WO2021236475A1 - Compounds that inhibit asparagine synthetase and their methods of use - Google Patents

Abstract

The invention is relevant to chemistry of organic compounds, pharmacology and medicine, and is related to treatment and/or prevention of a disease or condition, associated with over expressed or dysregulated glutamine-dependent asparagine synthetase, using a new family of chemical compounds having the ability to inhibit asparagine synthetase (ASMS). New chemical compounds with high inhibitory activity against asparagine synthase and promising for use in therapy. This invention also covers pharmaceutical compositions containing therapeutically effective amount of compound according to the invention and method of treatment and/or prevention of a disease or condition, associated with over-expressed or dysregulated glutamine dependent asparagine synthetase.

Description

COMPOUNDS THAT INHIBIT ASPARAGINE SYNTHETASE AND THEIR METHODS OF USE FIELD OF THE INVENTION This invention pertains to compounds that inhibit the activity of asparagine synthetase (ASNS), compositions containing these compounds, and methods of treating diseases in which ASNS is over-expressed or dysregulated. BACKGROUND Asparagine Synthetase or glutamine-dependent asparagine synthetase catalyzes the biosynthesis of asparagine (Asn) from aspartate through an ATP-dependent reaction. ASNS gene transcription and ASNS activity is highly responsive to cellular stress, in particular to intracellular amino acid depletion. Asparagine depletion leads to an adaptive cellular response in which uncharged tRNA activates the serine threonine kinase GCN2. GCN2 in turn phosphorylates the translation initiation factor eIF2a, resulting in increased translation of the transcription factor ATF4. ATF4 induces ASNS, which results in glutamine-dependent asparagine synthesis from aspartate (Figure 1) [Lomelino CL, Andring JT, McKenna R, Kilberg MS.J Biol Chem. 2017 Dec 8;292(49):19952-19958]. Sustained levels of asparagine suppress apoptosis and promote cellular adaptation to depletion of glutamine and other nonessential amino acids, while intracellular depletion of asparagine induces apoptosis even in the face of an abundant supply of glutamine and other nonessential amino acids. That suggests, ASNS plays an important role during tumor cell accumulation and progression by maintaining cell viability. Mining publicly available databases (kmplot.com/analysis/) reveals that ASNS gene expression is correlated with survival in various types of cancer including head-neck squamous cell carcinoma, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, and sarcoma (Figure 2). Most of the epidemiological and experimental evidence gathered thus far suggests a pro- cancer role of ASNS pointing to a metabolic advantage for high-ASNS cancer cells. Elevated ASNS protein expression is associated with resistance to asparaginase therapy. ASNS expression levels may also be inversely correlated with asparaginase efficacy in certain solid tumors (M.S. Kilberg et al. J Biol Chem.2017 Dec 8;292(49):19952-19958) The literature reports ASNS as a target in treating acute lymphoblastic leukemia (ALL) (N. Richards et al. Chem Biol.2006; 13(12): 1339–1347). Involvement of ASNS in sarcomas including rhabdomyosarcomas and leiomyosarcomas is reported in Elife. (2015) 4:e09436. doi: 10.7554/eLife.09436.022. Involvement of ASNS in human ovarian carcinomas is reported in Mol Cancer Ther. (2006) 5:2613–23. Involvement of ASNS in gastric cancers is reported in Scand J Gastroenterol. (2016) 51:1220–6. Involvement of ASNS in melanoma and epidermoid carcinoma is reported in Biotechnol Appl Biochem. (2016) 63:328–33. Involvement of ASNS in non-small cell lung cancer (NSCLC) is reported in Cancer Gene Ther. (2016) 23:287–94. Involvement of ASNS in breast cancer and a breast cancer metastatic behavior is reported in ChemBiol DrugDes. (2014) 84:578–84 and Nature. (2018) 554:378–81. Involvement of ASNS in prostate cancer is reported in Eur J Clin Invest. (2007) 37:126–33 and Am J Pathol. (2012) 180:895–903. Involvement of ASNS in colorectal cancers including colon carcinoma with mutated KRAS is reported in Neoplasia. (2016) 18:654–65. Together, these data support inhibition of ASNS activity as a viable strategy to control the growth, proliferation, and migration of cancer cells, eliminate them, or enhance their sensitivity to existing chemotherapy drugs or radiotherapy. This invention relates to new families of chemical compounds having the ability to inhibit ASNS, and promising for use in the treatment of diseases during which ASNS is over-expressed or dysregulated. SUMMARY OF THE INVENTION An object of the present invention is the development and providing a new family of chemical compounds having the ability to inhibit asparagine synthetase (ASNS), and promising for use in the treatment of diseases during which asparagine synthetase (ASNS) is over-expressed or dysregulated. The technical result of the invention is the development and production of new chemical compounds with high inhibitory activity against asparagine synthase and promising for use in therapy diseases during which asparagine synthetase (ASNS) is over-expressed or dysregulated. In some preferred embodiments, the claimed compounds inhibit glutamine-dependent asparagine synthetase with an IC50 of less than 10 nM. In one aspect, the invention provides compounds of Formula I,

wherein: R¹ is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀- alkyl; R² is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀- alkyl; wherein R¹ and R² may optionally be joined to form a substituted and unsubstituted -C₃-C₁₀- cycloalkyl; L is independently selected for each occurrence from the group consisting of a covalent chemical bond, -O-; R³ is selected from -H, halo, -OH, -C₁-C₁₀-alkyl, -C(O)-(C₁-C₁₀-alkyl), -C(O)O(C₁-C₁₀-alkyl), - (CH₂)n-C(O)OH, -(CH₂)n-C(O)NH(C₁-C₁₀-alkyl), -(CH₂)n-C(O)NH₂, -(CH₂)n-C(O)O(C₁-C₁₀-alkyl), - C(O)- C₁-C₁₀-alkyl or -C(O)NH(CH₂)nOC₁-C₁₀-alkyl, wherein n is selected independently from 0, 1, 2, 3, 4, 5; R⁴ is selected from -H or -C₆-C₁₀-aryl, wherein said -C₆-C₁₀-aryl is alternatively substituted with one, two, three, four, or five substituents independently selected for each occurrence from R^4a; R^4a is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀ alkyl, -O-C₁-C₁₀ alkyl, halo or -O-(CH₂)p-C(O)OH, wherein p is selected independently from 0, 1, 2, 3, 4, 5; M is -NH-, -NH-CH₂-; A is independently selected for each occurrence from the group consisting of -C₆-C₁₀-aryl, - (CH₂)m-C₆-C₁₀-aryl,

, , , ,

, wherein m is selected independently from 0, 1, 2, 3, 4, 5; wherein A may optionally be substituted with one, two, three, four, five or six substituents independently selected for each occurrence from the group consisting of halo, -C₁-C₁₀-alkyl, halogenated -C₁-C₁₀-alkyl, -O-C₁-C₁₀-alkyl or -C₆-C₁₀-aryl; R⁵ is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀- alkyl, -С(O)С(СH₃)₃, -C(O)-CH₂(C₁-C₅-alkyl)-NH₂ or -C(O)-CH₂(C₁-C₅-alkyl)-NH(C₁-C₅-alkyl); R⁶ is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀- alkyl, -С(O)С(СH₃)₃, -C(O)-CH₂(C₁-C₅-alkyl)-NH₂ or -C(O)-CH₂(C₁-C₅-alkyl)-NH(C₁-C₅-alkyl). In some embodiments R⁵ is independently selected for each occurrence from the group consisting of:

R⁶ is independently selected for each occurrence from the group consisting of:

In some embodiments A is independently selected for each occurrence from the group consisting of:

In another embodiment, the invention relates to compounds of Formula I having the following stereochemical orientation:

, wherein each of R¹, R², R³, R^4a, R⁵, R⁶, A, L and M is as defined and described in embodiments herein, and therapeutically acceptable salts, prodrugs, esters, amides, salts of prodrugs, salts of esters, and salts of amides thereof. In some embodiments of the invention, the present invention provides compounds having the following structures:

