Combination therapy for treating cancer

Abstract

The present application relates to combination therapies for the treatment of cancer. The present disclosure provides methods of treating cancer in a patient. The method comprises administering to the patient an effective amount of a compound having formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and an effective amount of an immunomodulator. Also provided herein are compositions and kits for performing the methods described herein. In another aspect, the method comprises administering to the patient an effective amount of a compound having formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and an effective amount of radiation therapy.

Description

[0001] This application is a divisional application of the invention patent application with application date of November 2, 2020, application number 202080074637.9, and invention name “Combination therapy for treating cancer”.

[0002] Related Applications

[0003] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 930,054, filed on November 4, 2019, which is incorporated herein by reference in its entirety for all purposes. Background Art

[0004] Arginase is a manganese metalloenzyme that catalyzes the conversion of L-arginine to urea and L-ornithine. There are two isoforms, arginase 1 and arginase 2. Although L-arginine is not an essential amino acid because it can be provided by protein turnover in healthy adults, under various physiological and pathological conditions (e.g., pregnancy, autoimmune diseases, cancer), the expression and secretion of arginase are increased, resulting in reduced L-arginine levels. Immune cells are particularly sensitive to reduced L-arginine levels. Tumors use a variety of immunosuppressive mechanisms to evade the immune system. One of these is to reduce L-arginine by increasing the level of circulating arginase, increasing arginase expression and secretion by tumor cells, and recruiting myeloid-derived suppressor cells that express and secrete arginase. Together, these mechanisms lead to reduced L-arginine in the tumor microenvironment and produce an immunosuppressive phenotype. Pharmacological inhibition of arginase activity has been shown to reverse low L-arginine-induced immunosuppression in animal models. However, many proteins and pathways are involved in cancer, and their research is progressing rapidly. Therefore, there is a need to provide patients with new cancer therapies. Summary of the invention

[0005] In some embodiments, the present disclosure provides a method of treating cancer in a patient, comprising administering to the patient an effective amount of a compound having Formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and an effective amount of an immunomodulator;

[0006]

[0007] n is 0 or 1;

[0008] R 1 is -H or -C(O)CH(R 1a )NHR 1b ;

[0009] R 1a Selected from -H, -(C1-C6)alkyl and CH2OR 1c ;

[0010] R1b is -H; or alternatively, R 1a and R 1b together with the atoms to which they are attached, form a 5-membered heterocyclic ring; and

[0011] R 1c It is H or -CH3.

[0012] In some embodiments, the present disclosure provides a method of treating cancer in a patient, comprising administering to the patient an effective amount of a compound of Formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and an effective amount of radiation therapy. In some embodiments, the method further comprises administering to the patient an effective amount of an immunomodulator.

[0013] In some embodiments, the radiation therapy is fractionated radiation therapy.

[0014] In some embodiments of Formula (Ia) or (Ib), R 1 is -H or -C(O)CH(R 1a )NH2; and R 1a Selected from -H or -(C1-C6)alkyl.

[0015] In some embodiments of Formula (Ia) or (Ib), the compound is represented by Formula (IIa) or (IIb):

[0016]

[0017] wherein n is 0 or 1; and R 2 Selected from -H or -(C1-C4)alkyl.

[0018] In some embodiments, the immunomodulator is an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the immune checkpoint inhibitor is selected from CTLA-4 receptor inhibitors, PD-1 receptor inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, NKG2A receptor inhibitors, and combinations thereof. In some embodiments, the immunostimulant is a TLR3 agonist.

[0019] In some embodiments, the immune checkpoint inhibitor is an antibody or an antigen-binding fragment thereof.

[0020] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 receptor antibody, an anti-PD-1 receptor antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an anti-NKG2A receptor antibody. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 receptor antibody, an anti-PD-L1 antibody, an anti-NKG2A receptor antibody, or a combination thereof.

[0021] In some embodiments, the immune checkpoint inhibitor is durvalumab, tremelimumab, monalizumab, or a combination thereof.

[0022] In some embodiments, the cancer is breast cancer, bladder cancer, head and neck cancer, non-small cell lung cancer, small cell lung cancer, colorectal cancer, gastrointestinal stromal tumors, gastroesophageal cancer, renal cell carcinoma, prostate cancer, liver cancer, colon cancer, pancreatic cancer, ovarian cancer, lymphoma (including non-Hodgkin's lymphoma), cutaneous T-lymphocytic tumor, or melanoma. In some embodiments, the cancer is a hematological malignancy, including multiple myeloma, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CMML), and diffuse large B-cell lymphoma (DLBCL).

[0023] In some embodiments, the compound of Formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and the immunomodulator are administered sequentially, separately, or simultaneously.

[0024] In some embodiments, the present disclosure provides a compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, for use in treating cancer in a patient, wherein the compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and an immunomodulator are administered to the patient sequentially, separately, or simultaneously.

[0025] In some embodiments, the present disclosure provides an immunomodulator for use in treating cancer, wherein the immune checkpoint inhibitor and a compound of Formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, are administered to the patient sequentially, separately or simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A , 1B , 1C, and 1D show the effects of arginase inhibitors ( Figure 1A and 1B )、Anti-PDL1( Figure 1A and 1C ), and the combination of arginase inhibitor (ARG inh) and anti-PDL1 ( Figure 1A and 1D ) resulted in a reduction in tumor volume over time.

[0027] Figure 2A , 2B , 2C, and 2D show the effects of arginase inhibitors ( Figure 2A ), combination of arginase inhibitor and anti-PDL1 ( Figure 2B ), combination of arginase inhibitor and anti-NKG2A ( Figure 2C ), and a combination of arginase inhibitors, anti-PDL1 and anti-NKG2A ( Figure 2D ) resulted in a reduction in tumor volume over time.

[0028] Figure 3A , 3B , 3C, 3D, 3E, and 3F show that in the MC38-ova study, vehicle ( Figure 3A ), arginase inhibitors ( Figure 3B , 3E and 3F), TLR3 agonists ( Figure 3C , 3E and 3F), and a combination of an arginase inhibitor and a TLR3 agonist ( Figure 3D , 3E and 3F) tumor volume reduction over time.

[0029] Figure 4A , 4B and 4C show immune cell changes in tumors using arginase inhibitors, anti-PDL1, and a combination of arginase inhibitors and anti-PDL1.

[0030] Figure 4D , 4E and 4F show increased CD8+ ( Figure 4A and 4B ) and CD103+( Figure 4F )T cell function.

[0031] Figure 5A and 5B The authors demonstrated increased production of IFNγ in tumor-draining lymph nodes using arginase inhibitors, anti-PDL1, and a combination of arginase inhibitors and anti-PDL1. Figure 5A ) and TNFα( Figure 5B )'s CD8+ T cell function.

[0032] Fig. 6A The antitumor activity of an arginase inhibitor (Compound 12) in combination with radiation therapy (RT) in the Lewis Lung syngeneic tumor model was demonstrated.

[0033] Figure 6B It was demonstrated that the combination of an arginase inhibitor (Compound 12) and radiation therapy (RT) reduced Lewis lung tumor volume at the end of the study (Day 19). DETAILED DESCRIPTION

[0034] The present disclosure relates to methods of treating cancer in a patient. In one embodiment, the method comprises administering to the patient a compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, in combination with an immunomodulator. The compound of formula (Ia) or (Ib), or any subclass or species thereof, can be used as an arginase inhibitor in therapy.

[0035] Unless otherwise modified by the term "complete", as in "intact antibody", the term "antibody" as used herein also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function (e.g., the ability to bind to antigens such as CTLA-4, PD1, PD-L1, or NKG2A). Typically, such fragments will contain an antigen binding domain.

[0036] The term "mAb" refers to a monoclonal antibody. The antibodies disclosed herein may include, but are not limited to, all-natural antibodies; bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain V region fragment (scFv); fusion polypeptides; and unconventional antibodies.

[0037] The terms "treat," "treating," and "treatment" include reducing or inhibiting the activity of an enzyme or protein associated with arginase or cancer in a subject, improving one or more symptoms of cancer in a subject, or slowing or delaying the progression of cancer in a subject. The terms "treat," "treating," and "treatment" also include reducing or inhibiting the growth of a tumor or the proliferation of cancerous cells in a subject.

[0038] The terms "inhibit," "inhibition," or "inhibiting" include a decrease in the baseline activity of a biological activity or process.

[0039] The term "pharmaceutical composition" includes a composition comprising an active ingredient and a pharmaceutically acceptable excipient, carrier or diluent, wherein the active ingredient is a compound of formula (Ia) or (Ib) (including any subclass or species thereof) as described herein, or a pharmaceutically acceptable salt thereof, or an immunomodulator. The term "pharmaceutically acceptable excipient, carrier or diluent" includes compounds, materials, compositions and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, as determined by those skilled in the art, within the scope of reasonable medical judgment. In some embodiments, the pharmaceutical composition is in a solid dosage form, such as a capsule, tablet, granule, powder, pouch, etc. In some embodiments, the pharmaceutical composition is in the form of a sterile injectable solution in one or more aqueous or non-aqueous non-toxic parenterally acceptable buffer systems, diluents, solubilizers, co-solvents or carriers. Sterile injectable preparations can also be sterile injectable aqueous or oily suspensions or suspensions in non-aqueous diluents, carriers or co-solvents, which can be prepared using one or more suitable dispersants or wetting agents and suspending agents according to known procedures. The pharmaceutical composition can be a solution for intravenous push / infusion, or a freeze-dried system (alone or with excipients) reconstituted with or without other excipients using a buffer system. Freeze-dried freeze-dried materials can be prepared by non-aqueous solvents or aqueous solvents. The dosage form can also be a concentrate for further dilution for subsequent infusion.

[0040] The term "patient" includes warm-blooded mammals, such as primates, dogs, cats, rabbits, rats and mice. In some embodiments, the subject is a primate, such as a human. In some embodiments, the patient suffers from cancer. In some embodiments, the cancer is breast cancer, bladder cancer, head and neck cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, colorectal cancer, gastrointestinal stromal tumors, gastroesophageal cancer, renal cell carcinoma, prostate cancer, liver cancer, colon cancer, pancreatic cancer, ovarian cancer, lymphoma (including non-Hodgkin's lymphoma), cutaneous T lymphocyte tumor or melanoma. In some embodiments, the cancer is a malignant blood disease, including multiple myeloma, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CMML) and diffuse large B-cell lymphoma (DLBCL).

[0041] The term "effective amount" includes an amount of a compound of Formula (Ia) or (Ib) (including any subclasses or species thereof) as described herein and / or an amount of an immunomodulator that will cause a biological or medical response in a subject, for example, to reduce or inhibit the activity of an enzyme or protein associated with arginase or cancer; to improve cancer symptoms; or to slow or delay the progression of cancer. In some embodiments, the term "effective amount" includes an amount of a compound of Formula (Ia) or (Ib) (including any subclasses or species thereof) and / or an immunomodulator that is effective in at least partially alleviating, inhibiting, and / or ameliorating cancer or inhibiting arginase, and / or reducing or inhibiting the growth of a tumor or the proliferation of cancerous cells in a subject.

[0042] Compound

[0043] In one embodiment, a compound having formula (Ia), or a pharmaceutically acceptable salt thereof, is administered to a subject in combination with an immunomodulator, wherein

[0044]

[0045] n is 0 or 1;

[0046] R 1 is -H or -C(O)CH(R 1a )NHR 1b ;

[0047] R 1a Selected from -H, -(C1-C6)alkyl and CH2OR 1c ;

[0048] R 1b is -H; or alternatively, R 1a and R 1b together with the atoms to which they are attached, form a 5-membered heterocyclic ring; and

[0049] R 1c It is H or -CH3.

[0050] In one embodiment, a compound having formula (Ia) is disclosed. In another embodiment, a pharmaceutically acceptable salt of a compound having formula (Ia) is disclosed.

[0051] In some embodiments of Formula (Ia), R 1 is -H or -C(O)CH(R 1a )NH2; and R 1a Selected from -H or -(C1-C6)alkyl.

[0052] In some embodiments of Formula (Ia), R 1 Yes -H.

[0053] In some embodiments of Formula (Ia), R 1 is -C(O)CH(R 1a )NHR 1b ; R 1a is -H; and R 1b Yes -H.

[0054] In some embodiments of Formula (Ia), R 1 is -C(O)CH(R 1a )NHR 1b ; R 1a is -(C1-C6)alkyl; and R 1b Yes -H.

[0055] In some embodiments of Formula (Ia), R 1 is -C(O)CH(R 1a )NHR 1b ; R 1a It is CH2OR 1c ; and R 1b Yes -H.

[0056] In some embodiments of Formula (Ia), R 1 is -C(O)CH(R 1a )NHR 1b ; and R 1a and R 1b Together with the atoms to which they are attached they form a 5-membered heterocyclic ring.

[0057] In any of the foregoing embodiments of Formula (Ia), the compound is represented by any of the following structural formulas:

[0058]

[0059] Where R 1 The same as defined in the above formula (Ia).

[0060] In one embodiment, a compound having formula (IIa), or a pharmaceutically acceptable salt thereof, is disclosed:

[0061]

[0062] wherein n is 0 or 1; and R 2 Selected from -H or -(C1-C4)alkyl.

[0063] In one embodiment, a compound having formula (IIa) is disclosed. In another embodiment, a pharmaceutically acceptable salt of a compound having formula (IIa) is disclosed.

[0064] In some embodiments of Formula (IIa), R 3 Yes -H.

[0065] In some embodiments of Formula (IIa), R 3 It is -(C1-C4)alkyl.

[0066] In any of the foregoing embodiments of Formula (IIa), the compound is represented by one of the following structural formulas:

[0067]

[0068] Where R 2 The same as defined in the above formula (IIa).

