24-hour post-induction. Cells were pelleted at 4°C, resuspended in Expi293 Expression medium + 10% DMSO, aliquoted, and kept frozen at -80°C until ready for use.

[0642] For the assay, concentration-response curves of test and reference compounds were added to 384-well plates using an Apricot liquid handler (SPT Labtech) and backfilled with DMSO to a final concentration of 0.3%. cAMP was measured using the cisbio cAMP Gs dynamic HTRF kit (PerkinElmer) according to manufacturer instructions. Aliquots of frozen hPTHIR cells were quickly thawed and washed with phosphate-buffered saline (Sigma- Aldrich) to remove media and DMSO. The cells were resuspended in kit-supplied Stimulation Buffer at 0.2 x 106cells / mL. 10 pl of the hPTHIR cell dilution were added to each well of the assay plate and incubated with the compounds for 1 hour in a 37°C, 0% CO2incubator. Following this incubation, the cells were lysed and accumulated cAMP was detected through the addition of kit-supplied lysis buffer containing d2 -reagent and Eu- cryptate antibody. The HTRF signal was quantified using a BMG PHERAstar FSX plate reader optimized for HTRF assays. The HTRF ratio was determined by dividing the signal output at 665 nm by that at 620 nm. Data were normalized to the signal produced by 1 pM PTH(l-34) (100% activation) and vehicle (0% activation).

[0643] A complete listing of the compounds, characterization data, and PTH1R assay data for the exemplary compounds is set forth in the table in Figure 1. GCGR

[0644] Assays were performed using Expi293F Inducible cells (Invitrogen) stably expressing hGCGR via a pcZeo TetO DNA plasmid. Cell lines were maintained in suspension in Expi293 Expression Medium (ThermoFisher Scientific) supplemented with 10 pg / mL Blasticidin and 10 μg / mL Zeocin and incubated at 37°C, 8% CO2, with shaking. To induce receptor expression, hGCGR cells were incubated in induction medium (Expi293 Expression Medium with 4 pg / mL Doxycycline (Millipore Sigma), 5 mM sodium butyrate (Millipore Sigma) and 100 ng / mL Pertussis toxin (Millipore Sigma) for 24 hours at 32°C, 5% CO2, with shaking. Assay-ready aliquots were prepared by harvesting cells 24-hour post-induction. Cells were pelleted at 4°C, resuspended in Expi293 Expression medium + 10% DMSO, aliquoted, and kept frozen at -80°C until ready for use.

[0645] For the assay, concentration-response curves of test and reference compounds were added to 384-well plates using an Apricot liquid handler (SPT Labtech) and backfilled with DMSO to a final concentration of 0.3%. cAMP was measured using the cisbio cAMP Gs dynamic HTRF kit (PerkinElmer) according to manufacturer instructions. Aliquots of frozen hGCGR cells were quickly thawed and washed with phosphate-buffered saline (Sigma- Aldrich) to remove media and DMSO. The cells were resuspended in kit-supplied Stimulation Buffer at 60,000 cells / mL. 10 pl of the hGCGR cell dilution were added to each well of the assay plate and incubated with the compounds for 1 hour in a 37 °C, 0% CO2incubator. Following this incubation, the cells were lysed and accumulated cAMP was detected through the addition of kit-supplied lysis buffer containing d2 -reagent and Eu- cryptate antibody. The HTRF signal was quantified using a BMG PHERAstar FSX plate reader optimized for HTRF assays. The HTRF ratio was determined by dividing the signal output at 665 nm by that at 620 nm. Data were normalized to the signal produced by 3 nM Glucagon (100% activation) and vehicle (0% activation).

[0646] Results of the assay are provided in Table 1 below:

[0647] Table 1. GCGR Assay Data

[0648] GCGR pEC50>7: +++; pEC50between 6 and 7: ++; and pEC50<6: +.