In some preferred embodiments, the compounds of Formulae (I), (Ia), or (Ib) inhibit glutamine- dependent asparagine synthetase with an IC50 of less than 10 nM. Furthermore, the invention implies a pharmaceutical composition for treatment and/or prevention of a disease or condition, associated with over-expressed or dysregulated glutamine- dependent asparagine synthetase, comprising therapeutically effective amount of at least compound according to invention and at least one pharmaceutically acceptable excipient. In some embodiments disease is a proliferative disease. In some preferred embodiments, the proliferative disease is acute lymphoblastic leukemia, rhabdomyosarcoma and leiomyosarcoma, colon carcinoma with mutated KRAS, ovarian carcinomas, gastric cancers, melanoma, epidermoid carcinoma, non-small cell lung cancer, breast cancer, prostate cancer, soft tissue sarcoma, acute lymphoblastic leukemia, sarcoma with a mutant form of protein Ras, breast cancer or castration-resistant prostate cancer. In some preferred embodiments, disease is a disease characterized by the dysregulation of ASNS. In some preferred embodiments, a pharmaceutical composition according to the invention additionally comprising a therapeutically effective amount of at least one chemotherapeutic agent. In some preferred embodiments, a chemotherapeutic agent is an asparaginase. In some embodiments, an asparaginase is recombinant L-asparaginase derived from Escherichia coli or Erwinia chrysanthemi. In some embodiments, pharmaceutically acceptable excipient is pharmaceutically acceptable carrier, adjuvant, solvent. The specified technical result is also achieved by using a compound according to the invention for preparing a drug for treatment and/or prevention of a disease or condition, associated with over-expressed or dysregulated glutamine-dependent asparagine synthetase. In some embodiments, a method of reducing, or ameliorating a disease or condition, and/or symptoms associated therewith, mediated by inhibition of glutamine-dependent asparagine synthetase is disclosed, comprising administering to an individual in need thereof a therapeutically effective amount of a compound having a structure of Formula (I), (Ia), or (Ib) or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, a method for treatment of a proliferative disease is disclosed, said method comprising administering an effective amount of a compound of Formula (I), (Ia), or (Ib) or a pharmaceutically acceptable salt or solvate thereof, to a subject in need thereof. In some preferred embodiments, the proliferative disease comprises one selected from the following group: soft tissue sarcoma, acute lymphoblastic leukemia, sarcoma with a mutant form of protein Ras, breast cancer, castration-resistant prostate cancer. The invention also includes a method of prevention and/or treatment of a disease or condition, associated with over-expressed or dysregulated glutamine-dependent asparagine synthetase comprising administration of the pharmaceutical composition according to the invention to subject. In some preferred embodiments, the proliferative disease is acute lymphoblastic leukemia, rhabdomyosarcoma and leiomyosarcoma, colon carcinoma with mutated KRAS, ovarian carcinomas, gastric cancers, melanoma, epidermoid carcinoma, non-small cell lung cancer, breast cancer, prostate cancer, soft tissue sarcoma, acute lymphoblastic leukemia, sarcoma with a mutant form of protein Ras, breast cancer or castration-resistant prostate cancer. In some preferred embodiments, disease is a disease characterized by the dysregulation of ASNS. In some preferred embodiments, the method of prevention and/or treatment of a disease or condition according to the invention additionally comprising administration of therapeutically effective amount of at least one chemotherapeutic agent. In some preferred embodiments, a chemotherapeutic agent is an asparaginase. In some embodiments, an asparaginase is recombinant L-asparaginase derived from Escherichia coli or Erwinia chrysanthemi. In some preferred embodiments, the subject is human. In some preferred embodiments, the method of prevention and/or treatment of a disease or condition of the subject, wherein the subject is on an asparagine-free diet. Certain compounds of this invention may exist in tautomeric forms, and this invention includes all such tautomeric forms of those compounds unless otherwise specified. Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i. e., the R and S configurations for each asymmetric center. Thus single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention. Thus, this invention encompasses each diastereomer or enantiomer substantially free of other isomers (>90%, and preferably >95%, free from other stereoisomers on a molar basis) as well as a mixture of such isomers. Particular optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes known to the skilled artisans. The compounds of the present invention are used in the treatment of diseases, disorders or symptoms mediated by asparagine synthetase inhibition. Examples of diseases, disorders or symptoms mediated by asparagine synthetase inhibition include, but are not limited to, proliferative diseases. Such diseases may include, for example, acute lymphoblastic leukemia, rhabdomyosarcoma and leiomyosarcoma, colon carcinoma with mutated KRAS, ovarian carcinomas, gastric cancers, melanoma and epidermoid carcinoma, non-small cell lung cancer, breast cancer, prostate cancer. Still another embodiment pertains to methods for the treatment of a subject having a disease characterized by the dysregulation of ASNS. Still another embodiment comprises methods of treating a proliferative disease, the method comprising administering an effective amount of a compound or salt according to Formulae I, Ia, Ib, to a subject in need thereof. In a preferred embodiment, the proliferative disease comprises one selected from the following group: soft tissue sarcoma, acute lymphoblastic leukemia, sarcoma with a mutant form of protein Ras, breast cancer, castration-resistant prostate cancer. Still another embodiment comprises methods of treating a subject having a disease characterized by dysregulation of the ASNS comprising administering thereto a therapeutically effective amount of a compound of Formulae I, Ia, Ib, II, IIa, IIb and one or more than one additional therapeutic agent. In another embodiment, the invention provides a method for killing a cancer cell comprising administering an amount of a compound of Formulae I, Ia, Ib, in combination with a chemotherapeutic agent or agents that exploit metabolic vulnerabilities in the cancer cell. Such chemotherapeutic agents can include for example asparaginase (ASNase). Such agents may be, but are not limited to, recombinant L-asparaginase derived from Escherichia coli and Erwinia chrysanthemi. Treatment results in, for example, tumor regression. Tumor regression can include, for example, killing a cancer cell or preventing the cancer cells’ invasion and metastasis. Cancer cells can include, for example, tumor cells, neoplastic cells, malignant cells, metastatic cells, and hyperplastic cells. It is contemplated and therefore within the scope of the present invention that any feature that is described above can be combined with any other feature that is described above. It is also contemplated and therefore within the scope of the present invention that negative provisos can be added to exclude any compound or remove any feature. BRIEF DESCRIPTION OF THE FIGURES Figure 1. The role of Asparagine Synthetase and its Inhibition in regulating amino acid metabolism in cancer cells. Figure 2. Kaplan-Meier plots showing the effect of Asparagine Synthetase expression on Survival in various types of cancer: a) head and neck squamous cell carcinoma; b) clear cell renal cell carcinoma; c) hepatocellular carcinoma; d) sarcoma. Figure 3. Results of in vivo test of the claimed compounds, mouse melanoma B16/F10. Figure 4. Results of in vivo test of the claimed compounds, mouse breast tumor model 4T1 (asparaginase and tested compound treatment) Figure 5. Results of in vivo test of the claimed compounds, mouse breast tumor model 4T1 (asparagine-free diet). Figure 6.2D NOESY Spectrum of Intermediate 10. ABBREVIATIONS AND DEFINITIONS Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. The use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. With reference to the use of the words "comprise" or "comprises" or "comprising" in this patent application (including the claims), Applicants note that unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that Applicants intend each of those words to be so interpreted in construing this patent application, including the claims below. For a variable that occurs more than one time in any substituent or in the compound of the invention or any other formulae herein, its definition on each occurrence is independent of its definition at every other occurrence. Combinations of substituents are permissible only if such combinations result in stable compounds. Stable compounds are compounds which can be isolated in a useful degree of purity from a reaction mixture. As used herein, the term "ASNS" refers to the eukaryotic enzyme which catalyzes the conversion of aspartate to asparagine in an ATP-dependent manner. By “inhibits” it is meant that a test compound decreases the synthetase activity of recombinant or wild-type human asparagine synthetase. Inhibitors can be identified by incubating the purified enzyme with a test compound and measuring the synthetase activity of the enzyme by spectrophotometric monitoring of inorganic pyrophosphate production. Variable moieties of compounds herein are represented by identifiers (capital letters with numerical and/or alphabetical superscripts, so-called “R-groups”) and may be specifically embodied. It is meant to be understood that proper valences are maintained for all combinations herein, that monovalent moieties having more than one atom are attached through their left ends, and that divalent moieties are drawn from left to right. It is also meant to be understood that a specific embodiment of a variable moiety may be the same or different as another specific embodiment having the same identifier. This patent application uses the terms "substituent" and "radical" interchangeably. The term "hydrogen" (alone or in combination with another term(s)) means a hydrogen radical, and may be depicted as -H. The term "hydroxy" (alone or in combination with another term(s)) means -OH. The term "carboxy" (alone or in combination with another term(s)) means –C(O)OH. The term "amino" (alone or in combination with another term(s)) means -NH₂. The term "halogen" or "halo" (alone or in combination with another term(s)) means a fluorine radical (which may be depicted as -F), chlorine radical (which may be depicted as -CI), bromine radical (which may be depicted as -Br), or iodine radical (which may be depicted as -I). If a substituent is further described as being "substituted", a non-hydrogen radical is in the place of hydrogen radical on a carbon, nitrogen, oxygen or sulfur of the substituent. Thus, for example, a substituted alkyl substituent is an alkyl substituent in which at least one non-hydrogen radical is in the place of a hydrogen radical on the alkyl substituent. To illustrate, monofluoroalkyl is alkyl substituted with a fluoro radical, and difluoroalkyl is alkyl substituted with two fluoro radicals. It should be recognized that if there are more than one substitution on a substituent, each non- hydrogen radical may be identical or different (unless otherwise stated). If a substituent is described as being "alternatively substituted", the substituent may be either (1) not substituted or (2) substituted. If a substituent is described as being alternatively substituted with up to a particular number of non-hydrogen radicals, that substituent may be either (1) not substituted; or (2) substituted by up to that particular number of non-hydrogen radicals or by up to the maximum number of substitutable positions on the substituent, whichever is less. Thus, for example, if a substituent is described as a heteroaryl alternatively substituted with up to 3 non- hydrogen radicals, then any heteroaryl with less than 3 substitutable positions would be alternatively substituted by up to only as many non-hydrogen radicals as the heteroaryl has substitutable positions. To illustrate, tetrazolyl (which has only one substitutable position) would be alternatively substituted with up to one non-hydrogen radical. To illustrate further, if an amino nitrogen is described as being alternatively substituted with up to 2 non-hydrogen radicals, then a primary amino nitrogen will be alternatively substituted with up to 2 non-hydrogen radicals, whereas a secondary amino nitrogen will be alternatively substituted with up to only 1 non-hydrogen radical. The term "alkyl", as used herein, means monovalent, saturated, straight or branched chain hydrocarbon moieties, such as C₁-alkyl, C2-alkyl, C3-alkyl (propyl or isopropyl), C4-alkyl (for example butyl, tert-butyl or isobutyl), C₅-alkyl (for example pentyl, isopentyl etc.), C₆-alkyl (for example hexyl, isohexyl etc.) and the like. The term "COOH”, as used herein, means a moiety with a substantially similar physical or chemical property that imparts similar biological properties to the compound having Formula (I). Examples of COOH bioisosteres include monovalent radicals derived from removal of one hydrogen atom from a molecule such as isothiazol-3(2H)-one 1,1-dioxide, isothiazolidin-3-one 1,1-dioxide, 1,2,4-oxadiazol-5(2H)-one, 1,2,5-thiadiazolidin-3-one 1,1-dioxide, 1,2,5-thiadiazol-3-ol, 1,2,4- oxadiazolidine-3,5-dione, 2H-tetraazole and the like. The term "cyclic moiety", as used herein, means benzene, cycloalkane, cycloalkyl, cycloalkene, cycloalkenyl, heteroarene, heteroaryl, heterocycloalkane, heterocycloalkyl, heterocycloalkene, heterocycloalkenyl, phenyl and spiroalkyl. The term "cycloalkane", as used herein, means saturated cyclic or bicyclic hydrocarbon moieties, such as C3-cycloalkane, C4-cycloalkane, C₅-cycloalkane, C₆-cycloalkane, C7-cycloalkane, C₈-cycloalkane, C₉-cycloalkane, C₁₀-cycloalkane, C₁₁-cycloalkane, C₁₂-cycloalkane and the like. The term "cycloalkyl", as used herein, means monovalent, saturated cyclic and bicyclic hydrocarbon moieties, such as C₃-cycloalkyl, C₄-cycloalkyl, C₅-cycloalkyl, C₆-cycloalkyl, C₇- cycloalkyl, C8-cycloalkyl, C9-cycloalkyl, C₁₀-cycloalkyl, C₁1-cycloalkyl, C₁2-cycloalkyl and the like. The term "heteroarene", as used herein, means furan, imidazole, isothiazole, isoxazole, 1,2,3- oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, oxazole, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, thiazole, 1,3,4-thiadiazole, thiophene, triazine and 1,2,3-triazole. The term "heteroaryl", as used herein, means furanyl, imidazolyl, isothiazolyl, isoxazolyl, 1,2,3-oxadiazoyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrazolyl, thiazolyl, 1,2,3-thiadiazoyl, 1,2,5-thiadiazolyl, 1,3,4- thiadiazolyl, thiophenyl, triazinyl and 1,2,3-triazolyl. The term, "compound," as used herein with respect to any compound conforming to Formula (I), (Ia), or (Ib), is meant to include all stereoisomers, geometric iosomers, tautomers, and isotopes of the structures depicted. All compounds, and pharmaceutically acceptable salts thereof, are also meant to include solvated or hydrated forms. Those skilled in the arts will recognize that compounds of this invention may contain asymmetrically substituted carbon atoms in the R or S configuration wherein the terms "R" and "S" are as defined in Pure Appl. Chem. (1976) 45, pp.13-10. Compounds having asymmetrically substituted carbon atoms with equal amounts of R and S configurations are racemic at those atoms. Atoms having excess of one configuration over the other are assigned the configuration in excess, preferably an excess of about 85%-90%, more preferably an excess of about 95%-99%, and still more preferably an excess greater than about 99%. Accordingly, this invention is meant to embrace racemic mixtures, relative and absolute diastereoisomers and the compounds thereof. Compounds of this invention may also contain carbon-carbon double bonds or carbon- nitrogen double bonds in the Z or E configuration, in which the term "Z" represents the larger two substituents on the same side of a carbon-carbon or carbon-nitrogen double bond and the term "E" represents the larger two substituents on opposite sides of a carbon-carbon or carbon-nitrogen double bond. The compounds of this invention may also exist as a mixture of "Z" and "E" isomers. The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Compositions are provided which comprise any one of the compounds described herein (or a prodrug, pharmaceutically acceptable salt, or other pharmaceutically acceptable derivatives thereof), and one or more pharmaceutically acceptable carriers or excipients. These compositions optionally further comprise one or more additional therapeutic agents. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. As described herein, the compositions of the present invention comprise a compound of the invention together with a pharmaceutically acceptable carrier, which, as used herein, includes any solvents, diluents, or another vehicle, dispersion or suspension aids, surface-active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. The "subject" is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human. The method of the invention comprises administering to a subject in need thereof a therapeutically effective amount of a compound of the invention. A “therapeutically effective amount” is that amount effective for detectable killing or inhibition of the growth or spread of cancer cells; the size or number of tumors; or other measures of the level, stage, progression, or severity of cancer. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular anticancer agent, its mode of administration, combination treatment with other therapies, and the like. The compound, or a composition containing the compound, may be administered using any amount and any route of administration effective for killing or inhibiting the growth of cancer cells. The term "prodrug” as used herein is intended to encompass a class of analogs of compounds of the present invention wherein a metabolically labile moiety is attached to said compound of the invention through an available NH, C(O)H, COOH, C(O)NH₂, OH or SH functionality. The prodrug- forming moieties are removed by metabolic processes and release the active compounds having the free NH C(O)H COOH C(O)NH₂ OH or SH group in vivo Prodrugs are useful for adjusting such pharmacokinetic properties of the compounds as solubility and/or hydrophobicity, absorption in the gastrointestinal tract, bioavailability, tissue penetration, and rate of clearance. Design and preparation of such prodrugs is known to those skilled in the art, and are described in: Various forms of prodrugs are well known in the art and are described in: a) The Practice of Medicinal Chemistry 4^th Edition, Camille G. Wermuth et al., (Academic Press, 2015), Ch.28. ; b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds. Ch.5, pp.113-191 (Harwood Academic Publishers, 1991). Said references are incorporated herein by reference, particularly as to the description of prodrugs. Furthermore, the term “prodrug” as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term “prodrug” refers to compounds that are transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. The terms "treating", "treat" or "treatment" and the like include preventative (e.g., prophylactic) and palliative treatment. GENERAL METHODS Compositions, Methods of Treatment and Administration Metabolites of compounds having Formula (I), (Ia), or (Ib) produced by in vitro or in vivo metabolic processes, may also have utility for treating diseases caused or exacerbated by over- expressed or unregulated ASNS. Examples of such metabolites may include, but are not limited to, N-oxides, glucuronates, sulfates, phosphates, dealkylation products and the like. Compounds having Formula (I), (Ia), or (Ib) may be radiolabeled with a radioactive isotope such as carbon (i.e.¹³C), hydrogen (i.e. ²H and ³H), nitrogen (i.e.¹⁵N), phosphorus (i.e.³²P), sulfur (i.e.³⁵S), iodine (i.e.¹²⁵I), fluorine (i.e.¹⁹F) and the like. Radioactive isotopes may be incorporated into the compounds having Formula (I), (Ia), or (Ib), by reacting the same and a radioactive derivatizing agent or by incorporating a radiolabeled intermediate into their syntheses. The radiolabeled compounds of Formula (I), (Ia), or (Ib) are useful for both prognostic and diagnostic applications and for in vivo and in vitro imaging. Additionally, introduction of isotopic variants may provide beneficial pharmacokinetic or pharmacodynamics properties, particularly in regard to replacement of hydrogen with deuterium. Compounds having Formula (I), (Ia), or (Ib) may exist as acid addition salts, basic addition salts or zwitterions. Salts of compounds having Formula I are prepared during their isolation or following their purification. Acid addition salts are those derived from the reaction of a compound having Formulae (I), (Ia), or (Ib) with an acid. Examples of suitable salts include, but are not limited to, acetate, adipate, alginate, bicarbonate, citrate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, formate, fumarate, glycerophosphate, glutamate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactobionate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phosphate, picrate, propionate, succinate, tartrate, thiocyanate, trichloroacetic, trifluoroacetic, para-toluenesulfonate and undecanoate. Basic addition salts of compounds are those derived from the reaction of the compounds having Formula (I), (Ia), or (Ib), the bicarbonate, carbonate, hydroxide or phosphate of cations such as, but not limited to, lithium, sodium, potassium, calcium and magnesium. Any composition described herein can be administered to any part of the host's body for subsequent delivery to a target cell. A composition can be delivered to, without limitation, joints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, or the peritoneal cavity of a mammal. In terms of routes of delivery, a composition can be administered by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, intrarectal, intravaginal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal administration, or by gradual perfusion over time. In a further example, an aerosol preparation of a composition can be given to a host by inhalation. The dosage required will depend on the route of administration, the nature of the formulation, the nature of the patient's illness, the patient's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending clinicians. Suitable dosages are in the range of 0.001-1,000 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of cellular targets and the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art. Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50- , 100-, 150-, or more fold). Encapsulation of the compounds in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery. The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years). For example, a compound can be administered once a week (for, for example, 4 weeks to many months or years); once a month (for, for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer. It is also noted that the frequency of treatment can be variable. For example, the present compounds can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly. An effective amount of any composition provided herein can be administered to an individual in need of treatment. The term "effective" as used herein refers to any amount that induces a desired response while not inducing significant toxicity in the patient. Such an amount can be determined by assessing a patient's response after administration of a known amount of a particular composition. In addition, the level of toxicity, if any, can be determined by assessing a patient's clinical symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular composition administered to a patient can be adjusted according to a desired outcome as well as the patient's response and level of toxicity. Significant toxicity can vary for each particular patient and depends on multiple factors including, without limitation, the patient's disease state, age, and tolerance to side effects. Therapeutically effective amounts of a compound having Formula (I), (Ia), or (Ib) depend on patient, disease treated and severity thereof, composition comprising treatment, time of administration, route of administration, duration of treatment, potency, rate of clearance and whether or not another drug is co-administered. The amount of a compound having Formula (I), (Ia), or (Ib) used to make a composition to be administered daily to a patient in a single dose or in divided doses is from about 0.0001 to about 200 mg/kg body weight. Single dose compositions contain these amounts or a combination of submultiples thereof. Compounds having Formula (I), (Ia), or (Ib) may be administered with or without an excipient. Excipients include, for example, encapsulators and additives such as absorption accelerators, antioxidants, binders, buffers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. After formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, the compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by transdermal patch, powders, ointments, or drops), sublingually, buccally, as an oral or nasal spray, or the like. Excipients for preparation of compositions comprising a compound having Formula (I), (Ia), or (Ib) to be administered orally include, for example, agar, alginic acid, aluminum hydroxide, benzyl alcohol, benzyl benzoate, 1,3-butylene glycol, carbomers, castor oil, cellulose, cellulose acetate, cocoa butter, corn starch, corn oil, cottonseed oil, cross-povidone, diglycerides, ethanol, ethyl cellulose, ethyl laureate, ethyl oleate, fatty acid esters, gelatin, germ oil, glucose, glycerol, groundnut oil, hydroxypropylmethyl celluose, isopropanol, isotonic saline, lactose, magnesium hydroxide, magnesium stearate, malt, mannitol, monoglycerides, olive oil, peanut oil, potassium phosphate salts, potato starch, povidone, propylene glycol, Ringer's solution, safflower oil, sesame oil, sodium carboxymethyl cellulose, sodium phosphate salts, sodium lauryl sulfate, sodium sorbitol, soybean oil, stearic acids, stearyl fumarate, sucrose, surfactants, talc, tragacanth, tetrahydrofurfuryl alcohol, triglycerides, water and mixtures thereof. Excipients for preparation of compositions comprising a compound having Formula (I), (Ia), or (Ib) to be administered ophthalmically or orally include, for example, 1,3-butylene glycol, castor oil, corn oil, cottonseed oil, ethanol, fatty acid esters of sorbitan, glycerol, isopropanol, olive oil, polyethylene glycols, propylene glycol, sesame oil, water and mixtures thereof. Excipients for preparation of compositions comprising a compound having Formula (I), (Ia), or (Ib) to be administered osmotically include, for example, chlorofluoro-hydrocarbons, ethanol, water and mixtures thereof. Excipients for preparation of compositions comprising a compound having Formula (I), (Ia), or (Ib) to be administered parenterally include, for example, 1,3-butanediol, castor oil, corn oil, cottonseed oil, dextrose, germ oil, groundnut oil, liposomes, oleic acid, olive oil, peanut oil, Ringer's solution, safflower oil, sesame oil, soybean oil, U.S.P. or isotonic sodium chloride solution, water and mixtures thereof. Excipients for preparation of compositions comprising a compound having Formula (I), (Ia), or (Ib) to be administered rectally or vaginally include, for example, cocoa butter, polyethylene glycol, wax and mixtures thereof. Assays Asparagine synthetase binding affinity of compounds of the present invention may be determined using a variety of known methods. One such method is a sensitive and quantitative in vitro assay as described by N.G.J. Richards. et al. Chem Biol. 2006 Dec; 13(12): pp. 1339–1347. This assay was performed as follows: The activity of wild type human Asparagine synthetase (hASNS) in the presence of a tested compound was assayed by measuring the rate of inorganic pyrophosphate (PPi) production by a continuous assay employing the Sigma Pyrophosphate reagent (Catalog Number: P7275). The reagent couples the production of inorganic pyrophosphate to NADH consumption. The assay mixture contained 5 mM ATP, 10 mM L-aspartate, 100 mM NH₄Cl and 10 mM MgCl₂ dissolved in 100 mM EPPS buffer, pH 8, and either 10 nM, 30 nM, 50 nM or 80 nM of the tested compound (1 mL total volume). Reactions were initiated at 25°C by the addition of 8 μL of a solution containing hASNS to a final concentration of 10 nM hASNS. NADH consumption was monitored spectrophotometrically at 340 nm over a period of 40 min. All kinetic assays were performed in duplicates and repeated two times. The curve figures represent the mean values of duplicate experiments. It was assumed that the binding of the nine inhibitors would be competitive with respect to ATP, according to the following kinetic model:

