Derivatives of the aplysia proline-aplysia isoleucine peptide

Abstract

Provided herein are peptide analogs, pharmaceutical compositions comprising such compounds, and methods of treating cancer with such compounds.

Description

Technical Field

[0001] This article provides dolaproine-dolaisoleuine peptide analogs from sea hares, pharmaceutical compositions containing such compounds, and methods for treating cancer with such compounds.

[0002] background

[0003] Cancer is the second leading cause of death after coronary artery disease. In the United States, cancer accounts for nearly a quarter of all deaths. Globally, millions die from cancer each year. According to the American Cancer Society, in the United States alone, cancer causes more than half a million deaths annually, with more than 1.5 million new cases diagnosed each year. While deaths from heart disease have declined significantly, deaths from cancer are generally on the rise. Unless new drugs are discovered, cancer is projected to become the leading cause of death by the early next century.

[0004] Several cancers are clearly leading killers worldwide. Lung, prostate, breast, colon, pancreatic, ovarian, and bladder cancers, in particular, represent the leading causes of cancer death. With few exceptions, metastatic cancers are fatal. Furthermore, even those patients who initially survive primary cancer often experience drastically altered lives. Many cancer patients experience severe anxiety due to concerns about potential recurrence or treatment failure. Many patients experience physical weakness after treatment. In addition, many cancer patients experience recurrence.

[0005] Promising new cancer therapies include sulphurin and synthetic sulphurin analogues such as olistatin (US Patent Nos. 5,635,483, 5,780,588, 6,323,315, and 6,884,869; Shnyder et al., (2007) Int. J. Oncol. 31: 353-360; Otani, M. et al., Jpn. J. Cancer Res. 2000, 91, 837-844; PCT International Publication Nos. WO 01 / 18032A3, WO 2005 / 039492, WO 2006 / 132670, and WO 2009 / 095447; Fennell, BJ et al., J. Antimicrob. Chemther. 2003, 51, 833-841). Tail slugin and olistatin have been shown to interfere with microtubule dynamics, thereby disrupting cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and possess anticancer (US Patent No. 5,663,149) and antifungal activities (Pettit et al. (1998) Antimicrob. Agents and Chemother. 42: 2961-2965). Unfortunately, despite initial high interest, tail slugin 10 showed poor results as a single agent in phase II clinical trials (Shnyder (2007), ibid.). Some compounds in the olistatin family have shown greater promise as clinical candidates with improved efficacy and pharmaceutical properties compared to salicylate (Pettit et al., (1995) Anti-Cancer Drug Des. 10: 529-544; Pettit et al., (1998) Anti-Cancer Drug Des. 13: 243-277; Shnyder (2007), ibid.). Various synthetic analogs of this structural type have been described (US Patent Nos. 6,569,834; 6,124,431; and Pettit et al., (2011) J.Nat.Prod. 74: 962-968).

[0006] Orestatins possess several properties that make them attractive for drug development. First, these compounds are highly potent. Second, their preparation is straightforward due to the peptide scaffold. Third, they exhibit favorable pharmacokinetic and metabolic profiles compared to peptides in general or other specific cancer drug classes.

[0007] Despite their significant advantages, new cancer therapies with the desired drug properties are still needed.

[0008] Overview

[0009] This article provides a novel sea hare proline-sea hare isoleucine peptide analogue. Therefore, this article provides compounds of formula (I):

[0010]

[0011] in

[0012] R 1 and R 2 Each is independently -H or alkyl;

[0013] X is -O-, -NR z -, -S-, or does not exist;

[0014] Where R Z It is -H or alkyl;

[0015] R 3 It is a group of the following formula:

[0016] Where R 15 and R 16 Each can be independently -H, -OH, -NH2, -SH, -N3, alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH2, -alkyl-SH, or -alkyl-N3;

[0017] R 4 It is a group of the following formula:

[0018] Where R 17 and R 18 Each of the following can be independently -H, -OH, -NH2, -SH, -N3, -CO2H, alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH2, -alkyl-SH, -alkyl-N3, or -alkyl-CO2H

[0019] R 5 It is sec-butyl or isobutyl;

[0020] R 6 It is -H or alkyl;

[0021] R 7 and R 8 Each can be independently -H, alkyl, or -CO2R. a CONR b R c substituted or unsubstituted phenyl groups, or substituted or unsubstituted heterocycles;

[0022] Where R a It is -H or alkyl;

[0023] R b and R cEach is independently H or alkyl;

[0024] R 9 It is -H or alkyl; or R 9 With R 4 Together with the atoms they are connected to, they form substituted or unsubstituted heterocyclic alkyl rings;

[0025] R 10 It is -H or alkyl;

[0026] R 11 It is -H or alkyl;

[0027] R 12 It is -H or alkyl;

[0028] R 13 It is -H or alkyl; and

[0029] R 14 It is -H, -OH or alkyl;

[0030] The premise is that X does not exist and R 15 R 16 R 17 and R 18 When each is a methyl group, R 8 It is not a substituted or unsubstituted phenyl group, or a substituted or unsubstituted heterocyclic group;

[0031] Or its pharmaceutically acceptable salt.

[0032] This article also provides pharmaceutical compositions comprising an effective amount of at least one compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0033] This article also provides methods for treating subjects who have or are diagnosed with cancer, including administering to a subject in need of such treatment an effective amount of at least one compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0034] This article also provides the use of at least one compound of formula (I), or a pharmaceutically acceptable salt thereof, for treating cancer in subjects requiring such treatment.

[0035] This article also provides the use of at least one compound of formula (I), or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating cancer in subjects requiring such treatment.

[0036] This article also provides a kit containing at least one compound of formula (I), or a pharmaceutically acceptable salt thereof, for the treatment of cancer in subjects requiring such treatment, along with instructions for use.

[0037] This article also provides an article comprising at least one compound of formula (I), or a pharmaceutically acceptable salt thereof, for the treatment of cancer in subjects requiring such treatment.

[0038] Brief description of the attached figures

[0039] Figure 1 In vitro tubulin polymerization data are shown for tubulin treated with Examples 2, 4, and 6. Untreated (buffered) tubulin shows baseline levels of tubulin polymerization. Tubulin stabilizer (paclitaxel) and tubulin destabilizer (control) were used as controls. All compounds were used at a final concentration of 10 μM.

[0040] Figure 2 In vitro tubulin polymerization data are shown for tubulin treated with Examples 1, 3, and 5. Untreated (buffered) tubulin shows baseline levels of tubulin polymerization. Tubulin stabilizer (paclitaxel) and tubulin destabilizer (control) were used as controls. All compounds were used at a final concentration of 10 μM.

[0041] Figure 3 In vitro tubulin polymerization data for tubulin treated with Examples 8, 9, and 12 are shown. Untreated (buffered) tubulin shows baseline levels of tubulin polymerization. Tubulin stabilizer (paclitaxel) and tubulin destabilizer (control) were used as controls. All compounds were used at a final concentration of 10 μM.

[0042] Figure 4 In vitro tubulin polymerization data are shown for tubulin treated with Examples 7, 10, and 11. Untreated (buffered) tubulin shows baseline levels of tubulin polymerization. Tubulin stabilizer (paclitaxel) and tubulin destabilizer (control) were used as controls. All compounds were used at a final concentration of 10 μM.

[0043] Figure 5 In vitro tubulin polymerization data for tubulin treated with Examples 19, 20, and 21 are shown. Untreated (buffered) tubulin shows baseline levels of tubulin polymerization. Tubulin stabilizer (paclitaxel) and tubulin destabilizer (control) were used as controls. All compounds were used at a final concentration of 10 μM.

[0044] Figure 6In vitro tubulin polymerization data are shown for tubulin treated with Examples 13, 16, and 22. Untreated (buffered) tubulin shows baseline levels of tubulin polymerization. Tubulin stabilizer (paclitaxel) and tubulin destabilizer (control) were used as controls. All compounds were used at a final concentration of 10 μM.

[0045] Figure 7 In vitro tubulin polymerization data for tubulin treated with Examples 14, 15, 18, 20, and 23 are shown. Untreated (buffered) tubulin shows baseline levels of tubulin polymerization. Tubulin stabilizer (paclitaxel) and tubulin destabilizer (control) were used as controls. All compounds were used at a final concentration of 10 μM.

[0046] Figure 8 In vitro tubulin polymerization data for tubulin treated with Examples 17, 19, 25, and 26 are shown. Untreated (buffered) tubulin shows baseline levels of tubulin polymerization. Tubulin stabilizer (paclitaxel) and tubulin destabilizer (control) were used as controls. All compounds were used at a final concentration of 10 μM.

[0047] Figure 9 The results of Examples 1, 2, and 3, and paclitaxel, in in vitro cytotoxicity assays using PC3 cells, as described in Example B1, are shown. Data are plotted as the percentage of survival compared to untreated control wells, relative to the concentration of the test compound.

[0048] Figure 10 The results of Examples 1, 2, and 3, and paclitaxel, in in vitro cytotoxicity assays using HCT15 cells, as described in Example B1, are shown. Data are plotted as the percentage of survival compared to untreated control wells, relative to the concentration of the test compound.

[0049] Figure 11 The results of Examples 1, 2, and 3, and paclitaxel, in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as the percentage of survival compared to untreated control wells, contrasting the test compound concentrations.

[0050] Figure 12 The results of Examples 4, 5, and 6 in in vitro cytotoxicity assays using PC3 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0051] Figure 13The results of Examples 4, 5, and 6 in in vitro cytotoxicity assays using HCT15 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0052] Figure 14 The results of Examples 4, 5, and 6 in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as percentage survival compared to untreated control wells at test compound concentrations.

[0053] Figure 15 Results from Examples 7, 8, and 9 in in vitro cytotoxicity assays using PC3 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0054] Figure 16 The results of Examples 7, 8, and 9 in in vitro cytotoxicity assays using HCT15 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0055] Figure 17 Results from Examples 7, 8, and 9 in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0056] Figure 18 Results from Examples 10, 11, and 12 in in vitro cytotoxicity assays using PC3 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0057] Figure 19 Results from Examples 10, 11, and 12 in in vitro cytotoxicity assays using HCT15 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0058] Figure 20 Results from Examples 10, 11, and 12 in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as percentage survival compared to untreated control wells at test compound concentrations.

[0059] Figure 21Results from Examples 13, 14, and 15 in in vitro cytotoxicity assays using PC3 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0060] Figure 22 Results from Examples 13, 14, and 15 in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0061] Figure 23 Results from Examples 16, 21, and 22 in in vitro cytotoxicity assays using PC3 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0062] Figure 24 Results from Examples 16, 21, and 22 in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0063] Figure 25 Results from Examples 17, 18, and 19 in in vitro cytotoxicity assays using PC3 cells, as described in Example B1, are shown. Data are plotted as percentage survival compared to untreated control wells at test compound concentrations.

[0064] Figure 26 Results from Examples 17, 18, and 19 in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0065] Figure 27 The results of Examples 20 and 23 in in vitro cytotoxicity assays using PC3 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells, relative to test compound concentrations.

[0066] Figure 28 The results of Examples 20 and 23 in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells, relative to test compound concentrations.

[0067] Figure 29Results from Examples 25 and 26, in in vitro cytotoxicity assays using PC3 cells, are shown as described in Example B1. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations.

[0068] Figure 30 The results of Examples 25 and 26 in in vitro cytotoxicity assays using HCC-1954 cells, as described in Example B1, are shown. Data are plotted as survival percentages compared to untreated control wells at test compound concentrations. Invention Details

[0070] For the sake of simplicity, the contents of publications (including patents) cited in this specification are incorporated herein by reference.

[0071] The terms “contains,” “comprising,” and “including” as used in this article are used in their open, non-limiting sense.

[0072] To provide a more concise description, the term “about” is not used before some quantitative expressions in this document. It should be understood that, whether or not the term “about” is explicitly used, each quantity in this document represents an actual given numerical value, and also represents an approximation of a given numerical value that can be reasonably inferred based on ordinary techniques in the art, including equivalents and approximations of such given numerical values ​​due to experimental and / or measurement conditions.

[0073] The term "alkyl" itself, or as part of other terms, refers to a saturated C1-C alkyl group containing an ortho-, secondary-, tertiary-, or cyclic carbon atom. 12 Hydrocarbons. Specifically, alkyl groups are those having 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (tBu), n-pentyl, isopentyl, tert-pentyl, and n-hexyl, isohexyl. In some embodiments, the alkyl group has n, sec, or tertiary carbon atoms and no cyclic carbon atoms.

[0074] The term "alkenyl" itself, or as part of other terms, refers to a C2-C group containing an ortho-, secondary-, tertiary-, or cyclic carbon atom. 12 Hydrocarbons that are unsaturated at at least one position, i.e., carbon-carbon, sp 2 Double bond. Specific alkenyl groups are those having 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Examples include, but are not limited to: vinyl (-CH=CH2), allyl (-CH2CH2=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2). In some embodiments, the alkenyl group has a normal, secondary, or tertiary carbon atom and no cyclic carbon atom.

[0075] The term "alkynyl" itself, or as part of other terms, refers to a C2-C group containing a normal, secondary, tertiary, or cyclic carbon atom. 12 Hydrocarbons having at least one unsaturated site, namely a carbon-carbon, sp triple bond. Specifically, alkynyl groups are those having 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Examples include, but are not limited to, ethynyl (-C≡CH) and 2-propynyl (-CH₂C≡CH). In some embodiments, the alkynyl group has a normal, secondary, or tertiary carbon atom and no cyclic carbon atom.

[0076] The term "alkoxy" refers to -O-alkyl, where O is the connection point to the other parts of the molecule, and alkyl is as defined above.

[0077] The term "heterocyclic alkyl" refers to a saturated or partially saturated monocyclic, fused, bridged, or spirocyclic polycyclic ring structure, each ring containing 3 to 12 ring atoms selected from carbon atoms and up to three heteroatoms (selected from nitrogen, oxygen, and sulfur). Specific heterocyclic alkyl groups are those having 3-8 or 5-7 ring atoms per ring structure. The ring structure may optionally contain oxo groups on up to two carbon or sulfur ring atoms. Exemplary entities with suitable connecting moiety forms include:

[0078]

[0079]

[0080] The term "heteroaryl" refers to a monocyclic, fused bicyclic, or fused polycyclic aromatic heterocycle (the ring structure containing ring atoms selected from carbon atoms and up to four heteroatoms (selected from nitrogen, oxygen, and sulfur), with each heterocycle containing 3 to 12 ring atoms. Specific heteroaryl groups are those having 3-8 or 5-7 ring atoms per ring structure. Exemplary examples of heteroaryl groups include the following entities (in the form of appropriate linking terms):

[0081]

[0082] As used herein, the terms “heterocyclic,” “heterocyclic,” or “heterocyclic group” include “heterocyclic alkyl” and “heteroaryl” groups as defined above.

[0083] Those skilled in the art should recognize that the types of heterocyclic groups, heteroaryl groups, and heterocyclic alkyl groups listed or shown above are not exhaustive, and other types within the scope of these defined terms may also be selected.

[0084] The term "halogen" refers to chlorine, fluorine, bromine, or iodine. The term "halogenated" refers to chlorine, fluorine, bromine, or iodine.

[0085] The term "substituted" indicates that a particular group or part of it contains one or more substituents. The term "unsubstituted" indicates that a particular group does not contain any substituents. The term "optionally substituted" indicates that a particular group is either unsubstituted or substituted by one or more substituents. When using the term "substituted" to describe a structural system, substitution can occur at any position allowed by any valence state in the system.

[0086] Any formula described herein is intended to represent compounds having the structure shown in the formula, and certain variants or forms. Specifically, any compound of any formula shown herein may have an asymmetric center and thus exist in different enantiomeric forms. All optical isomers and enantiomers of the general formula compound, and mixtures thereof, are considered to be within the scope of the formula. Therefore, any formula described herein is intended to represent racemic mixtures, one or more enantiomeric forms, one or more diastereomeric forms, one or more racked isomeric forms, and mixtures thereof. Furthermore, certain structures may exist as geometric isomers (i.e., cis and trans isomers), tautomers, or racked isomers. Additionally, any formula shown herein is also intended to represent hydrates, solvates, and any of the amorphous and polymorphic forms of such compounds, and mixtures thereof, even if such forms are not explicitly listed. In some embodiments, the solvent is water and the solvate is a hydrate.

[0087] Any chemical formulas given herein are intended to represent both the unlabeled and isotopically labeled forms of the compounds. Isotopically labeled compounds have the structure described by the given chemical formulas, except that one or more atoms are replaced by atoms having a chosen atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, respectively, for example... 2 H, 3 H, 11 C 13 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F, 36 Cl and 125 I. These isotope-labeled compounds can be used for metabolic studies (preferably using...) 14 C) Reaction kinetic studies (using, for example) 2 H or 3 H), including in the tissue distribution testing of drugs or substrates using detection or imaging techniques (such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)), or in the treatment of patients with radiation. Specifically, 18 F or11 C-labeled compounds are particularly preferred for PET or SPECT studies. Additionally, heavy isotopes such as deuterium (i.e., 2 H) Substitution can provide certain therapeutic advantages due to higher metabolic stability (e.g., prolonged in vivo half-life or reduced dose requirement). The isotope-labeled compounds and their prodrugs described herein can generally be prepared by the methods disclosed in the following schemes or examples and preparations, wherein a non-isotope-labeled reagent is replaced with an readily available isotope-labeled reagent.

[0088] When referring to any of the formulas shown herein, the selection of a particular part from possible substances for a specific variable is not intended to limit the selection of the same substance for variables appearing elsewhere. In other words, unless otherwise stated, in cases where more than one variable appears, the selection of a substance from a particular list is independent of the selection of a substance for the same variable elsewhere in the formula.

[0089] When using a class of substituents in this paper, the nomenclature "C" is used. i-j "(where j > i)" represents an embodiment of any composition, use, or method described herein that independently achieves each number of carbon atoms from i to j (inclusive). For example, the term C 1-3 The embodiments containing one carbon atom (C1), two carbon atoms (C2), and three carbon atoms (C3) are represented independently.

[0090] Term C n-m Alkyl groups refer to aliphatic chains, whether straight or branched, where the total number of carbons N in the chain satisfies n ≤ N ≤ m, where m > n.

[0091] Use AutoNOM TM The software generates the chemical names listed in this article. If there is a discrepancy between the chemical structure and the listed name for that structure, the structure shall prevail.

[0092] Based on the above explanatory considerations regarding allocation and naming, it should be understood that this document explicitly references a group that is chemically meaningful and, unless otherwise stated, independently references embodiments of that group, and references each of the possible embodiments of the subgroups of that group that are explicitly stated.

[0093] In some implementations, R 1 and R 2 Each is independently -H or alkyl, such as C 1-6 Alkyl group. In some embodiments, R 1 and R 2 Each is independently -H or methyl. In some embodiments, R 1 and R 2 Each is independently an alkyl group. In some embodiments, R 1 and R2 Both are methyl groups. In some embodiments, R 1 and R 2 Both are -H.

[0094] In some implementations, X is absent. In other implementations, X is -O-. In some implementations, R 1 and R 2 Each is independently an alkyl group, and X is absent. In some embodiments, R 1 and R 2 All are methyl groups, and X is absent. In other embodiments, R 1 and R 2 Both are -H, and X is -O-. In some implementations, X is -NR. z -, where R z It is -H or alkyl. In some embodiments, R z It is -H. In some implementations, X is R. z It is an alkyl group, for example, C10. 1-6 Alkyl or methyl.

[0095] In some implementations, R 3 yes Where R 15 and R 16 Each of these can be independently -H, -OH, -NH2, -SH, -N3, alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH2, -alkyl-SH, or -alkyl-N3. 15 and R 16 Each can be independently -H, alkyl, or -(CH2). 0-6 C≡CH、-(CH2) 0-6 CH=CH2、-(CH2) 0-6 OH, -(CH2) 0-6 NH2、-(CH2) 0-6 SH, or -(CH2) 0-6 N3. In some implementations, R 15 and R 16 Each is independently -H, -OH, or an alkyl group. In some embodiments, R 15 and R 16 Each is independently -H, -OH, or methyl. In some embodiments, R 15 It is -OH, and R 16 It is hydrogen. In some implementations, R 15 It is -OH, and R 16 It is a methyl group.

[0096] In some implementations, R 3It is in the R stereochemical configuration relative to the rest of the molecule. In other embodiments, R 3 It is in the S stereochemical configuration relative to the rest of the molecule. In some embodiments, R 3 The group itself contains one or more chiral centers, and these stereocenters are each independently in the R or S configuration.

[0097] In some implementations, R 4 yes Where R 17 and R 18 Each of these is independently -H, -OH, -NH2, -SH, -N3, -CO2H, alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH2, -alkyl-SH, -alkyl-N3, or -alkyl-CO2H. In other embodiments, R 4 yes Where R 17 It is -H, -OH, -NH2, -SH, -N3, -CO2H, alkyl, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH2, -alkyl-SH, -alkyl-N3 or -alkyl-CO2H, and R 18 It is -H, -OH, -NH2, -SH, -N3, -CO2H, alkenyl, alkynyl, -alkyl-OH, -alkyl-NH2, -alkyl-SH, -alkyl-N3, or -alkyl-CO2H. In other embodiments, R 17 and R 18 Each can be independently -H, alkyl, or -(CH2). 0-6 C≡CH、-(CH2) 0-6 CH=CH2、-(CH2) 0-6 OH, -(CH2) 0-6 NH2、-(CH2) 0-6 SH, or -(CH2) 0-6 N3. In some implementations, R 17 and R 18 Each is independently -H, -OH, -NH2, -SH, -N3, -CO2H, alkyl, -alkyl-NH2, or -alkyl-N3. In some embodiments, R 17 and R 18 Each can be independently -H, -OH, -NH2, -SH, -N3, -CO2H, methyl, -CH2NH2, or -CH2N3.