[0649] GIPR

[0650] Cell-based functional activation of GIPR was measured using the CisBio cAMP Gs dynamic kit (PerkinElmer). Prior to the day of the assay, Expi293F Inducible cells (Invitrogen) stably overexpressing the human GIP receptor under a Tet repressor system were induced with 4 μg / mL doxycycline, 5 mM sodium butyrate, and 100 ng / mL Pertussis toxin at 37°C. After 20 hours of induction, cells were harvested, aliquoted, and cryopreserved. Concentration response curves of test and reference compounds were acoustically dispensed to a 384-well assay plate using an Echo 650 (Beckman Coulter). On the day of the assay, cells are rapidly thawed and resuspended in IX Cisbio stimulation buffer and plated at 2,500 cells / well, 10 p L / wcll into the assay plate containing compounds. Following a 1-hour incubation at 37°C and 0% CO2, the reaction was terminated by addition of the Cisbio kit- supplied lysis buffer containing d2-reagent and Eu-cryptate antibody. Following a 1-hour incubation at room temperature in the dark, HTRF-FRET values were measured using the BMG ClarioStar Plus plate reader according to manufacturer instructions. Data were normalized to the signal produced by 3 nM GIP 1-42 peptide (100% activation) and vehicle (0% activation).

[0651] Results of the GIPR Assay are provided in Table 2.

[0652] Table 2: GIPR Assay

[0653]

[0654] GIPR pEC50>7: +++; pEC50between 6 and 7: ++; and pEC50<6: +.

[0655] GLP1R

[0656] Cell-based functional activation of GLP1R was measured using the CisBio cAMP Gs dynamic kit (PerkinElmer). Prior to the day of the assay, Expi293F Inducible cells (Invitrogen) stably overexpressing the human GLP1 receptor under a Tet repressor system were induced with 4 μg / mL doxycycline, 5 mM sodium butyrate, and 100 ng / mL Pertussis toxin at 37°C. After 20 hours of induction, cells were harvested, aliquoted, and cryopreserved. Concentration response curves of test and reference compounds were acoustically dispensed to a 384-well assay plate using an Echo 650 (Beckman Coulter). On the day of the assay, cells are rapidly thawed and resuspended in IX Cisbio stimulation buffer and plated at 2,000 cells / well, 10 p L / wcll into the assay plate containing compounds.

[0657] Following a 1-hour incubation at 37°C and 0% CO2, the reaction was terminated by addition of the Cisbio kit-supplied lysis buffer containing d2-reagent and Eu-cryptate antibody. Following a 1-hour incubation at room temperature in the dark, HTRF-FRET values were measured using the BMG ClarioStar Plus plate reader according to manufacturer instructions. Data were normalized to the signal produced by 10 nM Danuglipron (100% activation) and vehicle (0% activation).

[0658] Results: For GLP1R, all compounds are + (pEC50>7: +++; pEC50between 6 and 7: ++; and pEC50<6:+).

[0659] INCORPORATION BY REFERENCE

[0660] All of the U.S. patents and U.S. and PCT patent application publications cited herein are hereby incorporated by reference.

[0661] EQUIVALENTS

[0662] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWe claim:

1. A compound according to Formula (I)or a pharmaceutically acceptable salt thereof; wherein:R1is (C1-C6)alkyl;R2is hydrogen or (C1-C6)alkyl optionally substituted with hydroxy or 4- to 7- membered heterocylcylalkyl;R3is (C1-C6)alkyl optionally substituted with (C3-C8)cycloalkyl or phenyl;R4aand R4bare independently hydrogen or (C1-C6)alkyl, wherein the (C1-C6)alkyl is optionally substituted with one or more instances of fluoro, hydroxy, or 4- to 7-membered heterocycloalkyl; or R4aand R4btaken together with the atoms to which they are attached form taken together with the nitrogen atom to which they are attached form a 4- to 7- membered heterocycloalkyl; andR5aand R5bare independently hydrogen or (C1-C6)alkyl, wherein the (C1-C6)alkyl is optionally substituted with one or more instances of fluoro, hydroxy, or 4- to 7-membered heterocycloalkyl; or R5aand R5btaken together with the nitrogen atom to which they are attached form taken together with the atoms to which they are attached form a 4- to 7- membered heterocycloalkyl; wherein at least one of R4aand R4bR5aand R5bis hydrogen, and at least one of R4aand R4bR5aand R5bis not hydrogen; provided that the compound is not:

2. The compound of claims 1, having the structure of Formula (la) or lb):or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1 or 2, wherein R1is methyl.

4. The compound of any one of claims 1 to 3, wherein R2is hydrogen.

5. The compound of any one of claims 1 to 3, wherein R2is methyl.

6. The compound of any one of claims 1 to 3, wherein R2is (C1-C6)alkyl substituted with hydroxy.

7. The compound of claim 6, wherein R2is 2-hydroxy-2-methylpropyl.

8. The compound of any one of claims 1 to 3, wherein R2is methyl substituted with 4- to7-membered heterocycloalkyl.

9. The compound of any one of claims 1 to 3 or 8, wherein the 4- to 7 -membered heterocycloalkyl is oxetanyl, tetrahydrofuranyl, or tetrahydropyranyl.