Scheme 1a. The ks were estimated by fitting the curve to the following equation: [PPi] = v_sst + (v₀ – v_ss)*(1 – e^-kt)/k where [PPi] is the concentration of inorganic pyrophosphate formed at time t, vo and vss are the initial and steady-state rates, respectively, and k is the apparent first-order rate constant for isomerization of EI to EI* k₅, k₆ and K_i were estimated by fitting the plots of k vs [I] using the following equation:

Ki* was calculated as K_i* = K_ik₆/(k₆+k₅) The following assays are used to demonstrate cytotoxicity of the compounds of the present invention against cancer and primary cells. Cell viability was assessed by reduction of resazurin or MTT dye by mitochondria in metabolically active (live) cells. Mycoplasma-free A172, A375, A549, HCT116, HT29, Fibroblast, MCF7, MDAMB231, MEF, MWF, RKO, SW620, MV-4-11, OPM2, Jurkat cell lines (purchased from the American Tissue and Cell Culture (ATCC®)), were cultured in T75 flasks containing in either asparagine-containing RPMI- 1640 medium (0.05 g/l of L-asparagine) (Paneko, Russia), asparagine-free DMEM medium (Paneko, Russia) or asparagine-free RPMI-1640 medium (Paneko, Russia). Cell cultures were incubated at 37°C and 5% CO₂. Cells were checked daily, and media was changed every 2-3 days according ATCC® specifications for each particular cell line. For the MTT assay cells were seeded in 96 well clear plates (Corning 3598) at 2.5x10⁵ cell/mL for A172, HCT116 and SW620. Cells were obtained from ATCC. Adherent cells were left for one day for attachment, while for suspension cells compounds were added on the same day. The compounds were added in triplicates, in concentrations -50, 10, 2, 0.4, 0.1, 0.02, 0.003, 0.0006 uM. The cells were incubated with compounds for 72 hours at 37^OC and 5% CO₂, after which MTT reagent (Sigma, M5655) was added to the final concentration of 0.5 mg/ml. The plates were then incubated at 37^OC and 5% CO2 for 4 hours, centrifuged for 12 minutes at 550 rcf and 4^oC and the supernatant was dispensed to a biohazard bag. 100 ul DMSO was added to each well, the solutions were mixed intensively for 1 hour at RT, and the absorbance was measured at 570 nM. Wells without addition of MTT reagent were used as blank, and their average value substracted from all values. The control values were taken as 100%, and other values calculated relative to control. IC50 (half maximal inhibitory concentration) was determined by fitting the concentration curve using a four-parameter variable slope in GraphPad Prism 8.0. For the resazurin assay cells were seeded in 96 well black plates with clear bottom (Corning 3904) in the following concentrations: 5x10⁵ cell/ml for OPM, MV-4-11, Jurkat; 2.5x10⁵ cell/mL for A172, A375, A549, HT29, MCF7, SW620, RKO; 1.25x10⁵ cells/ml for HCT116, MDA-MB-231, Human Postnatal Fibroblasts immortalized with hTERT (Fibro), MEF. All cells were obtained from ATCC, except Human Postnatal Fibroblasts that were obtained from a donor. Adherent cells were left for one day for attachment, while for suspension cells compounds were added on the same day. The compounds were added in triplicates, in concentrations -50, 10, 2, 0.4, 0.1, 0.02, 0.003, 0.0006 uM. The cells were incubated with compounds for 72 hours at 37^OC and 5% CO2, after which resazurin reagent (Sigma, R7017) was added to the final concentration of 40 uM. The plates were then incubated at 37^OC and 5% CO₂ for 4-6 hours, and fluorescence measured with excitation 560 nm, and emission 590 nm. Wells without addition of cells were used as blank, and their average value subtracted from all values. The control values were taken as 100%, and other values calculated relative to control. IC50 (half maximal inhibitory concentration) was determined by fitting the concentration curve using a four-parameter variable slope in GraphPad Prism 8.0. To test the ability of compounds to inhibit cell growth in asparagine-containing medium, cells were treated with a tested compounds with addition of non-toxic concentration Asparaginase (Lens- Farm, Russia). To determine a non-toxic concentration of Asparaginase with the highest sensitization effect to the tested compound, cells were titrated with Asparaginase in the presence of increasing concentrations of the tested compound.0.025 IU/ml of Asparaginase was used for determination of IC50. Two murine cancers models (B16F10 melanoma and 4T1 mammary tumor) were used to demonstrate the ability of compounds of the present invention to inhibit tumor growth in vivo. For the melanoma B16-F10 model 6-12 week old C57BL6 mice were inoculated subcutaneously with 0.5x10⁶ B16-F10 cells (ATCC), growing in the logarithmic growth phase and randomized into 3 groups: 1) control, 2) Asparaginase (50 IU/kg), and 3) a combination of Asparaginase (50 IU/Kg) with a tested compound (15 mg/kg). The compounds were co-administered intravenously, once a day for the duration of the study. Tumor volume was measured biweekly by calipers. For the mammary tumor 4T1 model two treatment regimens were used: I) co-administration of a tested compound with Asparaginase and II) administration of a tested compound in combination with asparagine-free diet. In both models 6-12 week old BALB/C mice were inoculated with 1x10⁶ 4T1 cells (ATCC) in 100 ul of RPMI medium into mammary fat pads. In the I) model mice were randomized to the following groups: 1) control, 2) Asparaginase (1mg/kg), 3) a tested compound (75 mg/kg), and 4) a combination of Asparaginase (1mg/kg) and a tested compound (50 mg/kg). The animals were treated 48 hours post-inoculation, for 5 days with compounds daily, by intravenous injection, with a subsequent break for two days and additional 5 days of treatment. In the II) model mice were fed with asparagine free-diet (asparagine-free chow was supplied by Research Diets, Inc. USA ), and animals were randomized to 1) a group fed with only asparagine- free chow (control), and two groups fed with asparagine-free chow and treated with a tested compound in a dose of 2) 25 mg/kg and 3) 50 mg/kg. Tumor volume was measured bi-weekly by calipers. Results When tested in an hASNS biochemical assay based on the assay conditions described above, the inhibitory activity K_i* of many compounds of the present invention were below 100 nM . In some embodiments Ki* were below 10 nM. In some embodiments Ki* were below 1 nM. Table 1 shows inhibitory constants (Ki*, in nΜ) for representative compounds where the data was obtained as described above. Table1. hASNS Biochemical Assay.

When tested in an MTT or resazurin assay based on the assay conditions described above, the inhibitory activity (IC50) of the compounds of the present invention were below 100 µM. In many cases, IC50’s were below 10 µM. In some embodiments, IC50’s were below 1 µM. In some embodiments, IC₅₀’s were below 0.1 µM. Table 2 shows the IC₅₀, in μΜ for representative compounds where the data was obtained as described above. The values given indicate the ability of the compounds to enter the cell and/or induce cell death in asparagine-free media (DMEM). Table 2. MTT/resazurin Assay in DMEM (asparagine-free medium)

When tested in an MTT or resazurin assay based on the assay conditions described above, the inhibitory activity (IC₅₀) of the compounds of the present invention were below 100 µM. In many cases, IC50’s were below 10 µM. In some embodiments, IC50’s were below 1 µM. In some embodiments, IC₅₀’s were below 0.1 µM. Table 3 shows the IC₅₀, in μΜ for representative compounds where the data was obtained as described above. The values given indicate the ability of the compounds induce cell death when co-administrated with 0.025 U/ml asparaginase in the asparagine-containing media (RPMI). Table 3. MTT/resazurin Assay (PRMI media + 0.025 U/ml asparaginase)

When tested in vivo based on the assay conditions described above, the following result for the selected compounds of the present invention were obtained (Figure 3-5). General Methods for Preparation of Compounds The compounds of the present invention can be prepared in a variety of ways known to one of ordinary skill in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods as hereinafter described below, together with synthetic methods known in the art of synthetic organic chemistry or variations thereon as appreciated by one of ordinary skill in the art. The present compounds can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one of ordinary skill in the art by routine optimization procedures. One skilled in the art of organic synthesis understands that vulnerable moieties such as C(O)OH, C(O) and C(O)H, NH, C(O)NH₂, OH and SH moieties may be protected and deprotected, as necessary. Protecting groups for C(O)OH moieties include, but are not limited to, allyl, benzoylmethyl, benzyl, benzyloxymethyl, tert-butyl, ethyl, methyl, 2,2,2-trichloroethyl, and the like. Protecting groups for C(O) and C(O)H moieties include, but are not limited to, 1,3-dioxylketal, diethylketal, dimethylketal, 1,3-dithianylketal, O-methyloxime, O-phenyloxime and the like. Protecting groups for NH moieties include, but are not limited to, acetyl, benzoyl, benzyl (pheriylmethyl), benzylidene, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), 3,4- dimethoxybenzyloxycarbonyl, diphenylmethyl, diphenylphosphoryl, formyl, methanesulfonyl, para- methoxybenzyloxycarbonyl, phenylacetyl, phthaloyl, succinyl, trichloroethoxycarbonyl, triethylsilyl, trifluoroacetyl, trimethylsilyl, triphenylmethyl, triphenylsilyl, para-toluenesulfonyl and the like. Protecting groups for OH and SH moieties include, but are not limited to, acetyl, allyl, allyloxycarbonyl, benzyloxycarbonyl (Cbz), benzoyl, benzyl, tert-butyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, 3,4-dimethoxybenzyl, 3,4-dimethoxybenzyloxycarbonyl, 1,1-dimethyl-2-propenyl, diphenylmethyl, methanesulfonyl, methoxyacetyl, 4-methoxybenzyloxycarbonyl, para- methoxybenzyl, methoxycarbonyl, methyl, para-toluenesulfonyl, 2,2,2-trichloroethoxycarbonyl, 2,2,2- trichloroethyl, triethylsilyl, trifluoroacetyl, 2-(trimethylsilyl)ethoxycarbonyl, 2-trimethylsilylethyl, triphenylmethyl, 2-(triphenylphosphonio)ethoxycarbonyl and the like. A discussion of protecting groups is provided in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 5th Ed., John Wiley & Sons, New York (2014) pp. 26-468, 554-684, 686-836, 837-894, 895-1193. ISBN: 978-1-118-90507-4 The compounds of the present invention may contain asymmetric or chiral centers, and therefore, exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds as well as mixtures thereof, including racemic mixtures, form part of the present invention. In addition, the present invention contemplates all geometric and positional isomers. For example, if the compound contains a double bond, both the cis and trans forms (designated as Z and E, respectively), as well as mixtures, are contemplated. Mixture of stereoisomers, such as diastereomeric mixtures, can be separated into their individual stereochemical components on the basis of their physical chemical differences by known methods such as chromatography and/or fractional crystallization. Enantiomers can also be separated by converting the enantiomeric mixture into a diasteromeric mixture by reaction with an appropriate optically active compound (e.g., amino acid), separating the diastereomers and converting (e.g., de-protecting) the individual diastereomers into the corresponding pure enantiomers. The compounds of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water (hydrate), ethanol, and the like. The present invention contemplates and encompasses both the solvated and unsolvated forms. It is also possible that compounds of the present invention may exist in different tautomeric forms. All tautomers of compounds of the present invention are contemplated. Those skilled in the art will recognize that the compound names and structures contained herein may be based on a particular tautomer of compound. While the name or structure for only a particular tautomer may be used, it is intended that all tautomers are encompassed by the present invention, unless stated otherwise. EXAMPLES The invention will now be further described with reference to the following illustrative examples in which, unless stated otherwise: (i) temperatures are given in degrees Celsius (°C.); operations are carried out at room temperature (rt) or ambient temperature, that is, in a range of 18- 25°C.; (ii) organic solutions were dried over anhydrous sodium or magnesium sulfate unless otherwise stated; evaporation of organic solvent was carried out using a rotary evaporator under reduced pressure (2-30 mm Hg) with a bath temperature of up to 60°C; or by centrifugal evaporation in a Genevac DD4 or HT4 apparatus; (iii) column chromatography means flash chromatography on silica gel, performed on prepacked columns utilizing ISCO Combiflash^® or Analogix IntelliFlash^® automated chromatography equipment; thin layer chromatography (TLC) was carried out on silica gel plates; (iv) in general, the course of reactions was followed by TLC or liquid chromatography/mass spectroscopy (LC/MS) and reaction times are given for illustration only; (v) final products have satisfactory proton nuclear magnetic resonance (NMR) spectra and/or mass spectra data; (vi) yields are given for illustration only and are not necessarily those which can be obtained by diligent process development; preparations were repeated if more material was required; (vii) when given, NMR data is in the form of delta values for major diagnostic protons, given in part per million (ppm) relative to tetramethylsilane (TMS) as an internal standard, and were obtained on a Varian 400 MHz instrument in deuterated dimethyl sulfoxide (DMSO-d₆) as solvent unless otherwise indicated; (viii) chemical symbols have their usual meanings; (ix) in the event that the nomenclature assigned to a given compound does not correspond to the compound structure depicted herein, the structure will control; (x) solvent ratio was given in volume:volume (v/v) terms; (xi) HPLC purification refers to purification on a Agilent 1260 HPLC instrument on a C₁₈ reverse phase HPLC Column with dimension 20 mm/100 or 50 mm/250 with a gradient elution, using as a mobile phase various ratios of H₂O/MeCN with 0.05% formic acid unless otherwise stated and used according to the manufacturer's instructions, obtained from Agilent Technologies, Inc.5301 Stevens Creek Blvd Santa Clara, CA 95051. United States;.; (xii) mass spectra were acquired when samples were separated using reverse-phase liquid chromatography (LC) and detected by atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) in positive and negative ionization mode; values for m/z are given; generally, only ions which indicate the parent mass are reported; and unless otherwise stated, the mass ion quoted is (M+H)⁺ or (M-H)-; The following abbreviations have the meanings indicated: AcOH means acetic acid; DCM mean dichloromethane; DIAD means diisopropylazodicarboxylate; DIEA or DIPEA means diisopropylethylamine; DMAP means N,N-dimethylaminopyridine; DMA means N,N-dimethylacetamide; DMF means N,N-dimethylformamide; DMSO means dimethylsulfoxide; dppf means 1,1'-bis(diphenylphosphino)ferrocene; DTAD means di-tert-butyl azodicarboxylate; EDCI means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; EtOAc means ethyl acetate; EtOH mean ethanol; equiv. means equivalent HATU means (7-azabenzotriazol-1-yl)-N,N'N'N'-tetramethyluronium hexafluorophosphate; HCl means hydrochloric acid; HOBT means 1-hydroxybenzotriazole; IPA means isopropyl alcohol; mCPBA means meta-chloroperbenzoic acid; MeOH means methanol; NCS means N-chlorosuccinimide; NIS means N iodosuccinimide; NMP means N-methyl-2-pyrrolidone; PTSA means para-toluenesulfonic acid; PyBOP means benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; rt means room temperature TBTU means 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate TEA means triethylamine; TEMPO means 2,2,6,6-tetramethylpiperidin-1-yl)oxyl TFA means trifluoroacetic acid; TFE means trifluoroethanol THF means tetrahydrofuran; NCS means N-chlorosuccinimide; PPh₃ means triphenylphosphine. X-Phos means 2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl. Compounds of Formula I wherein R⁴ is selected from substituted or unsubstituted phenyl were prepared according to Scheme 1.