[0098] In some implementations, R 4 With R 9 Together with the atoms they are attached to, they form substituted or unsubstituted heterocyclic alkyl rings. In some embodiments, R 4 With R9 Together with the atoms they are attached to, they form a 5- to 7-membered heterocyclic alkyl ring, which may be unsubstituted or substituted with one or more groups selected from -OH, -NH2, -SH, and -N3. In some embodiments, the heterocyclic alkyl ring is a pyrrole ring, which may be unsubstituted or substituted with one or more groups selected from -OH, -NH2, -SH, and -N3.

[0099] In some implementations, R 4 It is in the R stereochemical configuration relative to the rest of the molecule. In other embodiments, R 4 It is in the S stereochemical configuration relative to the rest of the molecule. In some embodiments, R 4 The group itself contains one or more chiral centers, and these stereocenters are each independently in the R or S configuration.

[0100] In some implementations, R 5 It is sec-butyl. In other embodiments, R 5 It is isobutyl. In some embodiments, R 5 It is in the R stereochemical configuration relative to the rest of the molecule. In other embodiments, R 5 It is in the S stereochemical configuration relative to the rest of the molecule. In some embodiments, R 5 The chiral center within the group is in the R configuration, and in other embodiments, the center is in the S configuration.

[0101] In some implementations, R 6 Yes -H. In other implementations, R 6 It is an alkyl group, for example, C 1-8 Alkyl, C 1-4 Alkyl, methyl, or ethyl.

[0102] In some implementations, R 7 and R 8 Each can be independently -H, alkyl, or -CO2R. a or -CONR b R c ;where R a It is -H or alkyl, for example, C 1-6 Alkyl or methyl; and R b and R c Each is independently -H or alkyl, for example, C 1-6 Alkyl or methyl.

[0103] In some implementations, R 7 and R 8Each is independently a substituted or unsubstituted phenyl or a substituted or unsubstituted heterocyclic group, wherein the phenyl or heterocyclic group may be substituted by one or more groups selected from halogen, oxo, hydroxyl, amino, alkyl, and alkoxy groups. In some other embodiments, R 7 It is an unsubstituted 3- to 8-membered heterocyclic ring. In some other embodiments, R 7 It is a substituted 3- to 8-membered heterocyclic ring. In some other embodiments, R 8 It is a phenyl group that is optionally substituted with a halogen.

[0104] In some implementations, R 7 It is in the R stereochemical configuration relative to the rest of the molecule. In other embodiments, R 7 It is in the S stereochemical configuration relative to the other parts of the molecule.

[0105] In some implementations, R 8 It is in the R stereochemical configuration relative to the rest of the molecule. In other embodiments, R 8 It is in the S stereochemical configuration relative to the other parts of the molecule.

[0106] In some implementations, R 7 It is -CO2R a -CONR b R c ; tetrazolyl or thiazolyl, wherein R a It is -H or alkyl, such as C 1-6 Alkyl or methyl; and R b and R c Each is independently -H or alkyl, for example, C 1-6 Alkyl or methyl; and R 8 It is a phenyl group that is optionally substituted with a halogen.

[0107] In some implementations, R 9 Yes -H. In other implementations, R 9 It is an alkyl group, for example, C 1-8 Alkyl, C 1-4 Alkyl, methyl, or ethyl. In some embodiments, R 9 It is -H or methyl. In some embodiments, R 9 It is a methyl group.

[0108] In some implementations, R 10 Yes -H. In other implementations, R 10 It is an alkyl group, for example, C 1-8 Alkyl, C 1-4 Alkyl, methyl, or ethyl. In some embodiments, R 10 It is -H or methyl. In some embodiments, R10 It is a methyl group.

[0109] In some implementations, R 11 Yes -H. In other implementations, R 11 It is an alkyl group, for example, C 1-8 Alkyl, C 1-4 Alkyl, methyl, or ethyl. In some embodiments, R 11 It is -H or methyl. In some embodiments, R 11 It is a methyl group.

[0110] In some implementations, R 12 Yes -H. In other implementations, R 12 It is an alkyl group, for example, C 1-8 Alkyl, C 1-4 Alkyl, methyl, or ethyl. In some embodiments, R 12 It is -H or methyl. In some embodiments, R 12 It is a methyl group.

[0111] In some implementations, R 13 Yes -H. In other implementations, R 13 It is an alkyl group, for example, C 1-8 Alkyl, C 1-4 Alkyl, methyl, or ethyl. In some embodiments, R 13 It is -H or methyl. In some embodiments, R 13 It is a methyl group.

[0112] In some implementations, R 14 Yes -H. In some implementations, R 14 It is an alkyl group, for example, C 1-6 Alkyl, methyl, or ethyl. In some embodiments, R 14 It is -OH.

[0113] In some implementations, R 14 It is in the R stereochemical configuration relative to the rest of the molecule. In other embodiments, R 14 It is in the S stereochemical configuration relative to the other parts of the molecule.

[0114] In some implementations, R 7 It is -CO2R a , where R a It is -H or alkyl, such as C 1-6 Alkyl or methyl; R 8 It is phenyl; and R 14 Yes -H. In some implementations, R 7 Yes - CONR b Rc , where R b and R c Each is independently -H or alkyl, such as C 1-6 Alkyl or methyl; R 8 It is phenyl; and R 14 Yes -H. In some implementations, R 7 It is an alkyl group, for example, C10. 1-6 Alkyl or methyl; R 8 It is phenyl; and R 14 It is -OH. In some embodiments, R 7 It is methyl, R 8 It is a phenyl group, and R 14 It is -OH. In some embodiments, R 7 and R 14 Both are -H, and R 8 It is a pyridyl, piperidinyl, unsubstituted phenyl, or phenyl substituted with a halogen such as fluorine, chlorine, or bromine. In some embodiments, R 7 It is -CO2R a , where R a It is -H or alkyl, for example, C 1-6 Alkyl or methyl; R 8 It is -H or alkyl, for example, C 1-6 Alkyl or methyl; and R 14 It is an alkyl group, for example, C 1-6 Alkyl, methyl, or ethyl. In some embodiments, R 7 It is -CO2R a , where R a It is -H or alkyl, for example, C 1-6 Alkyl or methyl; R 8 It is -H or alkyl, for example, C 1-6 Alkyl or methyl; and R 14 It is -OH.

[0115] In some implementations,

[0116] R 1 and R 2 Each is independently -H or C 1-6 alkyl;

[0117] X is -O-, or does not exist;

[0118] R 3 yes Where R 15 and R 16 Each can be independently -H, -OH, or C. 1-6 alkyl;

[0119] R4 yes Where R 17 It is -OH, -NH2, -SH, -N3, -CO2H, -C 1-6 Alkyl-NH2, alkynyl, alkenyl, or -C 1-6 Alkyl-N3; and R 18 Is it -H or C? 1-6 alkyl;

[0120] R 5 It is sec-butyl;

[0121] R 6 It is -H;

[0122] R 7 It is -H, C 1-6 Alkyl, -CO2R a -CONR b R c , tetrazolyl or thiazolyl; wherein R a Is it -H or C? 1-6 Alkyl; and R b and R c Each is either -H or C 1-6 alkyl;

[0123] R 8 It is -H, C 1-6 Alkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted heterocyclic compounds;

[0124] R 9 It is -H;

[0125] R 10 R 11 R 12 and R 13 Each is C independently 1-6 Alkyl; and

[0126] R 14 It is H, C 1-6 Alkyl or -OH.

[0127] In some implementations,

[0128] R 1 and R 2 Each is independently either -H or methyl;

[0129] X is -O-, or does not exist;

[0130] R 3 yes Where R 15 and R 16 Each can be independently -H, -OH, or methyl;

[0131] R 4 yes Where R 17 It is -OH, -NH2, -SH, -N3, -CO2H, aminomethyl, alkynyl, alkenyl, or azidomethyl; and R 18 It is -H or methyl;

[0132] R 5 It is sec-butyl;

[0133] R 6 It is -H;

[0134] R 7 It is -H, methyl, -CO2R a 、or -CONR b R c ;where R a It is -H or methyl; and R b and R c Each is either -H or methyl;

[0135] R 8 It is -H, methyl, ethyl, pyridyl, piperidinyl, unsubstituted phenyl, or halogen-substituted phenyl;

[0136] R 9 It is -H;

[0137] R 10 R 11 R 12 and R 13 Each is a methyl group; and

[0138] R 14 It is -H, methyl, or -OH.

[0139] In some implementations,

[0140] R 1 and R 2 Each is independently -H or C 1-6 alkyl;

[0141] X does not exist;

[0142] R 3 yes Where R 15 and R 16 Each can be independently -H, -OH, or C. 1-6 alkyl;

[0143] R 4 yes Where R 17 It is -N3, and R 18It is -H or methyl;

[0144] R 5 It is sec-butyl;

[0145] R 6 It is -H;

[0146] R 7 It is -H, C 1-6 Alkyl, -CO2R a -CONR b R c , tetrazolyl or thiazolyl; wherein R a Is it -H or C? 1-6 Alkyl; and R b and R c Each is either -H or C 1-6 alkyl;

[0147] R 8 It is -H, C 1-6 Alkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted heterocyclic compounds;

[0148] R 9 It is -H;

[0149] R 10 R 11 R 12 and R 13 Each is C independently 1-6 Alkyl; and

[0150] R 14 It is H, C 1-6 Alkyl or -OH.

[0151] In some implementations,

[0152] R 1 and R 2 Each is independently -H or C 1-6 alkyl;

[0153] X is -O-;

[0154] R 3 yes Where R 15 and R 16 Each can be independently -H, -OH, or C. 1-6 alkyl;

[0155] R 4 yes Where R 17 It is -N3, and R 18 It is -H or methyl;

[0156] R5 It is sec-butyl;

[0157] R 6 It is -H;

[0158] R 7 It is -H, C 1-6 Alkyl, -CO2R a -CONR b R c , tetrazolyl or thiazolyl; wherein R a Is it -H or C? 1-6 Alkyl; and R b and R c Each is either -H or C 1-6 alkyl;

[0159] R 8 It is -H, C 1-6 Alkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted heterocyclic compounds;

[0160] R 9 It is -H;

[0161] R 10 R 11 R 12 and R 13 Each is C independently 1-6 Alkyl; and

[0162] R 14 It is H, C 1-6 Alkyl or -OH.

[0163] In some embodiments of equation (I), wherein,

[0164] R 1 and R 2 Each of them is a methyl group;

[0165] X does not exist;

[0166] R 3 It is a group of the following formula:

[0167] Where R 15 and R 16 Each is a methyl group;

[0168] R 4 It is a group of the following formula:

[0169] Where R 17 It is -N3, -NH2, -OH, -SH, and R 18 It is -H or methyl;

[0170] R5 It is sec-butyl;

[0171] R 6 It is -H;

[0172] R 7 It is -CO2R a or CONR b R c ,

[0173] Where R a Is it -H or C? 1-6 alkyl;

[0174] R b and R c Each is independently H or C 1-6 alkyl;

[0175] R 8 It is phenyl;

[0176] R 9 It is -H;

[0177] R 10 R 11 R 12 and R 13 Each is independently a methyl group; and

[0178] R 14 Yes, it's -H.

[0179] In some embodiments of equation (I), wherein,

[0180] R 1 and R 2 Each is -H;

[0181] X is -O-;

[0182] R 3 It is a group of the following formula:

[0183] Where R 15 and R 16 Each is a methyl group;

[0184] R 4 It is a group of the following formula:

[0185] Where R 17 It is -N3, and R 18 It is -H or methyl;

[0186] R 5 It is sec-butyl;

[0187] R 6 It is -H;

[0188] R 7 It is -CO2R a or CONR b R c ,

[0189] Where R a Is it -H or C? 1-6 alkyl;

[0190] R b and R c Each is independently H or C 1-6 alkyl;

[0191] R 8 It is phenyl;

[0192] R 9 It is -H;

[0193] R 10 R 11 R 12 and R 13 Each is independently a methyl group; and

[0194] R 14 Yes, it's -H.

[0195] It should be understood that any variable group definition provided herein can be used in combination with any other variable group definition provided herein, thereby taking into account all possible combinations and substitutions of the variable groups provided herein that are chemically feasible.

[0196] In some embodiments, the compounds of formula (I) are selected from the group consisting of:

[0197] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0198] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0199] (S)-2-(dimethylamino)-N-((S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolid-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide;

[0200] (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0201] (2S)-2-(dimethylamino)-N-((2S)-3-hydroxy-1-(((3R,4S5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide;

[0202] (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0203] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0204] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0205] (S)-N-((S)-3-amino-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolid-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino)-1-oxopropyl-2-yl)-2-(dimethylamino)-3-methylbutyramide;

[0206] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0207] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0208] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0209] (S)-2-((S)-2-(aminooxy)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxohep-4-yl)-N,3-dimethylbutyramide;

[0210] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropionylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine;

[0211] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine;

[0212] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid;

[0213] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutyrylamino)-N,3-dimethylbutyramide;

[0214] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-2-((S)-2-(dimethylamino)-3-hydroxypropionylamino)-N,3-dimethylbutyramide;

[0215] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-mercapto-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0216] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-mercapto-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid;

[0217] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid;

[0218] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid;

[0219] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropionylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0220] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxybutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0221] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester;

[0222] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid;

[0223] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0224] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0225] (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenylethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0226] (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenylethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0227] (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0228] (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0229] (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenylethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0230] (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenylethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0231] (S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethylamino)propyl)pyrrolidone-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0232] (S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0233] (S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenylethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0234] (S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenylethyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0235] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpentan-4-ynylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine methyl ester;

[0236] (2S,3S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0237] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0238] (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0239] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-valine methyl ester;

[0240] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-6-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylhexanoylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine methyl ester;

[0241] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,4S)-4-azido-1-(dimethyl-L-valine)-N-methylpyrrolidine-2-carbamoyl)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine methyl ester;

[0242] (S)-3-((S)-2-(dimethylamino)-3-methylbutyrylamino)-4-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylprop-2-yl)amino)-2-methyl-3-oxopropyl)pyrrolid-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino)-4-oxobutyric acid;

[0243] (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0244] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-serine methyl ester;

[0245] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-isoleucine methyl ester;

[0246] (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0247] (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide;

[0248] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)propionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine methyl ester;

[0249] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine methyl ester;

[0250] (2S,3S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide;

[0251] ((2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino)-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide;

[0252] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine tert-butyl ester;

[0253] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine;

[0254] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine tert-butyl ester;

[0255] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxohep-4-yl)-N-methylbutyramide;

[0256] (2S,3S)-3-azido-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolid-1-yl)-5-methyl-1-oxohep-4-yl)-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide;

[0257] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazo-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide;

[0258] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine tert-butyl ester;

[0259] (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide; and

[0260] (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazo-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohep-4-yl)-N-methylbutyramide;

[0261] And its pharmaceutically acceptable salts.

[0262] The present invention also provides pharmaceutically acceptable salts of compounds of formula (I), preferably those described above and the specific compounds shown herein, pharmaceutical compositions comprising said salts, and methods of using said salts.

[0263] "Pharmaceutically acceptable salt" is intended to refer to a salt of the free acid or base of the compounds described herein that is non-toxic, biologically tolerable, or biologically suitable for administration to a subject. See also SMBerge et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmaceutically effective and suitable for contact with subject tissues without excessive toxicity, irritation, or anaphylactic reactions. The compounds described herein may have sufficiently acidic groups, sufficiently basic groups, or both types of functional groups, and therefore react with a variety of inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include acid addition salts such as sulfates, pyrosulfates, bisulfates, sulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propynylates, oxalates, malonates, succinates, octanoates, sebacic acid salts, fumarates, maleates, 1,4-butynediate, 1,6-hexynediate, benzoates, chlorobenzoates, methylbenzoates, and diethylbenzoates. Nitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methanesulfonates, propylsulfonates, benzenesulfonates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, and mandelates, as well as salts with inorganic bases such as sodium, potassium, magnesium, calcium, aluminum, etc., or organic bases such as methylamine, ethylamine, ethanolamine, lysine, ornithine, etc., and salts with various amino acids or amino acid derivatives such as acetylleucine, ammonium salts, etc.

[0264] For therapeutic purposes, pharmaceutical compositions including the compounds described herein may also include one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are substances that are non-toxic and biologically suitable for administration to a subject. Such excipients facilitate the formulation and administration of the compounds described herein and are compatible with the active ingredient. Examples of pharmaceutically acceptable excipients include stabilizers, lubricants, surfactants, diluents, antioxidants, binders, colorants, emulsifiers, or flavoring agents. In a preferred embodiment, the pharmaceutical composition is a sterile composition.

[0265] The pharmaceutical compositions described herein can be formulated as solutions, emulsions, suspensions, or dispersants in suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, suppositories, powders for reconstitution, or capsules together with a solid carrier, according to conventional methods in the art for the preparation of various dosage forms. For topical use, the compounds described herein are preferably formulated as creams or ointments or similar carriers suitable for topical administration. The pharmaceutical compositions and compounds described herein can be administered via suitable routes of delivery in the methods of the invention, such as oral, nasal, parenteral, rectal, topical, ocular, or inhalation.

[0266] As used herein, the terms “treatment” or “treatment” are intended to refer to the administration of the compound described herein to a subject for the purpose of producing a therapeutic benefit. Treatment includes reversing, alleviating, reducing, inhibiting the development of one or more symptoms of a disease, disorder, or symptom, or cancer, or reducing their severity. The term “subject” refers to a mammalian patient, such as a human, who requires the treatment.

[0267] In the treatment methods described herein, "effective amount" refers to an amount or dose sufficient to provide the desired therapeutic benefit to a subject requiring such treatment. The effective amount or dose of the compound described herein can be determined using conventional methods (such as modeling, dose escalation, or clinical trials), taking into account conventional factors such as the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the infection, the subject's health status, condition, and weight, and the attending physician's judgment. Exemplary doses range from about 1 μg to 2 mg of the active compound per kilogram of subject body weight per day, preferably about 0.05 to 100 mg / kg / day, or about 1 to 35 mg / kg / day, or about 0.1 to 10 mg / kg / day. The total dose can be administered in single or separate dose units (e.g., BID, TID, QID).

[0268] The compounds described herein may be used in combination with other active ingredients in cancer treatment in pharmaceutical compositions or methods. These other active ingredients may be administered separately from the compounds described herein or may be included together with the compounds described herein in the pharmaceutical composition. For example, these other active ingredients are known or found to be effective in treating cancer, including those active against other cancer-related targets, such as, but not limited to, velcade, rituximab, methotrexate, herceptin, vincristine, prednisone, irinotecan, and combinations thereof. Such combinations may be used to increase potency, reduce one or more side effects, or reduce the required dosage of the compounds of the present invention.

[0269] The compounds described herein can be used in combination with other active ingredients in cancer treatment for pharmaceutical compositions or methods. These other active ingredients may be administered separately from the compounds described herein or may be included together with the compounds described herein in the pharmaceutical compositions described herein. For example, these other active ingredients are known or found to be effective in treating cancer, including those active against other cancer-related targets, such as, but not limited to, velcade, rituximab, methotrexate, herceptin, vincristine, prednisone, irinotecan, and combinations thereof. Such combinations can be used to increase potency, reduce one or more side effects, or reduce the required dosage of the compounds of the present invention.

[0270] The compounds of formula (I) will now be described with reference to illustrative synthetic schemes used in the general preparation methods described herein and subsequent specific examples. Those skilled in the art will recognize that, to obtain the various compounds described herein, the starting materials can be appropriately selected to utilize the final desired substituents via suitable reaction schemes with or without protection to generate the desired products. Alternatively, it may be necessary or desirable to employ suitable groups at the final desired substituent positions that can be carried by the reaction scheme and, where appropriate, replaced by the desired substituents. Furthermore, those skilled in the art will recognize that protecting groups can be used to protect certain functional groups (amino, carboxyl, or side-chain groups) from the reaction conditions, and that such groups can be removed under standard conditions, where appropriate. The reactions described in Scheme A are preferably carried out at temperatures ranging from about room temperature to the reflux temperature of the organic solvent used. Unless otherwise stated, variables as defined above can be referenced to formula (I).