[0069] In one embodiment, a compound of formula (Ib), or a pharmaceutically acceptable salt thereof, is administered to a subject in combination with an immune checkpoint inhibitor, wherein

[0070]

[0071] n is 0 or 1;

[0072] R 1 is -H or -C(O)CH(R 1a )NHR 1b ;

[0073] R 1a Selected from -H, -(C1-C4)alkyl and CH2OR 1c ;

[0074] R 1b is -H; or alternatively, R 1a and R 1b together with the atoms to which they are attached, form a 5-membered heterocyclic ring; and

[0075] R 1c It is H or -CH3.

[0076] In one embodiment, a compound having formula (Ib) is disclosed. In another embodiment, a pharmaceutically acceptable salt of a compound having formula (Ib) is disclosed.

[0077] In some embodiments of Formula (Ib), R 1 Yes -H.

[0078] In some embodiments of Formula (Ib), R 1 is -C(O)CH(R 1a )NHR 1b ; R 1a is -H; and R 1b Yes -H.

[0079] In some embodiments of Formula (Ib), R 1 is -C(O)CH(R 1a )NHR 1b ; R 1a is -(C1-C6)alkyl; and R 1b Yes -H.

[0080] In some embodiments of Formula (Ib), R 1 is -C(O)CH(R 1a )NHR 1b ; R 1a It is CH2OR 1c ; and R 1b Yes -H.

[0081] In some embodiments of Formula (Ib), R 1 is -C(O)CH(R 1a )NHR 1b ; and R 1a and R 1b Together with the atoms to which they are attached they form a 5-membered heterocyclic ring.

[0082] In any of the foregoing embodiments of Formula (Ib), the compound is represented by any of the following structural formulas:

[0083]

[0084] Where R 1 The same as defined in the above formula (Ib).

[0085] In one embodiment, a compound having formula (IIb), or a pharmaceutically acceptable salt thereof, is disclosed:

[0086]

[0087] wherein n is 0 or 1; and R 2 Selected from -H or -(C1-C4)alkyl.

[0088] In one embodiment, a compound having formula (IIb) is disclosed. In another embodiment, a pharmaceutically acceptable salt of a compound having formula (IIb) is disclosed.

[0089] In some embodiments of Formula (IIb), R 2 Yes -H.

[0090] In some embodiments of Formula (IIb), R 2 It is -(C1-C4)alkyl.

[0091] In some embodiments of Formula (IIb), the compound is represented by one of the following structural formulas:

[0092]

[0093] Where R 2 The same as defined in the above formula (IIb).

[0094] In some embodiments, compounds of formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), and (IIa2) (including any subclasses and species thereof) are converted into compounds of formula (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses and species thereof) via intramolecular cyclization, and vice versa. In other words, it is an interconversion process. Compounds of formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), and (IIa2) (including any subclasses and species thereof), and compounds of formula (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses and species thereof), are each partially or completely converted into another according to conditions such as temperature, pressure, humidity, pH, and / or medium (e.g., solvent) composition. This is illustrated in the following scheme:

[0095]

[0096] Where R 1 The same as defined in the above formulae (Ia) and (Ib).

[0097] In some embodiments, a compound of Table 1, or a pharmaceutically acceptable salt thereof is disclosed:

[0098] Table 1

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] The term "C1-C4 alkyl" includes non-cyclic alkyl moieties having 1 to 4 carbon atoms, and "C1-C6 alkyl" includes non-cyclic alkyl moieties having 1 to 6 carbon atoms. Examples of C1-C4 alkyl moieties include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

[0106] The term "pharmaceutically acceptable salt" includes acid addition salts or base addition salts that retain the biological effectiveness and properties of the compounds of Formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses or species thereof) and the compounds of Table 1, and that are generally not biologically or otherwise undesirable. In many cases, the compounds of Formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses or species thereof) and the compounds of Table 1 are capable of forming acid and / or basic salts due to the presence of basic and / or carboxyl groups or groups similar thereto.

[0107] Pharmaceutically acceptable acid addition salts can be formed using inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide / hydrogen iodide Iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthoate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, palmoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, stearate, succinate, subsalicylate, sulfate / hydrogensulfate, tartrate, toluenesulfonate and trifluoroacetate. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid, sulfosalicylic acid, and the like.

[0108] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, ammonia and salts of ammonium and metals from columns I to XII of the periodic table. In certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0109] Pharmaceutically acceptable salts of compounds of Formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses or species thereof) and compounds of Table 1 can be synthesized from basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of an appropriate base (e.g., Na + , Ca 2+ Mg 2+ or K + The invention relates to the preparation of the present invention in the form of a hydroxyl radical ... Lists of additional suitable salts can be found, for example, in the following: "Remington's Pharmaceutical Sciences," 20th edition, Mack Publishing Company, Easton, Pa., (1985); Berge et al., "J. Pharm. Sci., 1977, 66, 1-19, and Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, Weinheim, Germany, 2002).

[0110] Any formula given herein is also intended to represent unlabeled and isotopically labeled forms of compounds having Formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses or species thereof) and compounds of Table 1. Isotopically labeled compounds have the structure depicted by the formula given herein, except that one or more atoms are replaced by atoms of the same element but with a different mass number. Examples of isotopes that can be incorporated into compounds having Formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses or species thereof) and compounds of Table 1 and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 15 N. 35 S. 36 Cl and 125 I. Isotopically labeled compounds of Formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses or species thereof) and compounds of Table 1 can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples, using an appropriate isotopically labeled reagent in place of the non-labeled reagent previously employed.

[0111] Compounds of Formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses or species thereof) and compounds of Table 1 may have different isomeric forms. The terms "optical isomer", "stereoisomer" or "diastereomer" refer to any of the various stereoisomeric configurations that may exist for a given compound of Formula (Ia), (Ia1), (Ia2), (IIa), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2) (including any subclasses or species thereof) and compounds of Table 1. It should be understood that substituents may be attached to chiral centers of carbon atoms and, therefore, the disclosed compounds include enantiomers, diastereomers and racemates. The term "enantiomer" includes pairs of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemic mixture. Where appropriate, the term is used to refer to a racemic mixture. The term "diastereomer" includes stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is specified according to the Cahn-Ingold-Prelog RS system. When a compound is a pure enantiomer, the stereochemistry at each chiral center can be specified by R or S. Depending on the direction (right-handed or left-handed) of rotating plane polarized light at the sodium D line wavelength, the unknown split compound of absolute configuration can be specified as (+) or (-). Certain compounds of Formula (Ia), (Ia1), (Ia2), (IIa1), (IIa2), (Ib), (Ib1), (Ib2), (IIb), (IIb1), and (IIb2), including any subclasses or species thereof, and compounds of Table 1 contain one or more asymmetric centers or axes, and may therefore give rise to enantiomers, diastereomers, or other stereoisomeric forms, which, with respect to absolute stereochemistry, may be defined as (R)- or (S)-. The present disclosure is intended to include all such possible isomers, including racemic mixtures, optically pure forms, and intermediate mixtures. Optically active (R)- and (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques well known in the art, such as chiral HPLC.

[0112] Immunomodulators

[0113] As used herein, "immunomodulator" refers to an agent that enhances an immune response (e.g., an anti-tumor immune response). An immunomodulator can be an antibody or an antigen-binding fragment thereof, a protein, a peptide, a DNA or RNA fragment, a small molecule, or a combination thereof. In some embodiments, an immunomodulator is an immune checkpoint inhibitor. In some embodiments, an immunomodulator is an immunostimulant. As used herein, "immune checkpoint inhibitor" refers to an agent (i.e., immune checkpoint agent) that inhibits blocking the immune system, such as attacking a protein or peptide of a cancer cell. In some embodiments, the immune checkpoint agent that blocks the immune system prevents the generation and / or activation of T cells. In some embodiments, an immune checkpoint inhibitor is a cytotoxic T lymphocyte-associated protein 4 (CTLA-4), a programmed cell death protein 1 (PD1), or a programmed death ligand 1 (PD-L1). PD-L1 and PD1 form a ligand-receptor pair bound to the cell surface, which, in healthy individuals, suppresses the immune response to prevent an overreaction of the immune system. In some embodiments, cancer cells hijack the normal PD-L1 / PD1 immune checkpoint mechanism by overexpressing the ligand PD-L1 (which binds to the PD1 on effector CD8 T cells), thereby preventing T cells from establishing an immune response to cancer cells and / or tumors. PD-L1 is expressed at high frequencies in a wide range of cancers. Tumor PD-L1 overexpression is associated with poor prognosis in a variety of cancers (see, for example, Hamid et al., Expert Opin Biol Ther [Expert Review of Biological Therapy] 13 (6): 847-861, 2013). As used herein, "immunostimulant" means a substance that stimulates the immune system without any antigen-specificity in an immune response by inducing activation of any component of the immune system or increasing its activity. In some embodiments, the immunostimulant is a toll-like receptor 3 (TLR3) agonist, such as polyinosinic acid: polycytidylic acid, which is also referred to as poly I: C or poly (I: C).

[0114] A review describing immune checkpoint pathways and blocking such pathways with immune checkpoint inhibitor compounds is provided by Pardoll in Nature Reviews Cancer (April 2012), pp. 252-264. Immune checkpoint inhibitor compounds show anti-tumor activity by blocking one or more endogenous immune checkpoint pathways that downregulate anti-tumor immune responses. Typically, inhibition or blocking of immune checkpoint pathways involves inhibiting the interaction of checkpoint receptors and ligands with immune checkpoint inhibitor compounds to reduce or eliminate signals and result in a reduction in anti-tumor responses.

[0115] As used herein, "immune checkpoint inhibitors" refer to agents (i.e., immune checkpoint agents) that inhibit proteins or peptides that block the immune system, such as attacking cancer cells. Immune checkpoint inhibitors can be antibodies or antigen-binding fragments thereof, proteins, peptides, small molecules, or combinations thereof. In some embodiments, immune checkpoint agents that block the immune system prevent the generation and / or activation of T cells. In some embodiments, immune checkpoint agents are cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD1), programmed death ligand 1 (PD-L1), or inhibitory receptors (such as NKG2A) that recognize HLA-E and are expressed by NK cells and T cell subsets. PD-L1 and PD1 form a cell surface-bound ligand-receptor pair that, in healthy individuals, suppresses immune responses to prevent overreaction of the immune system. In some embodiments, cancer cells hijack normal PD-L1 / PD1 immune checkpoint mechanisms by overexpressing the ligand PD-L1 (which binds to PD1 on effector CD8 T cells), thereby preventing T cells from establishing immune responses to cancer cells and / or tumors. PD-L1 is expressed at high frequencies in a wide range of cancers. Tumor PD-L1 overexpression is associated with poor prognosis in a variety of cancers (see, e.g., Hamid et al., Expert Opin Biol Ther 13(6):847-861, 2013).

[0116] In some embodiments of the present disclosure, immune checkpoint inhibitor compounds inhibit the signal transduction interaction between immune checkpoint receptors and the corresponding ligands of immune checkpoint receptors. Immune checkpoint inhibitor compounds can work by blocking the activation of immune checkpoint pathways, which is achieved by inhibiting (antagonizing) immune checkpoint receptors (some examples of receptors include CTLA-4, PD-1, and NKG2A) or by inhibiting the ligands of immune checkpoint receptors (some examples of ligands include PD-L1 and PD-L2). In such embodiments, the effect of immune checkpoint inhibitor compounds is to reduce or eliminate the downregulation of certain aspects of the anti-tumor response of the immune system in the tumor microenvironment.

[0117] In some embodiments, the immune checkpoint inhibitor inhibits the CTLA-4 pathway or the PD-L1 / PD1 pathway. In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the immune checkpoint inhibitor comprises an antibody that inhibits CTLA-4, PD1, or PD-L1. Immune checkpoint inhibitors, immune checkpoint inhibitors, and examples thereof are provided, for example, in WO 2016 / 062722.

[0118] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody or a derivative or antigen-binding fragment thereof. In an embodiment, the anti-CTLA-4 antibody selectively binds to the CTLA-4 protein or a fragment thereof. Examples of anti-CTLA-4 antibodies and their derivatives and fragments are described in, for example, US 6,682,736; US 7,109,003; US 7,123,281; US ​​7,411,057; US 7,807,797; US 7,824,679; US 8,143,379; US 8,491,895, and US 2007 / 0243184. In some embodiments, the anti-CTLA-4 antibody is tremelimumab or ipilimumab.

[0119] The immune checkpoint receptor cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is expressed on T cells and is involved in signaling pathways that reduce the level of T cell activation. CTLA-4 is believed to downregulate T cell activation through competitive binding and sequestration of CD80 and CD86. In addition, CTLA-4 has been shown to be involved in enhancing T Reg Immunosuppressive activity of cells.

[0120] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody or a derivative or antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody or a derivative or antigen-binding fragment thereof selectively binds to a PD-L1 protein or a fragment thereof. Examples of anti-PD-L1 antibodies and derivatives and fragments thereof are described in, for example, WO 01 / 14556, WO 2007 / 005874, WO2009 / 089149, WO 2011 / 066389, WO 2012 / 145493; US 8,217,149, US 8,779,108; US2012 / 0039906, US2013 / 0034559, US2014 / 0044738, and US2014 / 0356353. In some embodiments, the anti-PD-L1 antibody is MEDI4736 (durvalumab), MDPL3280A, 2.7A4, AMP-814, MDX-1105, atezolizumab (MPDL3280A), or BMS-936559.