1.11 Scheme 1. 3‐Hydroxy‐4‐arylpyrrolidine‐2‐carboxamides of Formula 1.11 were prepared from the corresponding aldehydes of Formula 1.1 according to Scheme 1. A benzaldehyde of Formula 1.1 was reacted with nitromethane under basic conditions, such as, for example, cyclopentylamine, in acetic acid to afford condensation product 1.2. Mannich-type addition of nitrostyrene 1.2 with dioxanone enamine such as, for example 2,2-dimethyl-5-morpholino-4H-1,3-dioxin in an inert solvent to a suitable temperature, conveniently acetonitrile at 45°C, resulted in formation of product 1.3. Treatment of the resulting 1.3 with an acid, such as oxalic acid followed by hydrogenation in the presence of Raney nickel provided the annulated pyrrolidine of Formula 1.4. The reactivity of the resulting secondary amino group in pyrrolidine necessitates protection of the pyrrolidine nitrogen. Suitable protecting groups will be known to those skilled in the art; tert-butyloxycarbonyl is particularly convenient due to relative ease of subsequent cleavage. Hydrolysis of N-protected pyrrolidines of Formula 1.6 of under mild acidic conditions, such as, aqueous acetic acid resulted in acetal cleavage and provided the tri-substituted pyrrolidines of Formula 1.7. Mono-oxidation of the primary hydroxy group to the carboxylic acid may be achieved with suitable oxidizing agents, such as, for example, sodium chlorite and sodium hypochlorite in the presence of TEMPO to provide an acid of Formula 1.8. Pyrrolidine carboxamides of Formula 1.10 were most conveniently prepared from acids of Formula 1.8 and the appropriate primary or secondary amines of Formula 1.9 by condensation. Suitable conditions include a coupling agent such as TBTU and a base, such as, DIEA in an inert solvent, preferably acetonitrile. Those skilled in the arts will recognize that many methods exist to form the requisite C(O)-N bond involving various selections of the coupling agents and bases according to known methods, and the present example should not be limiting. Removal of the protecting group from the pyrrolidine nitrogen in the amides 1.10 under appropriate conditions would provide products of Formula 1.11. Compounds of Formula I, wherein R⁴ is selected from substituted or unsubstituted phenyl were prepared according to Scheme 2 in an analogous fashion to Scheme 1.

R

Scheme 2. Compounds of the present invention according to Formulae I, Ia, Ib l were most conveniently prepared from the appropriate secondary amine of Formula 4.1 by amide coupling reaction according to Scheme 3. Suitable conditions include a coupling agent such as TBTU and a base, such as, DIPEA in an inert solvent, preferably DMF. Those skilled in the arts will recognize that many methods exist to form the requisite C(O)-N bond involving various selections of the coupling agents and bases according to known methods, and the present example should not be limiting. R

Scheme 3. Synthesis of Intermediates Preparation of Intermediate 1.2,2-Dimethyl-5-morpholino-4H-1,3-dioxin The synthesis of Intermediate 1 was previously described in 1) EP1783123, 2007, A1 and 2) Miltz, Wolfgang; Steglich, Wolfgang Synthesis, 1990, # 9, p.750 – 751. The mixture of 2,2-dimethyl-[1,3]dioxan-5-one (40 g; 0.308 mmol) and morpholine (32 mL; 0.307 mol) was dissolved in benzene (350 ml) and refluxed with Dean-Stark trap for 5 h. Then volatiles were removed under reduced pressure and the residue was subjected to flash chromatography (neutral Al2O3; Hex/EA 4:1) to give the title product as a yellow oil (58.2 g, 95%). Preparation of Intermediate 2.1-Fluoro-4-(2-nitrovinyl)benzene. The mixture of 4-fluorobenzaldehyde (30 g; 0.246 mol), cyclopentylamine (3 mL), acetic acid (150 mL) and nitromethane (150 ml) and refluxed for 8 h. The volatiles were removed under reduced pressure and the reaction mixture was treated with water (300 ml) and neutralized with solid NaHCO3. The solid material was collected by filtration, washed with water, hexane and air dried to provide the title product (32.4 g, 79%). Preparation of Intermediate 3. rac-4‐[(4R*)‐4‐[(1R*)‐1‐(4‐fluorophenyl)‐2‐nitroethyl]‐2,2‐ dimethyl‐2,4‐dihydro‐1,3‐dioxin‐5‐yl]morpholine. A stirred solution of Intermediate 1 (77.28 g; 0.388 mol) and Intermediate 2 (77.79 g; 0.465 mol) in acetonitrile (720 mL) was heated at 40-45 ºC for 12 hours. The solvent was removed in vacuo, and the residue was treated with MeOH (150 mL). The formed white crystals were collected by filtration, washed with cold MeOH and air-dried to afford the title product (102.8 g, 72%). Preparation of Intermediate 4. rac-(4R*)‐4‐[(1R*)‐1‐(4‐fluorophenyl)‐2‐nitroethyl]‐2,2‐ dimethyl‐1,3‐dioxan‐5‐one. Intermediate 3 (101.8 g; 0.278 mol) was dissolved in acetonitrile (1.1 L) at slight heating. Then a solution of oxalyc acid dihydrate (35.02 g; 0.278 mol; 1.0 eq.) in water (450 mL) was added and the reaction mixture was stirred at 40-42ºC for 6 hours. A second portion of oxalyc acid dihydrate (3.16 g; 25 mmol; 0.1 eq.) was added and the reaction mixture was stirred at 40-42ºC for 4 hours, then allowed to stand overnight at rt. Upon reaction completion, the mixture was treated with NaHCO₃ (4.6 g), NaCl (30 g) and ethyl acetate (100 mL) and the mixture was stirred at rt for 15 min. The organic phase was separated and concentrated in vacuo without drying. The residue was extracted with 200 ml of dichloromethane, organic phase was dried over Na₂SO₄ and concentrated in vacuo. Purification by flash-chromatography (Hexane/Ethyl acetate 4:1) afforded pure product as a light- yellow oil (77.2 g, 93%). Preparation of Intermediate 5. rac-(4aS*,7R*,7aS*)‐7‐(4‐fluorophenyl)‐2,2‐ dimethylhexahydro‐2H‐[1,3]dioxino[5,4‐b]pyrrole. A solution of intermediate 4 (77 g; 0.259 mol) in methanol (1.2 L) was hydrogenated in 2 L Parr steel autoclave over Raney nickel (150 g) under 10 bar at rt. Upon reaction completion, the reaction mixture was filtered through Celite^®. The filtrate was concentrated in vacuo, and the residue was dissolved in dichloromethane (150 mL) and dried over Na₂SO₄. The solvent was removed in vacuo to afford the title product as a brown oil (56.2 g, 86%). Intermediate 6. rac-tert‐butyl (4aS*,7R*,7aS*)‐7‐(4‐fluorophenyl)‐2,2‐dimethyl‐hexahydro‐ 2H‐[1,3]dioxino[5,4‐b]pyrrole‐5‐carboxylate. To a solution of intermediate 5 in dichloromethane (400 mL) was added a solution of K₂CO₃ (42.04 g; 0.304 mol) in water (250 mL). To the resulting mixture, a solution of di-tert-butyl dicarbonate (48.75 g; 0.224 mol) in dichloromethane (150 mL) was added dropwise at stirring over 20 min at rt. The mixture allowed to stand overnight, the organic layer was separated and dried over Na₂SO₄. The solvent was removed in vacuo, and the residue was purified by flash-chromatography on silica gel, eluting with hexane-ethyl acetate 4:1 to afford the title product as a light-brown oil (55.0 g, 70%). Preparation of Intermediate 7. rac-tert‐butyl (2R*,3R*,4S*)‐4‐(4‐fluorophenyl)‐3‐hydroxy‐2‐ (hydroxymethyl)pyrrolidine‐1‐carboxylate. To a stirred solution of Intermediate 6 (55 g; 0.157 mol) in acetic acid (207 mL) was added 89 mL of water. The resulting mixture was stirred at 45ºC for 5 h. Then the mixture was diluted with water (800 mL) and extracted with ethyl acetate (3×250 mL). The combined organic extracts were washed with 10% Na2CO3 and dried over Na2SO4. The solvent was removed in vacuo and the residue was treated with hexane (100 mL). The solid material was collected by filtration and dried at 60ºC for 2 h to provide the title product as a white solid (42.18 g, 87%). Preparation of Intermediate 8. rac-(2S*,3R*,4S*)‐1‐[(tert‐butoxy)carbonyl]‐4‐(4‐ fluorophenyl)‐3‐hydroxypyrrolidine‐2‐carboxylic acid. To a solution of intermediate 7 (42.1 g; 0.135 mol) and TEMPO (1.56 g; 10 mmol) in acetonitrile (680 mL) was added phosphate buffer (pH 6.7; CM 0.67; 590 mL). The mixture was heated to 40-45 ºC and then solutions of sodium chlorite (80% purity; 36.7 g; ~0.32 mol) in water (160 mL) and bleach (6.3 mL) in water (63 mL) were simultaneously added from two dropping funnels over 15 min. The resulting reaction mixture was stirred at 40-45ºC overnight. Upon cooling to room temperature, Na2SO3 (33.6 g) and NaCl (122 g) were added, and pH of the aqueous layer was adjusted to 4 by adding 4M HCl. The organic layer was separated, and the aqueous layer was extracted with THF (3 x 100 mL). The combined organic extracts were concentrated in vacuo and the residue was dissolved in the mixture of dichloromethane/THF (4:1) (300 mL). The residual water layer was discarded, and the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The solid residue was treated with MTBE (80 mL) and collected by filtration, washed with MTBE and dried at 50 ºC to provide the title product as a white solid (26.7 g, 61%). 1H NMR (400 MHz, DMSO, 50°C) δ 7.33 (m, 2H), 7.10 (m, 2H), 4.43 (m, 1H), 4.21 (m, 1H), 3.79 (m,1H), 3.33 (m, 1H), 3.21 (m, 1H), 1.38 (s, 4H), 1.36 (s ,5H). Preparation of Intermediate 8.1. rac-(2S*,3R*,4S*)-1-[(tert-butoxy)carbonyl]-3-hydroxy-4- phenylpyrrolidine-2-carboxylic acid Intermediate 8.1 was prepared as in preparation of Intermediate 2 by replacing 4- fluorobenzaldehyde for benzaldehyde followed by the procedures for preparation of Intermediate 3- 8. Preparation of Intermediate 8.2. rac-(2S*,3R*,4S*)-1-[(tert-butoxy)carbonyl]-4-(4- chlorophenyl)-3-hydroxypyrrolidine-2-carboxylic acid Intermediate 8.2 was prepared as in preparation of Intermediate 2 by replacing 4- fluorobenzaldehyde for 4-chlorobenzaldehyde followed by the procedures for preparation of Intermediate 3-8. Preparation of Intermediate 8.3. rac-(2S*,3R*,4S*)-1-[(tert-butoxy)carbonyl]-4-(3- fluorophenyl)-3-hydroxypyrrolidine-2-carboxylic acid Intermediate 8.3 was prepared as in preparation of Intermediate 2 by replacing 4- fluorobenzaldehyde for 3-fluorobenzaldehyde followed by the procedures for preparation of Intermediate 3-8. Preparation of Intermediate 8.4. rac-(2S*,3R*,4S*)-1-[(tert-butoxy)carbonyl]-3-hydroxy-4- (2-methoxyphenyl)pyrrolidine-2-carboxylic acid Intermediate 8.4 was prepared as in preparation of Intermediate 2 by replacing 4- fluorobenzaldehyde for 2-methoxybenzaldehyde followed by the procedures for preparation of Intermediate 3-8. Preparation of Intermediate 8.5. rac-(2S*,3R*,4S*)-1-[(tert-butoxy)carbonyl]-3-hydroxy-4- (2-methylphenyl)pyrrolidine-2-carboxylic acid. Intermediate 8.5 was prepared as in preparation of Intermediate 2 by replacing 4- fluorobenzaldehyde for 2-methylbenzaldehyde followed by the procedures for preparation of Intermediate 38 Preparation of intermediate 8.6. rac-(2S*,3R*,4S*)-4-[2-(benzyloxy)phenyl]-1-[(tert- butoxy)carbonyl]-3-hydroxypyrrolidine-2-carboxylic acid. Intermediate 8.6 was prepared as in preparation of Intermediate 2 by replacing 4- fluorobenzaldehyde for 2-(phenylmethoxy)benzaldehyde followed by the procedures for preparation of Intermediate 3-8. Preparation of Intermediate 9. rac-tert‐butyl (2S*,3R*,4S*)‐2‐[(diphenylmethyl)carbamoyl]‐ 4‐(4‐fluorophenyl)‐3‐hydroxypyrrolidine‐1‐carboxylate. A mixture of Intermediate 8 (1.00 g; 3.1 mmol), TBTU (1.14 g; 3.6 mmol) and DIPEA (1.14 mL; 6.4 mmol) in acetonitrile (20 mL) was stirred at rt for 20 minutes. Then benzhydrylamine (0.62 g; 3.4 mmol) was added, and the reaction mixture was stirred at rt overnight. The solvent was removed in vacuo, the residue was partitioned between dichloromethane and 10% K2CO3, and the obtained emulsion was filtered. The organic layer of the filtrate was separated, washed with 10% citric acid, water and dried over Na₂SO₄. The solvent was removed in vacuo, and the residue was treated with hexane /MTBE (1:1). The formed solid was collected by filtration and air-dried to provide the title product as an off-white solid (1.32 g, 87%). APCI-MS m/z calcd for C29H₃1FN2O4490, found 391 [M-boc+H]⁺; ¹H NMR (DMSO-d6, 400 MHz, 50°C) TBA 8.53 (m, 1H), (7.45–7.15 m, 12H), 7.10 (m, 2H), 6.14 (m, 1H), 5.32 (m, 1H), 4.41 (m, 1H), 3.79 (m, 1H), 3.20 (m, 2H), 1.38 (s, 3H), 1.18 (s, 6H). Preparation of Intermediate 10. rac-(2S*,3R*,4S*)‐N‐(diphenylmethyl)‐4‐(4‐fluorophenyl)‐ 3‐hydroxypyrrolidine‐2‐carboxamide hydrochloride. Intermediate 9 (1.32 g, 2.7 mmol) was treated with 18% HCl in dioxane at rt for 30 minutes. All volatiles were removed in vacuo, and the residue was triturated with diethyl ether. The formed precipitate was collected by filtration, washed with ether and air-dried to provide the title product as an amorphous white solid (1.13 g, 98%). APCI-MS m/z calcd for C₂₄H₂₃FN₂O₂390, found 391 [M+H]⁺; ¹H NMR (DMSO-d6, 400 MHz, 50°C) 10.47 (br. s, 1H), 9.23 (d, J = 8.2 Hz, 1H), 8.91 (br. s, 1H), 7.44 (m, 2H), 7.36–7.19 (m, 10H), 7.16 (m, 2H), 6.14 (d, J = 8.1 Hz, 1H), 4.58 (m, 1H), 4.40 (m, 1H), 3.78 (m, 1H), 3.49 (m, 1H), 3.33 (m, 1H). The relative configuration of Intermediate 10 was determined using 2D NOESY experiment (Figure 6). Preparation of Intermediates 10.1- 10.23. Intermediates 10.1 – 10.8 were prepared as in preparation of Intermediate 10 by replacing diphenylmethanamine with Reagent 2 as per the Table 4. Intermediates 10.9 – 10.23 were prepared as in preparation of Intermediate 10 by replacing Intermediate 8 for Intermediate 8.1-8.6 and replacing diphenylmethanamine for Reagent 2 as per the Table 4. Table 4.