[0271] Option A

[0272]

[0273] According to Scheme A, the preparation of the compound of formula (I) begins with the protected acid form of (A) labeled with isoleucine (Dil) from the sea hare (see Pettit et al., (1994) J. Org. Chem. 59: 1796-1800). Compound (A) shows a tert-butyl ester protecting group, but those skilled in the art can choose a suitable alternative. Coupling with a nitrogen-protected valine or isoleucine derivative (B) is achieved under standard peptide coupling conditions, wherein PG is a suitable amino protecting group, such as Boc (tert-butoxycarbonyl) or fluorenylmethoxycarbonyl (Fmoc) group. For example, the reaction is carried out in the presence of diethyl cyanophosphonate (DEPC), PyBrOP, PyBOP, BOP, diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), HBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethylureon hexafluorophosphate), HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylureon hexafluorophosphate), or combinations thereof. The reaction is generally carried out in the presence of a tertiary amine base, such as diisopropylethylamine. Suitable solvents include dichloromethane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl acetate, etc. The amino protecting group on the resulting dipeptide (C) is removed under suitable deprotection conditions. For example, if PG is a Boc group, compound (C) is treated with trifluoroacetic acid to form a free amine (D). If PG is an Fmoc group, compound (C) is treated with piperidine or diethylamine to produce compound (D). Under the peptide coupling conditions described above, compound (D) is then coupled to an amino acid derivative (E) (in a protected form if desired) to generate a tripeptide (F). The carboxyl protecting group is removed by acid treatment to provide a free acid (G).

[0274] Option B

[0275]

[0276] Referring to Scheme B, under the peptide coupling conditions described above, an amino-protected sea hare proline (Dap) named (H) (see Pettit et al., (1994) J. Org. Chem. 59: 6287-6295) is coupled to an amine (J) (which is prepared using methods known in the art). As described in Scheme A, the resulting dipeptide (K) is protected to provide compound (L).

[0277] Option C

[0278]

[0279] According to Scheme C, the acid (G) and amine (L) are coupled under the peptide coupling conditions described above to provide a compound of formula (I). In cases where the reaction product is the protected form of formula (I), suitable deprotection conditions are employed to obtain the target compound. Example

[0280] The following examples are provided to illustrate, but do not limit, the compositions, uses, and methods provided herein. The compounds were prepared using the general methods described above.

[0281] The following chemical abbreviations are used in the examples: Dov (valine from sea hare); Abu (2-aminobutyric acid); Dil (isoleucine from sea hare); Dpr (2,3-diaminopropionic acid); Su (succinimide); Dab (2,4-diaminobutyric acid); Dap (proline from sea hare); Bzl (benzyl); and Tr (triphenylmethyl).

[0282] Obtain the LCMS residence time on an Aquity UPLC BeH C8 1.7μm 2.1x50mm column at 40°C using one of the following methods (shown):

[0283] Method A: 0-0.50 min: isocratic 80 water / 10 acetonitrile / 10 water 1% formic acid; 0.50-3.50 min: linear gradient 80 water / 10 acetonitrile / 10 water 1% formic acid to 0 water / 90 acetonitrile / 10 water 1% formic acid; 3.50-3.99 min isocratic 0 water / 90 acetonitrile / 10 water 1% formic acid; 3.99-4.00 min linear gradient 0 water / 90 acetonitrile / 10 water 1% formic acid to 80 water / 10 acetonitrile / 10 water 1% formic acid.

[0284] Method B: 0-0.50 min: isocratic 85% water / 5% acetonitrile / 10% water with 1% formic acid; 0.50-1.60 min: linear gradient 85% water / 5% acetonitrile / 10% water with 1% formic acid to 0% water / 98% acetonitrile / 2% water with 1% formic acid; 1.60-1.80 min isocratic 0% water / 98% acetonitrile / 2% water with 1% formic acid; 1.80-1.90 min linear gradient 0% water / 98% acetonitrile / 10% water with 1% formic acid to 85% water / 10% acetonitrile / 5% water with 1% formic acid; 1.90-2.00 min isocratic 85% water / 5% acetonitrile / 10% water with 1% formic acid.

[0285] Example 1

[0286] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methyl (3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy Methyl 2-methylpropionamide (2-methylpropionamide)-3-phenylpropionate

[0287]

[0288] Diisopropylethylamine (DIEA; 9.13 mL, 51.3 mmol) was added to a stirred suspension of Boc-Dap-OH dicyclohexylamine salt (8.00 g, 17.1 mmol) and H-Phe-OMe HCl salt (4.42 g, 20.5 mmol) at room temperature in CH2Cl2 (20 mL), followed by diethyl pyrocarbonate (DEPC; 5.15 mL, 34.2 mmol). After 10 hours, analysis by liquid chromatography / mass spectrometry (LCMS) showed the reaction was complete. Using 2% to 10% MeOH in CH2Cl2 as the eluent, the solution was passed through silica gel (40 μm silica gel). Rapid chromatography on a 3.0 x 17.0 cm plate was used to separate Boc-Dap-Phe-OMe. A total of 7.45 g of Boc-Dap-Phe-OMe (16.61 mmol, 97% yield) was obtained.

[0289] Trifluoroacetic acid (TFA; 10 mL) was added to a stirred solution of Boc-Dap-Phe-OMe (4.67 g, 10.4 mmol) at room temperature in CH2Cl2 (10 mL). After 10 hours, analysis by LCMS showed the reaction was complete. The result was confirmed by using Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a column (150x30 mm) with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.52 g of TFA salt of H-Dap-Phe-OMe (6.23 mmol, 59%) was obtained.

[0290] DIEA (2.17 mL, 12.2 mmol) was added to a stirred solution of Fmoc-Ser(Bzl)-OH (2.82 g, 6.76 mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) at room temperature. The solution was cooled to 0 °C and stirred for 20 min. DIEA (2.17 mL, 12.2 mmol) was added to the reaction mixture. The solution was cooled to 0 °C and stirred for 20 min. 2-Chloro-1-methylpyridinium iodide (CMPI; 2.76 g, 10.8 mmol) was added to the reaction mixture, and the reaction mixture was allowed to reach room temperature. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was washed with 0.1 M HCl (150 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. Using 18% to 90% EtOAc in hexane as the eluent, the sample was passed through silica gel (40 μm silica gel). Fmoc-Ser(Bzl)-Dil-OtBu was separated by rapid chromatography on a 3.0 x 17.0 cm plate. A total of 3.75 g of Fmoc-Ser(Bzl)-Dil-OtBu (5.69 mmol, 84% yield) was obtained.

[0291] Piperidine (4 mL) was added to a stirred solution of Fmoc-Ser(Bzl)-Dil-OtBu (1.79 g, 2.72 mmol) at room temperature in MeCN (5 mL). After 5 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was extracted with hexane and the MeCN layer was concentrated under vacuum to produce crude H-Ser(Bzl)-Dil-OtBu, which did not require further purification.

[0292] DIEA (1.45 mL, 8.16 mmol) was added to a stirred suspension of crude H-Ser(Bzl)-Dil-OtBu and Dov (0.790 g, 5.44 mmol) at room temperature in DMF (10 mL), followed by HATU (2.07 g, 5.44 mmol). After 5 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a column (150x30 mm) with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.882 g of TFA salt of Dov-Ser(Bzl)-Dil-OtBu (1.30 mmol, 48%) was obtained.

[0293] TFA (4 mL) was added to a 5 mL solution of Dov-Ser(Bzl)-Dil-OtBu (0.288 g, 0.425 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were vacuum evaporated to produce a crude Dov-Ser(Bzl)-Dil-OH TFA salt that required no further purification.

[0294] DIEA (1.45 mL, 1.70 mmol) was added to a stirred suspension of crude Dov-Ser(Bzl)-Dil-OH TFA salt and H-Dap-Phe-OMe TFA salt (0.163 g, 0.467 mmol) at room temperature in DMF (10 mL), followed by HATU (0.323 g, 0.850 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.217 g of TFA salt of Dov-Ser(Bzl)-Dil-Dap-Phe-OMe (0.228 mmol, 54%) was obtained.

[0295] Hydrogenation was carried out under reflux conditions on a stirred suspension of Dov-Ser(Bzl)-Dil-Dap-Phe-OMe TFA salt (0.217 g, 0.228 mmol) and palladium supported on activated carbon (10% Pd-based, 0.174 g) at room temperature. After 48 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat and the filtrate was concentrated. Hydrogenation was performed using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a column (150 x 30 mm) with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.104 g of the TFA salt of the title compound was obtained (0.121 mmol, 47%). LCMS RT = 2.32 min (Method A); ESI-MS m / z 748.72 [M+H] + ;HRMS m / z748.4846[C 39 H 65 N5O9+H] + .

[0296] Example 2

[0297] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)- 3-Methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3- Methyl methoxy-2-methylpropionamide (MDMA)-3-phenylpropionate

[0298]

[0299] DIEA (2.17 mL, 12.2 mmol) was added to a stirred solution of EtOAc (15 mL) containing Fmoc-Thr(Bzl)-OH (2.92 g, 6.76 mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) at room temperature. The solution was cooled (0 °C) and stirred for 20 min. DIEA (2.17 mL, 12.2 mmol) was added to the reaction mixture. The solution was cooled (0 °C) and stirred for 20 min. CMPI (2.76 g, 10.8 mmol) was added to the reaction mixture and the reaction mixture was allowed to reach room temperature. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was washed with 0.1 M HCl (100 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. Using 18% to 90% EtOAc in hexane as the eluent, the mixture was passed through silica gel (40 μm silica gel). Fmoc-Thr(Bzl)-Dil-OtBu was separated by rapid chromatography on a 3.0 x 17.0 cm plate. A total of 3.71 g of Fmoc-Thr(Bzl)-Dil-OtBu (5.51 mmol, 82% yield) was obtained.

[0300] Piperidine (4 mL) was added to a stirred solution of Fmoc-Thr(Bzl)-Dil-OtBu (3.71 g, 5.51 mmol) at room temperature in MeCN (5 mL). After 5 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was extracted with hexane and the MeCN layer was concentrated under vacuum to produce crude H-Thr(Bzl)-Dil-OtBu, which did not require further purification.

[0301] DIEA (2.95 mL, 16.5 mmol) was added to a stirred suspension of crude H-Thr(Bzl)-Dil-OtBu and Dov (1.60 g, 11.0 mmol) at room temperature in DMF (20 mL), followed by HATU (4.19 g, 11.0 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 1.09 g of TFA salt of Dov-Ser(Bzl)-Dil-OtBu was obtained (1.58 mmol, 29%).

[0302] TFA (4 mL) was added to a 5 mL solution of Dov-Thr(Bzl)-Dil-OtBu TFA salt (0.331 g, 0.478 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude Dov-Thr(Bzl)-Dil-OH TFA salt, which did not require further purification.

[0303] DIEA (0.341 mL, 1.92 mmol) was added to a stirred suspension of crude Dov-Thr(Bzl)-Dil-OH TFA salt and H-Dap-Phe-OMe TFA salt (0.183 g, 0.526 mmol) at room temperature in DMF (10 mL), followed by HATU (0.364 g, 0.957 mmol). After 24 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.290 g of TFA salt of Dov-Thr(Bzl)-Dil-Dap-Phe-OMe (0.300 mmol, 63%) was obtained.

[0304] Hydrogenation was performed on a suspension of Dov-Thr(Bzl)-Dil-Dap-Phe-OMe TFA salt (0.290 g, 0.300 mmol) and palladium supported on activated carbon (10% Pd-based, 0.232 g) in MeOH (5 mL) under stirring at room temperature. After 24 hours, analysis by LC-MS showed the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat, and the filtrate was concentrated. Hydrogenation was carried out using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a column (150 x 30 mm) with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.129 g of the TFA salt of the title compound was obtained (0.147 mmol, 43%). LCMS RT = 2.40 min (Method A); ESI-MS m / z 762.75 [M+H] + HRMS m / z 762.5009[C 40 H 67 N5O9+H] + .

[0305] Example 3

[0306] (S)-2-(dimethylamino)-N-((S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((S)-2- ((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidine-1-yl)- 5-Methyl-1-oxohept-4-yl)(methyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide

[0307]

[0308] DIEA (11.4 mL, 64.1 mmol) was added to a stirred suspension of Boc-Dap-OH dicyclohexylamine salt (10.0 g, 21.4 mmol) and 2-(2-pyridyl)ethylamine (3.83 mL, 32.0 mmol) at room temperature in CH2Cl2 (20 mL), followed by DEPC (4.83 mL, 32.0 mmol). After overnight stirring, the reaction was complete as shown by LCMS analysis. Elution was performed using 2% to 10% MeOH / 1% NEt3 in CH2Cl2 as the elution buffer, followed by elution with silica gel (40 μm silica gel). Boc-Dap-2-(2-pyridyl)ethylamine was separated by rapid chromatography on a 3.0 x 17.0 cm plate. A total of 7.42 g of Boc-Dap-2-(2-pyridyl)ethylamine (19.0 mmol, 89% yield) was obtained.

[0309] TFA (10 mL) was added to a stirred solution of Boc-Dap-2-(2-pyridyl)ethylamine (7.42 g, 19.0 mmol) at room temperature in 10 mL of CH₂Cl₂. After stirring overnight, the reaction was confirmed to be complete by LCMS analysis. The reaction was confirmed by using a Phenomenex Synergi 10 μM Max-RP... The crude oily substance was purified by preparative RP-HPLC using a column (150x30 mm) with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 4.50 g of TFA salt of H-Dap-(2-pyridyl)ethylamine (11.1 mmol, 59%) was obtained.

[0310] TFA (4 mL) was added to a 5 mL solution of Dov-Ser(Bzl)-Dil-OtBu (0.287 g, 0.423 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were vacuum evaporated to produce a crude Dov-Ser(Bzl)-Dil-OH TFA salt that required no further purification.

[0311] DIEA (0.302 mL, 1.70 mmol) was added to a stirred suspension of crude Dov-Ser(Bzl)-Dil-OH TFA salt and H-Dap-2-(2-pyridyl)ethylamine TFA salt (0.136 g, 0.466 mmol) at room temperature in DMF (10 mL), followed by HATU (0.322 g, 0.847 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.258 g of the TFA salt of Dov-Ser(Bzl)-Dil-Dap-2-(2-pyridyl)ethylamine (0.288 mmol, 68%) was obtained.

[0312] Hydrogenation was carried out in a suspension of Dov-Ser(Bzl)-Dil-Dap-2-(2-pyridyl)ethylamine TFA salt (0.258 g, 0.330 mmol), ammonium formate (0.062 g, 0.991 mmol), and palladium supported on activated carbon (10% Pd-based, 0.100 g) in MeOH (5 mL) under stirring at room temperature. After 12 hours, the reaction was confirmed to be complete by LC-MS analysis. The crude reaction mixture was filtered on a diatomaceous earth mat, and the filtrate was concentrated. Hydrogenation was performed using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.014 g of the TFA salt of the title compound was obtained (0.017 mmol, 5%). LCMS RT = 1.76 min (Method A); ESI-MS m / z 691.56 [M+H] + HRMS m / z 691.4755[C 36 H 62 N6O7+H] + .

[0313] Example 4

[0314] (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-3- Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propane (1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide

[0315]

[0316] TFA (4 mL) was added to a 5 mL solution of Dov-Thr(Bzl)-Dil-OtBu (0.357 g, 0.618 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were then vacuum evaporated to produce a crude Dov-Thr(Bzl)-Dil-OH TFA salt that required no further purification.

[0317] DIEA (0.441 mL, 2.47 mmol) was added to a stirred suspension of crude Dov-Thr(Bzl)-Dil-OH TFA salt and H-Dap-2-(2-pyridyl)ethylamine TFA salt (0.198 g, 0.680 mmol) at room temperature in DMF (10 mL), followed by the addition of HATU (0.470 g, 1.24 mmol) and HOBt (0.189 g, 1.24 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a column (150x30 mm) with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.356 g of the TFA salt of Dov-Thr(Bzl)-Dil-Dap-2-(2-pyridyl)ethylamine (0.392 mmol, 63%) was obtained.

[0318] Hydrogenation was performed in a suspension of Dov-Thr(Bzl)-Dil-Dap-2-(2-pyridyl)ethylamine TFA salt (0.356 g, 0.392 mmol), ammonium formate (0.085 g, 1.35 mmol), and palladium supported on activated carbon (10% Pd-based, 0.175 g) at room temperature with stirring in MeOH (5 mL). After 12 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat, and the filtrate was concentrated. The concentration was determined by electrochemical analysis using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.010 g of the TFA salt of the title compound was obtained (0.012 mmol, 3%). LCMS RT = 1.72 min (Method A); ESI-MS m / z 705.56 [M+H] +728.33 [M+Na] + HRMS m / z 705.4917[C 37 H 64 N6O7+H] + .

[0319] Example 5

[0320] (2S)-2-(dimethylamino)-N-((2S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((2S)-2- ((1R , 2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidine-1-yl)- 5-Methyl-1-oxohept-4-yl)(methyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide

[0321]

[0322] The title compound was obtained as a byproduct of the hydrogenation reaction in Example 3. A total of 0.010 g of the TFA salt of the title compound (0.001 mmol) was obtained. LCMS RT = 1.83 min (Method A); ESI-MS m / z 697.76 [M+H] + ;HRMS m / z697.5226[C 36 H 68 N6O7+H] + .

[0323] Example 6

[0324] (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-3- Methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino) (propyl)pyrrolidone-1-yl)-5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0325]

[0326] The title compound was obtained as a byproduct of the hydrogenation reaction in Example 4. A total of 0.010 g of the TFA salt of the title compound (0.012 mmol) was obtained. LCMS RT = 1.81 min (Method A); ESI-MS m / z 711.43 [M+H] + HRMS m / z 711.5389[C 37 H 70 N6O7+H] + .

[0327] Example 7

[0328] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(di) (methylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)- Methyl 3-methoxy-2-methylpropionamide (3-phenylpropionate)

[0329]

[0330] DIEA (0.880 mL, 4.93 mmol) was added to a stirred solution of (2S,3S)-Fmoc-Abu(3-N3)-OH (1.00 g, 2.74 mmol) and H-Dil-OtBu hydrochloride (0.810 g, 2.74 mmol) at room temperature. The solution was cooled (0 °C) and stirred for 20 min. DIEA (0.880 mL, 4.93 mmol) was added to the reaction mixture. The solution was cooled (0 °C) and stirred for 20 min. CMPI (1.12 g, 4.38 mmol) was added to the reaction mixture, and the reaction mixture was allowed to reach room temperature. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was washed with 0.1 M HCl (100 mL x 2) followed by brine (20 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and vacuum concentrated to produce crude Fmoc-Abu(3-N3)-Dil-OtBu (1.12 g, 1.84 mmol) which did not require further purification.

[0331] Piperidine (2 mL) was added to a stirred solution of Fmoc-Abu(3-N3)-Dil-OtBu (1.00 g, 1.65 mmol) at room temperature in 10 mL of MeCN. After 5 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was extracted with hexane and the MeCN layer was concentrated under vacuum to produce crude H-Abu(3-N3)-Dil-OtBu, which did not require further purification.

[0332] DIEA (0.880 mL, 4.94 mmol) was added to a stirred suspension of crude H-Abu(3-N3)-Dil-OtBu and Dov (0.478 g, 3.29 mmol) at room temperature in DMF (10 mL), followed by HATU (1.25 g, 3.29 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.670 g of TFA salt of Dov-Abu(3-N3)-Dil-OtBu (1.07 mmol, 65%) was obtained.

[0333] TFA (5 mL) was added to a 5 mL solution of Dov-Abu(3-N3)-Dil-OtBu TFA salt (0.289 g, 0.425 mmol) at room temperature. After 12 hours, analysis by LCMS showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude Dov-Abu(3-N3)-Dil-OH TFA salt, which did not require further purification.

[0334] DIEA (1.18 g, 1.60 mL, 9.11 mmol) was added to a stirred suspension of crude Dov-Abu(3-N3)-Dil-OH TFA salt (1.04 g, 1.82 mmol) and H-Dap-Phe-OMe TFA salt (1.59 g, 3.44 mmol) at room temperature in DMF (10 mL), followed by HATU (1.74 g, 4.56 mmol). After 10 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Gemini NX-C1810μm analyzer. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid. A total of 935 mg of the formate of the title compound was obtained (1.12 mmol, 49%). LCMS RT = 1.09 min (Method A); ESI-MS m / z 787.53 [M+H] + HRMS m / z 787.5072[C 40 H 66 N8O8+H] + .

[0335] Example 8

[0336] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-amino-2-((S)-2-(dimethylamino) (N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy Methyl 2-methylpropionamide (2-methylpropionamide)-3-phenylpropionate

[0337]

[0338] DIEA (1.08 mL, 6.08 mmol) was added to a stirred solution of EtOAc (10 mL) containing Fmoc-Dpr(Boc)-OH (1.44 g, 3.38 mmol) and H-Dil-OtBu hydrochloride (1.00 g, 3.38 mmol) at room temperature. The solution was cooled to (0 °C) and stirred for 20 min. Additional DIEA (1.08 mL, 6.08 mmol) was added to the reaction mixture, and the solution was stirred at 0 °C for 20 min. CMPI (1.38 g, 5.41 mmol) was added to the reaction mixture, and the reaction mixture was warmed to room temperature. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was washed with 0.1 M HCl (100 mL x 2), followed by brine (20 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. Using 18% to 90% EtOAc in hexane as the eluent, the solution was passed through silica gel (40 μm silica gel). Fmoc-Dpr(Boc)-Dil-OtBu was separated by rapid chromatography on a 3.0 x 17.0 cm plate. A total of 1.27 g of Fmoc-Dpr(Boc)-Dil-OtBu (1.90 mmol, 56% yield) was obtained.