[0121] The immune checkpoint receptor programmed death 1 (PD-1) is expressed by activated T cells upon prolonged exposure to antigens. Engagement of PD-1 with its known binding ligands (PD-L1 and PD-L2) occurs primarily within the tumor microenvironment and results in downregulation of anti-tumor specific T cell responses. Both PD-L1 and PD-L2 are known to be expressed on tumor cells. Expression of PD-L1 and PD-L2 on tumors is associated with reduced survival outcomes.

[0122] In some embodiments, the anti-PD-L1 antibody is MEDI4736, also known as durvalumab. In some embodiments, the anti-PD-L1 antibody comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NO: 1-8. MEDI4736 is an anti-PD-L1 antibody that is selective for PD-L1 polypeptides and blocks the binding of PD-L1 to PD-1 and CD80 receptors. MEDI4736 can relieve PD-L1-mediated inhibition of human T cell activation in vitro, and can further inhibit tumor growth in a xenograft model via a T cell-dependent mechanism. MEDI4736 is further described, for example, in US 8,779,108. The fragment crystallizable (Fc) domain of MEDI4736 contains triple mutations in the constant domain of the IgG1 heavy chain that reduce binding to the complement component C1q and Fcγ receptors responsible for mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0123] In some embodiments, MEDI4736 or its antigen-binding fragment comprises a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. In some embodiments, MEDI4736 or its antigen-binding fragment for use comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises an amino acid sequence of SEQ ID NO: 1, and the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, MEDI4736 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2, and CDR3 sequence defined by Kabat of SEQ ID NO: 3-5, and wherein the light chain variable region comprises a CDR1, CDR2, and CDR3 sequence defined by Kabat of SEQ ID NO: 6-8. One of ordinary skill in the art will be able to easily identify Chothia's, Abm's, or other CDR definitions known to one of ordinary skill in the art. In some embodiments, MEDI4736 or an antigen-binding fragment thereof comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H90PT antibody as described in WO 2011 / 066389.

[0124] In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody or a derivative or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody selectively binds to the PD-1 protein or a fragment thereof. In some embodiments, the anti-PD1 antibody is nivolumab, pembrolizumab, or pidilizumab.

[0125] NKG2A receptor is an inhibitory receptor that is combined with HLA-E and expressed on tumor-infiltrating cytotoxic NK and CD8 T lymphocytes. By expressing HLA-E, cancer cells can protect themselves from being killed by NKG2A+ immune cells. HLA-E is often upregulated on cancer cells of many solid tumors or malignant hematological diseases. Monalizumab (IPH2201) is a humanized IgG4 that blocks the combination of NKG2A and HLA-E, thereby allowing the activation of NK cells and cytotoxic T cell responses. Examples of anti-NKG2A antibodies and derivatives and fragments thereof are described in WO 2016 / 041947, the contents of which are hereby incorporated by reference in their entirety (including but not limited to sequence listing).

[0126] In some embodiments of the present disclosure, the immune checkpoint inhibitor compound is an organic small molecule (molecular weight less than 1000 Daltons), a peptide, a polypeptide, a protein, an antibody, an antibody fragment, or an antibody derivative. In some embodiments, the immune checkpoint inhibitor compound is an antibody. In some embodiments, the antibody is a monoclonal antibody, particularly a human or humanized monoclonal antibody.

[0127] Monoclonal antibodies, antibody fragments and antibody derivatives for blocking immune checkpoint pathways can be prepared by any of several methods known to those of ordinary skill in the art, including but not limited to somatic cell hybridization techniques and hybridoma methods. Hybridoma production is described in Antibodies, A Laboratory Manual [Antibodies: Laboratory Manual], Harlow and Lane, 1988, Cold Spring Harbor Publications [Cold Spring Harbor Press], New York. Human monoclonal antibodies can be identified and isolated by screening phage display libraries of human immunoglobulin genes by methods described in, for example, U.S. Patent Nos. 5223409, 5403484, 5571698, 6582915 and 6593081. Monoclonal antibodies can be prepared using the general method described in U.S. Patent No. 6331415 (Cabilly).

[0128] As an example, human monoclonal antibodies can be obtained using XenoMouse TM (Abgenix, Fremont, CA) or hybridomas from B cells of the XenoMouse. The XenoMouse is a murine host with functional human immunoglobulin genes as described in U.S. Pat. No. 6,162,963 (Kucherlapati).

[0129] Methods for preparing and using immune checkpoint antibodies are described in the following illustrative publications. The preparation and therapeutic use of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 7,229,628 (Allison), 7,311,910 (Linsley), and 8,017,144 (Korman). The preparation and therapeutic use of anti-PD-1 antibodies are described in U.S. Patent No. 8,008,449 (Korman) and U.S. Patent Application No. 2011 / 0271358 (Freeman). The preparation and therapeutic use of anti-PD-L1 antibodies are described in U.S. Patent No. 7,943,743 (Korman). The preparation and therapeutic use of anti-TIM-3 antibodies are described in U.S. Patent Nos. 8,101,176 (Kuchroo) and 8,552,156 (Tagayanagi). The preparation and therapeutic use of anti-LAG-3 antibodies are described in U.S. Patent Application No. 2011 / 0150892 (Thudium) and International Publication No. WO 2014 / 008218 (Lonberg). The preparation and therapeutic use of anti-KIR antibodies are described in U.S. Patent No. 8119775 (Moretta). The preparation of antibodies that block inhibitory pathways regulated by BTLA (anti-BTLA antibodies) is described in U.S. Patent No. 8563694 (Mataraza).

[0130] In some embodiments of the present disclosure, the immune checkpoint inhibitor compound is a CTLA-4 receptor inhibitor, a PD-1 receptor inhibitor, a LAG-3 receptor inhibitor, a TIM-3 receptor inhibitor, a BTLA receptor inhibitor, or a KIR receptor inhibitor. In some embodiments, the immune checkpoint inhibitor compound is an inhibitor of PD-L1 or an inhibitor of PD-L2.

[0131] In some embodiments of the present disclosure, the immune checkpoint inhibitor compound is an inhibitor of the PD-L1 / PD-1 pathway or the PD-L2 / PD-1 pathway. In some embodiments, the inhibitor of the PD-L1 / PD-1 pathway is MEDI4736.

[0132] In some embodiments of the present disclosure, the immune checkpoint inhibitor compound is an anti-CTLA-4 receptor antibody, an anti-PD-1 receptor antibody, an anti-LAG-3 receptor antibody, an anti-TIM-3 receptor antibody, an anti-BTLA receptor antibody, an anti-KIR receptor antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.

[0133] In some embodiments of the present disclosure, the anti-CTLA-4 receptor antibody is ipilimumab or tremelimumab. In some embodiments, the anti-PD-1 receptor antibody is lambrolizumab, pidilizumab, or nivolumab. In some embodiments, the anti-KIR receptor antibody is lirilumab.

[0134] Radiation therapy

[0135] Radiation therapy, also known as high-dose ionizing radiation, is used in combination with a compound of Formula (Ia) or Formula (Ib), or a pharmaceutically acceptable salt thereof, for the treatment of cancer. In some embodiments, the methods of the invention comprise administering radiation therapy to a patient in combination with a compound of Formula (Ia) or Formula (Ib), or a pharmaceutically acceptable salt thereof, and an immunomodulator.

[0136] Radiation therapy can be x-rays, gamma rays, or charged particles. Radiation therapy can be external beam radiation therapy or internal radiation therapy (also called brachytherapy). Whole body radiation therapy using radioactive materials such as radioactive iodine can also be used. External beam radiation therapy includes 3D conformational radiation therapy, intensity modulated radiation therapy, image-guided radiation therapy, tomotherapy, stereotactic radiosurgery, proton therapy, or other charged particle beams.

[0137] Radiation therapy can be x-rays, gamma rays, or charged particles. Radiation therapy can be external beam radiation therapy or internal radiation therapy (also called brachytherapy). Whole body radiation therapy using radioactive materials such as radioactive iodine can also be used. External beam radiation therapy includes 3D conformational radiation therapy, intensity modulated radiation therapy, image-guided radiation therapy, tomotherapy, stereotactic radiosurgery, proton therapy, or other charged particle beams.

[0138] In some embodiments, the radiotherapy is fractionated radiotherapy. In one embodiment, the fractionated radiotherapy includes 2 to 14 parts. In another embodiment, the fractionated radiotherapy includes 2 to 7 parts. In another embodiment, the fractionated radiotherapy includes 3 to 6 parts. In one embodiment, the fractionated radiotherapy includes 2, 3, 4, 5, 6, or 7 parts. In one embodiment, the fractionated radiotherapy includes 5 parts. In some embodiments, these radiotherapy parts are administered in multiple consecutive days. In one embodiment, the radiotherapy can include more than one dose in a day and / or multiple doses in multiple consecutive days. In one mode, these radiotherapy parts are administered on the 1st day, the 2nd day, the 3rd day, the 4th day, and the 5th day.

[0139] Combination therapy

[0140] The present disclosure provides methods for treating cancer, wherein a compound of formula (Ia) or (Ib) (including any subclass or species thereof), or a pharmaceutically acceptable salt thereof, and an immunomodulator are administered in combination. In another aspect, the present disclosure provides methods for treating cancer, wherein a compound of formula (Ia) or (Ib) (including any subclass or species thereof), or a pharmaceutically acceptable salt thereof, and radiation therapy (optionally with an immunomodulator) are administered in combination.

[0141] In some embodiments, the compound of formula (Ia) or (Ib) or its pharmaceutically acceptable salt and the immunomodulator are administered separately, sequentially or simultaneously. In some embodiments, the compound of formula (Ia) or (Ib) or its pharmaceutically acceptable salt and radiotherapy are administered on the same day or on different days. In one embodiment, radiotherapy is used before the treatment of the compound of formula (Ia) or (Ib) or its pharmaceutically acceptable salt and / or immunomodulator. In another embodiment, radiotherapy is used after the treatment of the compound of formula (Ia) or (Ib) or its pharmaceutically acceptable salt and / or immunomodulator. In another embodiment, radiotherapy is administered simultaneously with the treatment of the compound of formula (Ia) or (Ib) or its pharmaceutically acceptable salt and / or immunomodulator.

[0142] In some embodiments, the compound of formula (Ia) or (Ib) is selected from the compounds listed in Table 1, i.e., compounds 1 to 33, and the immunomodulator is selected from durvalumab, tremelimumab, monalizumab, or a combination thereof.

[0143] In some embodiments, administration of a combination of a compound of Formula (Ia) or (Ib) and an immunomodulator results in an additive and / or synergistic effect. As used herein, the term "synergistic" refers to a combination therapy (e.g., a combination of MEDI4736 or an antigen-binding fragment thereof and an arginase inhibitor as described herein) that is more effective than the additive effects of a single therapy.

[0144] In some embodiments, compared with the method of applying only one agent, the method provided herein (e.g., applying a combination of a compound and an immunomodulator with formula (Ia) or (Ib)) advantageously enhances antigen presentation, and / or promotes T cell activation, thereby providing a safer and more effective treatment for the patient. In some embodiments, compared with the method of applying an immunomodulator alone or the compound with formula (Ia) or (Ib) alone, the method provided herein causes CD8+T cells, NK cells, and / or CD103+ dendritic cells to increase. In some embodiments, compared with the method of applying only one agent, the method provided herein causes the patient's interferon-γ (IFNγ) level to increase. In some embodiments, compared with the method of applying only one agent, the method provided herein causes the patient's interleukin-2 (IL-2) level to increase.

[0145] SEQ ID NOs: 1-8 correspond to the amino acid sequence of MEDI4736, which is an anti-PD-L1 antibody as described in the Examples herein. SEQ ID NO: 3 corresponds to the amino acid sequence of the light chain variable region of MEDI4736. SEQ ID NO: 4 corresponds to the amino acid sequence of the heavy chain variable region of MEDI4736. SEQ ID NOs: 5-10 correspond to the CDRs of MEDI4736.

[0146] SEQ ID NOs: 9-16 correspond to the amino acid sequence of tremelimumab, an anti-CTLA-4 antibody as described in the Examples herein.

[0147] SEQ ID NOs: 17-24 correspond to the amino acid sequences of monalizumab, which is an anti-NKG2A antibody as described in the Examples herein.

[0148] All references cited herein, including patents, patent applications, articles, textbooks, etc. and references cited therein (to the same extent as if they had not already been cited), are hereby incorporated by reference in their entirety.

[0149] Examples

[0150] Aspects of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain compounds and intermediates of the present disclosure and methods of using the compounds of the present disclosure. It should be clear to those skilled in the art that many modifications can be made to materials and methods without departing from the scope of the present disclosure.

[0151] Unless otherwise stated:

[0152] (i) Unless otherwise stated, all syntheses were carried out at ambient temperature (i.e. in the range of 17°C to 25°C) and under an atmosphere of an inert gas such as nitrogen;

[0153] (ii) evaporation by rotary evaporation or in vacuo using a Genevac device or a Biotage v10 evaporator and work-up procedure after removal of residual solids by filtration;

[0154] (iii) Automation Teledyne Isco Rf or Teledyne Isco Use pre-filled RediSep Rf Gold TM Silica columns (20-40 μm, spherical particles), GraceResolv TM column( Purification was performed by flash chromatography on silica) or Silicycle columns (40-63 μm).

[0155] (iv) Preparative chromatography was performed on a Gilson preparative HPLC instrument equipped with UV acquisition; alternatively, preparative chromatography was performed on a Waters automated purification HPLC-MS instrument equipped with MS and UV triggered acquisition;

[0156] (v) Chiral preparative chromatography was performed on a Gilson instrument (233 injector / fraction collector, 333 and 334 pumps, 155 UV detector) with UV acquisition or on a Varian Prep Star instrument (2 SD1 pumps, 325 UV detector, 701 fraction collector) with the pumps running with Gilson 305 injection; alternatively, chiral preparative chromatography was performed on a Waters preparative 100 SFC-MS instrument with MS and UV triggered acquisition or a Thar MultiGram III SFC instrument with UV acquisition.