Preparation of Intermediate 10.24. rac-(2R*,3S*,4S*)-N-(diphenylmethyl)-3-fluoro-4-(4- fluorophenyl)pyrrolidine-2-carboxamide hydrochloride

To a -78 °C cooled solution of Intermediate 9 (7.4 g, 15 mmol) in dichloromethane (50 mL) was added (diethylamino)sulfur trifluoride (3.9 mL, 30 mmol). The reaction mixture was stirred for 3 h, then remove cold bath and stir 1 h at room temperature. The mixture was cooled in ice bath and sodium bicarbonate solution was added and the mixture was allowed to warm to room temperature. The organic layer was separated, dried over sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with dichloromethane. The purified product was treated with treated with 18% HCl/dioxane at rt for 30 minutes. All volatiles were removed in vacuo, and the residue was triturated with diethyl ether. The formed precipitate was collected by filtration, washed with ether and air-dried to provide the title product as white solid (5.2 g, 82%). LCMS (APCI) m/z: calcd for C₂₄H₂₂F₂N₂O 392, found 393 [M+H]⁺. Preparation of Intermediate 10.25. rac-(2R*,3S*,4S*)-N-(diphenylmethyl)-3-fluoro-4-(2- methylphenyl)pyrrolidine-2-carboxamidehydrochloride

Intermediate 10.25 was prepared as in preparation of Intermediate 10.24 by replacing of Intermediate 9 with tert-butyl (2S,3R,4S)-2-[(diphenylmethyl)carbamoyl]-3-hydroxy-4-(2- methylphenyl)pyrrolidine-1-carboxylate. LCMS (APCI) m/z: calcd for C25H₂5FN2O 388, found 389 [M+H]⁺. Preparation of Intermediate 10.26. rac-(2S*,3R*,4S*)-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-methoxypyrrolidine-2-carboxamide.

To a stirred and cooled to 5°C solution of intermediate 9 (7.4 g, 15 mmol) in dry DMF (50 ml) was added sodium hydride suspension in mineral oil (1.1g, 22.5 mmol) in portions, and the mixture was stirred for 15 min. Then a solution of iodomethane (1.5 mL, 22.5 mmol) solution in dry DMF (5 ml) was added dropwise. Upon reaction completion (TLC monitored), the mixture was carefully poured into ice-cold water (100 ml). Ethyl acetate (100 mL) was added thereto, and the layers were separated. The aqueous layer was extracted with EtOAc (100 ml). The combined organic layers were washed with brine (2x100 ml), dried over sodium sulphate and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography on silica (Hexane/EA = 9/1). The purified product was treated with treated with 18% HCl/dioxane at rt for 30 minutes. All volatiles were removed in vacuo, and the residue was triturated with diethyl ether. The formed precipitate was collected by filtration, washed with ether and air-dried to provide the title product as white solid (3.6 g, 61%). LCMS (APCI) m/z: calcd for C₂₅H₂₅FN₂O₂ 404, found 405 [M+H]⁺. Syntheses of Example Compounds Example 1. (2S)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared according to Scheme 4.

Scheme 4 To a stirred solution of (2S)-N-(diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride (317 mg, 100 µmol), (2S)-2-{[(tert-butoxy)carbonyl]amino}propanoic acid (190 mg, 1 eq.) and DIPEA (500 µL, 3.5 eq.) in dry DMF (10 mL), TBTU (435 mg, 1.2 eq.) was added in one portion. The resulting mixture was stirred at rt for 45 min. The reaction mixture was subjected to HPLC purification (Instrument: Agilent 1260; Column: Phenomenex Luna С18250х21.2; Mobile phase: A 0.1% formic acid in water – В 0.1% formic acid in acetonitrile; Gradient А-В 0-100%; UV detection at 215254275 nm), to provide tert-butyl N-[(2S)-1-[(2S)-2-[(diphenylmethyl)carbamoyl]pyrrolidin-1-yl]-1-oxopropan- 2-yl]carbamate, which was treated with 10% HCL in trifluoroethanol (10 mL) and evaporated to dryness to afford (2S)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride as a white solid (325 mg, 87%). APCI-MS m/z calcd for C₂₁H₂₅N₃O₂351, found 352 [M+H]⁺; 1H NMR (400 MHz, DMSO-d6) δ 8.90 (2x d, J = 8.2 Hz, 1H), 8.06 (br s, 3H), 7.55 – 7.03 (m, 10H), 6.06 (2x d, J = 8.7 Hz, 1H), 4.57 (2x dd, J = 8.2, 4.0 Hz, 1H), 4.31 – 3.92 (m, 1H), 3.65 (m, 1H), 3.57 – 3.32 (m, 2H), 2.20 – 2.04 (m, 1H), 2.04 – 1.67 (m, 3H), 1.34 (2x d, J = 6.9 Hz, 3H). Example 17. (2R,3S,4R)-1-[(2S)-2-aminobutanoyl]-N-benzhydryl-4-(4-fluorophenyl)-3- hydroxy-pyrrolidine-2-carboxamide. The title compound was prepared according to Scheme 5,6.

Scheme 5. To a stirred solution of Intermediate 10 (480 mg, 1 eq.), (2S)-2-(9H-fluoren-9- ylmethoxycarbonylamino)butanoic acid (366 mg, 1 eq.) and DIPEA (465 µL, 2.5 eq.) in dry DMF (9 mL), TBTU (435 mg, 1.2 eq.) was added in one portion. The resulting mixture was stirred at rt for 15 min. The reaction mixture was subjected to HPLC purification (detector UV 210 nm, flow rate 20 mL/min, Luna C18(2) 250×21.2 mm column, eluting with A: acetonitrile modified with 0.1% formic acid; and B: 0.1% formic acid in water (gradient of acetonitrile 60% to 70% at 0 - 30 min), to provide (9H‐fluoren‐9‐yl)methyl N‐[(2S)‐1‐[(2S,3R,4S)‐2‐[(diphenylmethyl)carbamoyl]‐4‐(4‐fluorophenyl)‐3‐ hydroxypyrrolidin‐1‐yl]‐1‐oxobutan‐2‐yl]carbamate isomer A (retention time: 24.9 min) and 9H- fluoren-9-ylmethyl (9H‐fluoren‐9‐yl)methyl N‐[(2S)‐1‐[(2R,3S,4R)‐2‐[(diphenylmethyl)carbamoyl]‐4‐ (4‐fluorophenyl)‐3‐hydroxypyrrolidin‐1‐yl]‐1‐oxobutan‐2‐yl]carbamate isomer B (retention time: 26.6 min) as a white foam (390 mg, 50% yield).