10. The compound of claim 9, wherein the 4- to 7-membered heterocycloalkyl is oxetan- 3-yl, tetrahydrofuran-3-yl, or tetrahydropyran-4-yl.

11. The compound of any one of claims 1 to 10, wherein R3is methyl optionally substituted with (C3-C8)cycloalkyl or phenyl.

12. The compound of any one of claims 1 to 11, wherein R3is (C1-C6)alkyl optionally substituted with phenyl.

13. The compound of any one of claims 1 to 11, wherein R3is (C1-C6)alkyl optionally substituted with cyclopropyl, cyclobutyl, or cyclopentyl.

14. The compound of any one of claims 1 to 10, wherein R3is n-butyl.

15. The compound of any one of claims 1 to 14, wherein at least two of R4a, R4b, R5aand R5bare hydrogen.

16. The compound of claim 1, wherein three of R4a, R4b, R5aand R5bare hydrogen.

17. The compound of any one of claims 1 to 15, wherein at least two of R4aand R4btaken together with the nitrogen atom to which they are attached form azetidine.

18. The compound of any one of claims 1 to 16, wherein one of R4aand R4b, or R5aand R5bis (C1-C6)alkyl optionally substituted with fluoro, hydroxy, or 4- to 7-membered heterocycloalkyl.

19. The compound of claim 18, wherein one of R4aand R4bis 2,2,2-trifluoroethyl.

20. The compound of claim 18, wherein one of R4aand R4bis 2-hydroxy-2-methylpropyl.

21. The compound of claim 18, wherein the 4- to 7-membered heterocycloalkyl is 3- oxetanyl.

22. The compound of claim 18, wherein the 4- to 7-membered heterocycloalkyl is tetrahydrofuran-2-yl.

23. The compound of any one of claims 1 to 22, wherein one of R4aand R4bis methyl, ethyl, isopropyl, or n-butyl.

24. The compound of any one of claims 1 to 22, wherein one of R4aand R4bis methyl.

25. The compound of any one of claims 1 to 24, wherein one of R5aand R5bis methyl.

26. A compound according to Formula (II):or a pharmaceutically acceptable salt thereof; wherein:R1is (C1-C6)alkyl;R2is hydrogen or (C1-C6)alkyl;R3is (C1-C6)alkyl; andR4a, R4b, R5aand R5bare each independently hydrogen or (C1-C6)alkyl; wherein at least one of R4aand R4bR5aand R5bis not hydrogen.

27. The compound of claim 24, having the structure of Formula (Ila) or (Uh) :or a pharmaceutically acceptable salt thereof.

28. The compound of claim 26 or 27, wherein R1is methyl.

29. The compound of any one of claims 26 to 28, wherein R2is hydrogen.

30. The compound of any one of claims 26 to 28, wherein R2is methyl.

31. The compound of any one of claims 26 to 30, wherein R3is n-butyl.

32. The compound of any one of claims 26 to 31, wherein R4ais methyl.

33. The compound of any one of claims 26 to 32, wherein R4b, R5aand R5bare each hydrogen.

34. A compound having a structure selected from the following table:

35. A compound according to Formula (III):or a pharmaceutically acceptable salt thereof, wherein:X is N or CH;Y is hydrogen or NH2;R6is:R7is hydrogen, orR6and R7taken together with the atom to which they are attached form:R8is (C1-C6)alkyl;R9and R10are independently (C1-C6)alkyl; andR11is (C1-C6)alkyl; andR12is (C1-C6)alkyl optionally substituted with heterocycloalkyl.

36. A compound according to claim 33, having the structure of Formula (Illa), (Illb),(IIIc) or (Illd):or a pharmaceutically acceptable salt thereof.