[0157] (vi) the yield, when any, need not be the maximum achievable;

[0158] (vii) In general, the structure of the final product having Formula I was confirmed by nuclear magnetic resonance (NMR) spectroscopy; NMR chemical shift values ​​were measured on the delta scale [proton magnetic resonance spectra were determined using a Bruker Avance III 600 (600 MHz), Bruker Avance 400 (400 MHz), Bruker Avance 300 (300 MHz) or Bruker DRX 500 (500 MHz) instrument]; measurements were performed at ambient temperature unless otherwise stated; the following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublets; dt, doublet of triplet; bs, broad signal.

[0159] (viii) In general, the final product of Formula I is also characterized by mass spectrometry (LCMS or UPLC) after liquid chromatography; UPLC is performed as follows: using a Waters UPLC equipped with a Waters SQ mass spectrometer (column temperature 40°C, UV = 220-300nm or 190-400nm, mass spectrum = ESI with positive / negative switching) at a flow rate of 1 mL / min using a solvent system of 97% A+3% B to 3% A+97% B for 1.50 min (total run time including equilibration back to starting conditions, etc. is 1.70 min), wherein A = 0.1% formic acid or 0.05% trifluoroacetic acid in water (for acid treatment) or 0.1% ammonium hydroxide in water (for base treatment), and B = acetonitrile. For acid analysis, the column used was Waters Acquity HSS T3 (1.8 μm, 2.1 x 50 mm); for base analysis, the column used was Waters Acquity BEH C18 (1.7 μm, 2.1 x 50 mm). Alternatively, UPLC was performed as follows: using a Waters UPLC equipped with a Waters SQ mass spectrometer (column temperature 30°C, UV = 210-400 nm, mass spectrum = ESI with positive / negative switching) at a flow rate of 1 mL / min using a solvent gradient from 2% to 98% B for 1.5 min (total run time with equilibration back to starting conditions was 2 min), where A = 0.1% formic acid in water, and B = 0.1% formic acid in acetonitrile (for acid treatment); or A = 0.1% ammonium hydroxide in water, and B = acetonitrile (for base treatment). For acid analysis, the column used was Waters Acquity HSS T3 (1.8 μm, 2.1 x 30 mm), and for base analysis, the column used was Waters Acquity BEH C18 (1.7 μm, 2.1 x 30 mm); LCMS was performed as follows: using a Waters Alliance HT (2795) equipped with a Waters ZQ ESCi mass spectrometer and a Phenomenex Gemini-NX C18 (5 μm, 110A, 2.1 x 50 mm column), at a flow rate of 1.1 mL / min, 95% A to 95% B for 4 min, holding for 0.5 min, where A = 0.1% formic acid and B = 0.1% formic acid in acetonitrile (for acid treatment); or A = 0.1% ammonium hydroxide in water, and B = acetonitrile (for base treatment).In addition, LCMS was performed as follows: Shimadzu UFLC equipped with a Shimadzu LCMS-2020 mass spectrometer and a Waters HSS C18 (1.8 μm, 2.1× 50 mm) or Shim-pack XR-ODS (2.2 μm, 3.0×50 mm) or Phenomenex Gemini-NX C18 (3 μm, 3.0× 50 mm) column was used at a flow rate of 0.7 mL / min (for Waters HSS C18 column), 1.0 mL / min (for Shim-pack XR-ODS column) or 1.2 mL / min (for Phenomenex Gemini-NX C18) at a temperature of 95% A to 95% B for 2.2 min, hold 0.6 min, where A = 0.1% formic acid or 0.05% trifluoroacetic acid in water (for acid treatment), or 0.1% ammonium hydroxide or 6.5 mM ammonium carbonate in water (for base treatment), and B = acetonitrile. Unless otherwise stated, the reported molecular ions correspond to [M+H]+; for molecules with multiple isotopic patterns (Br, Cl, etc.), unless otherwise stated, the reported values ​​are those obtained for the lowest isotopic mass.

[0160] (ix) Ion exchange purification was usually performed using an SCX-2 (Biotage) column.

[0161] (x) intermediate purity is assessed by thin layer chromatography, mass spectrometry, LCMS, UPLC / MS, HPLC (high performance liquid chromatography) and / or NMR analysis;

[0162] (xi) The following abbreviations are used:-

[0163] EtOH: Ethanol

[0164] EtOAc: Ethyl acetate

[0165] LDA: Lithium diisopropylamide

[0166] MeOH: Methanol

[0167] TFA: trifluoroacetic acid

[0168] MeCN: Acetonitrile

[0169] LCMS: Liquid chromatography-mass spectrometry

[0170] rt or RT: room temperature

[0171] aq: aqueous solution

[0172] THF: Tetrahydrofuran

[0173] KHMDS: Potassium bis(trimethylsilyl)amide

[0174] DCM: Dichloromethane

[0175] DMF: Dimethylformamide

[0176] HATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo

[0177] [4,5-b]pyridinium 3-oxide hexafluorophosphate)

[0178] BOC: tert-Butoxycarbonyl

[0179] DTNB: 5,5′-dithiobis(2-nitrobenzoic acid)

[0180] TNB: 2-nitro-5-thiobenzoic acid

[0181] HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

[0182] The preparation of compounds 1 to 9 in Table 1 is illustrated in the following Examples 1 to 9. In addition, the preparation of compounds 10 to 33 in Table 1 is described as Examples 7 to 30 in WO 2019 / 159120, the contents of which are hereby incorporated by reference in their entirety.

[0183] Example 1: (2R, 4S)-4-amino-2-(4-dihydroxyborylbutyl)piperidine-2-carboxylic acid

[0184]

[0185] Intermediate 1: (R)-2-Benzyl 1-(tert-butyl)-4-oxopiperidine-1,2-dicarboxylate

[0186] At 0 ° C, N, N'-diisopropylcarbodiimide (3.49 mL, 22.4 mmol) and DMAP (0.249 g, 2.04 mmol) were added to a stirred solution of (R)-1-(tert-butoxycarbonyl)-4-oxopiperidine-2-carboxylic acid (4.955 g, 20.37 mmol) and benzyl alcohol (2.11 mL, 20.4 mmol) in DCM (150 mL). The reaction was stirred for 17 h while slowly warming to room temperature. The reaction mixture was filtered and the filtrate was concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 50% EtOAc in hexane) to give (R)-4-oxopiperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (intermediate 1, 6.50 g, yield 96%), which was a colorless oil and a mixture of rotamers. 1H NMR (500MHz, CDCl3) δ1.40(5H,br s),1.49(4H,br s),2.43-2.65(2H,m),2.70-2.92(2H,m),3.53-3.74(1H,m),3.94-4.10(1H,m),4.89(0.5H,br s),5.10-5.23(2.5H,m),7.31-7.41(5H,m); m / z: (ES + )[M+Na] + =356.

[0187] Intermediate 2: (2R,4R)-4-hydroxypiperidin-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester

[0188] At 0 ° C, sodium borohydride (0.738g, 19.5mmol) was added in batches to a stirred solution of (R)-4-oxopiperidin-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (intermediate 1, 6.504g, 19.51mmol) in a mixture of MeOH / THF (1:20, 105mL). The mixture was stirred for 5h, then carefully quenched with 1M HCl (aqueous solution) (15mL-gas evolution), and warmed to room temperature. The mixture was diluted with water (25mL) and EtOAc (50mL). Each phase was separated, and the aqueous phase was extracted with EtOAc (4x 20mL). The combined organic matter was washed with a saturated NaCl aqueous solution, dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 45% EtOAc in hexanes) to give 2-benzyl 1-(tert-butyl) (2R,4R)-4-hydroxypiperidine-1,2-dicarboxylate (Intermediate 2, 3.84 g, 59% yield) as a 5:1 mixture of diastereomers (the major diastereomer is the title compound) and as a mixture of rotamers as a colorless gum. 1HNMR(500MHz,CDCl3)δ1.38(5H,br s),1.40-1.44(2H,m),1.46(5.4H,br s),1.60-1.69(1.4H,m),1.70-1.79(0.2H,m),1.83-1.97(1.2H,m),2.48(1H,br dd),2.92-3.08(1H,m),3.28-3.46(0.2H,m),3.54-3.70(1H,m),3.76-3.84(0.1H,m),3.87-3.96(0.1H,m),4.00(0.5H,br d),4.15(0.5H,s),4.65-4.74(0.1H,m),4.87(0.6H,br d),5.08(0.5H,br d),5.13-5.26(2.4H,m),7.31-7.40(6H,m); m / z: (ES + )[M+Na] + =358.

[0189] Intermediate 3: 2-Benzyl 1-(tert-butyl)-(2R,4R)-4-((methylsulfonyl)oxy)piperidine-1,2-dicarboxylate

[0190] Methanesulfonic anhydride (3.59 g, 20.6 mmol) was added portionwise to a stirred solution of (2R,4R)-4-hydroxypiperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (Intermediate 2, 3.84 g, 11.5 mmol; 5:1 mixture of diastereomers) and triethylamine (3.35 mL, 24.0 mmol) in DCM (50 mL) at 0°C. The cooling bath was allowed to stop and the reaction was allowed to warm to room temperature. After 6 h, the reaction was diluted with DCM (50 mL) and washed sequentially with 1 M HCl (aq) (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness to give crude (2R,4R)-2-benzyl 1-(tert-butyl)-4-((methylsulfonyl)oxy)piperidine-1,2-dicarboxylate (Intermediate 3, 4.73 g, 100% yield) as a light orange gum and a mixture of diastereomers. The crude material was used directly without further purification. m / z: (ES + )[M+Na] + =436.

[0191] Intermediate 4: (2R,4S)-4-azidopiperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester

[0192] Sodium azide (3.72 g, 57.2 mmol) was added to a stirred solution of (2R, 4R)-4-((methylsulfonyl)oxy)piperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (Intermediate 3, 4.73 g, 11.4 mmol; mixture of diastereomers) in DMF (50 mL), and the reaction was warmed to 60 ° C for 20 h. The mixture was allowed to cool to room temperature, filtered and the filtrate was diluted with water (400 mL) and EtOAc (40 mL). The phases were separated and the aqueous phase was extracted with EtOAc (4 x 40 mL). The combined organics were washed with saturated aqueous NaCl solution (2 x 40 mL), dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 30% EtOAc in hexanes) to give diastereomerically pure 2-benzyl 1-(tert-butyl) (2R,4S)-4-azidopiperidine-1,2-dicarboxylate (Intermediate 4, 2.58 g, 63% yield) as a colorless gum and as a mixture of rotamers. 1 H NMR(500MHz,CDCl3)δ1.39(4H,br s),1.46(5H,br s),1.63-1.73(1H,m),1.74-1.87(1H,m),1.95(1H,ddd),2.50-2.61(1H,m),3.03-3.44 (1H,m),3.73-3.89(0.5H,m),3.90-4.01(1.5H,m),4.58-4.74(0.5H,m),4.88(0.5H,br s),5.15-5.34(2H,m),7.30-7.43(5H,m); m / z: (ES + )[M-Boc] + =261.

[0193] Intermediate 5: (4S)-4-azido-2-(but-2-en-1-yl)piperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester

[0194] (2R, 4S) -4- azidopiperidine -1,2- dicarboxylic acid 2- benzyl 1- (tert-butyl) ester (intermediate 4, 1.94g, 5.38mmol) and crotyl bromide (0.977mL, 8.07mmol) were dissolved in THF (30mL), and the solution was cooled to -78 ° C. KHMDS solution (1M in 2-methyltetrahydrofuran, 7.0mL, 7.0mmol) was added dropwise over 10min. The reaction mixture was slowly warmed to room temperature and stirred for a total of 18h. The crude reaction mixture was quenched with saturated NH4Cl aqueous solution and then diluted with saturated NaCl aqueous solution and EtOAc (50mL). Each phase was separated, and the aqueous layer was extracted with EtOAc (3x 30mL). The combined organic matter was washed with saturated NaCl aqueous solution, dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (2% to 30% EtOAc in hexanes) to afford 2-benzyl 1-(tert-butyl) (4S)-4-azido-2-(but-2-en-1-yl)piperidine-1,2-dicarboxylate (Intermediate 5, 2.106 g, 94% yield) as a mixture of cis / trans diastereomers and as a mixture of rotamers and E / Z olefins. 1 H NMR(500MHz,CD2Cl2)δ1.40-1.42(4H,m),1.43(5H,br s),1.49-1.58(1H,m),1.59-1.66(0.6H,m),1.67-1.74(3.4H,m),1.86(0.5H,dd),1.89-2.05(2H,m),2.07 -2.19(1H,m),2.42(0.5H,dd),2.58-2.69(1H,m),2.71-2.83(0.5H,m),3.01-3.16(0.5H,m),3.21(0.5H,br dd),3.31-3.44(0.5H,m),3.61-3.77(1.5H,m),3.97-4.07(0.5H,m),5.10-5 .27(2H,m),5.36-5.45(1H,m),5.51-5.74(2H,m),7.32-7.47(5H,m); m / z: (ES + )[M-Boc] + =315.