Scheme 6. To a stirred solution of 9H-fluoren-9-ylmethyl (9H‐fluoren‐9‐yl)methyl N‐[(2S)‐1‐[(2R,3S,4R)‐ 2‐[(diphenylmethyl)carbamoyl]‐4‐(4‐fluorophenyl)‐3‐hydroxypyrrolidin‐1‐yl]‐1‐oxobutan‐2‐ yl]carbamate (390 mg) in dry DMF (4 mL), DBU (400 µL) was added. The resulting mixture was stirred at r. for 30 min. The crude product was purified by preparative HPLC (detector UV 210 nm, flow rate 20 mL/min, Luna C18(2) 250×21.2 mm column, mobile phase acetonitrile with 0.1% formic acid and water with 0.1% formic acid, (gradient of acetonitrile 20-40-97% at 0-22-27 min) to provide (2R,3S,4R)-1-[(2S)-2-aminobutanoyl]-N-benzhydryl-4-(4-fluorophenyl)-3-hydroxy-pyrrolidine-2- carboxamide as a white solid (225 mg, 85%). mp 180-181 °C; rotation value [α]²⁰ _D (10g/L, DCM) = 4.8; APCI-MS m/z calcd for C28H₃0FN3O3475, found 476 [M+H]⁺; 1H NMR (DMSO-d6, 400 MHz) 8.81 (d, J = 8.2 Hz, 1/2H), 8.47 (d, J = 8.2 Hz, 1/2H), 7.38–7.16 (m, 12H), 7.11 (m, 2H), 6.13 (t, J = 8.0 Hz, 1H), 5.55 (br. s, 1/2H), 5.28 (br. s, 1/2H), 4.83 (d, J = 7.7 Hz 1/2H) 461 (d J = 78 Hz 1/2H) 442 (m 1H) 413 (t J = 90 Hz 1/2H) 381 (dd J = 116 89 Hz, 1/2H), 3.53 (m, 1H), 3.39 (m, 1/2H) 3.28 (t, J = 10.9 Hz, 1/2H), 3.14 (dd, J = 7.8, 5.3 Hz, 1H), 1.62 – 1.46 (m, 1/2H), 1.29 (m, 1H), 1.08 (m, 1/2H), 0.85 (t, J = 7.4 Hz, 3/2H), 0.50 (t, J = 7.4 Hz, 3/2H). Example 2. (2RS,3SR,4RS)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)-3-hydroxy-4- phenylpyrrolidine-2-carboxamide. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.9. APCI-MS m/z calcd for C₂₇H₂₉N₃O₃444, found 445 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.19 – 8.45 (m, 1H), 8.12 (br s, 3H), 7.54 – 6.91 (m, 15H), 6.12 (m, 1H), 5.83 – 5.19 (br s, 1H), 5.12 – 3.39 (m, 6H), 1.57 – 0.76 (m, 3H). Example 3. (2S)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z calcd for C26H₃4N4O3451, found 452 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 8.3 Hz, 1H), 8.38 (d, J = 8.2 Hz, 1H), 7.91 (br s, 3H), 7.45 – 7.10 (m, 10H), 6.05 (2x d, J = 8.3 Hz, 1H), 4.48 (2x dd, J = 8.2, 4.6 Hz, 1H), 4.35 (t, J = 7.9 Hz, 1H), 3.79 – 3.32 (m, 3H), 2.21 – 1.69 (m, 5H), 1.40 – 1.14 (m, 3H), 0.89 (m, 6H). Example 4. (2RS,3SR,4RS)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)-3-hydroxy-4-(2- methylphenyl)pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.22. APCI-MS m/z calcd for C28H₃1N3O3458, found 459 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.26 – 8.42 (m, 1H), 7.95 (br s, 3H), 7.58 – 6.93 (m, 16H), 6.29 – 5.94 (m, 1H), 5.92 – 5.18 (m, 1H), 5.13 – 3.27 (m, 8H), 2.39 – 2.11 (m, 3H), 1.56 – 0.69 (m, 3H). Example 5. (2RS,3SR,4SR)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)-3-fluoro-4-(2- methylphenyl)pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.25 APCI-MS m/z calcd for C28H₃0FN3O2460, found 461 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.10 – 8.65 (m, 1H), 8.12 (br s, 3H), 7.44 – 6.86 (m, 14H), 6.09 (2x d, J = 8.4 Hz, 1H), 5.62 – 4.89 (m, 1H), 4.68 (m, 1H), 4.44 – 3.31 (m, 5H), 2.36 – 2.07 (m, 3H), 1.58 – 1.27 (m, 3H). Example 6. (2S,3R,4S)‐1‐[(2S)‐2‐aminopropanoyl]‐N‐(diphenylmethyl)‐4‐(4‐fluorophenyl)‐3‐ hydroxypyrrolidine‐2‐carboxamide. The title compound was prepared as in preparation of Example 17 by replacing (2S)-2-(9H- fluoren-9-ylmethoxycarbonylamino)butanoic acid with (2S)-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)propanoic acid and Isomer B with isomer A. APCI-MS m/z calcd for C₂₇H₂₈FN₃O₃462, found 463 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.66 (2x d, J = 8.3 Hz, 1H), 7.53 – 6.94 (m, 14H), 6.11 (2x d, J = 8.0 Hz, 1H), 5.33 (br s, 1H), 4.67 (2x d, J = 8.2 Hz, 1H), 4.41 (2x d, J = 8.5 Hz, 1H), 4.14 – 3.83 (m, 1H), 3.74 – 3.21 (m, 4H), 1.14 – 0.97 (m, 3H). Example 7. (2R,3S,4R)‐1‐[(2S)‐2‐aminopropanoyl]‐N‐(diphenylmethyl)‐4‐(4‐fluorophenyl)‐3‐ hydroxypyrrolidine‐2‐carboxamide. The title compound was prepared as in preparation of Example 17 by replacing (2S)-2-(9H- fluoren-9-ylmethoxycarbonylamino)butanoic acid with (2S)-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)propanoic acid. APCI-MS m/z calcd for C27H₂8FN3O3462, found 463 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (2x d, J = 8.4 Hz, 1H), 7.57 – 7.15 (m, 12H), 7.11 (m, 2H), 6.13 (t, J = 8.1 Hz, 1H), 5.44 (2x br s, 1H), 4.72 (2x d, J = 7.8 Hz, 1H), 4.51 – 4.35 (m, 1H), 4.21 – 3.23 (m, 5H), 1.17 – 0.66 (m, 3H). Example 8. (2RS,3SR,4RS)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10. APCI-MS m/z calcd for C₂₇H₂₈FN₃O₃462, found 463 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.27 – 8.48 (m, 1H), 8.34 – 7.86 (br s, 3H), 7.57 – 6.85 (m, 15H), 6.12 (m, 1H), 5.60 (br s, 1H), 5.11 – 3.34 (m, 6H), 1.51 – 0.79 (m, 3H). Example 9. (2RS,3SR,4SR)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)-3-fluoro-4-(4- fluorophenyl)pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.24. APCI-MS m/z calcd for C₂₇H₂₇F₂N₃O₂464, found 465 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.93 (m, 1H), 8.00 (br s, 3H), 7.53 – 6.87 (m, 14H), 6.08 (2x d, J = 8.3 Hz, 1H), 5.76 – 3.32 (m, 7H), 1.55 – 0.91 (m, 3H). Example 10. (rac-(2S*,3R*,4S*)-1-(1-aminocyclopropanecarbonyl)-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with 1-{[(tert-butoxy)carbonyl]amino}cyclopropane-1- carboxylic acid. APCI-MS m/z calcd for C28H₂8FN3O3 474 found 475 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (br s, 3H), 7.54 – 7.04 (m, 15H), 6.12 (d, J = 8.2 Hz, 1H), 5.44 (br s, 1H), 4.71 (br s, 1H), 4.42 (br s, 1H), 4.04 (br s, 1H), 3.69 – 3.31 (m, 3H), 1.60 – 0.67 (m, 4H). Example 11. (2R,3S,4R)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)-3-hydroxy-4-(2- methoxyphenyl)pyrrolidine-2-carboxamide. The title compound was prepared as in preparation of Example 17 by replacing Intermediate 10 with Intermediate 10.21 and (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid with (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanoic acid. APCI-MS m/z calcd for C₂₈H₃₁N₃O₄474 found 475 [M+H]⁺. Example 12. (2S,3R,4S)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)-3-hydroxy-4-(2- methoxyphenyl)pyrrolidine-2-carboxamide. The title compound was prepared as in preparation of Example 17 by replacing Intermediate 10 with Intermediate 10.21 and (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoic acid with (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanoic acid and Isomer B with Isomer A. APCI-MS m/z calcd for C28H₃1N3O4474 found 475 [M+H]⁺. Example 13. ((2RS,3SR,4RS)-1-[(2S)-2-aminopropanoyl]-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-methoxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.26. APCI-MS m/z calcd for C28H₃0FN3O3476 found 477 [M+H]⁺ Example 14. (2RS,3SR,4RS)-N-(diphenylmethyl)-4-(4-fluorophenyl)-3-hydroxy-1-[(2S)-2- (methylamino)propanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-{[(tert-butoxy)carbonyl](methyl)amino}propanoic acid. APCI-MS m/z calcd for C₂₈H₃₀FN₃O₃476, found 477 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.30 – 8.39 (m, 3H), 7.74 – 6.89 (m, 14H), 6.12 (2x d, J = 7.4 Hz, 1H), 5.84 – 5.31 (m, 1H), 4.95 – 4.62 (m, 1H), 4.62 – 4.32 (m, 1H), 4.32 – 3.74 (m, 2H), 3.74 – 3.28 (m, 4H), 2.47 (m, 3H), 1.34 (m, 3H). Example 15. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10. APCI-MS m/z calcd for C₂₈H₃₀FN₃O₃476, found 477 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.33 – 8.50 (m, 1H), 8.08 (br s, 3H), 7.65 – 6.92 (m, 14H), 6.12 (2x d, J = 8.2 Hz, 1H), 5.97 – 5.23 (m, 1H), 5.03 – 4.59 (m, 1H), 4.58 – 3.27 (m, 5H), 2.16 – 1.30 (m, 2H), 0.99 – 0.46 (m, 3H). Example 16. (2S,3R,4S)-1-[(2S)-2-aminobutanoyl]-N-(diphenylmethyl)-4-(4-fluorophenyl)-3- hydroxypyrrolidine-2-carboxamide. The title compound was prepared as in preparation of Example 17 by replacing Isomer B with Isomer A. mp 186-190 °C; rotation value [α]²⁰D (10g/L, DCM) = 11.2; APCI-MS m/z calcd for C₂₈H₃₀FN₃O₃476, found 477 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.63 (2x d, J = 8.1 Hz, 1H), 7.55 – 6.93 (m, 14H), 6.11 (2x d, J = 8.0 Hz, 1H), 5.38 (2x d, J = 5.8 Hz, 1H), 4.68 (m, 1H), 4.57 – 4.27 (m, 1H), 4.19 (br s, 1H), 4.12 – 3.79 (m, 1H), 3.79 – 3.27 (m, 4H), 1.71 – 1.28 (m, 2H), 0.85 (m, 3H). Example 18. rac-(2S*,3R*,4S*)-1-(2-amino-2-methylpropanoyl)-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with 2-{[(tert-butoxy)carbonyl]amino}-2-methylpropanoic acid. APCI-MS m/z calcd for C28H₃0FN3O3476, found 477 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.63 (br s, 1H), 8.04 (br s, 3H), 7.56 – 7.02 (m, 14H), 6.11 (d, J = 8.3 Hz, 1H), 5.37 (br s, 1H), 4.74 (br s, 1H), 4.46 (br s, 1H), 4.18 (br s, 1H), 3.78 – 3.31 (m, 2H), 1.50 (s, 6H). Example 19. (2RS,3SR,4RS)-1-[(2S)-2-aminopropanoyl]-4-(4-chlorophenyl)-N- (diphenylmethyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.10. APCI-MS m/z calcd for C₂₇H₂₈ClN₃O₃478, found 479 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.40 – 8.52 (m, 1H), 8.07 (br s, 3H), 7.69 – 6.93 (m, 14H), 6.29 – 6.04 (m, 1H), 5.62 (br s, 1H), 5.05 – 4.59 (m, 1H), 4.59 – 4.31 (m, 1H), 4.22 – 3.74 (m, 2H), 3.75 – 3.35 (m, 3H), 1.48 – 0.78 (m, 3H). Example 20. (2RS,3SR,4RS)-1-[(2S)-2-amino-3-methylbutanoyl]-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutanoic acid. APCI-MS m/z calcd for C₂₉H₃₂FN₃O₃490, found 491 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.29 – 8.51 (m, 1H), 8.06 (br s, 3H), 7.57 – 6.83 (m, 14H), 6.12 (m, 1H), 5.60 (m, 1H), 5.16 – 4.61 (m, 1H), 4.61 – 3.75 (m, 4H), 3.75 – 3.31 (m, 3H), 2.35 – 1.72 (m, 1H), 1.12 – 0.47 (m, 6H). Example 21. (2RS,3SR,4RS)-1-[(2S)-2-aminopentanoyl]-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-{[(tert-butoxy)carbonyl]amino}pentanoic acid. APCI-MS m/z calcd for C29H₃2FN3O3490, found 491 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.18 – 8.50 (m, 1H), 7.97 (br s, 3H), 7.48 – 6.99 (m, 14H), 6.41 – 5.98 (m, 1H), 5.82 – 5.22 (m, 1H), 5.00 – 4.56 (m, 1H), 4.56 – 3.29 (m, 5H), 1.90 – 1.49 (m, 2H), 1.49 – 1.29 (m, 2H), 0.97 – 0.42 (m, 3H). Example 22. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-methoxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.26 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-{[(tert-butoxy)carbonyl]amino}butanoic acid. APCI-MS m/z calcd for C29H₃2FN3O3490, found 491 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.45 – 8.69 (m, 1H), 8.04 (br s, 3H), 7.53 – 7.03 (m, 14H), 6.14 (2x d, J = 8.5 Hz, 1H), 5.36 – 4.81 (m, 1H), 4.45 – 3.29 (m, 6H), 3.23 – 3.13 (4x s, 3H), 1.75 (m, 2H), 0.99 – 0.46 (m, 3H). Example 23. (2RS,3SR,4RS)-1-[(2S)-2-aminopropanoyl]-N-[bis(4-fluorophenyl)methyl]-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.3. APCI-MS m/z calcd for C₂₇H₂₆F₃N₃O₃498, found 499 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.23 – 8.58 (m, 1H), 8.10 (br s, 3H), 7.58 – 7.24 (m, 6H), 7.24 – 7.00 (m, 6H), 6.12 (2x d, J = 8.2 Hz, 1H), 5.61 (4x br s, 1H), 4.90 – 4.57 (m, 1H), 4.57 – 3.30 (m, 6H), 1.55 – 0.80 (m, 3H). Example 24. (2RS,3SR,4RS)-1-[(2S)-2-aminopropanoyl]-N-[bis(3-fluorophenyl)methyl]-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.5. APCI-MS m/z calcd for C27H₂6F3N3O3498, found 499 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.28 – 8.67 (m, 1H), 8.05 (br s, 3H), 7.35 m, 4H), 7.25 – 6.92 (m, 8H), 6.18 m, 1H), 5.69 (br s, 1H), 5.20 – 3.71 (m, 4H), 3.71 – 3.36 (m, 3H), 1.63 – 0.69 (m, 3H). Example 25. (2RS,3SR,4RS)-1-[(2S)-2-aminopropanoyl]-N-[bis(2-fluorophenyl)methyl]-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.6. APCI-MS m/z calcd C27H₂6F3N3O3498, found 499 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.31 – 8.45 (m, 1H), 8.06 (br s, 3H), 7.80 – 6.81 (m, 12H), 6.57 (m, 1H), 5.95 – 5.29 (m, 1H), 5.12 – 4.60 (m, 1H), 4.60 – 4.32 (m, 1H), 4.32 – 3.75 (m, 2H), 3.75 – 3.28 (m, 3H), 1.47 – 1.28 (m, 3H). Example 26. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-N-[bis(4-fluorophenyl)methyl]-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.3 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-{[(tert-butoxy)carbonyl]amino}butanoic acid. APCI-MS m/z calcd C₂₈H₂₈F₃N₃O₃512, found 513 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.26 – 8.56 (m, 1H), 8.00 (br s, 3H), 7.34 (m, 6H), 7.11 (m, 4H), 6.13 (m, 1H), 5.94 – 5.31 (m, 1H), 5.02 – 4.56 (m, 1H), 4.56 – 4.29 (m, 1H), 4.29 – 3.70 (m, 2H), 3.70 – 3.29 (m, 3H), 1.75 (m, 2H), 0.99 – 0.45 (m, 3H). Example 27. 2-{2-[(3S,4R,5S)-1-[(2S)-2-aminopropanoyl]-5-[(diphenylmethyl)carbamoyl]-4- hydroxypyrrolidin-3-yl]phenoxy}acetic acid. Example 27 was prepared according to Scheme 7.

Scheme 7 APCI-MS m/z calcd C29H₃1N3O6518, found 519 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.71 (2x d, J = 7.9 Hz, 1H), 7.64 – 7.07 (m, 12H), 6.88 (m, 2H), 6.10 (d, J = 8.1 Hz, 1H), 4.94 – 3.36 (m, 6H), 1.57 – 0.76 (m, 3H). Example 28. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-2-[(diphenylmethyl)carbamoyl]-4-(4- fluorophenyl)pyrrolidin-3-yl acetate hydrochloride. The title compound was prepared according to Scheme 8.

Scheme 8 APCI-MS m/z calcd C30H₃2FN3O4518, found 519 [M+H]⁺; 1H NMR (400 MHz, DMSO-d₆) δ 8.92 (4x d, J = 8.8 Hz, 1H), 8.06 (br s, 3H), 7.61 – 7.00 (m, 14H), 6.33 – 6.05 (m, 1H), 5.77 – 5.37 (m, 1H), 4.83 (4x d, J = 7.8 Hz, 1H), 4.35 – 3.32 (m, 5H), 1.79 (m, 2H), 1.44 (4x s, 3H), 1.11 – 0.46 (m, 3H). Example 29. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-N-[bis(4-fluorophenyl)methyl]-4-(4- fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.19 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z calcd C26H₃1F3N4O4521, found 522 [M+H]⁺. Example 30.2RS,3SR,4RS&)-4-(3-fluorophenyl)-N-[2-(4-fluorophenyl)ethyl]-3-hydroxy-1- [(2S)-3-methyl-2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.17 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z calcd C28H₃6F2N4O4531, found 532 [M+H]⁺; 1H NMR (400 MHz, DMSO-d₆) δ 8.98 – 8.20 (m, 3H), 8.20 – 7.56 (m, 1H), 7.55 – 6.81 (m, 8H), 5.66 – 5.19 (m, 1H), 4.94 – 4.08 (m, 4H), 4.08 – 3.19 (m, 6H), 2.84 – 2.60 (m, 2H), 2.48 (m, 3H), 2.00 (m, 1H), 1.36 (m, 3H), 0.98 – 0.69 (m, 6H). Example 31.2-{[(2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-2-[(diphenylmethyl)carbamoyl]- 4-(4-fluorophenyl)pyrrolidin-3-yl]oxy}acetic acid. The title compound was prepared according to Scheme 9.

Scheme 9 APCI-MS m/z calcd C₃₀H₃₂FN₃O₅534, found 535 [M+H]⁺; 1H NMR (400 MHz, DMSO-d6) δ 9.42 – 8.79 (m, 1H), 7.99 (br s, 3H), 7.54 – 7.03 (m, 14H), 6.11 (m, 1H), 5.29 – 3.30 (m, 9H), 1.76 (m, 2H), 0.96 – 0.48 (m, 3H). Example 32. (2RS,3SR,4RS)-N-[(3,5-difluorophenyl)methyl]-4-(3-fluorophenyl)-3-hydroxy-1- [(2S)-3-methyl-2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.19 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C₂₇H₃₃F₃N₄O₄535, found 536 [M+H]⁺. Example 33. (2RS,3SR,4RS&)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- (9H-fluoren-9-yl)-3-hydroxy-4-phenylpyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.8 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C32H₃6N4O4541, found 542 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.76 – 8.50 (m, 1H), 8.50 – 8.20 (m, 1H), 8.06 – 7.69 (m, 5H), 7.69 – 7.09 (m, 12H), 6.09 (m, 1H), 5.66 – 5.31 (m, 1H), 4.99 – 3.26 (m, 7H), 2.09 (m, 1H), 1.37 (m, 3H), 1.11 – 0.73 (m, 6H). Example 34. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- (diphenylmethyl)-3-hydroxy-4-phenylpyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.9 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C32H₃8N4O4543, found 544 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.18 – 8.20 (m, 2H), 7.79 (br s, 3H), 7.46 – 7.07 (m, 15H), 6.28 – 5.94 (m, 1H), 5.68 – 5.24 (m, 1H), 4.91 – 3.30 (m, 7H), 2.17 – 1.74 (m, 1H), 1.68 – 1.28 (m, 3H), 0.98 – 0.39 (m, 6H). Example 35. (2RS,3SR,4RS&)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-4- (4-chlorophenyl)-N-[1-(2-fluorophenyl)cyclopropyl]-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.12 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C28H₃4ClFN4O4545, found 546 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.29 – 8.24 (m, 2H), 7.90 (br s, 3H), 7.60 (m, 1H), 7.53 – 7.12 (m, 5H), 7.03 (m, 2H), 5.75 – 5.00 (m, 1H), 4.98 – 3.38 (m, 8H), 2.10 – 1.61 (m, 1H), 1.48 – 1.29 (m, 3H), 1.25- 1.15 (m, 4H), 1.11 – 0.28 (m, 6H). Example 36. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-N-(diphenylmethyl)-4-(4- fluorophenyl)-3-[(methylcarbamoyl)methoxy]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared according to Scheme 10.

Scheme 10 APCI MS / l d C H FN O 547 f d 548 [M H] ¹H NMR (400 MHz, DMSO-d6) δ 9.42 – 8.82 (m, 1H), 7.95 (br s, 3H), 7.61 – 6.99 (m, 14H), 6.99 – 6.61 (m, 1H), 6.25 – 5.97 (m, 1H), 5.33 – 4.82 (m, 1H), 4.60 – 3.34 (m, 8H), 2.39 (m, 3H), 1.76 (m, 2H), 0.99 – 0.79 (m, 3H). Example 37. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-N-[bis(3,4-difluorophenyl)methyl]-4- (4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.8 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-{[(tert-butoxy)carbonyl]amino}butanoic acid . APCI-MS m/z C₂₈H₂₆F₅N₃O₃548, found 549 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.44 – 8.63 (m, 1H), 7.92 (br s, 3H), 7.38 (m, 6H), 7.23 – 6.98 (m, 4H), 6.39 – 6.08 (m, 1H), 5.97 – 5.44 (m, 1H), 4.99 – 4.32 (m, 2H), 4.31 – 3.29 (m, 5H), 1.76 (m, 2H), 0.97 – 0.43 (m, 3H). Example 38. methyl 2-{[(2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-2- [(diphenylmethyl)carbamoyl]-4-(4-fluorophenyl)pyrrolidin-3-yl]oxy}acetate hydrochloride. The title compound was prepared according to Scheme 11.