[0339] Piperidine (2 mL) was added to a stirred solution of Fmoc-Thr(Bzl)-Dil-OtBu (1.27 g, 1.90 mmol) at room temperature in 10 mL of MeCN. After 5 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was extracted with hexane and the MeCN layer was concentrated under vacuum to produce crude H-Dpr(Boc)-Dil-OtBu, which did not require further purification.

[0340] DIEA (1.02 mL, 5.70 mmol) was added to a stirred suspension of crude H-Dpr(Boc)-Dil-OtBu and Dov (0.552 g, 3.80 mmol) at room temperature in DMF (10 mL), followed by HATU (1.45 g, 3.80 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.598 g of TFA salt of Dov-Dpr(Boc)-Dil-OtBu was obtained (0.871 mmol, 46%).

[0341] TFA (5 mL) was added to a 10 mL solution of Dov-Dpr(Boc)-Dil-OtBu TFA salt (1.52 g, 2.65 mmol) at room temperature. After 10 hours, analysis by LCMS showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude Dov-Dpr-Dil-OHTFA salt, which did not require further purification.

[0342] DIEA (0.460 mL, 2.64 mmol) was added to a stirred suspension of Dov-Dpr-Dil-OH TFA salt (1.00 g, 1.89 mmol) and Fmoc-OSu (0.891 g, 2.64 mmol) at room temperature in CH2Cl2 (10 mL). After 12 hours, the reaction was complete as shown by LCMS analysis. Volatile organic compounds were evaporated under vacuum. The volatile organic compounds were analyzed using Phenomenex Synergi 10 μM Max-RP. The crude oily substance was purified by preparative RP-HPLC using a 150x30mm column and 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 1.49 g of formate enriched with Dov-Dpr(Fmoc)-Dil-OH was obtained.

[0343] DIEA (0.970 g, 1.30 mL, 7.514 mmol) was added to a suspension of Dov-Dpr(Fmoc)-Dil-OH formate (1.20 g, 1.75 mmol) and H-Dap-Phe-OMe TFA salt (0.982 g, 2.13 mmol) in DMF (10 mL) at 23 °C with stirring. HATU (1.43 g, 3.76 mmol) was then added. After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C1810μm analyzer. The crude oil was purified by preparative RP-HPLC using a column (150x30 mm) with 10% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 1.01 g of Dov-Dpr(Fmoc)-Dil-Dap-Phe-OMe (1.04 mmol, 56%) was obtained.

[0344] Piperidine (5 mL) was added to a stirred solution of Dov-Dpr(Fmoc)-Dil-Dap-Phe-OMe (1.01 g, 1.04 mmol) at 23 °C in acetonitrile (10 mL). After 3 hours, LCMS analysis showed that the reaction was complete. Hexane was added to the crude reaction solution. The acetonitrile layer was concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C1810μL microscope. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 413 g of the formate of the title compound was obtained (0.521 mmol, 50%). LCMS RT = 2.10 min (Method A); ESI-MS m / z 747.84 [M+H] + HRMS m / z 747.5008[C 39 H 66 N6O8+H] + .

[0345] Example 9

[0346] (S)-N-((S)-3-amino-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy- 2-Methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4- (methyl)amino)-1-oxopropyl-2-yl)-2-(dimethylamino)-3-methylbutyramide

[0347]

[0348] DIEA (0.511 mL, 2.87 mmol) was added to a stirred suspension of crude Dov-Dpr(Fmoc)-Dil-OH and H-Dap-2-(2-pyridyl)ethylamine TFA salt (0.230 g, 0.789 mmol) at room temperature in DMF (10 mL), followed by HATU (0.545 g, 1.43 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (25 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.150 g of TFA salt of Dov-Dpr(Fmoc)-Dil-Dap-2-(2-pyridyl)ethylamine (0.146 mmol, 20%) was obtained.

[0349] Piperidine (2 mL) was added to a stirred solution of Dov-Dpr(Fmoc)-Dil-Dap-2-(2-pyridyl)ethylamine TFA salt (0.150 g, 0.146 mmol) at room temperature. After 5 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was extracted with hexane and the MeCN layer was concentrated under vacuum. The final concentration was determined using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.089 g of the TFA salt of the title compound was obtained (0.111 mmol, 75%). LCMS RT = 1.52 min (Method A); ESI-MS m / z 690.67 [M+H] + ;HRMS m / z690.4917[C 36 H 63 N7O6+H] + .

[0350] Example 10

[0351] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-2-((S)-2-(dimethyl) (amino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methyl methyl oxy-2-methylpropionamide (2-methylpropionamide)-3-phenylpropionate

[0352]

[0353] DIEA (0.792 mL, 4.44 mmol) was added to a stirred solution of N-Boc-4-azido-alanine dicyclohexylamine salt (1.02 g, 2.47 mmol) and H-Dil-OtBu hydrochloride (0.730 g, 2.74 mmol) at room temperature. The solution was cooled (0 °C) and stirred for 20 min. DIEA (0.792 mL, 4.44 mmol) was added to the reaction mixture. The solution was cooled (0 °C) and stirred for 20 min. CMPI (1.01 g, 3.95 mmol) was added to the reaction mixture, and the reaction mixture was allowed to reach room temperature. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was washed with 0.1 M HCl (100 mL x 2) followed by brine (20 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum to produce crude N-Boc-4-azido-Ala-Dil-OtBu (1.08 g, 2.29 mmol) which did not require further purification.

[0354] TFA (3 mL) was added to a 5 mL solution of N-Boc-4-azido-Ala-Dil-OtBu (1.08 g, 2.29 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were vacuum evaporated to produce the crude H-4-azido-Ala-Dil-OH TFA salt, which required no further purification.

[0355] DIEA (1.36 mL, 7.63 mmol) was added to a stirred suspension of Dov (0.739 g, 5.09 mmol) at room temperature in 10 mL of DMF, followed by HATU (1.95 g, 5.09 mmol). After 10 minutes, crude H-4-azido-Ala-Dil-OH was added to the reaction mixture. The reaction was complete after 6 hours, as shown by LCMS analysis. The reaction was confirmed by Phenomenex Synergi 10 μ Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.130 g of TFA salt of Dov-4-azido-Ala-Dil-OH (0.261 mmol, 10%) was obtained.

[0356] DIEA (0.140 mL, 0.783 mmol) was added to a stirred suspension of crude Dov-4-azido-Ala-Dil-OH TFA salt (0.130 g, 0.261 mmol) and H-Dap-Phe-OMe TFA salt (0.205 g, 0.588 mmol) at room temperature in DMF (10 mL), followed by HATU (0.198 g, 0.522 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.015 g of the TFA salt of the title compound was obtained (0.017 mmol, 7%). LCMS RT = 2.36 min (Method A); ESI-MS m / z 773.48 [M+H] + HRMS m / z 773.4916[C 39 H 64 N8O8+H] +.

[0357] Example 11

[0358] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-azido-2-((S)-2-(dimethyl) (amino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methyl Methyl oxy-2-methylpropionamide (2-methylpropionamide)-3-phenylpropionate

[0359]

[0360] DIEA (0.759 mL, 4.26 mmol) was added to a stirred solution of N-Boc-4-azido-homalanine dicyclohexylamine salt (1.01 g, 2.37 mmol) and H-Dil-OtBu hydrochloride (0.700 g, 2.37 mmol) at room temperature. The solution was cooled (0 °C) and stirred for 20 min. DIEA (0.759 mL, 4.26 mmol) was added to the reaction mixture. The solution was cooled (0 °C) and stirred for 20 min. CMPI (0.967 g, 3.79 mmol) was added to the reaction mixture, and the reaction mixture was allowed to reach room temperature. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was washed with 0.1 M HCl (100 mL x 2) followed by brine (20 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and vacuum concentrated to produce crude N-Boc-4-azido-high-Ala-Dil-OtBu (1.09 g, 2.25 mmol) that did not require further purification.

[0361] TFA (3 mL) was added to a 5 mL solution of N-Boc-4-azido-highAla-Dil-OtBu (1.09 g, 2.25 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were vacuum evaporated to produce the crude H-4-azido-highAla-Dil-OH TFA salt, which required no further purification.

[0362] DIEA (1.20 mL, 6.73 mmol) was added to a stirred suspension of Dov (0.652 g, 4.49 mmol) in DMF (10 mL) at room temperature, followed by HATU (1.71 g, 4.49 mmol). After 10 minutes, crude H-4-azido-highAla-Dil-OH TFA salt was added to the reaction mixture. The reaction was complete after 6 hours, as shown by LCMS analysis. The reaction was confirmed by Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.127 g of Dov-4-azido-highAla-Dil-OH TFA salt (0.223 mmol, 10%) was obtained.

[0363] DIEA (0.140 mL, 0.783 mmol) was added to a stirred suspension of crude Dov-4-azido-highAla-Dil-OH TFA salt (0.127 g, 0.223 mmol) and H-Dap-Phe-OMe TFA salt (0.198 mmol, 0.568 mmol) at room temperature in DMF (10 mL), followed by HATU (0.198 g, 0.522 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.015 g of the TFA salt of the title compound was obtained (0.017 mmol, 7%). LCMS RT = 2.38 min (Method A); ESI-MS m / z 787.49 [M+H] + HRMS m / z 787.5078[C 40 H 66 N8O8+H] + .

[0364] Example 12

[0365] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-amino-2-((S)-2-(dimethylamino) (N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy Methyl 2-methylpropionamide (2-methylpropionamide)-3-phenylpropionate

[0366]

[0367] DIEA (1.08 mL, 6.08 mmol) was added to a stirred solution of EtOAc (10 mL) containing Fmoc-Dab(Boc)-OH (1.54 g, 3.38 mmol) and H-Dil-OtBu hydrochloride (1.00 g, 3.38 mmol) at room temperature. The solution was cooled (0 °C) and stirred for 20 min. Additional DIEA (1.08 mL, 6.08 mmol) was added to the reaction mixture, and the solution was stirred at 0 °C for 20 min. Then, CMPI (1.38 g, 5.41 mmol) was added to the reaction mixture, and the reaction mixture was warmed to room temperature. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was washed with 0.1 M HCl (100 mL x 2), followed by brine (20 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. Using 18% to 90% EtOAc in hexane as the eluent, the solution was passed through silica gel (40 μm silica gel). Fmoc-Dab(Boc)-Dil-OtBu was separated by rapid chromatography on a 3.0 x 17.0 cm plate. A total of 1.18 g of Fmoc-Dpr(Boc)-Dil-OtBu (1.73 mmol, 51% yield) was obtained.

[0368] Piperidine (2 mL) was added to a stirred solution of Fmoc-Dab(Boc)-Dil-OtBu (1.18 g, 1.73 mmol) at room temperature in 10 mL of MeCN. After 5 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was extracted with hexane and the MeCN layer was concentrated under vacuum to produce crude H-Dab(Boc)-Dil-OtBu, which did not require further purification.

[0369] DIEA (0.925 mL, 5.19 mmol) was added to a stirred suspension of crude H-Dab(Boc)-Dil-OtBu and Dov (0.502 g, 3.46 mmol) at room temperature in DMF (10 mL), followed by HATU (1.32 g, 3.46 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.410 g of TFA salt of Dov-Dab(Boc)-Dil-OtBu (0.583 mmol, 34%) was obtained.

[0370] TFA (2 mL) was added to a 10 mL solution of Dov-Dab(Boc)-Dil-OtBu TFA salt (0.240 g, 0.419 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude Dov-Dab-Dil-OH TFA salt, which did not require further purification.

[0371] DIEA (0.080 mL, 0.447 mmol) was added to a stirred suspension of Dov-Dab-Dil-OH TFA salt (0.175 g, 0.406 mmol) and Fmoc-OSu (0.151 g, 0.447 mmol) at room temperature in CH2Cl2 (5 mL). After 12 hours, analysis by LCMS showed that the reaction was complete. Volatile organic compounds were evaporated under vacuum. The crude reaction mixture was dissolved in EtOAc and washed with 0.1 M HCl (100 mL x 2) followed by washing with brine (20 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum to produce crude Dov-Dab(Fmoc)-Dil-OH, which did not require further purification.

[0372] DIEA (0.291 mL, 1.67 mmol) was added to a stirred suspension of crude Dov-Dab(Fmoc)-Dil-OH (0.363 g, 0.556 mmol) and H-Dap-Phe-OMeTFA salt (0.194 g, 0.420 mmol) at room temperature in DMF (10 mL), followed by HATU (0.424 g, 1.11 mmol). After 10 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (30 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex Synergi 10 μM Max-RP. The crude oily substance was purified by preparative RP-HPLC using a 150x30mm column and 10% to 90% MeCN in 0.2% aqueous formic acid as the eluent. A total of 449 mg of formate enriched Dov-Dab(Fmoc)-Dil-Dap-Phe-OMe was obtained.

[0373] Piperidine (5 mL) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-Dap-Phe-OMe formate (0.449 g) at room temperature in 10 mL of MeCN. After 10 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was extracted with hexane and the MeCN layer was concentrated under vacuum. The final product was analyzed using a Phenomenex Gemini NX C1810μ Max-RP microscope. The crude oil was purified by RP-HPLC on a 150x30mm column using 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 43.0 g of the formate of the title compound was obtained (0.053 mmol, 13%). LCMS RT = 1.24 min (Method B); ESI-MS m / z 761.57 [M+H] + HRMS m / z 761.5165[C 40 H 68 N6O8+H] + .

[0374] Example 13

[0375] (S)-2-((S)-2-(aminooxy)-3-methylbutyryloxy)-N-((3R,4S,5S)-3-methoxy-1- ((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrole Alkyl-1-yl)-5-methyl-1-oxohept-4-yl)-N,3-dimethylbutyramide

[0376]

[0377] DIEA (0.65 mL, 3.7 mmol) was added to a solution of H-Dil-OtBu hydrochloride (0.60 g, 2.03 mmol) and Fmoc-Val-OH (0.829 g, 2.44 mmol) stirred in EtOAc (3 mL). The reaction mixture was cooled to 0 °C and stirred for 20 min, after which DIEA (0.65 mL, 3.7 mmol) was added. The reaction mixture was then cooled (0 °C) for another 20 min, after which CMPI (0.83 g, 3.7 mmol) was added. After 8 hours, analysis by LCMS showed that the reaction was complete. The reaction mixture was washed with 1 M HCl (150 mL x 2) and brine (50 mL). The organic phase was dried on Na2SO4, filtered, and concentrated under vacuum. Using 18% to 90% EtOAc in hexane as the eluent, the mixture was passed through silica gel (40 μm silica gel). Fmoc-Val-Dil-OtBu was separated by rapid chromatography on a 3.0 x 17.0 cm plate. A total of 1.1 g of Fmoc-Val-Dil-OtBu (1.9 mmol, 93% yield) was obtained.

[0378] TFA (5 mL) was added to a stirred suspension of Fmoc-Val-Dil-OtBu (0.883 g, 1.52 mmol) and Boc-Dap-2-(2-pyridyl)ethylamine (0.451 g, 1.52 mmol) at room temperature in CH2Cl2 (10 mL). After 8 hours, analysis by LCMS showed the reaction was complete. The volatile organic compounds were vacuum evaporated to produce crude Fmoc-Val-Dil-OH and H-Dap-2-(2-pyridyl)ethylamine TFA salts, which did not require further purification for use.

[0379] DIEA (1.10 mL, 6.08 mmol) was added to a stirred suspension of crude H-Dap-2-(2-pyridyl)ethylamine TFA salt and Fmoc-Val-Dil-OH at room temperature in EtOAc (2 mL), followed by DEPC (0.92 mL, 6.08 mmol). After 15 hours, the reaction was complete as shown by LCMS analysis. The reaction was washed with saturated NaHCO3 solution (50 mL) followed by water (50 mL x 2). The organic fraction was filtered through a magnesium sulfate pad and concentrated under vacuum. Eluent was obtained by passing through silica gel (40 μm silica gel) using 5% to 10% MeOH in CH2Cl2. The resulting viscous oil was purified by rapid chromatography on a 23x123mm (23x123mm) plate. A total of 0.888 g of Fmoc-Val-Dil-Dap-2-(2-pyridyl)ethylamine (1.11 mmol, 73% yield) was obtained.

[0380] Piperidine (5 mL) was added to a stirred solution of Fmoc-Val-Dil-Dap-2-(2-pyridyl)ethylamine (1.75 g, 2.19 mmol) at room temperature in CH2Cl2 (5 mL). After 8 hours, analysis by LCMS showed that the reaction was complete. The volatile organic compounds were vacuum evaporated to produce crude H-Val-Dil-Dap-2-(2-pyridyl)ethylamine, which did not require further purification for use.

[0381] DIEA (0.797 mL, 5.02 mmol) was added to a stirred suspension of crude H-Val-Dil-Dap-2-(2-pyridyl)ethylamine and N-Boc-N-hydroxyVal-OH (0.390 g, 1.67 mmol) at room temperature in DMF (5 mL), followed by HATU (1.28 g, 3.34 mmol). After 8 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (50 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum to yield 0.563 g of crude N-Boc-N-hydroxyVal-Val-Dil-Dap-2-(2-pyridyl)ethylamine (0.712 mmol, 43%), which did not require further purification for use.

[0382] TFA (2 mL) was added to a solution of N-Boc-N-hydroxyVal-Val-Dil-Dap-2-(2-pyridyl)ethylamine (0.563 g, 0.712 mmol) in DMF (2 mL) at room temperature. After 2 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were evaporated under vacuum. The volatile organic compounds were analyzed using a Phenomenex Synergi 10 μMax-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.209 g of the TFA salt of the title compound was obtained (0.302 mmol, 43%). LCMS RT = 1.70 min (Method A); ESI-MS m / z 691.53 [M+H] + ;HRMS m / z691.4755[C 36 H 62 N6O7+H] + .

[0383] Example 14

[0384] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxypropionyl) (amino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methyl propionyl)-L-phenylalanine

[0385]

[0386] HCO₂NH₄ (0.808 g, 12.8 mmol) and palladium-supported activated carbon (10% Pd-based, 0.250 g) were added to a stirred solution of H-Ser(Bzl)-OH (0.500 g, 2.56 mmol) and paraformaldehyde (1.15 g, 38.4 mmol) at room temperature. After 72 hours, analysis by LC-MS showed the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat and the filtrate was concentrated. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.168 g of TFA salt of N,N-dimethylSer(Bzl)-OH was obtained (0.498 mmol, 19%).

[0387] Piperidine (15 mL) was added to a stirred solution of Fmoc-Val-Dil-OtBu (13.3 g, 22.8 mmol) at room temperature in CH2Cl2 (20 mL). After 8 hours, analysis by LCMS showed that the reaction was complete. The volatile organic compounds were vacuum evaporated to produce crude H-Val-Dil-OtBu, which did not require further purification.

[0388] DIEA (1.25 mL, 6.99 mmol) was added to a stirred suspension of crude H-Val-Dil-OtBu and N,N-dimethylSer(Bzl)-OH TFA salt (0.780 g, 3.49 mmol) at room temperature in DMF (10 mL), followed by HATU (1.77 g, 4.66 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.220 g of TFA salt of N,N-dimethylSer(Bzl)-Val-Dil-OtBu (0.325 mmol, 14%) was obtained.

[0389] TFA (2 mL) was added to a 5 mL solution of N,N-dimethyl Ser(Bzl)-Val-Dil-OtBu TFA salt (0.220 g, 0.325 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude N,N-dimethyl Ser(Bzl)-Val-Dil-OH TFA salt, which did not require further purification.

[0390] DIEA (0.207 mL, 1.30 mmol) was added to a stirred suspension of crude N,N-dimethyl Ser(Bzl)-Val-Dil-OH TFA salt (0.166 g, 0.477 mmol) at room temperature in DMF (5 mL), followed by HATU (0.330 g, 0.868 mmol). After 4 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.201 g of TFA salt of N,N-dimethylSer(Bzl)-Val-Dil-Dap-Phe-OMe (0.211 mmol, 49%) was obtained.

[0391] 10% Pd / C (10 mg) was added to a suspension of N,N-dimethyl Ser(Bzl)-Val-Dil-Dap-Phe-OMe TFA salt (0.201 g, 0.211 mmol) and NH4HCO2 (2.00 g, 31.7 mmol) at room temperature with stirring in MeOH (6 mL) and water (1.0 mL). The solution was stirred vigorously at room temperature. After 10 hours, LCMS analysis showed that the reaction was complete and that phenylalanine methyl ester was hydrolyzed. The reaction mixture was filtered on a diatomaceous earth mat and the filtrate was concentrated. The solution was analyzed using Phenomenex Synergi 10 μ Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 63.0 mg of the title compound in TFA salt (0.074 mmol, 31%) was obtained. LCMS RT = 2.01 min (Method A); ESI-MS m / z 734.61 [M+H] +HRMS m / z 734.4715[C 38 H 63 N5O9+H] + .

[0392] Example 15

[0393] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)-3-hydroxy) (butyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2- Methylpropionyl)-L-phenylalanine

[0394]

[0395] HCO₂NH₄ (3.01 g, 47.8 mmol) and palladium-supported activated carbon (10% Pd-based, 1.00 g) were added to a stirred solution of H-Thr(Bzl)-OH (2.00 g, 9.56 mmol) and paraformaldehyde (9.87 g, 95.6 mmol) at room temperature. After 72 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat and the filtrate was concentrated. The concentration was determined by using Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 1.54 g of TFA salt of N,N-dimethylThr(Bzl)-OH was obtained (4.38 mmol, 19%).