[0195] Intermediate 6: (2R,4S)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2- 2-Benzyl 1-(tert-butyl)piperidin-1,2-dicarboxylate

[0196] Bis(1,5-cyclooctadiene)diiridium(I)dichloride (50mg, 0.074mmol) and bis(diphenylphosphino)methane (57mg, 0.15mmol) are added to an oven-dried round-bottom flask. The flask is sealed and purged with N2. The solid is dissolved in DCM (9mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.32mL, 2.2mmol) is slowly added to the solution. The reaction is stirred at room temperature for 10min. (4S)-4-azido-2-(but-2-ene-1-yl)piperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (intermediate 5, 616mg, 1.49mmol) is added to the reaction as a solution in DCM (3mL), and the reaction mixture is stirred at room temperature for 66h. The reaction mixture was cooled to 0 ° C and carefully quenched with MeOH (1 mL) and water (5 mL). The layers were separated and the aqueous layer was extracted with DCM (3 x 15 mL). The combined organic matter was dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 15% EtOAc in hexane) to give diastereoisomerically pure (2R, 4S) -4- azido -2- (4- (4,4,5,5- tetramethyl -1,3,2- dioxaborolane -2- bases) butyl) piperidine -1,2- dicarboxylic acid 2- benzyl 1- (tert-butyl) ester (intermediate 6, 261 mg, 32% yield), which was transparent, colorless jelly. 1 H NMR(500MHz, CDCl3)δ0.79(2H,t),1.25(12H,s),1.29-1.35(1H,m),1.36-1 .39(1H,m),1.41(9H,s),1.42-1.46(2H,m),1.57-1.68(1H,m),1.85-1.94( 3H,m),1.95-2.01(1H,m),2.05(1H,dd),2.92-3.11(1H,m),3.49-3.72(1H, m),3.98-4.03(1H,m),5.09(1H,d),5.18(1H,d),7.29-7.42(5H,m); m / z: (ES + )[M+Na] + =565.

[0197] Intermediate 7: (2R,4S)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-di Oxaborolan-2-yl)butyl)piperidine-2-carboxylic acid

[0198] Pd / C (10% wt, 50 mg, 0.047 mmol) was added to a solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 6, 268 mg, 0.494 mmol) in EtOAc (3 mL). The suspension was stirred for 17 h at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times). The reaction mixture was diluted with MeOH (5 mL), filtered through celite and concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 45% MeOH in DCM) to afford (2R,4S)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 7, 156 mg, 74% yield) as a white dry film. 1 H NMR(500MHz,CD2Cl2)δ0.71(2H,t),1.07-1.16(1H,m),1.19(14H,s),1.31-1.37(2H,m),1.40(9H,s),1.80-1.96(1H,m),2.02(3H,br d),2.33(1H,br s),3.00(1H,br s),3.53(1H,br s),3.92(1H,br s),8.60(3H,br s); m / z: (ES + )[M+H] + =427.

[0199] Example 1: (2R, 4S)-4-amino-2-(4-dihydroxyborylbutyl)piperidine-2-carboxylic acid

[0200] Trifluoroacetic acid (0.53 mL, 6.9 mmol) was added dropwise to a stirred solution of (2R, 4S)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 7, 146 mg, 0.342 mmol) in DCM (2 mL) at room temperature. After 2 h, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1 M HCl(aq) (3.0 mL, 3.0 mmol) and Et2O (3 mL). Phenylboronic acid (125 mg, 1.03 mmol) was added and the clear biphasic solution was stirred at room temperature for 4 h. The mixture was diluted with Et2O (20 mL) and water (5 mL), and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 mL) to give (2R,4S)-4-amino-2-(4-boronobutyl)piperidine-2-carboxylic acid (62 mg, 74% yield) as a white solid. 1 H NMR(500MHz,D2O)δ0.71-0.82(2H,m),1.10-1.30(2H,m),1.33-1.44(2H,m),1.45-1.55(1H,m),1.62(1H,dd),1.77(1 H,ddd),1.84-1.93(1H,m),2.01-2.08(1H,m),2.18(1H,ddd),3.07(1H,td),3.22(1H,dt),3.28-3.39(1H,m); m / z: (ES + )[M+H] + =245.

[0201] Example 2: (2R,4S)-4-((S)-2-amino-3-methylbutyramido)-2-(4-dihydroxyborylbutyl)piperidin-2- Formic acid

[0202]

[0203] Intermediate 8: (2R,4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2- 2-Benzyl 1-(tert-butyl)piperidin-1,2-dicarboxylate

[0204] Zinc (270 mg, 4.14 mmol) and AcOH (1.20 mL, 20.9 mmol) were added to a stirred solution of (2R, 4S)-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (intermediate 6, 748 mg, 1.38 mmol) in THF (10 mL). The rapidly stirred mixture was heated to 30 ° C for 18 h. The mixture was cooled to room temperature, diluted with DCM (30 mL), and filtered through diatomaceous earth. The filter cake was washed with DCM, and the filtrate was concentrated to dryness. The resulting residue was distributed between EtOAc (40 mL) and saturated NaHCO3 aqueous solution. Each phase was separated, and the organic matter was washed with saturated NaHCO3 aqueous solution and saturated NaCl aqueous solution. The organics were dried over MgSO4, filtered and concentrated to dryness to give 2-benzyl 1-(tert-butyl)-(2R,4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 8, 713 mg, 100% yield) as a clear, colorless gum. The crude material was used directly without further purification. 1 H NMR(500MHz, CDCl3)δ0.79(2H,t),1.24(12H,s),1.32-1.38(2H,m),1.39-1.47(13H,m),1.68(2H,br t),1.83-1.99(4H,m),2.93(1H,td),2.97-3.07(1H,m),3.96-4.10(1H,m),5.06-5.23(2H,m),7.30-7.41(5H,m); m / z: (ES + )[M+H] + =517.

[0205] Intermediate 9: (2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutyramido)-2-(4- 2-Benzyl 1-(tert-butyl)piperidin-1,2-dicarboxylate Ester

[0206] N,N-diisopropylethylamine (0.12 mL, 0.63 mmol) was slowly added to a stirred solution of COMU (270 mg, 0.63 mmol) and Boc-Val-OH (137 mg, 0.631 mmol) in DMF (2 mL) at room temperature. The solution was stirred at room temperature for 30 min and then cooled to 0 °C. A solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 8, 310 mg, 0.60 mmol) in DMF (2 mL) and N,N-diisopropylethylamine (0.10 mL, 0.60 mmol) were added and the reaction was stirred for 17 h while slowly warming to room temperature. The reaction mixture was diluted with water (40 mL) and the resulting precipitate was collected by filtration. The solid was dissolved in EtOAc, dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (10% to 100% EtOAc in hexanes) to give 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 9, 184 mg, 43% yield) as a colorless film and a mixture of rotamers. 1 HNMR(500MHz, CDCl3)δ0.73-0.80(2H,m),0.85(3H,d),0.89(3H,d),1.22(12H,br s),1.23-1.29(2H,m),1.37-1.45(20H,m),1.62-1.74(1H,m),1.86-1.99(3H,m),2.00-2 .12(3H,m),2.97(1H,t),3.78(1H,t),3.94-4.06(1H,m),4.07-4.14(1H,m),5.00(1H,br s),5.05-5.24(2H,m),6.05(1H,br d),7.28-7.36(5H,m); m / z: (ES + )[M+H] + =716.

[0207] Intermediate 10: (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)

[0208] butyl)piperidine-2-carboxylic acid

[0209] Pd / C (10% wt, 27 mg, 0.025 mmol) was added to a solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 9, 184 mg, 0.257 mmol) in EtOAc (2 mL). The suspension was stirred at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times) for 16 h. The reaction mixture was diluted with EtOAc (20 mL) and MeOH (20 mL), filtered through celite and concentrated to dryness. The resulting residue was purified by flash silica chromatography (20% to 100% EtOAc in hexanes followed by 10% MeOH in DCM) to afford (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 10, 116 mg, 72% yield) as a white solid and as a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.78(3H,brd),0.83-0.91(2H,m),0.94(3H,br d),1.20-1.25(12H,m),1.40(9H,br s),1.42-1.53(11H,m),1.51-1.66(1H,m),1.75-2.18(4H,m),2.19-2.34(1 H,m),2.88-3.06(1H,m),3.85-4.06(2H,m),4.07-4.26(1H,m),5.14(1H,br s),5.93(1H,br s),6.73(1H,br s),7.30-7.48(1H,m); m / z: (ES + )[M+H] + =627.

[0210] Example 2: (2R,4S)-4-((S)-2-amino-3-methylbutyramido)-2-(4-dihydroxyborylbutyl)piperidin-2- Formic acid

[0211] Trifluoroacetic acid (0.433 mL, 5.63 mmol) was added dropwise to a stirred solution of (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 10, 176 mg, 0.281 mmol) in DCM (2 mL) at room temperature. After 3 h, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1 M HCl(aq) (3.0 mL, 3.0 mmol) and EtO (3 mL). Phenylboronic acid (103 mg, 0.845 mmol) was added and the clear biphasic solution was stirred at room temperature for 4 h. The mixture is diluted with EtO (20mL) and water (5mL), and each layer is separated. The aqueous layer is washed with EtO. The aqueous layer is freeze-dried and purified by ion exchange chromatography (PoraPak Rxn CX 20cc columns). The desired product is eluted from the column using a MeOH solution (20mL) of 5% ammonia to obtain (2R, 4S) -4- ((S) -2- amino -3- methylbutyramide) -2- (4- dihydroxyboryl butyl) piperidine -2- carboxylic acid (Example 2, 89mg, yield 92%) as a white solid. 1 H NMR(500MHz,D2O)δ0.73-0.83(2H,m),0.88-0.96(6H,m),1.14-1.24(1H,m),1.25-1.35(1H,m),1.37-1.50(2H,m),1.64-1.76(1H, m),1.79-1.99(4H,m),2.01-2.09(1H,m),2.17(1H,dd),3.11(1H,d),3.16-3.24(1H,m),3.31(1H,dt),4.10-4.22(1H,m); m / z: (ES + )[M+H] + =344.

[0212] Example 3: (2R,4S)-4-(2-aminoacetamido)-2-(4-dihydroxyborylbutyl)piperidine-2-carboxylic acid

[0213]

[0214] Intermediate 11: (2R,4S)-4-(2-((tert-butoxycarbonyl)amino)acetamido)-2-(4-(4,4,5,5-tetra 2-Benzyl 1-(tert-butyl)piperidin-1,2-dicarboxylate

[0215] N,N-diisopropylethylamine (0.12 mL, 0.63 mmol) was slowly added to a stirred solution of COMU (270 mg, 0.63 mmol) and Boc-Gly-OH (110 mg, 0.63 mmol) in DMF (2 mL) at room temperature. The solution was stirred at room temperature for 30 min and then cooled to 0 °C. A solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 8, 310 mg, 0.60 mmol) in DMF (2 mL) and N,N-diisopropylethylamine (0.11 mL, 0.60 mmol) were added and the reaction was stirred for 17 h while slowly warming to room temperature. The reaction mixture was diluted with water (60 mL) and the pH was adjusted to about 5 with acetic acid. The aqueous phase was extracted with EtOAc (4x15mL). The combined organic matter was washed with saturated aqueous NaCl solution (2x 10mL), dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (10% to 100% EtOAc in hexane) to give (2R, 4S)-4-(2-((tert-butoxycarbonyl)amino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (intermediate 11, 204mg, yield 51%), which is a colorless film and a mixture of rotamers. 1 H NMR(500MHz,CDCl3)δ0.75(2H,br t),1.21(14H,s),1.24(2H,br d),1.38(9H,s),1.40(10H,s),1.71(1H,dd),1.81-1.91(1H,m),1.93-2.04(3H,m),2.86-3.04(1H,m),3.62(2H,br s),3.93-4.04(1H,m),4.06-4.15(1H,m),5.11(2H,s),5.14(1H,br s),6.27(1H,br s),7.28-7.40(5H,m); m / z: (ES + )[M+H] + =674.

[0216] Intermediate 12: (2R,4S)-1-(tert-butoxycarbonyl)-4-(2-((tert-butoxycarbonyl)amino)acetamido)- 2-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid

[0217] Pd / C (10% wt, 32 mg, 0.030 mmol) was added to a solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-(2-((tert-butoxycarbonyl)amino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 11, 204 mg, 0.303 mmol) in EtOAc (2 mL). The suspension was stirred at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times) for 16 h. The reaction mixture was diluted with EtOAc (20 mL) and MeOH (20 mL), filtered through celite, and the filtrate was concentrated to dryness. The resulting residue was purified by flash silica chromatography (25% to 100% EtOAc in hexanes) to give (2R,4S)-1-(tert-butoxycarbonyl)-4-(2-((tert-butoxycarbonyl)amino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 12, 117 mg, 66% yield) as a white solid and as a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.78(2H,t),1.17-1.29(13H,m),1.40(10H,br s),1.45(9H,s),1.48-1.57(2H,m),1.76-2.01(3H,m),2.04-2.13(1H,m),2.98(1H,br t),3.47-3.66(1H,m),3.75(1H,s),3.90-4.06(2H,m),4.13-4.25(1H,m),5.41(1H,br s),5.92(1H,br s),6.73(1H,br s),7.65(1H,br s); m / z: (ES) + )[M+H] + =584.

[0218] Example 3: (2R,4S)-4-(2-aminoacetamido)-2-(4-dihydroxyborylbutyl)piperidine-2-carboxylic acid

[0219] Trifluoroacetic acid (0.31 mL, 4.0 mmol) was added dropwise to a stirred solution of (2R,4S)-1-(tert-butoxycarbonyl)-4-(2-((tert-butoxycarbonyl)amino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 12, 117 mg, 0.201 mmol) in DCM (2 mL) at room temperature. After 3 h, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1 M HCl(aq) (3.0 mL, 3.0 mmol) and Et2O (3 mL). Phenylboronic acid (73 mg, 0.60 mmol) was added and the clear biphasic solution was stirred at room temperature for 4 h. The mixture was diluted with Et2O (20 mL) and water (5 mL), and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 mL) to give (2R,4S)-4-(2-aminoacetamido)-2-(4-dihydroxyborylbutyl)piperidine-2-carboxylic acid (Example 3, 61 mg, 100% yield) as a white solid. 1 H NMR(500MHz,D2O)δ0.72-0.84(2H,m),1.14-1.25(1H,m),1.26-1.34(1H,m),1.41(2H,quin),1.72(1H,dtd),1.79-1.94 (2H,m),1.96-2.08(2H,m),2.10(1H,dd),3.14-3.25(1H,m),3.30-3.36(1H,m),3.37(2H,s),4.08-4.19(1H,m); m / z: (ES + )[M+H] + =302.