37. The compound of claim 35 or 36, wherein X is CH2.

38. The compound of claim 35 or 36, wherein X is N.

39. The compound of any one of claims 35 to 38, wherein Y is hydrogen.

40. The compound of any one of claims 35 to 38, wherein Y is NH2.

41. The compound of any one of claims 35 to 40, wherein R8is hydrogen.

42. The compound of any one of claims 35 to 40, wherein R8is (C1-C6)alkyl.

43. The compound of claim 42, wherein R8is n-butyl.

44. The compound of any one of claims 35 to 43, wherein R9and R10are each methyl.

45. The compound of any one of claims 35 to 44, wherein R11is methyl.

46. The compound of any one of claims 35 to 45, wherein R12is methyl.

47. The compound of any one of claims 35 to 45, wherein R12is 3-(oxetanyl)methyl.

48. The compound of any one of claims 35 to 45, wherein R12is 3-(tetrahydrofurayl)methyl.

49. A compound according to Formula (IV):or a pharmaceutically acceptable salt thereof, wherein:R13and R14are each independently (C1-C6)alkyl; andR15and R16are each independently (C1-C6)alkyl.

50. The compound of claim 49, having the structure of Formula (IVa), (IVb), (IVc), or (IVd):or a pharmaceutically acceptable salt thereof.

51. The compound of claim 49 or 50, wherein R14is n-butyl.

52. The compound of any one of claims 49 to 51, wherein R13is methyl.

53. The compound of any one of claims 49 to 52, wherein R15and R16are each methyl.

54. A compound having the structure of Formula (V):or a pharmaceutically acceptable salt thereof, wherein:R17is:R18is hydrogen; orR17and R18taken together with the atom to which they are attached form:R19is (C1-C6)alkyl;R20is (C1-C6)alkyl or (C3-C8)cycloalkyl, each of which is optionally substituted with one or more fluoro, hydroxy, (C1-C6)alkoxy, and (C1-C6)thioalkoxy;R21and R22are each independently (C1-C6)alkyl or R21and R22taken together with the atom to which they are attached form (C3-C8)cycloalkyl; andR23and R24are each independently (C1-C6)alkyl.

55. The compound of claim 54, having the structure of Formula (Va), (Vb), (Vc), (Vd), (Vf), (Vg), or (Vh):or a pharmaceutically acceptable salt thereof.

56. The compound of claim 54 or 55, whereinR19is n-butyl.

57. The compound of any one of claims 54 to 56, wherein R20is methyl or cyclopropyl.

58. The compound of any one of claims 54 to 57, wherein R21and R22taken together with the atom to which they are attached form cyclopropyl.

59. The compound of any one of claims 50 to 54, wherein R23and R24are each methyl.

60. A compound of Formula (VI):or a pharmaceutically acceptable salt thereof, wherein:R25and R26are independently (C1-C6) alkyl; andR27is (C1-C6)alkyl optionally substituted with one or more fluorine atoms.

61. The compound of claim 60, having the structure of Formula (Via) or (VIb):or a pharmaceutically acceptable salt thereof.

62. The compound of claim 60 or 61, wherein R25is methyl.

63. The compound of any one of claims 60 to 62, wherein R26is methyl.

64. The compound of any one of claims 60 to 63, wherein R27is 2,2,2-trifluoroethyl.

65. A compound of Formula (VII):or a pharmaceutically acceptable salt thereof, wherein:R28and R29are independently (C1-C6) alkyl; andR30is (C1-C6)alkyl optionally substituted with (C3-C8)cycloalkyl.

66. The compound of claim 65, having the structure of Formula (Vila) or (Vllb):

67. The compound of claim 65 or 66, wherein R28is methyl.

68. The compound of any one of claims 65 to 67, wherein R29is methyl.

69. The compound of any one of claims 65 to 68, wherein R30is methyl substituted with (C3-C8)cycloalkyl.

70. The compound of any one of claims 65 to 69, wherein R30is cyclopentylmethyl.

71. A compound having the structure:or a pharmaceutically acceptable salt thereof.

72. A pharmaceutical composition, comprising a compound of any one of claims 1 to 71, or a pharmaceutically acceptable salt thereof; and at least one pharmaceutically acceptable excipient.

73. A method for treating or preventing osteoporosis, fracture, osteomalacia, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia or tumoral calcinosis, comprising administering to a subject in need thereof a therapeutically amount of a compound of any one of claims 1 to 71, or a pharmaceutically acceptable salt thereof.

74. A method of: a) reducing of HbAlc; b) treating or preventing type 2 diabetes, hyperglycemia, impaired glucose tolerance, or non-insulin dependent diabetes, and / or obesity; c) reducing body weight and / or food intake, and / or inducing satiety; and / or d) treating or preventing Alzheimer’s disease, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and / or cardiovascular diseases; comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 71 , or a pharmaceutically acceptable salt thereof.

Images