[0396] DIEA (1.82 mL, 10.2 mmol) was added to a stirred suspension of crude H-Val-Dil-OtBu and N,N-dimethylThr(Bzl)-OH TFA salt (1.21 g, 5.10 mmol) at room temperature in DMF (20 mL), followed by HATU (2.59 g, 6.80 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (15 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.161 g of TFA salt of N,N-dimethylThr(Bzl)-Val-Dil-OtBu (0.234 mmol, 7%) was obtained.

[0397] TFA (2 mL) was added to a 5 mL solution of N,N-dimethylThr(Bzl)-Val-Dil-OtBu TFA salt (0.220 g, 0.325 mmol) at room temperature. After 10 hours, analysis by LCMS showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude N,N-dimethylThr(Bzl)-Val-Dil-OH TFA salt, which did not require further purification.

[0398] DIEA (0.147 mL, 0.927 mmol) was added to a stirred suspension of crude N,N-dimethylThr(Bzl)-Val-Dil-OH TFA salt (0.118 g, 0.339 mmol) at room temperature in DMF (5 mL), followed by HATU (0.235 g, 0.618 mmol). After 4 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.186 g of TFA salt of N,N-dimethylThr(Bzl)-Val-Dil-Dap-Phe-OMe (0.193 mmol, 62%) was obtained.

[0399] 10% Pd / C (100 mg) was added to a suspension of N,N-dimethylThr(Bzl)-Val-Dil-Dap-Phe-OMe TFA salt (0.186 g, 0.193 mmol) and NH4HCO2 (2.00 g, 31.7 mmol) at room temperature with stirring. The solution was stirred vigorously at room temperature. After 72 hours, LCMS analysis showed that the reaction was complete and that phenylalanine methyl ester was hydrolyzed. The reaction mixture was filtered on a diatomaceous earth mat and the filtrate was concentrated. The solution was analyzed using Phenomenex Synergi 10 μ Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 63.0 mg of the title compound in TFA salt (0.073 mmol, 38%) was obtained. LCMS RT = 2.04 min (Method A); ESI-MS m / z 748.58 [M+H] +HRMS m / z 748.4854[C 39 H 65 N5O9+H] + .

[0400] Example 16

[0401] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,55)-4-((2S,3S)-3-azido-2-((S)-2-(di) (methylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)- 3-Methoxy-2-methylpropionamide)-3-phenylpropionic acid

[0402]

[0403] Lithium hydroxide monohydrate (9.6 mg, 0.229 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-OMe TFA salt (60 mg, 0.067 mmol) at room temperature in MeOH (0.1 mL) and THF (0.1 mL). After 12 hours, analysis by LCMS showed the reaction was complete. The reaction was confirmed by using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 55.0 mg of the title compound in TFA salt (0.062 mmol, 93%) was obtained. LCMS RT = 2.25 min (Method A); ESI-MS m / z 773.45 [M+H] + HRMS m / z 773.49119[C 39 H 64 N8O8+H] + .

[0404] Example 17

[0405] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylprop-2- (1-(amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4- (base)-2-((2S) , 3R)-2-(dimethylamino)-3-hydroxybutyrylamino)-N , 3-Dimethylbutyramide

[0406]

[0407] Ammonium chloride (5.72 mg, 0.107 mmol), DIEA (28.6 μL, 0.160 mmol), and EDCI (30.3 mg, 0.107 mmol) were added to a stirred solution of N,N-dimethylThr-Val-Dil-Dap-Phe-OH TFA salt (40 mg, 0.053 mmol) at room temperature in CH2Cl2 (10 mL). After 12 hours, analysis by LC-MS showed that the reaction was complete. The reaction was confirmed by LC-MS analysis using a Phenomenex Synergi 10 μL Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 28.0 mg of the title compound in TFA salt (0.037 mmol, 70%) was obtained. LCMS RT = 2.14 min (Method A); ESI-MS m / z 747.61 [M+H] + ;HRMS m / z747.5017[C 39 H 66 N6O8+H] + .

[0408] Example 18

[0409] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenylprop-2- (1-(amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4- (S)-2-((S)-2-(dimethylamino)-3-hydroxypropionylamino)-N,3-dimethylbutyramide

[0410]

[0411] Ammonium chloride (1.00 mg, 0.019 mmol), DIEA (3.21 μL, 0.018 mmol), and EDCI (5.09 mg, 0.018 mmol) were added to a stirred solution of N,N-dimethyl Ser-Val-Dil-Dap-Phe-OH TFA salt (12 mg, 0.016 mmol) at room temperature in CH2Cl2 (10 mL). After 12 hours, analysis by LC-MS showed that the reaction was complete. The reaction was confirmed by LC-MS analysis using a Phenomenex Synergi 10 μL Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 10.0 mg of the title compound in TFA salt (0.014 mmol, 83%) was obtained. LCMS RT = 2.07 min (Method A); ESI-MS m / z 733.63 [M+H] + ;HRMS m / z733.4866[C 38 H64 N6O8+H] + .

[0412] Example 19

[0413] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methyl (3-Mercapto-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy Methyl 2-methylpropionamide (2-methylpropionamide)-3-phenylpropionate

[0414]

[0415] DIEA (2.17 mL, 12.2 mmol) was added to a stirred solution of EtOAc (15 mL) containing Fmoc-Cys(Trt)-OH (3.96 g, 6.76 mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) at room temperature. The solution was cooled (0 °C) and stirred for 20 min. An additional DIEA (2.17 mL, 12.2 mmol) was added to the reaction mixture, and the solution was stirred at 0 °C for 20 min. Then, CMPI (2.76 g, 10.8 mmol) was added to the reaction mixture, and the reaction mixture was allowed to reach room temperature. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was washed with 0.1 M HCl (100 mL x 2), followed by brine (20 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. Using 18% to 90% EtOAc in hexane as the eluent, the solution was passed through silica gel (40 μm silica gel). Fmoc-Cys(Trt)-Dil-OtBu was separated by rapid chromatography on a 3.0 x 17.0 cm plate. A total of 4.73 g of Fmoc-Cys(Trt)-Dil-OtBu (5.72 mmol, 85% yield) was obtained.

[0416] Piperidine (5 mL) was added to a stirred solution of Fmoc-Cys(Trt)-Dil-OtBu (2.61 g, 3.16 mmol) at room temperature in 10 mL of MeCN. After 5 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was extracted with hexane and the MeCN layer was concentrated under vacuum to produce crude H-Cys(Trt)-Dil-OtBu, which did not require further purification.

[0417] DIEA (1.69 mL, 9.47 mmol) was added to a stirred suspension of crude H-Cys(Trt)-Dil-OtBu and Dov (0.916 g, 6.31 mmol) at room temperature in DMF (15 mL), followed by HATU (2.40 g, 6.31 mmol). After 8 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 1.40 g of TFA salt of Dov-Cys(Trt)-Dil-OtBu (1.66 mmol, 52%) was obtained.

[0418] TFA (5 mL) was added to a 10 mL solution of Dov-Cys(Trt)-Dil-OtBu TFA salt (1.40 g, 1.66 mmol) at room temperature. After 10 hours, analysis by LCMS showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude Dov-Cys(Trt)-Dil-OH TFA salt, which did not require further purification.

[0419] DIEA (0.237 mL, 1.33 mmol) was added to a stirred suspension of Dov-Cys(Trt)-Dil-OH TFA salt (0.225 g, 285 mmol) and H-Dap-Phe-OMe TFA salt (0.139 mmol, 0.301 mmol) at room temperature in DMF (10 mL), followed by the addition of HOBt (0.102 g, 0.666 mmol) and HATU (0.253 g, 0.666 mmol). After stirring overnight, the reaction was complete as determined by LCMS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (50 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum, and were used without further purification.

[0420] TFA (10 mL) was added to a solution of Dov-Cys(Trt)-Dil-Dap-Phe-OMeTFA salt (0.568 g, 0.507 mmol) at room temperature. After 12 hours at 60 °C, the reaction was complete as shown by LC-MS analysis. Volatile organic compounds were evaporated under vacuum. The volatile organic compounds were analyzed using Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 10.0 mg of the disulfide-bridged dimer TFA salt of the title compound was obtained (0.011 mmol, 3%). LCMS RT = 2.50 min (Method A); ESI-MS m / z 764.60 [M+H] + HRMS m / z 763.4547z=2.

[0421] Example 20

[0422] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methyl (3-Mercapto-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy (2-methylpropionylamino)-3-phenylpropionic acid

[0423]

[0424] DIEA (8.78 mL, 55.2 mmol) was added to a stirred solution of Boc-Dap-OH dicyclohexylamine salt (6.47 g, 13.8 mmol) and H-Phe-OtBu HCl salt (3.91 g, 15.2 mmol) at room temperature in DCM (20 mL), followed by DEPC (3.12 mL, 20.7 mmol). After 8 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were evaporated under vacuum to give a crude product that required no further purification.

[0425] TFA (10 mL) was added to a stirred solution of Boc-Dap-Phe-OtBu (5.25 g, 10.7 mmol) at room temperature in CH2Cl2 (10 mL). After 12 hours, LCMS analysis showed that the reaction was complete, and the product was a mixture of free phenylalanine carboxylic acid and tBu ester. The reaction was confirmed by using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. 2.85 g of amber oil (5.65 mmol) of H-Dap-Phe-OtBu TFA salt and a total of 2.01 g of yellow oil (4.49 mmol) of H-Dap-Phe-OH TFA salt were obtained.

[0426] DIEA (0.219 mL, 1.23 mmol) was added to a stirred suspension of crude Dov-Cys(Trt)-Dil-OH TFA salt (0.208 g) and H-Dap-Phe-OMe TFA salt (0.144 g, 0.369 mmol) at room temperature in DMF (10 mL), followed by HATU (0.234 g, 0.615 mmol). After 10 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oily substance was purified by preparative RP-HPLC using a column (150x30 mm) with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.509 g of TFA salt enriched with Dov-Cys(Trt)-Dil-Dap-Phe-OtBu was obtained.

[0427] TFA (5 mL) was added to a room temperature solution enriched with Dov-Cys(Trt)-Dil-Dap-Phe-OtBu TFA salt (0.50 g). The solution was heated to 60 °C and stirred for 24 hours. After 24 hours, analysis by LCMS showed that the reaction was complete. Volatile organic compounds were evaporated under vacuum. The volatile organic compounds were then analyzed using a Phenomenex Synergi 10 μMax-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 56.0 mg of the title compound in TFA salt (0.065 mmol, 25%) was obtained. LCMS RT = 2.32 min (Method A); ESI-MS m / z 750.60 [M+H] + HRMS m / z 750.4460[C 38 H 63 N5O8S+H] + .

[0428] Example 21

[0429] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methyl (3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy (2-methylpropionylamino)-3-phenylpropionic acid

[0430]

[0431] DIEA (0.326 mL, 1.83 mmol) was added to a stirred suspension of crude Dov-Ser(Bzl)-Dil-OH TFA salt (0.232 g) and H-Dap-Phe-OtBu TFA salt (0.196 g, 0.389 mmol) at room temperature in DMF (10 mL), followed by HATU (0.348 g, 0.914 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.378 g of TFA salt (0.380 mmol, 98%) of Dov-Ser(Bzl)-Dil-Dap-Phe-OtBu was obtained.

[0432] TFA (2 mL) was added to a 5 mL solution of Dov-Ser(Bzl)-Dil-Dap-Phe-OtBu TFA salt (0.455 g, 0.517 mmol) at room temperature. After 12 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude Dov-Ser(Bzl)-Dil-Dap-Phe-OH TFA salt, which did not require further purification.

[0433] Hydrogenation was performed on a suspension of crude Dov-Ser(Bzl)-Dil-Dap-Phe-OH TFA salt and activated carbon-supported palladium (10% Pd-based, 10.0 mg) in MeOH (10 mL) at room temperature with stirring. After 12 hours, LC-MS analysis showed the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat, and the filtrate was concentrated. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a column (150 x 30 mm) with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.115 g of the TFA salt of the title compound was obtained (0.136 mmol, 28%). LCMS RT = 2.10 min (Method A); ESI-MS m / z 734.61 [M+H] + HRMS m / z 734.4708[C 38 H 63 N5O9+H] + .

[0434] Example 22

[0435] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)- 3-Methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3- Methoxy-2-methylpropionamide (3-phenylpropionic acid)

[0436]

[0437] DIEA (0.246 mL, 1.38 mmol) was added to a stirred suspension of crude Dov-Thr(Bzl)-Dil-OH TFA salt (0.180 g) and H-Dap-Phe-OtBu TFA salt (0.148 g, 0.294 mmol) at room temperature in DMF (10 mL), followed by HATU (0.262 g, 0.690 mmol). After 6 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.298 g of TFA salt (0.296 mmol, 78%) of Dov-Thr(Bzl)-Dil-Dap-Phe-OtBu was obtained.

[0438] TFA (2 mL) was added to a 5 mL solution of Dov-Thr(Bzl)-Dil-Dap-Phe-OtBu TFA salt (0.298 g, 0.296 mmol) at room temperature. After 10 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were vacuum evaporated to produce crude Dov-Thr(Bzl)-Dil-Dap-Phe-OH TFA salt, which did not require further purification.

[0439] Hydrogenation was performed on a suspension of crude Dov-Thr(Bzl)-Dil-Dap-Phe-OH TFA salt and activated carbon-supported palladium (10% Pd-based, 10.0 mg) in MeOH (10 mL) at room temperature with stirring. After 12 hours, LC-MS analysis showed the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat, and the filtrate was concentrated. The concentration was determined by using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 0.120 g of the TFA salt of the title compound was obtained (0.139 mmol, 47%). LCMS RT = 2.22 min (Method A); ESI-MS m / z 748.62 [M+H] + HRMS m / z 748.4842[C 39 H 65 N5O9+H] + .

[0440] Example 23

[0441] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-hydroxy (N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy- methyl 2-methylpropionamide (M2-3-phenylpropionate)

[0442]

[0443] Hydrogenation was performed in a suspension of N,N-dimethyl Ser(Bzl)-Val-Dil-Dap-Phe-OMe TFA salt (50.0 mg, 0.053 mmol) and activated carbon-supported palladium (10% Pd-based, 10 mg) at room temperature with stirring at 10 mL. After 10 hours, analysis by LC-MS showed the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat, and the filtrate was concentrated. The concentration was determined by electrophoresis using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 8.00 mg of the title compound in TFA salt (0.009 mmol, 18%) was obtained. LCMS RT = 2.38 min (Method A); ESI-MS m / z 748.57 [M+H] + ;HRMS m / z748.4849[C 39 H 65 N5O9+H] + .

[0444] Example 24

[0445] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((2S,3R)-2-(dimethylamino)- 3-Hydroxybutyrylamino)-N,3-Dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy Methyl 2-methylpropionamide (2-methylpropionamide)-3-phenylpropionate

[0446]

[0447] The title compound was prepared using a method similar to that described in the general synthetic scheme and examples.

[0448] Example 25

[0449] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethyl) (N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3- Methyl methoxy-2-methylpropionamide (MDMA)-3-phenylpropionate

[0450]

[0451] Trimethylphosphine (1 M, 0.022 mL, 0.022 mmol) in THF was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-OMe TFA salt (10 mg, 0.011 mmol) at room temperature. After 4 hours, analysis by LCMS showed that the reaction was complete, and H2O (0.05 mL) was added to the reaction mixture. The reaction was then analyzed using Phenomenex Synergi 10 μMax-RP. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 6.00 mg of the title compound in TFA salt (0.007 mmol, 62%) was obtained. LCMS RT = 2.12 min (Method A); ESI-MS m / z 761.63 [M+H] + HRMS m / z 761.5159[C 40 H 68 N6O8+H] + .

[0452] Example 26

[0453] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethyl) (N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3- Methoxy-2-methylpropionamide (3-phenylpropionic acid)

[0454]

[0455] Trimethylphosphine (1 M, 0.022 mL, 0.022 mmol) in THF was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-OH TFA salt (10 mg, 0.011 mmol) at room temperature. After 4 hours, analysis by LCMS showed that the reaction was complete, and H2O (0.05 mL) was added to the reaction mixture. The reaction was then analyzed using Phenomenex Synergi 10 μM Max-RP. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.00 mg of the title compound in TFA salt (0.002 mmol, 21%) was obtained. LC-MS / RT = 2.02 min (Method A); ESI-MS m / z 747.65 [M+H] + HRMS m / z 747.5008[C 39 H 66 N6O8+H] + .

[0456] Example 27

[0457] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)- 3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenylethylamino)propyl)pyrrolidine- 1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide

[0458]

[0459] DIEA (8.76 mL, 55.2 mmol) was added to a stirred solution of Boc-Dap-OH dicyclohexylamine salt (6.47 g, 13.8 mmol) and phenethylamine (3.914 g, 15.19 mmol) at 20 mL of CH₂Cl₂ at 25 °C, followed by DEPC (3.12 mL, 20.7 mmol). After 8 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were evaporated under vacuum to give a crude product that required no further purification. A total of 4.04 g of Boc-Dap-phenethylamine (10.3 mmol) was obtained. LCMS RT = 3.00 min (Method A); ESI-MS m / z 391.37 [M+H] + .

[0460] TFA (15.0 mL) was added to a stirred solution of Boc-Dap-phenylethylamine (4.04 g, 10.3 mmol) at 25 °C in CH2Cl2 (15.0 mL). After 14 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were evaporated under vacuum and analyzed using a Phenomenex Synergi 10 μM Max-RP analyzer. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.51 g of H-Dap-phenylethylamine in TFA salt (6.21 mmol) was obtained. LCMS RT = 1.72 min (Method A); ESI-MS m / z 291.29 [M+H] + .

[0461] DIEA (0.343 mL, 1.97 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-OH TFA salt (0.300 g, 0.526 mmol) and H-Dap-phenylethylamine TFA salt (0.191 g, 0.471 mmol) at room temperature, followed by the addition of HATU (0.501 g, 1.31 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 196 mg of the title compound in TFA salt (0.233 mmol, 49%) was obtained. LCMS RT = 2.45 min (Method A); ESI-MS m / z 729.55 [M+H] + HRMS m / z 729.5030[C 38 H 64 N8O6+H] + .

[0462] Example 28

[0463] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)- 1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxo (Propyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0464]

[0465] DIEA (11.4 mL, 0.064 mol) and DEPC (6.44 mL, 0.043 mol) were added to a stirred solution of Boc-Dap-OH dicyclohexylamine salt (10.0 g, 0.021 mol) and (1R, 2S)-(-)-norephedrine (3.87 g, 0.026 mol) at room temperature in CH2Cl2 (20 mL). After 14 hours, analysis by LCMS showed that the reaction was complete. 0.1 M HCl (20.0 mL) was added to the crude reaction mixture. The organic layer was separated, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum to produce the crude product. A total of 7.92 g of Boc-Dap-(1R, 2S)-(-)-norephedrine (18.8 mmol, 88%) was obtained. LCMS RT = 2.25 min (Method A); ESI-MS m / z 421.31 [M+H] + .

[0466] TFA (10 mL) was added to a stirred solution of Boc-Dap-(1R,2S)-(-)-norephedrine (7.92 g, 18.8 mmol) at room temperature in CH2Cl2 (10 mL). After 10 hours, analysis by LCMS showed the reaction was complete. The reaction was confirmed by using a Phenomenex Synergi 10 μM Max-RP... The crude oily substance was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 5.00 g of TFA salt of H-Dap-(1R,2S)-(-)-norephedrine was prepared (11.5 mmol, 61%). LCMS RT = 1.10 min (Method A); ESI-MS m / z 321.33 [M+H] + .

[0467] DIEA (0.351 mL, 1.97 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-OH TFA salt (0.300 g, 0.526 mmol) and H-Dap-(1R, 2S)-(-)-norephedrine TFA salt (0.211 g, 0.486 mmol) at room temperature, followed by the addition of HATU (0.501 g, 1.31 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 128 mg of the title compound in TFA salt (0.147 mmol, 30%) was obtained. LCMS RT = 1.95 min (Method A); ESI-MS m / z 759.65 [M+H] + HRMS m / z 759.5121[C 39 H 66 N8O7+H] + .

[0468] Example 29

[0469] (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenylethyl)amino) (1-(1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2- ((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0470]

[0471] DIEA (6.78 mL, 42.7 mmol) and DEPC (2.41 mL, 16.0 mmol) were added to a stirred solution of Boc-Dap-OH dicyclohexylamine salt (5.00 g, 10.7 mmol) and 2-(4-chlorophenyl)ethylamine (1.85 g, 11.7 mmol) at room temperature. After 10 hours, the reaction was complete as indicated by LCMS analysis. The volatile organic compounds were evaporated under vacuum to give a crude product that did not require further purification. A total of 4.25 g of Boc-Dap-2-(4-chlorophenyl)ethylamine (10.0 mmol, 94%) was obtained. LCMS RT = 3.10 min (Method A); ESI-MS m / z 425.32 [M+H] + .

[0472] TFA (15 mL) was added to a stirred solution of Boc-Dap-2-(4-chlorophenyl)ethylamine (4.25 g, 10.0 mmol) at room temperature in CH2Cl2 (15 mL). After 10 hours, analysis by LCMS showed the reaction was complete. The reaction was confirmed by using Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.27 g of the TFA salt of H-Dap-2-(4-chlorophenyl)ethylamine was prepared (5.18 mmol, 52%). LCMS RT = 2.05 min (Method A); ESI-MS m / z 325.24 [M+H] + .