[0220] Example 4: (2R,4S)-4-[[(2S)-2-aminopropionyl]amino]-2-(4-dihydroxyborylbutyl)piperidin-2-carboxylate acid

[0221]

[0222] Intermediate 13: (2R,4S)-4-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]amino]-2-[4-(4,4, 2-Benzyl 1-tert-butyl 5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]piperidine-1,2-dicarboxylate

[0223] N,N-Diisopropylethylamine (0.17 mL, 1.0 mmol) was slowly added to a stirred solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 8, 245 mg, 0.474 mmol), Boc-Ala-OH (108 mg, 0.571 mmol) and COMU (244 mg, 0.571 mmol) in DMF (1.5 mL) at 0°C. The reaction was stirred for 1.5 h while slowly warming to room temperature. The reaction mixture was diluted with water (20 mL) and EtOAc (20 mL) and the phases were separated. The aqueous phase was extracted with EtOAc (3 x 20 mL), and the combined organics were washed with saturated aqueous NaCl solution, dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (15% to 80% EtOAc in hexanes) to give (2R,4S)-4-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]piperidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester (Intermediate 13, 191 mg, 59% yield) as a pale yellow gum and a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.69-0.83(2H,m),1.22(12H,d),1.26(5H,td),1.38(9H,br s),1.40(9H,br d),1.42-1.49(3H,m),1.57-1.73(1H,m),1.83-1.98(3H,m),2.01-2.05(2H,m),2.91-3.03(1H,m),4.02(2H,br s),4.96(1H,br s),5.05-5.22(2H,m),6.21(1H,br s),7.27-7.36(5H,m); m / z: (ES + )[M+H] + =688.

[0224] Intermediate 14: (2R,4S)-1-tert-butoxycarbonyl-4-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl] [amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]piperidine-2-carboxylic acid

[0225] Pd / C (10% wt, 15 mg, 0.014 mmol) was added to a solution of (2R, 4S)-4-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]piperidine-1,2-dicarboxylic acid 2-benzyl 1-tert-butyl ester (Intermediate 13, 190 mg, 0.28 mmol) in EtOAc (2 mL). The suspension was stirred for 17 h at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times). The reaction mixture was diluted with EtOAc (15 mL) and MeOH (2 mL), filtered through celite and concentrated to dryness. The resulting residue was purified by flash silica chromatography (20% to 100% EtOAc in hexanes) to afford (2R,4S)-1-tert-butoxycarbonyl-4-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]piperidine-2-carboxylic acid (Intermediate 14, 134 mg, 81% yield) as a dry film and as a mixture of rotamers. 1 H NMR(500MHz,CDCl3)δ0.78(2H,br t),1.20-1.25(12H,m),1.29-1.36(5H,m),1.41(9H,s),1.44(11H,s),1.48-1.62(2H,m),1.77-2.01(3H,m),2.09(2H,br s),2.97(1H,br s),3.92-4.05(1H,m),5.07(1H,br s),5.48(1H,br s),6.71(1H,br s),7.61(1H,br s); m / z: (ES + )[M+H] + =598.

[0226] Example 4: (2R,4S)-4-[[(2S)-2-aminopropionyl]amino]-2-(4-dihydroxyborylbutyl)piperidin-2-carboxylate acid

[0227] Trifluoroacetic acid (0.34 mL, 4.4 mmol) was added dropwise to a stirred solution of (2R, 4S)-1-tert-butoxycarbonyl-4-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]piperidine-2-carboxylic acid (Intermediate 14, 130 mg, 0.22 mmol) in DCM (1 mL) at room temperature. After 3 h, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1 M HCl (aq) (2.0 mL, 2.0 mmol) and Et2O (2 mL). Phenylboronic acid (80 mg, 0.65 mmol) was added and the clear biphasic solution was stirred at room temperature for 3 h. The mixture was diluted with Et2O (20 mL) and water (5 mL), and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 mL). The obtained material was purified by reverse phase chromatography (RediSep Rf C18, 0 to 15% acetonitrile in water) to give (2R,4S)-4-[[(2S)-2-aminopropanoyl]amino]-2-(4-boronobutyl)piperidine-2-carboxylic acid (Example 4, 25 mg, 37% yield) as a white solid. 1 HNMR(500MHz,D2O)δ0.72-0.80(2H,m),1.12-1.23(1H,m),1.24-1.26(1H,m),1.27(3H,d),1.40(2H,quin),1.66-1.76(1H,m),1.78-1 .92(2H,m),1.93-1.99(1H,m),2.00-2.06(1H,m),2.10(1H,dd),3.18(1H,ddd),3.27-3.36(1H,m),3.53(1H,q),4.10(1H,tt); m / z: (ES + )[M+H] + =316.

[0228] Example 5: (2R,4S)-4-[[(2S)-2-aminobutyryl]amino]-2-(4-dihydroxyborylbutyl)piperidin-2-carboxylate acid

[0229]

[0230] Intermediate 15: (2R,4S)-4-((S)-2-((tert-butyloxycarbonyl)amino)butyramide)-2-(4-(4,4,5, 2-Benzyl 1-(tert-butyl)piperidin-1,2-dicarboxylate

[0231] N,N-diisopropylethylamine (0.165 mL, 0.94 mmol) was slowly added to a stirred solution of (2R,4S)-4-amino-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]piperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (Intermediate 8, 244 mg, 0.47 mmol), Boc-Abu-OH (96 mg, 0.47 mmol) and COMU (206 mg, 0.48 mmol) in DMF (3 mL) at 0 °C. The reaction was stirred for 16 h while slowly warming to room temperature. The crude reaction mixture was diluted with water (30 mL) and extracted with EtOAc (3 x 10 mL). The combined organics were washed sequentially with saturated aqueous NaHCO3 solution (20 mL) and saturated aqueous NaCl solution (15 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (15% to 60% EtOAc in hexanes) to give 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)butanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 15, 215 mg, 65% yield) as a clear gum and a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.71-0.79(2H,m),0.87(3H,brt),1.19(4H,br s),1.21(9H,s),1.36(5H,br s),1.38(8H,s),1.39-1.41(8H,m),1.48-1.58(2H,m),1.68(1H,br dd),1.72-1.81(1H,m),1.84-1.98(3H,m),1.99-2.02(1H,m),2.88-3.04(1H,m),3.89(1H,br d),3.95-4.07(2H,m),5.00(1H,br d),5.05-5.22(2H,m),6.20(1H,br s),7.27-7.36(5H,m); m / z: (ES + )[M+H] + =703.

[0232] Intermediate 16: (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)butanamide butyl)piperidine-2-carboxylic acid

[0233] Pd / C (10% wt, 16 mg, 0.015 mmol) was added to a solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)butyramido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 15, 215 mg, 0.31 mmol) in EtOAc (3 mL). The suspension was stirred at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times) for 24 h. The reaction mixture was diluted with EtOAc (10 mL) and MeOH (1 mL), filtered through celite and concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 100% EtOAc in hexanes) to afford (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)butanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 16, 147 mg, 78% yield) as a dry film and as a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.75(2H,br s),0.89(3H,br s),1.20(12H,s),1.24-1.33(2H,m),1.37(10H,br s),1.40(10H,s),1.42-1.61(4H,m),1.85(3H,br s),1.98(2H,br s),2.01-2.06(1H,m),2.95(1H,br s),3.98(2H,br s),5.27(0.5H,br s),5.68(0.5H,br s),6.74(0.5H,br s),7.52(0.5H,br s); m / z: (ES + )[M+H] + =612.

[0234] Example 5: (2R,4S)-4-[[(2S)-2-aminobutyryl]amino]-2-(4-dihydroxyborylbutyl)piperidin-2-carboxylate acid

[0235] Trifluoroacetic acid (0.37 mL, 4.8 mmol) was added dropwise to a stirred solution of (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)butanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 16, 147 mg, 0.24 mmol) in DCM (1 mL) at room temperature. After 2 h, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1 M HCl(aq) (2.0 mL, 2.0 mmol) and Et2O (2 mL). Phenylboronic acid (88 mg, 0.72 mmol) was added and the clear biphasic solution was stirred at room temperature for 4 h. The mixture was diluted with Et2O (5 mL) and water (2 mL), and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 mL). The obtained material was purified by reverse phase chromatography (RediSep Rf C18, 0 to 15% acetonitrile in water) to give (2R,4S)-4-[[(2S)-2-aminobutyryl]amino]-2-(4-boronobutyl)piperidine-2-carboxylic acid (Example 5, 37 mg, 47% yield) as a white solid. 1 HNMR(500MHz,D2O)δ0.76(2H,br t),0.87(3H,t),1.13-1.23(1H,m),1.24-1.34(1H,m),1.40(2H,quin),1.63(2H,dq),1 .67-1.74(1H,m),1.78-1.85(1H,m),1.86-1.96(2H,m),1.99-2.07(1H,m),2.13(1H,br dd),3.10-3.24(1H,m),3.25-3.41(2H,m),4.07-4.21(1H,m); m / z: (ES + )[M+H] + =330.

[0236] Example 6: (2R,4S)-4-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-2-(4-dihydroxyborylbutyl) Piperidine-2-carboxylic acid

[0237]

[0238] Intermediate 17: (2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2-(4- 2-Benzyl 1-(tert-butyl)piperidin-1,2-dicarboxylate Ester

[0239] N,N-Diisopropylethylamine (0.17 mL, 0.94 mmol) was added to a stirred solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 8, 244 mg, 0.47 mmol), Boc-Leu-OH (96 mg, 0.47 mmol) and COMU (206 mg, 0.48 mmol) in DMF (3 mL) at 0°C. The reaction was stirred for 16 h while slowly warming to room temperature. The crude reaction mixture was diluted with water (30 mL) and extracted with EtOAc (3 x 10 mL). The combined organics were washed sequentially with saturated aqueous NaHCO3 solution (20 mL) and saturated aqueous NaCl solution (15 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (15% to 60% EtOAc in hexanes) to give 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 17, 224 mg, 65% yield) as a white foam and a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.73-0.80(2H,m),0.89(3H,d),0.90(3H,d),1.22(12H,s),1.27(1H,br s),1.39(9H,s),1.40(9H,s),1.41-1.48(4H,m),1.55-1.64(2H,m),1.68(1H,br dd),1.85-1.98(3H,m),2.01(1H,br d),2.02-2.05(1H,m),2.93-3.02(1H,m),3.94-4.08(3H,m),4.83(1H,br d),5.05-5.22(2H,m),6.20(1H,br s),7.28-7.31(1H,m),7.33(4H,d); m / z: (ES + )[M+H] + =731.

[0240] Intermediate 18: (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-4-methyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)

[0241] butyl)piperidine-2-carboxylic acid

[0242] Pd / C (10% wt, 13 mg, 0.012 mmol) was added to a solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 17, 174 mg, 0.24 mmol) in EtOAc (2.5 mL). The suspension was stirred at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times) for 23 h. The reaction mixture was diluted with EtOAc (10 mL) and MeOH (1 mL), filtered through celite and concentrated to dryness. The resulting residue was purified by flash silica chromatography (15% to 100% EtOAc in hexanes) to afford (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 18, 149 mg, 98% yield) as a dry film and as a mixture of rotamers. 1 H NMR(500MHz,CDCl3)δ0.78(2H,br t),0.91(6H,br d),1.23(11H,s),1.27-1.36(2H,m),1.40(10H,s),1.43(11H,s),1.46-1.55(2H,m),1.60-1.73(2H,m),1. 78-1.96(3H,m),1.97-2.02(1H,m),2.05-2.14(1H,m),2.82-3.08(1H,m),3.92-4.08(2H,m),5.00(0.4H,br s),5.49(0.6H,br d),6.76(0.4H,br d),7.60(0.6H,br s); m / z: (ES + )[M+H] + =640.

[0243] Example 6: (2R,4S)-4-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-2-(4-dihydroxyborylbutyl) Piperidine-2-carboxylic acid

[0244] Trifluoroacetic acid (0.36 mL, 4.7 mmol) was added dropwise to a stirred solution of (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 18, 149 mg, 0.23 mmol) in DCM (1 mL) at room temperature. After 2 h, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1 M HCl(aq) (2.0 mL, 2.0 mmol) and Et2O (2 mL). Phenylboronic acid (85 mg, 0.70 mmol) was added and the clear biphasic solution was stirred at room temperature for 4 h. The mixture was diluted with Et2O (10 mL) and water (2 mL) and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 mL) to give (2R, 4S)-4-[[(2S)-2-amino-4-methyl-pentanoyl]amino]-2-(4-dihydroxyborylbutyl)piperidine-2-carboxylic acid (Example 6, 81 mg, 97% yield) as a white solid. 1 H NMR(500MHz,D2O)δ0.74-0.82(2H,m),0.89(3H,d),0.91(3H,d),1.15-1.25 (1H,m),1.24-1.34(1H,m),1.38-1.47(3H,m),1.47-1.54(1H,m),1.60(1H, dt),1.65-1.76(1H,m),1.79-1.98(3H,m),2.01-2.09(1H,m),2.14(1H,dd) ,3.15-3.23(1H,m),3.31(1H,dt),3.39(1H,t),4.10-4.17(1H,m); m / z: (ES + )[M-H2O+H] + =340.