Scheme 11 APCI-MS m/z calcd C31H₃4FN3O5548, found 549 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.59 – 8.46 (m, 1H), 8.09 (br s, 3H), 7.59 – 6.86 (m, 14H), 6.32 – 6.02 (m, 1H), 5.48 – 4.75 (m, 1H), 4.73 – 3.57 (m, 8H), 3.57 – 3.33 (m, 3H), 2.03 – 1.32 (m, 2H), 1.00 – 0.44 (m, 3H). Example 39. (2RS,3RS,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-4-(3- fluorophenyl)-3-hydroxy-N-{[3-(trifluoromethyl)phenyl]methyl}pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.14 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C27H₃2F4N4O4553, found 554 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.79 – 8.24 (m, 2H), 8.02 (br s, 3H), 7.83 – 6.92 (m, 8H), 5.80 – 5.40 (m, 1H), 5.12 – 4.07 (m, 6H), 4.06 – 3.78 (m, 1H), 3.78 – 3.29 (m, 3H), 2.21 – 1.74 (m, 1H), 1.55 – 1.14 (m, 3H), 0.99 – 0.58 (m, 6H). Example 40. (2RS,3RS,4RS)-N-(9H-fluoren-9-yl)-3-hydroxy-1-[(2S)-3-methyl-2-[(2R)-2- (methylamino)propanamido]butanoyl]-4-phenylpyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.8 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C33H₃8N4O4555, found 556 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.97 – 8.37 (m, 3H), 8.27 (t, J = 9.2 Hz, 1H), 8.00 – 7.06 (m, 13H), 6.08 (m, 1H), 5.65 – 5.27 (4x d,1H), 4.96 – 4.10 (m, 4H), 4.10 – 3.32 (m, 5H), 2.48 (4x s, 3H), 2.08 (m 1H) 164 130 (m 3H) 111 074 (m 6H) Example 41. (2RS,3RS,4RS)-N-(diphenylmethyl)-3-hydroxy-1-[(2S)-3-methyl-2-[(2R)-2- (methylamino)propanamido]butanoyl]-4-phenylpyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.9 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C₃₃H₄₀N₄O₄557, found 558 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.94 – 8.32 (m, 4H), 7.60 – 7.04 (m, 15H), 6.39 – 5.98 (m, 1H), 5.67 – 5.22 (m, 1H), 5.07 – 4.59 (m, 1H), 4.59 – 4.03 (m, 3H), 4.03 – 3.31 (m, 4H), 2.48 (m, 3H), 2.17 – 1.77 (m, 1H), 1.58 – 1.28 (m, 3H), 0.99 – 0.39 (m, 6H). Example 42. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- (diphenylmethyl)-3-hydroxy-4-(2-methylphenyl)pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.22 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C₃₃H₄₀N₄O₄557, found 558 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.02 – 8.25 (m, 2H), 7.97 (br s, 3H), 7.63 – 6.90 (m, 14H), 6.28 – 5.95 (m, 1H), 5.67 – 5.24 (m, 1H), 5.20 – 3.27 (m, 8H), 2.40 – 2.14 (m, 3H), 2.08 – 1.74 (m, 1H), 1.57 – 1.27 (m, 3H), 0.99 – 0.43 (m, 6H). Example 43. (2RS,3RS&,4RS&&)-4-(4-chlorophenyl)-N-[1-(2-fluorophenyl)cyclopropyl]-3- hydroxy-1-[(2S)-3-methyl-2-[(2R)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.12 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C29H₃6ClFN4O4559, found 560 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.87 – 8.12 (m, 4H), 7.88 – 6.84 (m, 8H), 5.69 – 5.12 (m, 1H), 4.97 – 3.26 (m, 8H), 2.48 (m, 3H), 2.12 – 1.58 (m, 1H), 1.36 (m, 3H), 1.29 – 0.33 (m, 10H). Example 44. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-2-[(diphenylmethyl)carbamoyl]-4-(4- fluorophenyl)pyrrolidin-3-yl 2,2-dimethylpropanoate hydrochloride. The title compound was prepared as in preparation of Example 28 by replacing acetic anhydride with di-tert-butyl dicarbonate. APCI-MS m/z for C₃₃H₃₈FN₃O₄560, found 561 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.53 – 8.59 (m, 1H), 7.96 (br s, 3H), 7.66 – 6.97 (m, 14H), 6.09 (2x d, J = 8.7 Hz, 1H), 5.79 – 5.04 (m, 1H), 5.07 – 4.63 (m, 1H), 4.56 – 3.28 (m, 5H), 2.05 – 1.42 (m, 2H), 0.98 – 0.36 (m, 13H). Example 45. (2RS,3RS,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- (diphenylmethyl)-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C₃₂H₃₇FN₄O₄561, found 562 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.72 (2x d, J = 8.3 Hz, 1H), 8.50 (2x d, J = 8.6 Hz, 1H), 7.92 (br s, 3H), 7.50 – 7.00 (m, 14H), 6.12 (2x d, J = 8.2 Hz, 1H), 5.74 – 5.24 (m, 1H), 5.15 – 4.87 (m, 1H), 4.86 – 3.73 (m, 6H), 3.47 – 3.25 (m, 1H), 1.92 (m, 1H), 1.47 – 1.27 (m, 3H), 0.96 – 0.40 (m, 6H). Example 46. (2RS,3RS,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- (diphenylmethyl)-4-(3-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.13 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C₃₂H₃₇FN₄O₄561, found 562 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.07 – 8.22 (m, 2H), 8.20 – 7.46 (m, 3H), 7.46 – 6.96 (m, 14H), 6.22 – 6.00 (m, 1H), 5.76 – 5.25 (4x t, 1H), 5.13 – 3.96 (m, 5H), 3.86 (m, 1H), 3.77 – 3.30 (m, 4H), 2.13 – 1.66 (m, 1H), 1.51 – 1.15 (m, 3H), 0.97 – 0.43 (m, 6H). Example 47. (2RS,3RS,4RS)-N-{[1,1'-biphenyl]-2-yl}-4-(3-fluorophenyl)-3-hydroxy-1-[(2S)- 3-methyl-2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.14 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C32H₃7FN4O4561, found 562 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.47 – 8.30 (m, 4H), 8.18 – 6.81 (m, 13H), 5.79 – 5.23 (m, 1H), 5.20 – 3.30 (m, 7H), 2.48 (m, 3H), 2.01 (m, 1H), 1.36 (m, 3H), 0.98 – 0.71 (m, 6H). Example 48. (2RS,3SR,4RS)-4-(3-fluorophenyl)-3-hydroxy-1-[(2S)-3-methyl-2-[(2S)-2- (methylamino)propanamido]butanoyl]-N-{[3-(trifluoromethyl)phenyl]methyl}pyrrolidine-2- carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.14 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C32H₃7FN4O4567, found 568 [M+H]⁺. Example 49. (2RS,3RS,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-4-(3- fluorophenyl)-3-hydroxy-N-{2-[4-(trifluoromethyl)phenyl]ethyl}pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.18 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z C32H₃7FN4O4567, found 568 [M+H]⁺. Example 50. (2RS,3RS,4RS)-N-{[1,1'-biphenyl]-2-yl}-4-(3-fluorophenyl)-3-hydroxy-1-[(2S)-3- methyl-2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}butanamido]-3-methylbutanoic acid. APCI-MS m/z C33H₃9FN4O4575, found 576 [M+H]⁺. Example 51. (2RS,3RS,4RS)-1-[(2S,3S)-2-[(2S)-2-aminopropanamido]-3-methylpentanoyl]- N-(diphenylmethyl)-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S,3S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylpentanoic acid. APCI-MS m/z for C₃₃H₃₉FN₄O₄575, found 576 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.01 – 8.33 (m, 2H), 7.93 (br s, 3H), 7.65 – 6.75 (m, 14H), 6.33 – 5.98 (m, 1H), 5.71 – 5.17 (m, 1H), 5.07 – 3.27 (m, 8H), 1.79 (m, 1H), 1.69 – 0.97 (m, 7H), 0.97 – 0.38 (m, 4H). Example 52. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- (diphenylmethyl)-4-(4-fluorophenyl)-3-methoxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.26 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C₃₃H₃₉FN₄O₄575, found 576 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.03 (2x d, J = 8.5 Hz, 1H), 8.48 (2x d, J = 8.5 Hz, 1H), 7.86 (br s, 3H), 7.56 – 6.99 (m, 14H), 6.15 (2x d, J = 8.4 Hz, 1H), 5.08 (2x d, J = 7.4 Hz, 1H), 4.71 – 3.16 (m, 10H), 2.18 – 1.69 (m, 1H), 1.34 (m, 3H), 0.96 – 0.44 (m, 6H). Example 53. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-amino-N-methylpropanamido]-3- methylbutanoyl]-N-(diphenylmethyl)-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-N- methylpropanamido]-3-methylbutanoic acid. APCI-MS m/z for C33H₃9FN4O4575, found 576 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.52 (m, 1H), 8.06 (br s, 3H), 7.54 – 6.55 (m, 14H), 6.33 – 5.90 (m, 1H), 5.34 (br s, 1H), 4.96 – 3.34 (m, 7H), 3.04 – 2.62 (m, 3H), 2.34 – 2.09 (m, 1H), 1.48 – 0.40 (m, 9H). Example 54. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-4-(4- chlorophenyl)-N-(9H-fluoren-9-yl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.11 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C32H₃5ClN4O4575, found 576 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 8.79 – 8.21 (m, 2H), 8.20 – 7.73 (m, 5H), 7.73 – 7.13 (m, 11H), 6.45 – 5.93 (m, 1H), 5.80 – 5.30 (m, 1H), 5.11 – 3.29 (m, 8H), 2.09 (m, 1H), 1.70 – 1.29 (m, 3H), 0.97 – 0.70 (m, 6H). Example 55. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-4-(4- chlorophenyl)-N-(9H-fluoren-9-yl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared according to Scheme 12 from Isomer B. C

Isomer A Scheme 12 APCI-MS m/z for C₃₂H₃₇ClN₄O₄577, found 578 [M+H]⁺; Example 56. (2S,3R,4S)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-4-(4- chlorophenyl)-N-(diphenylmethyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared according to Scheme 12 from Isomer A. APCI-MS m/z for C32H₃7ClN4O4577, found 578 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.04 – 8.20 (m, 2H), 7.98 (s, 3H), 7.48 – 7.03 (m, 14H), 6.10 (2x d, J = 8.2 Hz, 1H), 5.47 (2x d, J = 5.5 Hz, 1H), 4.96 – 4.57 (m, 1H), 4.57 – 4.23 (m, 2H), 4.23 – 3.96 (m, 1H), 3.87 (m, 1H), 3.70 (t, J = 9.6 Hz, 1H), 3.62 – 3.45 (m, 1H), 2.01 (m, 1H), 1.38 (m, 3H), 0.88 (m, 6H). Example 57. (2RS,3SR,4RS)-4-(3-fluorophenyl)-3-hydroxy-1-[(2S)-3-methyl-2-[(2S)-2- (methylamino)propanamido]butanoyl]-N-{2-[4-(trifluoromethyl)phenyl]ethyl}pyrrolidine-2- carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.18 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C29H₃6F4N4O4581, found 582 [M+H]⁺. Example 58. (2RS,3SR,4RS)-N-(2,2-diphenylethyl)-4-(3-fluorophenyl)-3-hydroxy-1-[(2S)-3- methyl-2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.15 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C₃₄H₄₁FN₄O₄589, found 590 [M+H]⁺; 1H NMR (400 MHz, DMSO-d6) δ 8.95 – 7.50 (m, 4H), 7.47 – 6.85 (m, 14H), 5.66 – 4.97 (m, 1H), 4.72 – 4.02 (m, 4H), 4.06 – 3.27 (m, 6H), 2.48 (m, 3H), 2.13 – 1.86 (m, 1H), 1.51 – 1.28 (m, 3H), 0.98 – 0.62 (m, 6H). Example 59. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopentanamido]-3-methylbutanoyl]-N- (diphenylmethyl)-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}pentanamido]-3-methylbutanoic acid . APCI-MS m/z for C₃₄H₄₁FN₄O₄589, found 590 [M+H]⁺; 1H NMR (400 MHz, DMSO-d6) δ 9.10 – 8.24 (m, 2H), 7.77 (br s, 3H), 7.53 – 6.97 (m, 14H), 6.36 – 5.99 (m, 1H), 5.67 – 5.22 (m, 1H), 5.20 – 3.27 (m, 8H), 1.99 (m, 1H), 1.68 (m, 2H), 1.33 (m, 2H), 0.98 – 0.39 (m, 9H). Example 60. (2RS,3SR,4RS)-4-(4-chlorophenyl)-N-(9H-fluoren-9-yl)-3-hydroxy-1-[(2S)-3- methyl-2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.11 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C₃₃H₃₇ClN₄O₄589, found 590 [M+H]⁺; 1H NMR (400 MHz, DMSO-d6) δ 9.10 – 8.15 (m, 4H), 8.01 – 7.10 (m, 13H), 6.08 (m, 1H), 5.74 – 5.37 (4x d, 1H), 5.04 – 4.11 (m, 4H), 4.11 – 3.77 (m, 2H), 3.77 – 3.51 (m, 2H), 3.51 – 3.29 (m, 1H), 2.53 - 2.44 (4x s, 3H), 2.23 – 1.95 (m, 1H), 1.62 – 1.29 (m, 3H), 1.11 – 0.75 (m, 6H). Example 61. tert-butyl N-[(2S)-1-[(2S,3R,4S)-2-[(diphenylmethyl)carbamoyl]-4-(4- fluorophenyl)-3-hydroxypyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate (Isomer A). The title compound was prepared as in preparation of Example 17 by replacing (2S)-2-(9H- fluoren-9-ylmethoxycarbonylamino)butanoic acid with (2S)-2-{[(tert-butoxy)carbonyl]amino}-3- methylbutanoic acid . APCI-MS m/z for C₃₄H₄₀FN₃O₅590, found 591 [M+H]⁺; 1H NMR (400 MHz, DMSO-d6) δ 8.44 (2x d, J = 8.1 Hz, 1H), 7.66 – 6.92 (m, 14H), 6.59 (br s, 1H), 6.09 (d, J = 8.0 Hz, 1H), 5.26 (d, J = 5.5 Hz, 1H), 4.74 (2x d, J = 7.5 Hz, 1H), 4.34 (q, J = 7.4 Hz, 1H), 4.28 – 3.82 (m, 2H), 3.83 – 3.36 (m, 2H), 2.04 – 1.83 (m, 1H), 1.34 (s, 9H), 0.86 (m, 6H). Example 62. (2RS,3SR,4RS)-1-[(2S)-2-aminobutanoyl]-2-[(diphenylmethyl)carbamoyl]-4-(4- fluorophenyl)pyrrolidin-3-yl N-(3-methoxypropyl)carbamate hydrochloride. The title compound was prepared according to Scheme 13.