[0473] DIEA (0.602 mL, 3.46 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-OH TFA salt (0.526 g, 0.923 mmol) and H-Dap-2-(4-chlorophenyl)ethylamine TFA salt (0.347 g, 0.792 mmol) at room temperature, followed by the addition of HATU (0.878 g, 2.30 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 596 mg of the title compound in TFA salt (0.680 mmol, 85%) was obtained. LCMS RT = 2.38 min (Method A); ESI-MS m / z 763.71 [M+H] + HRMS m / z 763.4633[C 38 H 63 N8O6Cl+H] + .

[0474] Example 30

[0475] (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenylethyl)amino) (1-(1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2- ((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0476]

[0477] DIEA (4.76 mL, 26.7 mmol) and DEPC (2.41 mL, 16.0 mmol) were added to a stirred solution of Boc-Dap-OH dicyclohexylamine salt (5.00 g, 10.7 mmol) and 2-(2-chlorophenyl)ethylamine (1.85 g, 11.7 mmol) at room temperature. After 10 hours, analysis by LCMS showed that the reaction was complete. The volatile organic compounds were evaporated under vacuum to give a crude product that did not require further purification. A total of 3.98 g of Boc-Dap-2-(2-chlorophenyl)ethylamine (9.37 mmol, 88%) was obtained. LCMS RT = 3.04 min (Method A); ESI-MS m / z 425.23 [M+H] + .

[0478] TFA (5 mL) was added to a stirred solution of Boc-Dap-2-(2-chlorophenyl)ethylamine (3.98 g, 9.37 mmol) at room temperature in CH2Cl2 (5 mL). After 10 hours, analysis by LCMS showed the reaction was complete. The reaction was confirmed by using Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.87 g of H-Dap-2-(2-chlorophenyl)ethylamine was prepared as a TFA salt (6.55 mmol, 70%). LCMS RT = 1.83 min (Method A); ESI-MS m / z 325.22 [M+H] + .

[0479] DIEA (0.515 mL, 2.96 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-OH TFA salt (0.450 g, 0.789 mmol) and H-Dap-2-(2-chlorophenyl)ethylamine TFA salt (0.320 g, 0.731 mmol) at room temperature, followed by the addition of HATU (0.751 g, 1.97 mmol). After 10 hours, the reaction was complete as shown by LC-MS analysis. The reaction was confirmed by using a Phenomenex Synergi 10 μM Max-RP... The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 253 mg of the title compound in TFA salt was obtained (0.289 mmol, 32%, based on RSM). LCMS RT = 1.26 min (Method B); ESI-MS m / z 763.60 [M+H] + HRMS m / z 763.4632[C 38 H 63 N8O6Cl+H] + .

[0480] Example 31

[0481] (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3- Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenethyloxy)propyl)pyrrolidine-1- 5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0482]

[0483] Trimethylphosphine in THF (1 M, 0.045 mL, 0.045 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-PE TFA salt (25 mg, 0.030 mmol) at room temperature. After 1 hour, analysis by LCMS showed that the reaction was complete. The reaction was confirmed by using a Phenomenex Synergi 10 μMax-RP... The crude reaction mixture was purified by preparative RP-HPLC using a 150x30mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 9.0 mg of the formate of the title compound was obtained (0.012 mmol, 41%). LCMS RT = 1.02 min (Method B); ESI-MS m / z 703.71 [M+H] + HRMS m / z 703.5117[C 38 H 66N6O6+H] + .

[0484] Example 32

[0485] (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-1- ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxo (propyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0486]

[0487] Trimethylphosphine (1 M, 0.046 mL, 0.046 mmol) in THF was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-norephedrine TFA salt (27 mg, 0.031 mmol) at room temperature. After 2 hours, analysis by LC-MS showed the reaction was complete. The reaction was confirmed by using a Phenomenex Synergi 10 μM Max-RP... The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous trifluoroacetic acid as the eluent. A total of 6.8 mg of the TFA salt of the title compound was obtained (0.008 mmol, 26%). LCMS RT = 1.95 min (Method B); ESI-MS m / z 733.72 [M+H] + HRMS m / z 733.5227[C 39 H 68 N6O7+H] + .

[0488] Example 33

[0489] (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenylethyl)amino)- 1-Methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2- ((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0490]

[0491] Trimethylphosphine (1 M, 0.2 mL, 0.2 mmol) in THF was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-2-(4-chlorophenyl)ethylamine TFA salt (117 mg, 0.133 mmol) at room temperature. After 2 hours, analysis by LCMS showed that the reaction was complete. The reaction was confirmed by using a Phenomenex Synergi 10 μM Max-RP... The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 9.0 mg of the title compound formate (0.011 mmol, 9%) was obtained. LCMS RT = 0.97 min (Method B); ESI-MS m / z 737.51 [M+H] + ;HRMS m / z737.4731[C 38 H 65 N6O6Cl+H] + .

[0492] Example 34

[0493] (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenylethyl)amino)- 1-Methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2- ((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0494]

[0495] Trimethylphosphine (1 M, 0.085 mL, 0.085 mmol) in THF was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-2-(2-chlorophenyl)ethylaminocarbamate (46 mg, 0.057 mmol) at room temperature. After 1 hour, the solution was purified by using Phenomenex Synergi 10 μM Max-RP. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 13.6 mg of the title compound formate (0.017 mmol, 30%) was obtained. LCMS RT = 1.03 min (Method B); ESI-MS m / z 737.57 [M+H] + ;HRMSm / z737.4731[C 38 H 65 N6O6Cl+H] + .

[0496] Example 35

[0497] (S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3-methoxy 1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(phenylethylamino)propyl)pyrrolidine-1-yl)- 5-Methyl-1-oxohept-4-yl)-N-methylbutyramide

[0498]

[0499] DIEA (0.360 mL, 2.07 mmol) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-OH TFA salt (0.450 g, 0.587 mmol) and H-Dap-phenylethylamine TFA salt (0.200 g, 0.495 mmol) at room temperature, followed by the addition of HATU (0.526 g, 1.38 mmol). After 10 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Synergi 10 μM Max-RP. The crude oily substance was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 276 mg of formate enriched Dov-Dab(Fmoc)-Dil-Dap-phenylethylamine was obtained (0.266 mmol, 54%). LCMS RT = 1.37 min (Method B); ESI-MS m / z 925.48 [M+H] + .

[0500] Piperidine (5 mL) was added to a stirred solution of Dov-Dpr(Fmoc)-Dil-Dap-phenylethylamine (0.276 g, 0.266 mmol) at room temperature in acetonitrile (10 mL). After 10 hours, LC-MS analysis showed the reaction was complete. Hexane was added to the crude reaction mixture. The acetonitrile layer was concentrated under vacuum. The concentration was determined using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 98.0 mg of the formate of the title compound was obtained (0.120 mmol, 45%). LCMS RT = 1.02 min (Method B); ESI-MS m / z 703.78 [M+H] + HRMS m / z 703.5117[C 38 H 66 N6O6+H] + .

[0501] Example 36

[0502] (S)-4-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-1- ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxo (propyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0503]

[0504] DIEA (0.376 mL, 2.11 mmol) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-OH TFA salt (0.459 g, 0.599 mmol) and H-Dap-(1R, 2S)-(-)-norephedrine TFA salt (0.225 g, 0.518 mmol) at room temperature, followed by the addition of HATU (0.536 g, 1.41 mmol). After 10 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Synergi 10 μM Max-RP. The crude oily substance was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 449 mg of enriched Dov-Dab(Fmoc)-Dil-Dap-(1R,2S)-(-)-norephedrine formate was obtained (0.420 mmol, 81%). LCMS RT = 1.35 min (Method B); ESI-MS m / z 955.74 [M+H] + .

[0505] Piperidine (5 mL) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-Dap-(1R,2S)-(-)-norephedrine formate (0.449 g, 0.420 mmol) at room temperature in acetonitrile (10 mL). After 10 hours, LC-MS analysis showed the reaction was complete. Hexane was added to the crude reaction mixture. The acetonitrile layer was concentrated under vacuum. The concentration was determined using a Phenomenex Synergi 10 μMax-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 53.0 mg of the title compound formate (0.068 mmol, 14%) was obtained. LC-MS / RT = 0.79 min (Method B); ESI-MS m / z 733.71 [M+H] + HRMS m / z 733.5227[C 39 H 68 N6O7+H] + .

[0506] Example 37

[0507] (S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((4-chlorophenylethyl)amino)-1-methyl (Oxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2- (dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0508]

[0509] DIEA (0.204 mL, 1.17 mmol) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-OH TFA salt (0.255 g, 0.333 mmol) and H-Dap-2-(4-chlorophenyl)ethylamine TFA salt (0.127 g, 0.290 mmol) at room temperature, followed by the addition of HATU (0.298 g, 0.781 mmol). After 10 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 190 mg of formate of Dov-Dab(Fmoc)-Dil-Dap-2-(4-chlorophenyl)ethylamine was obtained (0.177 mmol, 61%). LCMS RT = 1.49 min (Method B); ESI-MS m / z 959.62 [M+H] + .

[0510] Piperidine (5 mL) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-Dap-2-(4-chlorophenyl)ethylamine carbamate (0.190 g, 0.177 mmol) at room temperature in acetonitrile (5 mL). After 10 hours, LCMS analysis showed the reaction was complete. Hexane was added to the crude reaction mixture. The acetonitrile layer was concentrated under vacuum. The concentration was determined using a Phenomenex Synergi 10 μM Max-RP... The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 118 mg of the formate of the title compound was obtained (0.151 mmol, 85%). LC-MS / RT = 1.06 min (Method B); ESI-MS m / z 737.55 [M+H] + ;HRMSm / z 737.4729[C 38 H 65 N6O6Cl+H] + .

[0511] Example 38

[0512] (S)-4-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((2-chlorophenylethyl)amino)-1-methyl (Oxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-2-((S)-2- (dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0513]

[0514] DIEA (0.300 g, 400 μL, 2.29 mmol) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-OH TFA salt (0.306 g, 0.399 mmol) and H-Dap-2-(2-chlorophenyl)ethylamine TFA salt (0.170 g, 0.388 mmol) at room temperature, followed by the addition of HATU (360 mg, 0.944 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Gemini 10 μL Max-RP. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 115 mg of Dov-Dab(Fmoc)-Dil-Dap-2-(2-chlorophenyl)ethylamine was obtained as a white solid (0.120 mmol, 26%). LCMS RT = 1.40 min (Method B); ESI-MS m / z 959.75 [M+H] + .

[0515] Piperidine (2 mL) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-Dap-2-(2-chlorophenyl)ethylamine (0.115 g, 0.120 mmol) at room temperature in acetonitrile (10 mL). After 3 hours, analysis by LCMS showed that the reaction was complete. Hexane was added to the crude reaction mixture. The acetonitrile layer was concentrated under vacuum. The concentration was determined by using Phenomenex Synergi 10 μ Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 30.0 mg of the title compound was obtained as a white solid (0.041 mmol, 34%). LC-MS / RT = 1.15 min (Method B); ESI-MS m / z 737.68 [M+H] + HRMS m / z 737.4729[C 38 H65 N6O6Cl+H] + .

[0516] Example 39

[0517] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyryl) (amino)-N-methylpentan-4-ynylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methyl (Propionyl)-L-phenylalanine methyl ester

[0518]

[0519] DIEA (1.49 mL, 9.38 mmol) was added to a stirred solution of Boc-propargyl Gly-OH (1.00 g, 4.69 mmol) and H-Dil-OtBu hydrochloride (1.15 g, 3.90 mmol) at 25 °C. The solution was cooled to 0 °C and stirred for 20 min, and an additional portion of DIEA (1.49 mL, 9.38 mmol) was added, and the reaction mixture was stirred at 0 °C for 20 min. CMPI (1.80 g, 7.04 mmol) was then added to the reaction mixture, which was heated to room temperature and stirred for 12 h. The crude reaction mixture was washed with 0.1 M HCl (20 mL x 2), followed by brine (20 mL x 2). The organic fraction was dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum to give the crude product. A total of 2.05 g of Boc-propargyl Gly-Dil-OtBu (4.50 mmol, 96%) was obtained. LCMS RT = 3.46 minutes (Method A); ESI-MS m / z 455.42 [M+H] + .

[0520] TFA (6 mL) was added to a stirred solution of Boc-propargyl Gly-Dil-OtBu (2.05 g, 4.50 mmol) at 25 °C in CH2Cl2 (6 mL). After 14 hours, analysis by LCMS showed that the reaction was complete. The volatile organic compounds were concentrated under vacuum to obtain a crude product that did not require further purification. A total of 1.30 g of TFA salt of H-propargyl Gly-Dil-OH was obtained (3.16 mmol, 70%).

[0521] DIEA (2.33 mL, 13.1 mmol) was added to a stirred 25°C solution of Dov (1.27 g, 8.71 mmol) in DMF (10 mL), followed by HATU (3.32 g, 8.71 mmol). After 10 minutes, a solution of H-propargyl Gly-Dil-OH TFA salt (1.30 g, 3.16 mmol) in DMF was added to the reaction. The reaction was complete after 8 hours, as shown by LC-MS analysis. The reaction was confirmed using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 455 mg of the TFA salt of Dov-propargyl Gly-Dil-OH was obtained (0.844 mmol, 27% yield). LCMS RT = 1.65 min (Method A); ESI-MS m / z 425.95 [M+H] + .

[0522] DIEA (0.222 mL, 1.40 mmol) was added to a stirred solution of Dov-propargyl Gly-Dil-OH TFA salt (0.198 g, 0.367 mmol) and H-Dap-Phe-OMe TFA salt (0.156 g, 0.338 mmol) at room temperature, followed by the addition of HATU (0.355 g, 0.931 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried over magnesium sulfate, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Synergi 10 μM Max-RP. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 10.0 mg of the title compound in TFA salt (0.012 mmol, 3%) was obtained. LCMS RT = 2.50 min (Method A); ESI-MS m / z 756.44 [M+H] + ;HRMS m / z756.4902[C 41 H 65 N5O8+H] + .

[0523] Example 40

[0524] (2S,3S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenyl) (propyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxo (hept-4-yl)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0525]

[0526] Hoenil (0.045 mL, 0.258 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-OH formate (42.1 mg, 0.051 mmol), ammonium chloride (7.9 mg, 0.148 mmol), and TBTU (52.5 mg, 0.163 mmol) at room temperature. After 16 hours, analysis by LC-MS showed that the reaction was complete. The reaction was confirmed by using a Phenomenex Gemini-NX 10 μC-18 thermometer. The crude reaction mixture was purified by preparative RP-HPLC using a column (150 x 30 mm) with 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of 13.4 mg of the title compound (0.017 mmol, 33%) was obtained. LCMS RT = 1.05 min (Method B); ESI-MS m / z 772.61 [M+H] + HRMS m / z 772.5078[C 39 H 65 N9O7+H] + .

[0527] Example 41

[0528] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)- 3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino) (propyl)pyrrolidone-1-yl)-5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0529]

[0530] DIEA (343 μL, 1.97 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-OH TFA salt (0.300 g, 0.526 mmol) and H-Dap-2-(2-pyridyl)ethylamine TFA salt (0.287 g, 0.709 mmol) at 23 °C, followed by the addition of HATU (0.501 g, 1.31 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Gemini-NX 10 μC-18 micrometer. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 139 mg of the title compound (0.190 mmol, 29%) was obtained. LCMS RT = 0.916 min (Method B); ESI-MS m / z 730.64 [M+H] + HRMS m / z 730.4985[C 37 H 63 N9O6+H] + .

[0531] Example 42

[0532] (2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino) (1-O-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy (5-methyl-1-oxohep-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0533]

[0534] Hoenil (0.022 mL, 0.124 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-OH formate (24.0 mg, 0.029 mmol), tert-butylamine hydrochloride (7.9 mg, 0.072 mmol), and HATU (24.5 mg, 0.064 mmol) at room temperature. After 18 hours, analysis by LC-MS showed that the reaction was complete. The reaction was confirmed by using a Phenomenex Gemini-NX 10 μC-18... The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of 15.4 mg of the title compound (0.017 mmol, 59%) was obtained. LCMS RT = 1.27 min (Method B); ESI-MS m / z 828.8 [M+H] + HRMS m / z 828.5671[C 39 H 66 N6O8+H] + .

[0535] Example 43

[0536] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino) (N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy 2-methylpropionyl)-L-valine methyl ester

[0537]

[0538] DIEA (4.44 g, 6.00 mL, 34.4 mmol) was added to a stirred suspension of Boc-Dap-OH dicyclohexylamine salt (4.00 g, 8.54 mmol) and H-Val-OMe HCl salt (1.80 g, 10.7 mmol) at 20 mL of CH2Cl2. This was followed by the addition of HATU (2.15 g, 2.00 mL, 0.013 mol). After 10 hours, the reaction was complete as indicated by LCMS analysis. The volatile organic compounds were evaporated under vacuum to give a crude product that required no further purification. A total of 6.80 g of colorless oily Boc-Dap-Val-OMe (17.0 mmol, 80%) was obtained. LCMS RT = 1.33 min (Method B); ESI-MS m / z 401.6 [M+H] + .

[0539] The suspension of Boc-Dap-Val-OMe (6.80 g, 17.0 mmol) in 4.0 M HCl in dioxane (20 mL) was stirred at 23 °C. After 4 hours, analysis by LCMS showed that the reaction was complete. The volatile organic compounds were evaporated under vacuum to give the crude product, which did not require further purification. A total of 4.57 g of H-Dap-Val-OMe hydrochloride (13.6 mmol, 80%) was obtained. LCMS RT = 0.726 min (Method B); ESI-MS m / z 301.45 [M+H] + .

[0540] DIEA (0.222 g, 0.300 mL, 1.72 mmol) was added to a stirred suspension of Dov-Abu(3-N3)-Dil-OH TFA salt (0.144 g, 0.253 mmol) and H-Dap-Val-OMe HCl salt (0.332 g, 0.985 mmol) at 23 °C in DMF (10 mL), followed by HATU (0.240 g, 0.631 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Gemini-NX 10 μC-18 micrometer. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 129 mg of the title compound (0.175 mmol, 55%) as a pale yellow solid was obtained. LCMS RT = 1.15 min (Method B); ESI-MS m / z 739.75 [M+H] +HRMS m / z 739.5074 [C 36 H 66 N8O8+H] + .

[0541] Example 44

[0542] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-6-amino-2-((S)-2-(dimethylamino)1-3- (Methylbutyrylamino)-N-methylhexanoylamino1-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2- Methylpropionyl-L-phenylalanine methyl ester

[0543]

[0544] DIEA (4.44 g, 6.00 mL, 34.4 mmol) was added to a stirred solution of Boc-Lys(Fmoc)-OH (5.60 g, 12.0 mmol) and H-Dil-OtBu hydrochloride (3.06 g, 10.9 mmol) at 23 °C. The solution was cooled to 0 °C and stirred for 0.5 h. After 0.5 h, another DIEA (4.44 g, 6.00 mL, 34.4 mmol) was added to the reaction mixture and the reaction was stirred at 0 °C for 0.5 h. Then CMPI (4.20 g, 16.4 mmol) was added to the reaction mixture, which was slowly heated to room temperature and stirred for 10 h. The crude reaction mixture was washed with 1 M HCl (30 mL x 2) followed by brine (25 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. A total of 7.38 g of pale yellow solid Boc-Lys(Fmoc)-Dil-OtBu (10.4 mmol, 86%) was obtained. LCMS RT = 1.85 min (Method B); ESI-MS m / z 710.1 [M+H] + .

[0545] A suspension of Boc-Lys(Fmoc)-Dil-OtBu (7.38 g, 10.4 mmol) in 4.0 M HCl was stirred at 23 °C. After 10 hours, LCMS analysis showed that the reaction was complete. The volatile organic compounds were evaporated under vacuum to give a crude product that did not require further purification. A total of 7.05 g of a pale yellow HCl salt of H-Lys(Fmoc)-Dil-OH (0.012 mol, 89%) was obtained. LCMS RT = 1.20 min (Method B); ESI-MS m / z 554.54 [M+H] + .

[0546] Add DIEA (4.44 g, 6.00 mL, 0.034 mol) to the suspension of Dov-OH (2.50 g, 17.2 mmol) in DMF (20 mL) and stir for 23 °C. Then add HATU (4.82 g, 12.6 mmol). After 5 minutes, add H-Lys(Fmoc)-Dil-OH HCl salt (7.05 g, 0.012 mol) to the reaction mixture. After 4 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by using a Phenomenex Gemini NX-C18 10u The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 4.45 g of pale yellow solid, Dov-Lys(Fmoc)-Dil-OH (6.54 mmol, 78%), was obtained. LC-MS / RT = 1.21 min (Method B); ESI-MS m / z 681.68 [M+H] + .

[0547] DIEA (1.65 g, 2.20 mL, 12.7 mmol) was added to a stirred suspension of Dov-Lys(Fmoc)-Dil-OH (2.17 g, 3.19 mmol) and H-Dap-OMe TFA salt (1.78 g, 3.85 mmol) at 23 °C in DMF (10 mL), followed by HATU (1.94 g, 5.10 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by a Phenomenex Gemini 10 μM Max-RP. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 2.25 g of pale yellow solid, Dov-Lys(Fmoc)-Dil-Dap-Phe-OMe (2.23 mmol, 70%), was obtained. LCMS RT = 1.29 min (Method B); ESI-MS m / z 1011.77 [M+H] + .