[0245] Example 7: (2R,4S)-4-[[(2S,3S)-2-amino-3-methyl-pentanoyl]amino]-2-(4-dihydroxyborylbutyryl)- 2-(2-(4-(4-yl)piperidine-2-carboxylic acid)

[0246]

[0247] Intermediate 19: (2R,4S)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)-2- 2-Benzyl 1-(tert-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate Butyl) ester

[0248] N,N-diisopropylethylamine (0.24 mL, 1.4 mmol) was slowly added to a stirred solution of (2R,4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (Intermediate 8, 355 mg, 0.687 mmol), Boc-Ile-OH (159 mg, 0.687 mmol) and COMU (300 mg, 0.70 mmol) in DMF (4 mL) at 0°C. The reaction was stirred for 16 h while slowly warming to room temperature. The crude reaction mixture was diluted with water (30 mL) and the mixture was stirred for 10 min. The resulting precipitate was collected by filtration. The solid was dissolved in EtOAc (20 mL) and washed with saturated aqueous NaCl solution (5 mL), dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 60% EtOAc in hexanes) to give 2-benzyl 1-(tert-butyl) (2R,4S)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 19, 424 mg, 85% yield) as a white foam and a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.73-0.80(2H,m),0.82-0.90(6H,m),0.97-1.11(1H,m),1.21(12H,s),1.26-1.31(1H,m),1.32-1.36(1H,m),1.38(9H,br s),1.39(9H,s),1.41-1.43(2H,m),1.67(1H,br dd),1.75-1.84(2H,m),1.86-1.98(3H,m),2.00(1H,br d),2.03(1H,br d),2.92-3.02(1H,m),3.81(1H,t),3.95-4.09(2H,m),4.98(1H,br s),5.04-5.21(2H,m),6.07(1H,br d),7.28-7.36(5H,m); m / z: (ES) + )[M+H] + =730.

[0249] Intermediate 20: (2R,4S)-1-(tert-butoxycarbonyl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)- 3-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidin-2-yl acid

[0250] Pd / C (10% wt, 22 mg, 0.021 mmol) was added to a solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 19, 302 mg, 0.41 mmol) in EtOAc (4 mL). The suspension was stirred at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times) for 20 h. The reaction mixture was diluted with EtOAc (10 mL) and MeOH (1 mL), filtered through celite and concentrated to dryness. The resulting residue was purified by flash silica chromatography (15% to 100% EtOAc in hexanes followed by 0 to 50% The reaction mixture was purified by evaporation of 4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 20, 223 mg, 84% yield) as a dry film and a mixture of rotamers; and the boronic acid byproduct (2R,4S)-2-(4-dihydroxyborylbutyl)-1-(tert-butoxycarbonyl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)piperidine-2-carboxylic acid (30 mg, 13% yield) as a white solid and a mixture or rotamers. Intermediate 20: 1 H NMR(500MHz, CDCl3)δ0.72-0.81(2H,m),0.82-0.88(3H,m),0.89-0.94(3H,m),0.98- 1.11(1H,m),1.21-1.24(12H,m),1.26-1.31(1H,m),1.32-1.38(2H,m),1.40(10H,br s),1.42(10H,br s),1.45-1.55(1H,m),1.53-1.64(1H,m),1.76-1.97(4H,m),1.98-2.08(3H,m),2.83-3.10(1H,m),3.90-4.08(2H,m),5.15(0.5H,br d),5.81(0.5H,brs),6.74(0.5H,br s),7.48(0.5H,br s); m / z: (ES + )[M+H] + =640. Boric acid by-product: 1H NMR(500MHz, CDCl3)δ0.76-0.86(2H,m),0.86-0.96(6H,m),1.03-1.13(1H,m),1.43(9H,br s),1.44(12H,s),1.53-1.68(1H,m),1.78-1.90(2H,m),1.90-1.99(2H,m),1.99- 2.04(1H,m),2.07-2.21(1H,m),2.98-3.11(1H,m),3.43-3.61(1H,m),3.90(1H,br s),3.96-4.12(2H,m),5.19(1H,br s),5.53-5.76(1H,m),6.72(1H,br s); m / z: (ES + )[M+H] + =558.

[0251] Example 7: (2R,4S)-4-[[(2S,3S)-2-amino-3-methyl-pentanoyl]amino]-2-(4-dihydroxyborylbutyryl)- 2-(2-(4-(4-yl)piperidine-2-carboxylic acid)

[0252] Trifluoroacetic acid (0.62 mL, 8.1 mmol) was added dropwise to a stirred solution of (2R,4S)-1-(tert-butoxycarbonyl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 20, 223 mg, 0.35 mmol) and the boronic acid byproduct (2R,4S)-2-(4-dihydroxyboronobutyl)-1-(tert-butoxycarbonyl)-4-((2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentanamido)piperidine-2-carboxylic acid (30 mg, 0.05 mmol) in DCM (2 mL) at room temperature. After 2h, the solution was concentrated under reduced pressure, and the resulting residue was dissolved in 1M HCl (aqueous solution) (3.0mL, 3.0mmol) and Et2O (3mL). Phenylboronic acid (147mg, 1.21mmol) was added, and the transparent biphasic solution was stirred at room temperature for 3h. The mixture was diluted with Et2O (5mL) and water (1mL), and each layer was separated. The aqueous layer was washed with Et2O. The aqueous layer was freeze-dried and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using a 5% ammonia solution of MeOH (20mL) to obtain (2R, 4S) -4- [[(2S, 3S) -2- amino -3- methyl-pentanoyl] amino] -2- (4- dihydroxyboryl butyl) piperidine -2- carboxylic acid (Example 7, 126mg, yield 88%) as a white solid. 1H NMR(500MHz,D2O)δ0.78(2H,td),0.85-0.91(6H,m),1.11-1.25(2H,m),1.26-1.34(1H,m),1.37-1.49(3H,m),1.64-1.76(2H,m),1 .80-1.91(2H,m),1.92-1.99(1H,m),2.01-2.09(1H,m),2.17(1H,dd),3.15-3.24(2H,m),3.31(1H,dt),4.10-4.21(1H,m); m / z: (ES + )[M+H] + =358.

[0253] Example 8: (2R,4S)-4-[[(2S)-2-amino-3,3-dimethyl-butyryl]amino]-2-(4-dihydroxyborylbutyryl) 2-(2-(4-(4-yl)piperidine-2-carboxylic acid)

[0254]

[0255] Intermediate 21: (2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutyramido)-2- 2-Benzyl 1-(tert-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate Butyl) ester

[0256] N,N-Diisopropylethylamine (0.24 mL, 1.4 mmol) was slowly added to a stirred solution of 2-benzyl 1-(tert-butyl) (2R,4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 8, 355 mg, 0.687 mmol), Boc-tert-Leu-OH (159 mg, 0.687 mmol) and COMU (300 mg, 0.70 mmol) in DMF (4 mL) at 0°C. The reaction was stirred for 16 h while slowly warming to room temperature. The crude reaction mixture was diluted with water (30 mL) and the mixture was stirred for 10 min. The resulting precipitate was collected by filtration. The solid was dissolved in EtOAc (20 mL) and washed with saturated aqueous NaCl solution (5 mL), dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 55% EtOAc in hexanes) to afford 2-benzyl 1-(tert-butyl) (2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 21, 372 mg, 74% yield) as a white foam. 1H NMR(500MHz, CDCl3)δ0.73-0.81(2H,m),0.95(9H,s),1.24(12H,s),1.41(10H,s),1.42-1.46(12H,m), 1.68(1H,dd),1.87-1.95(1H,m),1.96-2.01(2H,m),2.02-2.05(2H,m),2.92-3.04(1H,m),3.69(1H,br d),3.98-4.12(2H,m),5.05-5.25(3H,m),5.50-5.62(1H,m),7.30-7.39(5H,m); m / z: (ES + )[M+H] + =730.

[0257] Intermediate 22: (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3,3- 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidin-2-yl acid

[0258] Pd / C (10% wt, 27 mg, 0.025 mmol) was added to a solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 21, 372 mg, 0.511 mmol) in EtOAc (4 mL). The suspension was stirred at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times) for 20 h. The reaction mixture was diluted with EtOAc (10 mL) and MeOH (1 mL), filtered through celite and concentrated to dryness. The resulting residue was purified by flash silica chromatography (40% to 100% EtOAc in hexanes followed by 0 to 40% The reaction mixture was purified by evaporation of 1% MeOH in EtOAc to afford (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 22, 265 mg, 81% yield) as a dry film and a mixture of rotamers; and the boronic acid byproduct (2R,4S)-2-(4-dihydroxyborylbutyl)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanamido)piperidine-2-carboxylic acid (32 mg, 11% yield) as a clear dry film and a mixture of rotamers. Intermediate 22: 11H NMR (500 MHz, CDCl3) δ 0.75 (2H, br t), 0.93 (9H, s), 1.20 (12H, s), 1.24 - 1.33 (2H, m), 1.36 (9H, s), 1.38 (9H, br s), 1.40 (3H, br s), 1.51 - 1.64 (1H, m), 1.72 - 1.82 (1H, m), 1.83 - 1.98 (3H, m), 2.03 (1H, br s), 2.79 - 3.06 (1H, m), 3.76 (0.4H, br d), 3.85 - 4.07 (2.6H, m), 5.42 (0.6H, br d), 6.39 - 6.71 (1H, m), 6.74 - 6.99 (0.4H, m); m / z: (ES + ) [M + H] + = 640. Borate by - product: 1 1H NMR (500 MHz, CDCl3) δ 0.77 - 0.86 (2H, m), 0.97 (9H, s), 1.42 (9H, br s), 1.43 (14H, br s), 1.52 - 1.67 (1H, m), 1.75 - 1.90 (2H, m), 1.91 - 2.04 (3H, m), 2.08 - 2.19 (1H, m), 2.97 - 3.14 (1H, m), 3.44 - 3.63 (1H, m), 3.87 (1H, br d), 3.97 - 4.11 (2H, m), 5.50 (1H, br s), 6.53 - 6.79 (1H, m); m / z: (ES + ) [M + H] + = 558.

[0259] Example 8: (2R,4S)-4-[[(2S)-2-amino-3,3-dimethyl-butyryl]amino]-2-(4-dihydroxyborylbutyryl) 2-(2-(4-(4-yl)piperidine-2-carboxylic acid)

[0260] Trifluoroacetic acid (0.73 mL, 9.4 mmol) was added dropwise to a stirred solution of (2R,4S)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 22, 265 mg, 0.414 mmol) and the boronic acid byproduct (2R,4S)-2-(4-dihydroxyboronobutyl)-1-(tert-butoxycarbonyl)-4-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanamido)piperidine-2-carboxylic acid (32 mg, 0.057 mmol) in DCM (2 mL) at room temperature. After 2h, the solution was concentrated under reduced pressure, and the resulting residue was dissolved in 1M HCl (aqueous solution) (3.0mL, 3.0mmol) and Et2O (3mL). Phenylboronic acid (172mg, 1.41mmol) was added, and the transparent biphasic solution was stirred at room temperature for 4h. The mixture was diluted with Et2O (5mL) and water (1mL), and each layer was separated. The aqueous layer was washed with Et2O. The aqueous layer was freeze-dried and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using a 5% ammonia solution of MeOH (20mL) to obtain (2R, 4S) -4- [[(2S) -2- amino -3,3- dimethyl - butyryl] amino] -2- (4- dihydroxyboryl butyl) piperidine -2- carboxylic acid (Example 8, 158mg, yield 94%) as a white solid. 1 H NMR(500MHz,D2O)δ0.74-0.81(2H,m),0.95(9H,s),1.15-1.25(1H,m),1.26-1.34(1H,m),1.38-1.48(2H,m),1.65-1.75(1H,m),1.80-1 .91(2H,m),1.96(1H,ddd),2.01-2.10(1H,m),2.19(1H,dd),3.05(1H,s),3.15-3.24(1H,m),3.30(1H,dt),4.10-4.23(1H,m); m / z: (ES) + )[M+H] + =358.

[0261] Example 9: (2R,4S)-4-[[(2R)-2-amino-3-methyl-butyryl]amino]-2-(4-dihydroxyborylbutyl) Piperidine-2-carboxylic acid

[0262]

[0263] Intermediate 23: (2R,4S)-4-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutyramido)-2-(4- 2-Benzyl 1-(tert-butyl)piperidin-1,2-dicarboxylate Ester

[0264] At 0 ° C, N, N-diisopropylethylamine (0.063 mL, 0.36 mmol) was slowly added to a stirred solution of HATU (61 mg, 0.16 mmol) and Boc-D-Val-OH (33 mg, 0.15 mmol) in DMF (1 mL). The solution was stirred for 10 min, and then a solution of (2R, 4S)-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylic acid 2-benzyl 1-(tert-butyl) ester (Intermediate 8, 75 mg, 0.15 mmol) in DMF (1 mL) was added. The reaction was stirred for 16 h while slowly warming to room temperature. The crude reaction was diluted with 0.1 M HCl (aqueous solution) (30 mL) and EtOAc. The phases were separated and the aqueous phase was extracted with EtOAc (3 x 15 mL). The combined organics were washed with saturated aqueous NaCl, dried over MgSO4, filtered and concentrated to dryness. The resulting residue was purified by flash silica chromatography (5% to 40% EtOAc in hexanes) to give 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 23, 74 mg, 71% yield) as a colorless film and a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.75(2H,t),0.84(3H,d),0.89(3H,d),1.21(12H,s),1.30-1.35(1H,m),1.37(9H,s),1.39(9H,s),1.42(4H,br s),1.69(1H,dd),1.81-1.91(1H,m),1.92-2.01(3H,m),2.02(2H,s),2.94-3.02(1H,m),3. 75(1H,dd),3.91-4.03(1H,m),5.03-5.19(3H,m),6.10(1H,d),7.28-7.39(5H,m); m / z: (ES + )[M+H] + =715.