Scheme 13 APCI-MS m/z for C₃₃H₃₉FN₄O₅591, found 592 [M+H]⁺. Example 63. (2R,3S,4R)-4-(4-chlorophenyl)-N-(diphenylmethyl)-3-hydroxy-1-[(2S)-3-methyl- 2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 55 by replacing of (2S)-2- [(2S)-2-{[(tert-butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid from Isomer B. APCI-MS m/z for C33H₃9ClN4O4591, found 592 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.09 – 8.40 (m, 4H), 7.61 – 7.03 (m, 14H), 6.11 (2x d, J = 8.2 Hz, 1H), 5.50 (2x d, J = 5.6 Hz, 1H), 4.83 (2x d, J = 7.7 Hz, 1H), 4.54 – 4.11 (m, 3H), 3.99 – 3.67 (m, 2H), 3.55 (m, 2H), 3.50 – 3.30 (m, 1H), 2.46 - 2.39 (2x s, 3H), 1.92 (2xm, 1H), 1.35 (m, 3H), 0.95 – 0.39 (m, 6H). Example 64. (2S,3R,4S)-4-(4-chlorophenyl)-N-(diphenylmethyl)-3-hydroxy-1-[(2S)-3-methyl- 2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 55 by replacing of (2S)-2- [(2S)-2-{[(tert-butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid from Isomer A. APCI-MS m/z for C₃₃H₃₉ClN₄O₄591, found 592 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.33 – 8.20 (m, 4H), 7.65 – 7.03 (m, 14H), 6.07 (2x d, J = 7.5 Hz, 1H), 5.49 (2x d, J = 5.4 Hz, 1H), 4.78 (2x d, J = 7.4 Hz, 1H), 4.62 – 4.24 (m, 2H), 4.15 (t, J = 9.1 Hz, 1H), 4.02 – 3.76 (m, 1H), 3.69 (t, J = 9.5 Hz, 1H), 3.54 (d, J = 7.6 Hz, 1H), 2.45 (s, 3H), 2.02 (m, 1H), 1.40 (m, 3H), 0.97 – 0.73 (m, 6H). Example 65. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- [bis(4-fluorophenyl)methyl]-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.3 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C32H₃5F3N4O4597, found 598 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.20 – 8.23 (m, 2H), 7.99 (br s, 2H), 7.55 – 7.22 (m, 6H), 7.22 – 6.93 (m, 6H), 6.53 – 5.97 (m, 1H), 5.79 – 5.25 (m, 1H), 5.20 – 3.28 (m, 8H), 2.14 – 1.65 (m, 1H), 1.57 – 1.16 (m, 3H), 1.00 – 0.40 (m, 6H). Example 66. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- [bis(4-fluorophenyl)methyl]-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.5 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C32H₃5F3N4O4597, found 598 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.15 – 8.21 (m, 2H), 7.91 (br s, 3H), 7.57 – 6.86 (m, 12H), 6.39 – 5.94 (m, 1H), 5.83 – 5.32 (m, 1H), 5.22 – 3.28 (m, 8H), 2.01 (m, 1H), 1.54 – 0.35 (m, 9H). Example 67. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- [bis(2-fluorophenyl)methyl]-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine2 carboxamide hydrochloride with Intermediate 106 and (2S) 2{[(tert butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C₃₂H₃₅F₃N₄O₄597, found 598 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.18 – 8.27 (m, 2H), 8.13 (br s, 3H), 7.85 – 6.91 (m, 12H), 6.77 – 6.32 (m, 1H), 5.7 - 5.0 (br s, 1H), 4.91 – 3.61 (m, 8H), 2.12 – 1.81 (m, 1H), 1.50 – 1.14 (m, 3H), 1.11 – 0.52 (m, 6H). Example 68. (2RS,3RS&,4RS)-N-(2,2-diphenylpropyl)-4-(3-fluorophenyl)-3-hydroxy-1-[(2S)- 3-methyl-2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.16 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C35H43FN4O4603, found 604 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.00 – 8.30 (m, 4H), 7.60 – 6.90 (m, 15H), 5.60 – 5.05 (m, 1H), 5.03 – 3.25 (m, 10H), 2.44 (m, 3H), 1.97 (m, 1H), 1.68 (m, 3H), 1.46 – 1.30 (m, 3H), 0.99 – 0.68 (m, 6H). Example 69. (2RS,3RS,4RS)-N-(1,3-diphenylpropan-2-yl)-4-(3-fluorophenyl)-3-hydroxy-1- [(2S)-3-methyl-2-[(2S)-2-(methylamino)propanamido]butanoyl]pyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.18 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl](methyl)amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C35H43FN4O4603, found 604 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.26 – 8.23 (m, 3H), 8.20 – 7.69 (m, 1H), 7.47 – 6.91 (m, 14H), 5.57 – 5.03 (m, 1H), 4.96 – 3.25 (m, 8H), 2.99 – 2.53 (m, 4H), 2.50-2.44 (m, 3H), 2.13 – 1.81 (m, 1H), 1.36 (m, 3H), 0.97 – 0.43 (m, 6H). Example 70. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-amino-4-methylpentanamido]-3- methylbutanoyl]-N-(diphenylmethyl)-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-4- methylpentanamido]-3-methylbutanoic acid. APCI-MS m/z for C₃₅H₄₃FN₄O₄603, found 604 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.60 (m, 1H), 7.96 (m, 1H), 7.47 – 6.95 (m, 14H), 6.12 (m, 1H), 5.39 (m, 1H), 5.19 – 4.54 (m, 1H), 4.52 – 4.25 (m, 2H), 4.25 – 4.02 (m, 1H), 4.02 – 3.30 (m, 3H), 2.06 – 1.45 (m, 2H), 1.47 – 1.14 (m, 2H), 0.99 – 0.35 (m, 12H). Example 71. (2RS,3RS,4RS)-1-[(2S)-2-[(2S)-2-aminobutanamido]-3-methylbutanoyl]-N- [bis(4-fluorophenyl)methyl]-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.3 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}butanamido]-3-methylbutanoic acid. APCI-MS m/z for C₃₃H₃₇F₃N₄O₄611, found 612 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d6) δ 8.81 (2x d, J =8.3 Hz, 1H), 8.51 (2x d, J = 8.7 Hz, 1H), 8.00 (br s, 3H), 7.53 – 7.22 (m, 6H), 7.22 – 6.96 (m, 6H), 6.14 (2x d, J = 8.2 Hz, 1H), 5.54 (2x d, J = 5.6 Hz, 1H), 5.23 – 3.29 (m, 8H), 2.02 (m, 1H), 1.92 – 1.62 (m, 2H), 0.98 – 0.33 (m, 9H). Example 72. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-2- [(diphenylmethyl)carbamoyl]-4-(4-fluorophenyl)pyrrolidin-3-yl methyl carbonate hydrochloride. The title compound was prepared according to Scheme 14.

Scheme 14 APCI-MS m/z for C₃₄H₃₉FN₄O₆619, found 620 [M+H]⁺; 1H NMR (400 MHz, DMSO-d6) δ 9.25 – 8.24 (m, 2H), 7.91 (br s, 3H), 7.49 – 6.96 (m, 14H), 6.24 – 5.95 (m, 1H), 5.62 – 5.14 (m, 1H), 4.98 – 3.29 (m, 11H), 2.05 (m, 1H), 1.71 – 1.13 (m, 3H), 1.01 – 0.44 (m, 6H). Example 73. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- [bis(4-methoxyphenyl)methyl]-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.4 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C34H41FN4O6621, found 622 [M+H]⁺; 1H NMR (400 MHz, DMSO-d₆) δ 8.82 – 8.28 (m, 2H), 8.04 (br s, 3H), 7.85 – 7.59 (m, 1H), 7.59 – 6.47 (m, 12H), 5.29 (br s, 1H), 4.91 – 3.38 (m, 13H), 2.01 (m, 1H), 1.61 – 1.10 (m, 3H), 1.12 – 0.51 (m, 6H). Example 74. (2RS,3SR,4RS)-1-[(2S)-2-[(2S)-2-aminopropanamido]-3-methylbutanoyl]-N- [bis(4-chlorophenyl)methyl]-4-(4-fluorophenyl)-3-hydroxypyrrolidine-2-carboxamide hydrochloride. The title compound was prepared as in preparation of Example 1 by replacing (2S)-N- (diphenylmethyl)pyrrolidine-2-carboxamide hydrochloride with Intermediate 10.2 and (2S)-2-{[(tert- butoxy)carbonyl]amino}propanoic acid with (2S)-2-[(2S)-2-{[(tert- butoxy)carbonyl]amino}propanamido]-3-methylbutanoic acid. APCI-MS m/z for C32H₃5Cl2FN4O4630, found 631 [M+H]⁺; 1H NMR (400 MHz, DMSO-d6) δ 9.20 – 8.23 (m, 1H), 7.95 (br s, 3H), 7.56 – 7.22 (m, 10H), 7.22 – 7.02 (m, 2H), 6.29 – 5.95 (m, 1H), 5.76 – 5.31 (m, 1H), 5.15 – 4.52 (m, 1H), 4.52 – 4.27 (m, 2H), 4.27 – 3.72 (m, 3H), 3.72 – 3.29 (m, 3H), 2.15 – 1.64 (m, 1H), 1.49 – 1.15 (m, 3H), 0.98 – 0.42 (m, 6H). Even though the invention has been described with reference to the disclosed embodiments, for specialists in this field it should be obvious that the specific details of the described experiments are given only to illustrate the present invention and they should not be construed as in any way limiting the scope of the invention. It should be understood that it is possible to implement various modifications without deviation from the essence of the present invention.

Claims

Claims. 1. A compound of Formula I, or a tautomer, a stereoisomer, an individual isomer, a mixture of isomers, or pharmaceutically acceptable salt or solvate thereof:

wherein: R¹ is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀- alkyl; R² is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀- alkyl; wherein R¹ and R² may optionally be joined to form a substituted and unsubstituted -C₃-C₁₀- cycloalkyl; L is independently selected for each occurrence from the group consisting of a covalent chemical bond, -O-; R³ is selected from -H, halo, -OH, -C₁-C₁₀-alkyl, -C(O)-(C₁-C₁₀-alkyl), -C(O)O(C₁-C₁₀-alkyl), -(CH₂)n-C(O)OH, -(CH₂)n-C(O)NH(C₁-C₁₀-alkyl), -(CH₂)n-C(O)NH₂, -(CH₂)n-C(O)O(C₁-C₁₀-alkyl), - C(O)- C₁-C₁₀-alkyl or -C(O)NH(CH₂)nOC₁-C₁₀-alkyl, wherein n is selected independently from 0, 1, 2, 3, 4, 5; R⁴ is selected from -H or -C₆-C₁₀-aryl, wherein said -C₆-C₁₀-aryl is alternatively substituted with one, two, three, four, or five substituents independently selected for each occurrence from R^4a; R^4a is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀ alkyl, -O-C₁-C₁₀ alkyl, halo or -O-(CH₂)p-C(O)OH, wherein p is selected independently from 0, 1, 2, 3, 4, 5; M is -NH-, -NH-CH₂-; A is independently selected for each occurrence from the group consisting of -C₆-C₁₀-aryl, - (CH₂)m-C₆-C₁₀-aryl,

, wherein m is selected independently from 0, 1, 2, 3, 4, 5; wherein A may optionally be substituted with one, two, three, four, five or six substituents independently selected for each occurrence from the group consisting of halo, -C₁-C₁₀-alkyl, halogenated -C₁-C₁₀-alkyl, -O-C₁-C₁₀-alkyl or -C₆-C₁₀-aryl; R⁵ is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀- alkyl, -С(O)С(СH₃)₃, -C(O)-CH₂(C₁-C₅-alkyl)-NH₂ or -C(O)-CH₂(C₁-C₅-alkyl)-NH(C₁-C₅-alkyl); R⁶ is independently selected for each occurrence from the group consisting of -H, -C₁-C₁₀- alkyl, -С(O)С(СH₃)₃, -C(O)-CH₂(C₁-C₅-alkyl)-NH₂ or -C(O)-CH₂(C₁-C₅-alkyl)-NH(C₁-C₅-alkyl).

2. The compound according to claim 1, wherein R⁵ is independently selected for each occurrence from the group consisting of:

R⁶ is independently selected for each occurrence from the group consisting of:

3. The compound according to claim 1, wherein A is independently selected for each occurrence from the group consisting of:

4. The compound according to claim 1, wherein, the compound of formula (I) is a compound having formula Ia or Ib:

wherein R¹, R², R³, R^4a, R⁵, R⁶, A, L and M are defined as in claim 1.

5. The compound according to claim 1, wherein the compound is selected from the following compounds:

F C

,

.

6. A pharmaceutical composition for treatment and/or prevention of a disease or condition, associated with over-expressed or dysregulated glutamine-dependent asparagine synthetase, comprising therapeutically effective amount of at least compound according to claim 1 and at least one pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6, wherein disease is a proliferative disease.

8. The pharmaceutical composition according to claim 7, wherein the proliferative disease is acute lymphoblastic leukemia, rhabdomyosarcoma and leiomyosarcoma, colon carcinoma with mutated KRAS, ovarian carcinomas, gastric cancers, melanoma, epidermoid carcinoma, non-small cell lung cancer, breast cancer, prostate cancer, soft tissue sarcoma, acute lymphoblastic leukemia, sarcoma with a mutant form of protein Ras, breast cancer or castration- resistant prostate cancer.

9. The pharmaceutical composition according to claim 6, wherein disease is a disease characterized by the dysregulation of ASNS.

10. The pharmaceutical composition according to claim 6, wherein additionally comprising a therapeutically effective amount of at least one chemotherapeutic agent.

11. The pharmaceutical composition according to claim 10, wherein a chemotherapeutic agent is an asparaginase.

12. The pharmaceutical composition according to claim 11, wherein an asparaginase is recombinant L-asparaginase derived from Escherichia coli or Erwinia chrysanthemi.

13. The pharmaceutical composition according to claim 6, wherein pharmaceutically acceptable excipient is pharmaceutically acceptable carrier, adjuvant, solvent.

14. Use of compound according to claim 1 for preparing a drug for treatment and/or prevention of a disease or condition, associated with over-expressed or dysregulated glutamine- dependent asparagine synthetase.

15. A method of prevention and/or treatment of a disease or condition, associated with over-expressed or dysregulated glutamine-dependent asparagine synthetase comprising administration of the pharmaceutical composition according to claim 6 to subject.

16. The method according to claim 15, wherein disease is a proliferative disease.

17. The method according to claim 16, wherein the proliferative disease is acute lymphoblastic leukemia, rhabdomyosarcoma and leiomyosarcoma, colon carcinoma with mutated KRAS, ovarian carcinomas, gastric cancers, melanoma, epidermoid carcinoma, non-small cell lung cancer, breast cancer, prostate cancer, soft tissue sarcoma, acute lymphoblastic leukemia, sarcoma with a mutant form of protein Ras, breast cancer or castration-resistant prostate cancer.

18. The method according to claim 15, wherein disease is a disease characterized by the dysregulation of ASNS.

19. The method according to claim 15, wherein additionally comprising administration of therapeutically effective amount of at least one chemotherapeutic agent.

20. The method according to claim 19, wherein a chemotherapeutic agent is an asparaginase.

21. The method according to claim 15, wherein the subject is human.

22. The method according to claim 15, wherein subject is on asparagine-free diet.