[0548] Piperidine (5 mL) was added to a suspension of Dov-Lys(Fmoc)-Dil-Dap-Phe-OMe (2.25 g, 2.23 mmol) in acetonitrile (20 mL) at 23 °C under stirring. After 2 hours, LCMS analysis showed the reaction was complete. Hexane was added to the crude reaction solution. The acetonitrile layer was concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C1810μm analyzer. The crude oil was purified by preparative RP-HPLC using a column (150 x 30 mm) with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 898 mg of the title compound (1.14 mmol, 51%) was obtained as a white solid. LCMS RT = 0.840 min (Method B); ESI-MS m / z 789.5 [M+H] + HRMS m / z 789.5482[C 42 H 72 N6O8+H] + .

[0549] Example 45

[0550] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,4S)-4-azido-1-(dimethyl-L-valine) (N-methylpyrrolidine-2-carbamate)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methyl L-phenylalanine methyl ester (-propionyl)

[0551]

[0552] DIEA (2.22 g, 3.0 mL, 17.2 mmol) was added to a suspension of cis-Fmoc-Pro(4-N3)-OH (2.00 g, 5.27 mmol) and H-Dil-OtBu HCl (1.49 g, 5.27 mmol) stirred at 23 °C. The solution was cooled to 0 °C and stirred for 0.5 h. Additional DIEA (2.22 g, 3.0 mL, 17.2 mmol) was added to the cooled reaction mixture, and the reaction mixture was stirred at 0 °C for 0.5 h. Then CMPI (2.03 g, 7.93 mmol) was added to the reaction mixture, which was slowly heated to room temperature and stirred for 10 h. The crude reaction mixture was washed with 1 M HCl (30 mL x 2) followed by brine (25 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The crude product was ready for use without further purification. A total of 3.13 g of a yellow oily substance, cis-Fmoc-Pro(4-N3)-Dil-OtBu (5.05 mmol, 76%), was obtained. LCMS RT = 1.73 min (Method B); ESI-MS m / z 621.46 [M+H] + .

[0553] Piperidine (10 mL) was added to a stirred solution of cis-Fmoc-Pro(4-N3)-Dil-OtBu (3.13 g, 4.04 mmol) at 23 °C in acetonitrile (20 mL). After 10 hours, analysis by LCMS showed that the reaction was complete. Hexane was added to the crude reaction mixture. The acetonitrile layer was concentrated under vacuum, and no further purification was required to use the crude product. A total of 1.57 g of clear oil of H-Pro(4-N3)-Dil-OtBu (3.95 mmol, 88%) was obtained. LCMS RT = 1.19 min (Method B); ESI-MS m / z 398.50 [M+H] + .

[0554] DIEA (1.84 g, 2.50 mL, 0.014 mol) was added to a DMF (10 mL) suspension containing Dov-OH (1.03 g, 7.12 mmol) and H-Pro(4-N3)-Dil-OtBu (1.57 g, 3.56 mmol) under stirring at 23 °C, followed by the addition of HATU (2.03 g, 5.33 mmol). After 3 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C1810μm analyzer. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 817 mg of Dov-Pro(4-N3)-Dil-OtBu (1.56 mmol, 44%) as a white solid was obtained. LCMS RT = 1.21 min (Method B); ESI-MS m / z 525.28 [M+H] + .

[0555] The suspension of Dov-Pro(4-N3)-Dil-OtBu (0.817 g, 1.56 mmol) in 3.0 HCl was stirred at 23 °C. After 10 hours, analysis by LCMS showed that the reaction was complete. The volatile organic compounds were evaporated under vacuum to give the crude product, which did not require further purification. A total of 704 mg of the HCl salt of Dov-Pro(4-N3)-Dil-OH (1.39 mmol, 89%) was obtained. LCMS RT = 0.676 min (Method B); ESI-MS m / z 469.44 [M+H] + .

[0556] DIEA (0.132 g, 0.200 mL, 1.02 mmol) was added to a stirred suspension of Dov-Pro(4-N3)-Dil-OH HCl salt (0.120 g, 0.256 mmol) and H-Dap-Phe-OMe TFA salt (0.143 g, 0.310 mmol) at 23 °C in DMF (10 mL), followed by HATU (0.156 g, 0.410 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C1810μm analyzer. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 37.0 mg of the title compound (0.046 mmol, 18%) as a yellow solid was obtained. LCMS RT = 1.13 min (Method B); ESI-MS m / z 799.43 [M+H] + HRMS m / z 799.5064[C 41 H 66 N8O8+H] + .

[0557] Example 46

[0558] (S)-3-((S)-2-(dimethylamino)-3-methylbutyrylamino)-4-((( 3 R,4S,5S)-3-methoxy-1- ((S)-2-((1R,2R)-1-methoxy-3-(((S)-1-methoxy-1-oxo-3-phenylprop-2-yl)amino)-2-methyl- 3-Oxopropyl)pyrrolidone-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino-4-oxobutyric acid

[0559]

[0560] DIEA (6.00 g, 8.10 mL, 46.41 mmol) was added to a stirred solution of Boc-Asp(OBzl)-OH (5.00 g, 15.5 mmol) and H-Dil-OtBuTFA salt (4.36 g, 15.5 mmol) at 23 °C. The reaction mixture was cooled to 0 °C and stirred for 0.5 h. After 0.5 h, another DIEA (6.00 g, 8.01 mL, 46.4 mmol) was added to the reaction mixture and stirred for 0.5 h. Then CMPI (5.93 g, 23.2 mmol) was added to the reaction mixture, which was slowly heated to room temperature and stirred for 10 h. The crude reaction mixture was washed with 1 M HCl (30 mL x 2) followed by brine (25 mL x 2). The organic fraction was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was ready for use without further purification. A total of 7.85 g of brown oily substance, Boc-Asp(OBzl)-Dil-OtBu (13.9 mmol, 90%), was obtained. LCMS RT = 1.72 min (Method B); ESI-MS m / z 565.3 [M+H] + .

[0561] A solution of Boc-Asp(OBzl)-Dil-OtBu (7.85 g, 13.9 mmol) in 4.0 M HCl was stirred at 23 °C. After 10 hours, LC-MS analysis showed that the reaction was complete. The volatile organic compounds were evaporated under vacuum to give a crude product that did not require further purification. A total of 7.85 g of H-Asp(OBzl)-Dil-OH enriched hydrochloride was obtained. LC-MS RT = 0.951 min (Method B); ESI-MS m / z 409.40 [M+H] + .

[0562] DIEA (6.96 g, 9.40 mL, 0.054 mol) and HATU (7.60 g, 19.9 mmol) were added to a stirred suspension of Dov-OH (3.00 g, 20.7 mmol) in DMF (20 mL) at 23 °C, followed by the addition of H-Asp(OBzl)-Dil-OH HCl salt (7.85 g). After 4 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C18 10 μL filter. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 2.00 g of white solid Dov-Asp(OBzl)-Dil-OH (3.74 mmol, 28%) was obtained. LCMS RT = 1.10 min (Method B); ESI-MS m / z 536.5 [M+H] + .

[0563] DIEA (0.970 g, 1.30 mL, 7.47 mmol) was added to a suspension of Dov-Asp(OBzl)-Dil-OH (1.00 g, 1.87 mmol) and H-Dap-Phe-OMe TFA salt (1.04 g, 2.25 mmol) in DMF (10 mL) at 23 °C with stirring. HATU (1.14 g, 2.99 mmol) was then added. After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by using a Phenomenex Gemini NX-C18 10 μL filter. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 1.25 g of orange solid Dov-Asp(OBzl)-Dil-Dap-Phe-OMe (1.44 mmol, 77%) was obtained. LCMS RT = 1.21 min (Method B); ESI-MS m / z 866.6 [M+H] + .

[0564] 10% Pd / C (50 mg) was added to a stirred suspension of Dov-Asp(OBzl)-Dil-Dap-Phe-OMe (0.525 g, 0.606 mmol) at 23 °C in MeOH (10 mL), followed by the addition of a hydrogen atmosphere. After 5 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was filtered on a diatomaceous earth mat, and volatile organic compounds were then evaporated. The volatile organic compounds were analyzed using a Phenomenex Gemini NX-C1810μm analyzer. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 6.00 mg of the title compound (0.008 mmol, 1%) as a white solid was obtained. LCMS RT = 1.04 min (Method B); ESI-MS m / z 776.43 [M+H]+ HRMS m / z 776.4794[C 40 H 65 N5O 10 +H] + .

[0565] Example 47

[0566] (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyryloxy)-3-hydroxy-N-((3R,4S,5S)-1- ((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxo (propyl)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0567]

[0568] DIEA (417 mg, 562 μL, 3.23 mmol) was added to a stirred solution of Dov-Dab(Fmoc)-Dil-OH TFA salt (561 mg, 0.883 mmol) and H-Dap-(1R, 2S)-(-)-norephedrine TFA salt (345 mg, 0.794 mmol) at 23 °C, followed by the addition of HATU (820 mg, 2.15 mmol). After 8 hours, analysis by LCMS showed the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was analyzed using a Phenomenex Gemini NX-C1810 μL... The crude oily substance was purified by preparative RP-HPLC using a column (150 x 30 mm) with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 326 mg of Dov-Thr(Bzl)-Dil-Dap-(1R,2S)-(-)-norephedrine (0.396 mmol, 37%) was obtained as a white solid. LCMS RT = 1.20 min (Method B); ESI-MS m / z 824.76 [M+H] + .

[0569] A suspension of Dov-Thr(Bzl)-Dil-Dap-(1R,2S)-(-)-norephedrine (326 mg, 0.396 mmol) in MeOH (10 mL) at 23 °C was loaded into a continuous-flow hydrogenation reactor using RaNi (CatCart) with elevated temperature (120 °C) and pressure (80 bar). After elution, analysis by LCMS showed that the reaction was complete. Volatile organic compounds were evaporated under vacuum to give the crude product, which was then purified using a Phenomenex Gemini NX-C18 10 μL filter. Preparative RP-HPLC purification was performed using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 15 mg of the title compound as a white solid (0.020 mmol, 5%) was obtained. LCMS RT = 1.17 min (Method B); ESI-MS m / z 734.5 [M+H] + HRMS m / z 734.5053[C 39 H 67 N5O8+H] + .

[0570] Example 48

[0571] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyryl) (amino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methyl (propionyl)-L-serine methyl ester

[0572]

[0573] DIEA (223 mg, 0.324 mL, 1.72 mmol) was added to a stirred suspension of Dov-Val-Dil-OH TFA salt (583 mg, 1.07 mmol) and H-Dap-Ser-OMe TFA salt (783 mg, 1.97 mmol) at 23 °C in DMF (10 mL), followed by HATU (1.03 g, 2.71 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by using a Phenomenex Gemini NX-C18 10 μL filter. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 704 mg of the title compound (1.01 mmol, 74%) as a pale yellow solid was obtained. LCMS RT = 0.917 min (Method B); ESI-MS m / z 700.43 [M+H] + HRMS m / z 700.4843[C 35 H 65 N5O9+H] + .

[0574] Example 49

[0575] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino) (N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy (2-methylpropionyl)-L-isoleucine methyl ester

[0576]

[0577] DIEA (7.30 g, 9.90 mL, 56.5 mmol) was added to a stirred suspension of Boc-Dap-OH dicyclohexylamine salt (6.62 g, 14.1 mmol) and H-Ile-OMe (3.08 g, 21.2 mmol) at 20 mL of CH₂Cl₂, followed by the addition of HATU (3.44 g, 3.20 mL, 0.021 mol). After 10 hours, the reaction was complete as indicated by LCMS analysis. The volatile organic compounds were evaporated under vacuum to give a crude product that required no further purification. A total of 8.56 g of brown oily Boc-Dap-Ile-OMe (20.7 mmol, 88%) was obtained. LCMS RT = 1.51 min (Method B); ESI-MS m / z 415.16 [M+H] + .

[0578] TFA (10 mL) was added to a suspension of Boc-Dap-Ile-OMe (6.62 g, 16.0 mmol) in CH2Cl2 (20 mL) at 23 °C with stirring. After 10 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were evaporated under vacuum. The volatile organic compounds were analyzed using a Phenomenex Gemini NX-C18 10 μL filter. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 3.55 g of H-Dap-Ile-OMe (8.29 mmol, 52%) as a yellow solid was obtained. LCMS RT = 0.691 min (Method B); ESI-MS m / z 315.16 [M+H] + .

[0579] DIEA (170 mg, 0.220 mL, 1.31 mmol) was added to a DMF (10 mL) suspension containing Dov-Abu(3-N3)-Dil-OH TFA salt (150 mg, 0.329 mmol) and H-Dap-Ile-OMe (207 mg, 0.657 mmol) under stirring at 23 °C, followed by the addition of HATU (251 mg, 0.657 mmol). After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by using a Phenomenex Gemini NX-C18 10 μL filter. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 90% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 148 mg of the title compound (0.197 mmol, 60%) was obtained as a pale yellow solid. LCMS RT = 1.43 min (Method B); ESI-MS m / z 753.48 [M+H] + HRMS m / z 753.5224[C 37 H 68 N8O8+H] + .

[0580] Example 50

[0581] ( 2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo- 3-Phenylacet-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl- 1-O-hept-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0582]

[0583] Trimethylphosphine (1M, 0.035mL, 0.035mmol) in THF was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-NH2 (13.4mg, 0.017mmol) at room temperature. After 3 hours, additional trimethylphosphine (1M, 0.020mL, 0.020mmol) was added. After another 1 hour, analysis by LCMS showed that the reaction was complete. The solution was diluted with water and DMF and allowed to stand for 30 minutes. The reaction was then analyzed using a Phenomenex Gemini-NX 10μC18 micrometer. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of 5.8 mg of the title compound (0.008 mmol, 45%) was obtained. LCMS RT = 0.85 min (Method B); ESI-MS m / z 746.6 [M+H] + HRMS m / z 746.5173[C 39 H 67 N7O7+H] + .

[0584] Example 51

[0585] (2S,3S)-3-amino-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino) (1-O-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy (5-methyl-1-oxohep-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyramide

[0586]

[0587] Trimethylphosphine (1M, 0.037mL, 0.037mmol) in THF was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-NHt-Bu (15.4mg, 0.019mmol) at room temperature. After 3 hours, additional trimethylphosphine (1M, 0.020mL, 0.020mmol) was added. After another 1 hour, analysis by LCMS showed that the reaction was complete. The reaction solution was diluted with water and DMF and allowed to stand for 30 minutes. The reaction was then analyzed using a Phenomenex Gemini-NX 10μC-18. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of 2.7 mg of the title compound (0.003 mmol, 18%) was obtained. LCMS RT = 0.85 min (Method B); ESI-MS m / z 746.6 [M+H] + HRMS m / z 802.5799[C 43 H 75 N7O7+H] + .

[0588] Example 52

[0589] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-N-methyl-2-((S)-3-methyl-2- (methylamino)butyrylamino)propionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methyl L-phenylalanine methyl ester (-propionyl)

[0590]

[0591] DIEA (1.35 mL, 7.74 mmol) was added to a suspension of Fmoc-MeVal-OH (1.03 g, 2.90 mmol) in DMF (10 mL) at 23 °C under stirring, followed by HATU (1.11 g, 2.90 mmol). After 5 minutes, H-4-azido-Ala-Dil-OH (0.610 g, 2.90 mmol) was added to the reaction mixture. After 10 hours, the reaction was complete as indicated by LCMS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (15 mL) and extracted with EtOAc (40 mL x 2). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Gemini NX-C18 10 μL filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 348 mg of yellow oil (0.535 mmol, 28%) of Fmoc-MeVal-4-azido-Ala-Dil-OH was obtained. LCMS RT = 1.80 min (Method B); ESI-MS m / z 651.3 [M+H] + .

[0592] DIEA (276 mg, 0.400 mL, 2.14 mmol) was added to a DMF (10 mL) suspension containing Fmoc-MeVal-4-azido-Ala-Dil-OH (348.00 mg, 0.535 mmol) and H-Dap-Phe-OMe (372.66 mg, 1.07 mmol) under stirring at 23 °C, followed by the addition of HATU (408 mg, 1.07 mmol). After 10 hours, LC-MS analysis showed complete reaction with deprotection of the Fmoc group. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex GeminiNX-C18 10 μL filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 5.00 mg of the title compound (0.006 mmol, 1%) was obtained. LCMS RT = 1.08 min (Method B); ESI-MS m / z 759.5 [M+H] + ;HRMS m / z759.4753[C 38 H 62 N8O8+H] + .

[0593] Example 53

[0594] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl) 2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy methyl 2-methylpropionyl)-L-phenylalanine methyl ester

[0595]

[0596] DIEA (3.30 g, 4.5 mL, 25.5 mmol) was added to a stirred suspension of Fmoc-MeVal-OH (2.46 g, 6.96 mmol) and H-Abu(3-N3)-Dil-OtBu (2.46 g, 6.38 mmol) at 23 °C in 10 mL of DMF, followed by HATU (3.65 g, 9.57 mmol). After 6 hours, LC-MS analysis showed the reaction was complete. The reaction was confirmed by using a Phenomenex GeminiNX-C18 10 μL filter. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 3.16 g of Fmoc-MeVal-Abu(3-N3)-Dil-OtBu formate (4.12 mmol, 65%) was obtained. LCMS RT = 2.08 min (Method A); ESI-MS m / z 722.7 [M+H] + .

[0597] TFA (10.0 mL) was added to a suspension of Fmoc-MeVal-Abu(3-N3)-Dil-OtBu formate (3.16 g, 4.12 mmol) in CH2Cl2 (5.0 mL) at 23 °C with stirring. After 14 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were evaporated under vacuum to obtain an oil. The oil was then analyzed using a Phenomenex Gemini NX-C18 10 μL filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. A total of 2.26 g of TFA salt of Fmoc-MeVal-Abu(3-N3)-Dil-OH was obtained (2.90 mmol, 66%). LCMS RT = 1.80 min (Method B); ESI-MS m / z 665.3 [M+H] + .

[0598] DIEA (0.083 mmol, 0.481 mmol) was added to a suspension of Fmoc-MeVal-Abu(3-N3)-Dil-OH TFA salt (80.0 mg, 0.103 mmol) and H-Dap-Phe-OMe TFA salt (83.9 mg, 0.182 mmol) in DMF (2 mL) at 23 °C with stirring. HATU (91.8 mg, 0.241 mmol) was then added. After 12 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was diluted with DMF and purified using a Phenomenex Gemini NX-C18 10 μL filter. Preparative RP-HPLC purification was performed using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 97.0 mg of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-OMe formate was obtained (0.093 mmol, 77%). LCMS RT = 1.90 min (Method B); ESI-MS m / z 995.6 [M+H] + .

[0599] Piperidine (5 mL) was added to a solution of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-OMe (97.0 mg, 0.093 mmol) in acetonitrile (10 mL) at 23 °C under stirring. After 5 hours, LCMS analysis showed the reaction was complete. Hexane (25 mL x 3) was added to the crude reaction solution to extract nonpolar byproducts. The acetonitrile layer was concentrated under vacuum and analyzed using a Phenomenex Gemini NX-C1810 μL microscope. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous NH4OH as the eluent. A total of 74.0 mg of the title compound as a white solid (0.096 mmol, 103%) was obtained. LCMS RT = 1.09 min (Method B); ESI-MS m / z 773.5 [M+H] + ;HRMS m / z773.4911[C 39 H 64 N8O8+H] + .

[0600] Example 54

[0601] (2S,3S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-amino-1-oxo-3-phenyl) (propyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxo (hept-4-yl)-3-azido-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide

[0602]

[0603] DIEA (11.0 g, 14.9 mL, 85.3 mmol) was added to a stirred suspension of Bod-Dap-OH dicyclohexylamine salt (10.0 g, 21.3 mmol) and H-Phe-NH2HCl salt (6.42 g, 32.0 mmol) at 23 °C in CH2Cl2 (20.0 mL), followed by DEPC (5.19 g, 4.80 mL, 0.032 mol). After 10 hours, the reaction was complete as shown by LCMS analysis. The crude reaction mixture was washed with H2O (25 mL x 2) followed by brine (25 mL x 2). The organic fraction was dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. Using 2% to 10% methanol in CH2Cl2 as the eluent, the mixture was analyzed by rapid chromatography (silica gel 40 μm, (Size) Purified crude orange oil. A total of 7.25 g of yellow oil of Boc-Dap-Phe-NH2 was obtained (16.7 mmol, 78%). LCMS RT = 1.28 min (Method B); ESI-MS m / z 434.19 [M+H] + .

[0604] TFA (10 mL) was added to a stirred suspension of Boc-Dap-Phe-NH2 (7.25 g, 16.7 mmol) at 23 °C in CH2Cl2 (10 mL). After 5 hours, analysis by LCMS showed that the reaction was complete. The volatile organic compounds were evaporated under vacuum to give a crude product that did not require further purification. A total of 6.00 g of yellow solid H-Dap-Phe-NH2 (13.4 mmol, 80%) was obtained. LCMS RT = 0.691 min (Method B); ESI-MS m / z 334.17 [M+H] + .