[0265] Intermediate 24: (2R,4S)-1-(tert-butoxycarbonyl)-4-((R)-2-((tert-butoxycarbonyl)amino)-3-methyl 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid

[0266] Pd / C (10% wt, 5 mg, 0.005 mmol) was added to a solution of 2-benzyl 1-(tert-butyl)-(2R,4S)-4-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-1,2-dicarboxylate (Intermediate 23, 69 mg, 0.10 mmol) in EtOAc (1 mL). The suspension was stirred at room temperature under a hydrogen atmosphere (balloon, evacuated the flask and backfilled with hydrogen 3 times) for 19 h. The reaction mixture was diluted with EtOAc (10 mL) and MeOH (1 mL), filtered through celite and concentrated to dryness. The resulting residue was purified by flash silica chromatography (40% to 100% EtOAc in hexanes followed by 0 to 40% MeOH in EtOAc) to afford (2R,4S)-1-(tert-butoxycarbonyl)-4-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 24, 40 mg, 66% yield) as a dry film and as a mixture of rotamers. 1 H NMR(500MHz, CDCl3)δ0.77(2H,t),0.91(6H,br d),1.20-1.24(13H,m),1.29(1H,br d),1.33-1.42(11H,m),1.43(10H,s),1.70-1.82(1H,m),1.83-1.90(1H,m),1. 91-2.02(4H,m),3.00(1H,t),3.83-3.94(1H,m),3.96-4.07(1H,m),4.13(1H,br s),5.40(1H,d),6.03-6.33(1H,m),6.90-7.22(1H,m); m / z: (ES + )[M+H] + =625.

[0267] Example 9: (2R,4S)-4-[[(2R)-2-amino-3-methyl-butyryl]amino]-2-(4-dihydroxyborylbutyl) Piperidine-2-carboxylic acid

[0268] Trifluoroacetic acid (0.10 mL, 1.3 mmol) was added dropwise to a stirred solution of (2R,4S)-1-(tert-butoxycarbonyl)-4-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)piperidine-2-carboxylic acid (Intermediate 24, 40 mg, 0.06 mmol) in DCM (1 mL) at room temperature. After 2 h, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1 M HCl(aq) (1.0 mL, 1.0 mmol) and Et2O (1 mL). Phenylboronic acid (24 mg, 0.20 mmol) was added and the clear biphasic solution was stirred at room temperature for 20 h. The mixture was diluted with Et2O (5 mL) and water (1 mL), and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 mL) to give (2R, 4S)-4-[[(2R)-2-amino-3-methyl-butyryl] amino]-2-(4-dihydroxyborylbutyl)piperidine-2-carboxylic acid (Example 9, 20 mg, 91% yield) as a white solid. 1 H NMR(500MHz,D2O)δ0.73-0.83(2H,m),0.87-0.95(6H,m),1.14-1.23(1H,m),1.24-1.33(1H,m),1.41(2H,quin),1.65-1.77(1H,m),1.79-1 .87(1H,m),1.88-1.98(3H,m),2.01-2.09(1H,m),2.15(1H,dd),3.14(1H,d),3.15-3.24(1H,m),3.31(1H,dt),4.11-4.20(1H,m); m / z: (ES + )[M-H2O+H] + =326.

[0269] Example 10. Compound 12 (arginase 1 inhibitor) in combination with anti-PDL1 or anti-PDL1 and anti-NKG2a or poly I:C Efficacy in preclinical models of cancer

[0270] method:

[0271] MC38-OVA: Mouse MC38 colorectal cancer cells (5x10 5Cells / mouse) were subcutaneously implanted in the right flank of 6-8 week old female C57BL / 6 mice. On the 6th day after transplantation, each group of mice was treated with vehicle (water), 30 mg / kg compound 12, 10 mg / kg anti-PDL1 (MEDI4736) or a combination. Compound 12 was prepared in water and orally administered twice a day. MEDI4736 was prepared in 1X PBS and intraperitoneally administered twice a week for 2 weeks. Tumor length and width were measured with a caliper and the formula (length x width) was used. 2 )*π / 6 to calculate the tumor volume, which was then reported as the calculated tumor volume.

[0272] To monitor immune cell changes, tumors were harvested four, ten, or fourteen days after treatment. The tumors were excised and mechanically minced. The tumors were then incubated for 40 min with a tumor dissociation enzyme mixture (Miltenyi Biotec) in a gentle MACS instrument (Miltenyi Biotec) at 37 ° C. Single cell suspensions were prepared and stained with a variety of immune cell markers to determine immune cell changes after treatment using multicolor flow cytometry. In addition, the functional response of CD8+ cytotoxic T cells was assessed by ex vivo re-stimulation with phorbol 12-myristate-13-acetate (PMA) and ionomycin before analysis.

[0273] CT.26WT: Mouse CT.26WT colorectal cancer cells (5 x 10 5 Cells / mouse) were subcutaneously implanted in the right flank of 6 to 8 week old female Balb / C mice. On the 6th day after implantation, each group of mice was treated with vehicle (water), 30mg / kg compound 12, 10mg / kg anti-PDL1 (MEDI4736) or combination. Another group was treated with 10mg / kg anti-NKG2A (monalizumab) in combination with compound 12 or MEDI4736, or a triple combination of compound 12, monalizumab and MEDI4736. Compound 12 was prepared in water and orally administered twice a day. MEDI4736 was prepared in 1X PBS and administered intraperitoneally twice a week for 2 weeks. Monalizumab was prepared in 1X PBS and administered intravenously twice a week for 1.5 weeks (3 doses). Tumor length and width were measured with a caliper, and the formula (length x width) was used. 2 )*π / 6 to calculate the tumor volume, which was then reported as the calculated tumor volume.

[0274] LL / 2: Mouse Lewis lung (LL / 2) cancer cells (1x 10 6Cells / mouse) were subcutaneously implanted in the right flank of 6-8 week old C57BL / 6 mice. On the 6th day after implantation, each group of mice was treated with vehicle (water), 30 mg / kg compound 12, 7.5 mg / kg TLR3 agonist (poly I:C) or a combination. Compound 12 was prepared in water and orally administered twice a day. Poly I:C was prepared in water and intraperitoneally administered 3 times a week. Tumor length and width were measured with a caliper and the formula (length x width) was used. 2 )*π / 6 to calculate the tumor volume, which was then reported as the calculated tumor volume.

[0275] result: like Figures 1A to 1D , Figures 2A to 2D ,and Figures 3A to 3F As shown in , compound 12 monotherapy was moderately effective in all three mouse syngeneic tumor models. Administration of anti-PDL1 alone also showed moderate efficacy in two colorectal cancer tumor models. The combination of agents showed significantly stronger efficacy: 87% tumor growth inhibition in MC38-OVA and 46% total tumor growth inhibition in the CT.26WT model (with one complete remission). Adding anti-NKG2a to compound 12+anti-PDL1 treatment resulted in 53% total tumor growth inhibition and 3 complete remissions. TLR3 agonists (poly I:C) showed moderate monotherapy activity, with a TGI of 76% on day 16. The combination of compound 12+poly I:C resulted in 87% tumor growth inhibition.

[0276] like Figures 4A to 4F As shown in, in vivo PD combination studies of ARG inhibitors with anti-PDL1 in the MC38-OVA model showed increases in multiple tumor immune cell populations (approximately 4-fold CD8+ T cells, 2-fold NK cells, 2-fold CD103+ DCs) and increased CD8 T cell activation. Figure 5A and 5B As shown in the results, ARG inhibition led to an increase in peripheral (draining LN) CD8+ T cells producing IFNg and TNFa. In the CT.26WT model, there was one complete remission.

[0277] Example 11. Compound 12 (arginase 1 inhibitor) combined with radiation therapy (RT) in preclinical models of cancer Benefits

[0278] method:1e6 LL / 2 murine lung cancer cells were implanted subcutaneously into the right flank of six to eight week old BL / 6 mice (Charles River Labs). On day 6 post-implantation, animals were randomized into four groups (n=12 / group) based on the average tumor volume per cage. Group 1 was anesthetized on days 6 and 9 for simulated irradiation and was administered vehicle (water) PO, BID from day 6. Group 2 was anesthetized on days 6 and 9 and given 5 Gy of tumor-targeted radiation. Group 3 was administered AZ8400 at 30 mg / kg PO bid from day 6, and Group 4 was administered AZ8400 at 30 mg / kg from day 6, and anesthetized and irradiated on days 6 and 9. Body weight and tumor volume were measured twice a week until tumors were >1500 mm3, at which time the animals were humanely sacrificed.

[0279] result:

[0280] like Figure 5A and 5B As shown in, in the mouse syngeneic lung tumor model LL / 2, the combination of compound 12 and radiation therapy exhibited significantly stronger efficacy, reaching approximately 60% tumor growth inhibition (p<0.05 compared to vehicle and monotherapy treatments), compared to compound 12 administered twice daily at 30 mg / kg and radiation therapy (5 Gy on days 6 and 9 post-implantation; approximately 30% tumor growth inhibition, p>0.05 compared to vehicle).

[0281]

[0282]

[0283]

Claims

1. A method for treating cancer in a patient, comprising administering to the patient an effective amount of a compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and an effective amount of an immunomodulator; n is 0 or 1; R 1 is -H or -C(O)CH(R 1a )NHR 1b ; R 1a Selected from -H, -(C1-C6)alkyl and CH2OR 1c ; R 1b is -H; or alternatively, R 1a and R 1b together with the atoms to which they are attached, form a 5-membered heterocyclic ring; and R 1c It is H or -CH3.

2. The method of claim 1, wherein R 1 is -H or -C(O)CH(R 1a )NH2; and R 1a Selected from -H or -(C1-C6)alkyl.

3. The method of claim 1 or 2, wherein the compound having formula (Ia) or (Ib) is represented by formula (IIa) or (IIb): wherein n is 0 or 1; and R 2 Selected from -H or -(C1-C4)alkyl.

4. The method of any one of claims 1 to 3, wherein the compound of formula (Ia) or (Ib) is selected from the compounds of Table 1.

5. The method of any one of claims 1 to 4, wherein the immunomodulator is an immune checkpoint inhibitor or an immunostimulatory agent.

6. The method of claim 5, wherein the immune checkpoint inhibitor is selected from the group consisting of a CTLA-4 receptor inhibitor, a PD-1 receptor inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a NKG2A receptor inhibitor, and a combination thereof.

7. The method of claim 5, wherein the immunostimulatory agent is a TLR3 agonist.

8. The method of any one of claims 1 to 6, wherein the immune checkpoint inhibitor is an antibody or an antigen-binding fragment thereof.

9. The method of any one of claims 1 to 6 and 8, wherein the immune checkpoint inhibitor is an anti-CTLA-4 receptor antibody, an anti-PD-1 receptor antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an anti-NKG2A receptor antibody.

10. The method of claim 9, wherein the immune checkpoint inhibitor is durvalumab, tremelimumab, monalizumab, or a combination thereof.

11. The method of any one of claims 1 to 10, wherein the cancer is breast cancer, bladder cancer, head and neck cancer, non-small cell lung cancer, small cell lung cancer, colorectal cancer, gastrointestinal stromal tumors, gastroesophageal cancer, renal cell carcinoma, prostate cancer, liver cancer, colon cancer, pancreatic cancer, ovarian cancer, melanoma, non-Hodgkin's lymphoma, cutaneous T-lymphocytic neoplasm, multiple myeloma, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CMML), and diffuse large B-cell lymphoma (DLBCL).

12. The method of any one of claims 1 to 11, wherein the compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and the immunomodulator are administered sequentially, separately or simultaneously.

13. A compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, for use in treating cancer in a patient, wherein the compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and an immunomodulator are administered to the patient sequentially, separately or simultaneously.

14. An immunomodulator for use in treating cancer, wherein the immune checkpoint inhibitor and the compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, are administered to the patient sequentially, separately or simultaneously.

15. A pharmaceutical product comprising i) a compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and ii) an immunomodulator; n is 0 or 1; R 1 is -H or -C(O)CH(R 1a )NHR 1b ; R 1a Selected from -H, -(C1-C6)alkyl and CH2OR 1c ; R 1b is -H; or alternatively, R 1a and R 1b together with the atoms to which they are attached, form a 5-membered heterocyclic ring; and R 1c It is H or -CH3.

16. The pharmaceutical product of claim 15, wherein the compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and the immunomodulator are present in a single dosage form.

17. The pharmaceutical product of claim 15, wherein the compound of formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and the immunomodulator are present in separate dosage forms.

18. A method of treating cancer in a patient, comprising administering to the patient an effective amount of a compound of Formula (Ia) or (Ib), or a pharmaceutically acceptable salt thereof, and an effective amount of radiation therapy; n is 0 or 1; R 1 is -H or -C(O)CH(R 1a )NHR 1b ; R 1a Selected from -H, -(C1-C6)alkyl and CH2OR 1c ; R 1b is -H; or alternatively, R 1a and R 1b together with the atoms to which they are attached, form a 5-membered heterocyclic ring; and R 1c It is H or -CH3.

19. The method of claim 18, wherein the compound of formula (Ia) or (Ib) is the same as defined in any one of claims 2 to 4.

20. The method of claim 18 or 19, wherein the radiation therapy is fractionated radiation therapy.

21. The method of any one of claims 18 to 20, further comprising administering to the patient an effective amount of an immunomodulator.

22. The method of any one of claims 18 to 21, wherein the immunomodulator is the same as defined in any one of claims 5 to 10.

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