[0605] DIEA (0.350 g, 0.500 mL, 2.74 mmol) was added to a stirred suspension of Fmoc-MeVal-Abu(3-N3)-Dil-OH TFA salt (456 mg, 0.586 mmol) and H-Dap-Phe-NH2TFA salt (457 mg, 1.02 mmol) at 23 °C in 10 mL of DMF, followed by HATU (0.520 g, 1.37 mmol). After 10 hours, the reaction was complete as shown by LC-MS analysis. The reaction was confirmed by using a Phenomenex Gemini NX-C18 10 μL filter. The crude reaction was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 526 mg of formate of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-NH2 was obtained (0.513 mmol, 75%). LCMS RT = 1.81 min (Method B); ESI-MS m / z 980.39 [M+H] + .

[0606] Piperidine (5 mL) was added to a 10 mL solution of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-NH2 (525 mg, 0.513 mmol) at 23 °C under stirring. After 2 hours, LCMS analysis showed the reaction was complete. Hexane (15 mL x 3) was added to the crude reaction mixture. The acetonitrile layer was concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C18 10 μL filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. A total of 354 mg of the title compound in TFA salt (0.406 mmol, 79%) was obtained. LCMS RT = 1.15 min (Method B); ESI-MS m / z 758.24 [M+H] + HRMS m / z 758.4915[C 38 H 63 N9O7+H] + .

[0607] Example 55

[0608] ((2S,3S)-3-azido-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(tert-butylamino) (1-O-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy (-5-methyl-1-oxohep-4-yl)-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide

[0609]

[0610] Add humic base (0.079 mL, 0.454 mmol) to a stirred solution of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-OH (111 mg, 0.113 mmol), tert-butylamine hydrochloride (31.5 mg, 0.287 mmol), and HATU (92.0 mg, 0.242 mmol) at room temperature. After 1 hour, analysis by LCMS showed that the reaction was complete. Dilute the crude reaction mixture with ethyl acetate and wash the organic fraction with 1N HCl and brine. Dry the organic fraction on magnesium sulfate, filter, and concentrate under reduced pressure. The crude yellow oily substance was dissolved in piperidine (2.0 mL) and acetonitrile (5.0 mL). After 1 hour, analysis by LCMS showed that the reaction was complete. Extract the acetonitrile layer with ethane (2x) and concentrate the acetonitrile layer under reduced pressure. The solution was analyzed using a Phenomenex Gemini-NX 10 μC-18. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of 50.3 mg of the title compound (0.062 mmol, 55%) was obtained. LCMS RT = 1.29 min (Method B); ESI-MS m / z 814.1 [M+H] + HRMS m / z 814.5541[C 42 H 71 N9O7+H] + .

[0611] Example 56

[0612] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl) 2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy 2-methylpropionyl)-L-phenylalanine tert-butyl ester

[0613]

[0614] DIEA (8.76 mL, 55.2 mmol) was added to a stirred solution of Boc-Dap-OH dicyclohexylamine salt (6.47 g, 13.8 mmol) and H-Phe-OtBu (3.91 g, 15.2 mmol) at 25 °C in DCM (20 mL), followed by DEPC (3.12 mL, 20.7 mmol). After 8 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were evaporated under vacuum to give a crude product that required no further purification. A total of 5.35 g of Boc-Dap-Phe-OtBu (10.9 mmol, 79%) was obtained. LCMS RT = 2.89 min (Method A); ESI-MS m / z 491.48 [M+H] + .

[0615] TFA (10.0 mL) was added to a stirred solution of Boc-Dap-Phe-OtBu (5.25 g, 10.7 mmol) at room temperature in CH2Cl2 (10.0 mL). After 12 hours, analysis by LCMS showed the reaction was complete. The reaction was confirmed by using a Phenomenex Synergi 10 μMax-RP... The crude oil was purified by preparative RP-HPLC using a 150 x 30 mm column with 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total of 2.85 g of TFA salt of H-Dap-Phe-OtBu was obtained (5.65 mmol, 68%). LC-MS / RT = 1.82 min (Method A); ESI-MS m / z 391.02 [M+H] + .

[0616] DIEA (319 mg, 430 μL, 2.47 mmol) was added to a DMF (10 mL) suspension containing Fmoc-MeVal-Abu(3-N3)-Dil-OH TFA salt (410 mg, 0.527 mmol) and H-Dap-Phe-OtBu TFA salt (482 mg, 0.956 mmol) under stirring at 23 °C, followed by the addition of HATU (470 mg, 1.23 mmol). After 10 hours, LCMS analysis showed complete reaction with 10% removal of Fmoc groups. The reaction was confirmed by using a Phenomenex Gemini NX-C1810 μL thermometer. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 26.0 mg of the title compound in white formate (0.030 mmol, 5%) was obtained. LCMS RT = 1.41 min (Method B); ESI-MS m / z 815.33 [M+H] + HRMS m / z 815.5383[C 42 H 70 N8O8+H] + .

[0617] Example 57

[0618] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-N-methyl-2-((S)-3-methyl) 2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy L-phenylalanine (-2-methylpropionyl)-L-phenylalanine

[0619]

[0620] DIEA (319 mg, 430 μL, 2.47 mmol) was added to a DMF (10 mL) suspension containing Fmoc-MeVal-Abu(3-N3)-Dil-OH TFA salt (410 mg, 0.527 mmol) and H-Dap-Phe-OtBu TFA salt (482 mg, 0.956 mmol) under stirring at 23 °C, followed by the addition of HATU (470 mg, 1.23 mmol). After 10 hours, LCMS analysis showed the reaction was complete. The reaction was confirmed by using a Phenomenex Gemini NX-C1810 μL... The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 648 mg of white formate (0.598 mmol, 97%) of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-OtBu was obtained. LCMS RT = 1.41 min (Method B); ESI-MS m / z 1037.41 [M+H] + .

[0621] TFA (5.00 mL) was added to a suspension of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-OtBu formate (648 mg, 0.598 mmol) in CH2Cl2 (5.00 mL) under stirring at 23 °C. After 2 hours, LCMS analysis showed the reaction was complete. The volatile organic compounds were evaporated under vacuum to give a brown oil. The oil was analyzed using a Phenomenex Gemini NX-C18 10 μL filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. A total of 502 mg of TFA salt of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-OH was obtained (0.458 mmol, 77%). LCMS RT = 1.81 min (Method B); ESI-MS m / z 981.22 [M+H] + .

[0622] Piperidine (5 mL) was added to a stirred solution of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-Phe-OH TFA salt (392 mg, 0.382 mmol) at 23 °C for acetonitrile (10 mL). After 2 hours, LCMS analysis showed the reaction was complete. Hexane (15 mL x 3) was added to the crude reaction mixture. The acetonitrile layer was concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C1810μL microscope. The crude oil was purified by preparative RP-HPLC using a 150x30mm column with 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. A total of 360 mg of the title compound in TFA salt was obtained. LCMS RT = 1.19 min (Method B); ESI-MS m / z 759.13 [M+H] + HRMS m / z 759.4755[C 38 H 62 N8O8+H] + .

[0623] Example 58

[0624] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino) (N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy 2-methylpropionyl)-L-phenylalanine tert-butyl ester

[0625]

[0626] DIEA (0.370 g, 0.500 mL, 2.87 mmol) was added to a stirred suspension of Dov-Abu(3-N3)-Dil-OH TFA salt (327 mg, 0.574 mmol) and H-Dap-Phe-OtBu TFA salt (0.340 g, 0.675 mmol) at 23 °C in DMF (10 mL), followed by HATU (0.550 g, 1.43 mmol). After 10 hours, analysis by LCMS showed that the reaction was complete. The reaction was confirmed by using a Phenomenex GeminiNX-C1810μF solution. The crude reaction was purified by preparative RP-HPLC using a 150x30mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 453 mg of the title compound formate (0.518 mmol, 72%) was obtained. LCMS RT = 1.42 min (Method B); ESI-MS m / z 828.94 [M+H] + ;HRMS m / z829.5536[C 43 H 72 N8O8+H] + .

[0627] Example 59

[0628] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)- 3-Methoxy-1-((S)-2-( ( 1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazole- 5-yl)ethyl)amino)propyl)pyrrolidone-1-yl)-5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0629]

[0630] DIEA (2.73 g, 3.7 mL, 21.1 mmol) was added to a suspension of Boc-Dap-OH dicyclohexylamine salt (2.48 g, 5.29 mmol) and (S)-2-phenyl-1-(1H-tetrazol-5-yl)ethylamine (1.00 g, 5.29 mmol) stirred at 23 °C in CH2Cl2 (20.0 mL), followed by DEPC (1.29 g, 1.20 mL, 7.93 mmol). After 10 hours, the reaction was complete as shown by LCMS analysis. Volatile organic compounds were evaporated under vacuum. The volatile organic compounds were analyzed using a Phenomenex Gemini NX-C18 10 μL filter. The crude oily substance was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 2.52 g of Boc-Dap-(S)-2-phenyl-1-(1H-tetrazol-5-yl)ethylamine formate (4.99 mmol, 94%) was obtained. LCMS RT = 1.35 min (Method B); ESI-MS m / z 459.2 [M+H] + .

[0631] TFA (5.00 mL) was added to a stirred suspension of Boc-Dap-(S)-2-phenyl-1-(1H-tetrazol-5-yl)ethylamine (2.52 g, 4.99 mmol) at 23 °C in CH2Cl2 (10 mL). After 5 hours, analysis by LCMS showed that the reaction was complete. The volatile organic compounds were evaporated under vacuum to give a crude product that did not require further purification. A total of 2.43 g of TFA salt of H-Dap-(S)-2-phenyl-1-(1H-tetrazol-5-yl)ethylamine was obtained (5.14 mmol, 84%). LCMS RT = 0.575 min (Method B); ESI-MS m / z 359.2 [M+H] + .

[0632] DIEA (0.35 g, 0.5 mL, 2.67 mmol) was added to a suspension of Dov-Abu(3-N3)-Dil-OH TFA salt (305 mg, 0.668 mmol) and H-Dap-(S)-2-phenyl-1-(1H-tetrazol-5-yl)ethylamine TFA salt (0.36 g, 1.002 mmol) stirred at 23 °C. HATU (509 mg, 1.34 mmol) was then added. After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C1810μm analyzer. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 47.0 mg of the title compound formate (0.056 mmol, 8%) was obtained. LCMS RT = 1.24 min (Method B); ESI-MS m / z 797.3 [M+H] + HRMS m / z 797.5139[C 39 H 64 N 12 O6+H] + .

[0633] Example 60

[0634] (2S , 3S)-3-azido-N-((3R) , 4S,S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2- methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazol-5-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl (-1-oxohep-4-yl)-N-methyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide

[0635]

[0636] DIEA (0.381 g, 0.500 mL, 2.97 mmol) was added to a suspension of Fmoc-MeVal-Abu(3-N3)-Dil-OH TFA salt (0.491 g, 0.631 mmol) and H-Dap-(S)-2-phenyl-1-(1H-tetrazol-5-yl)ethylamine TFA salt (0.532 g, 1.13 mmol) stirred at 23 °C. HATU (0.57 g, 1.486 mmol) was then added. After 10 hours, the reaction was complete as determined by LC-MS analysis. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The concentration was determined by using a Phenomenex Gemini NX-C18 10 μL filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous formic acid as the eluent. A total of 125 mg of formate of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-(S)-2-phenyl-1-(1H-tetrazol-5-yl)ethylamine was obtained (0.119 mmol, 16%). LCMS RT = 1.94 min (Method B); ESI-MS m / z 1005.35 [M+H] + .

[0637] Piperidine (5 mL) was added to a solution of Fmoc-MeVal-Abu(3-N3)-Dil-Dap-(S)-2-phenyl-1-(1H-tetrazol-5-yl)ethylamine (525 mg, 0.499 mmol) in acetonitrile (10 mL) at 23 °C with stirring. After 2 hours, LCMS analysis showed the reaction was complete. Hexane (x3) was added to the crude reaction solution. The acetonitrile layer was concentrated under vacuum. The concentration was determined using a Phenomenex Gemini NX-C18 10 μL filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. A total of 20.0 mg of the title compound in TFA salt (0.022 mmol, 5%) was obtained. LCMS RT = 1.37 min (Method B); ESI-MS m / z 783.42 [M+H] + ;HRMS m / z783.4979[C 38 H 62 N 12 O6+H] + .

[0638] Example 61

[0639] (2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)- 3-Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2- (1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide

[0640]

[0641] DIEA (2.15 g, 2.9 mL, 16.615 mmol) was added to a suspension of Boc-Dap-OH dicyclohexylamine salt (1.95 g, 4.154 mmol) and (S)-2-phenyl-1-(2-tetrazol-2-yl)ethylamine (1.00 g, 4.154 mmol) stirred at 23 °C in CH2Cl2 (20.0 mL), followed by DEPC (1.01 g, 0.9 mL, 0.006 mol). After 10 hours, the reaction was complete as indicated by LCMS analysis. The crude reaction mixture was washed with H2O (25 mL x 2) followed by brine (25 mL x 2). The organic fraction was dried on a MgSO4 pad, filtered, and concentrated under vacuum. A total of 1.65 g of yellow oily Boc-Dap-(S)-2-phenyl-1-(thiazol-2-yl)ethylamine (3.48 mmol, 84%) was obtained. LCMS RT = 1.59 minutes (Method B); ESI-MS m / z 475.2 [M+H] + .

[0642] TFA (10.0 mL) was added to a suspension of Boc-Dap-(S)-2-phenyl-1-(thiazol-2-yl)ethylamine (1.65 g, 3.49 mmol) stirred at 23 °C in CH2Cl2 (10.0 mL). After 4 hours, LCMS analysis showed the reaction was complete. Volatile organic compounds were evaporated under vacuum. The crude oily residue was dissolved in DMF (5 mL) and triethylamine (1 mL) to achieve pH 8. The solution was obtained using a Phenomenex Gemini NX-C18 10 μL filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous TFA as the eluent. A total of 935 mg of the TFA salt of H-Dap-(S)-2-phenyl-1-(thiazol-2-yl)ethylamine was obtained (1.92 mmol, 55%). LCMS RT = 1.04 min (Method B); ESI-MS m / z 375.0 [M+H] + .

[0643] DIEA (0.34 g, 0.5 mL, 2.65 mmol) was added to a suspension of Dov-Abu(3-N3)-Dil-OH TFA salt (302 mg, 0.661 mmol) and H-Dap-(S)-2-phenyl-1-(thiazolyl-2-yl)ethylamine TFA salt (247 mg, 0.661 mmol) stirred at 23 °C, followed by the addition of HATU (504 mg, 1.32 mmol). After 10 hours, analysis by LCMS showed that the reaction was complete. The crude reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic fractions were washed with brine, dried on a magnesium sulfate pad, filtered, and concentrated under vacuum. The solution was obtained by electrolysis using a Phenomenex GeminiNX-C1810μm filter. The crude oil was purified by preparative RP-HPLC using a 150x30 mm column with 10% to 90% MeCN in 0.1% aqueous TFA as the eluent. A total of 111 mg of the title compound in TFA salt (0.120 mmol, 18%) was obtained. LC-MS / RT = 1.33 min (Method B); ESI-MS m / z 812.2 [M+H] + HRMS m / z 812.4835[C 41 H 65 N9O6S+H] + .

[0644] Example 62

[0645] ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino) (N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy 2-methylpropionyl)-L-phenylalanine tert-butyl ester

[0646]

[0647] Trimethylphosphine in THF (1 M, 0.350 mL, 0.350 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-Ot-Bu formate (102.5 mg, 0.117 mmol) at room temperature. After 2 hours, analysis by LC-MS showed the reaction was complete. The reaction was confirmed by using a Phenomenex Gemini-NX 10 μC-18... The crude reaction mixture was purified by preparative RP-HPLC using a 150x30 mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 70.1 mg of the title compound formate (0.083 mmol, 70%) was obtained. LCMS RT = 1.22 min (Method B); ESI-MS m / z 803.3 [M+H]+ HRMS m / z 803.5642[C 43 H 74 N6O8+H] + .

[0648] Example 63

[0649] (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3- Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(1H-tetrazole-5- (1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide

[0650]

[0651] Trimethylphosphine in THF (1 M, 0.068 mL, 0.068 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-tetrazole carboxylate (19.4 mg, 0.023 mmol) at room temperature. After 2 hours, analysis by LCMS showed that the reaction was complete. The reaction was confirmed by using a Phenomenex Gemini-NX at 10 μC-18. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 9.9 mg of the title compound formate (0.012 mmol, 53%) was obtained. LCMS RT = 1.07 min (Method B); ESI-MS m / z 771.2 [M+H] + HRMS m / z 771.5233[C 39 H 66 N 10 O8+H] + .

[0652] Example 64

[0653] (2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-((3R,4S,5S)-3- Methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((( S )-2-Phenylacetyl-1-(thiazolyl-2-yl) Ethyl)amino)propyl)pyrrolidone-1-yl)-5-methyl-1-oxohep-4-yl)-N-methylbutyramide

[0654]

[0655] Trimethylphosphine in THF (1 M, 0.090 mL, 0.090 mmol) was added to a stirred solution of Dov-Abu(3-N3)-Dil-Dap-Phe-thiazocarbamate (37.2 mg, 0.043 mmol) at room temperature. After 1 hour, analysis by LCMS showed that the reaction was complete. The reaction was confirmed by using a Phenomenex Gemini-NX at 10 μC-18. The crude reaction mixture was purified by preparative RP-HPLC using a 150x30mm column with 5% to 95% MeCN in 0.1% aqueous formic acid as the eluent. A total of 16.8 mg of the formate of the title compound was obtained (0.020 mmol, 47%). LCMS RT = 1.15 min (Method B); ESI-MS m / z 786.2 [M+H] + ;HRMS m / z786.4940[C 41 H 67 N7O6S+H] + .

[0656] Example B1

[0657] In vitro cytotoxicity assay

[0658] The in vitro potency of compounds was measured by evaluating their cytotoxic activity against various cancer cell lines. This assay was performed in 96-well plates treated with clear tissue culture. The cell lines used were PC3 (human prostate cancer), HCC-1954 (human breast ductal carcinoma), and HCT15 (human colorectal adenocarcinoma, Pgp-expressing). Cells were seeded at approximately 1,000–1,500 cells / well in 50 μL of growth medium (RPMI-1640 + 10% heat-inactivated fetal bovine serum) and incubated overnight at 37°C and 5% CO2 to allow them to adhere. The next day, 50 μL of 2x stock solution of the carrier control (DMSO) or different concentrations of the compound were added to each well in triplicate. Additionally, control wells without cells or with only untreated cells were used. After adding the compound, the plates were incubated in a humidified tissue culture incubator at 37°C and 5% CO2 for 4–6 days to measure cytotoxicity. After 4–6 days, 20 μL of PrestoBlue was added to each well. TM Cell viability reagent (Life Technologies #A13261). Plates were incubated at 37°C for 1–2 hours. Biotek Synergy was used. TM The H4 microplate reader recorded fluorescence at 540 ex / 590 em. Representative data were plotted as the survival percentage compared to untreated control wells. Data for the compounds tested in this assay were plotted as the survival percentage compared to untreated control wells, such as... Figure 9-30 As shown.

[0659] Example B2

[0660] Assay for microtubule polymerization

[0661] The inhibitory effect of the compounds described herein on tubulin polymerization was evaluated on bovine brain tubulin. To evaluate compound activity, tubulin was seeded at approximately 400 μg / well in 100 μL universal tubulin buffer, and then treated in duplicate at a final concentration of 10 μM compound at the start of the assay. Following the addition of the test compound, the tubulin polymerization assay was typically performed at 37°C for 60 minutes. Tubulin polymerization was determined by absorbance spectroscopy analysis using optical density values ​​at 340 nm. To test the amount of polymerized tubulin, optical density values ​​at 340 nm per minute were acquired after the addition of the test compound. For analysis, the degree of tubulin polymerization in compound-treated tubulin was compared to the degree of polymerization in the control (buffer-treated tubulin). Specifically, the HTS-Tubulin Polymerization Assay Kit (Cytoskeleton Inc.; catalog number BK004P) was used for tubulin inhibition studies, employing the following sampling protocol:

[0662] 1. Before starting the experiment, preheat the photometer and 96-well plate to 37°C for 30 minutes. The hot plate is necessary for high polymerization activity and reproducible results.

[0663] 2. Enter all microplate reader parameters (absorbance at 340 nm, 37°C, one reading per minute) to prepare the spectrophotometer. Once the tubulin is dispensed into the 37°C wells, readings must begin immediately.

[0664] 3. Warm 500 μL of universal tubulin buffer to room temperature. Tubulin ligand dilution requires warm buffer.

[0665] 4. Paclitaxel was included as a control. 10 μL of paclitaxel was used per well, which brought the final concentration to 10 μM.

[0666] 5. Prepare cold test buffer: general microtubule protein buffer, 1 mM GTP, 10% glycerol.

[0667] 6. Resuspend 4 mg of tubulin in 1 mL of cold test buffer to achieve a final protein concentration of 4 mg / mL. Place the tube on ice and allow the protein to fully resuspend for 3 minutes.

[0668] 7. Prepare a 10X concentration of the selected compound in the test buffer.

[0669] 8. Transfer 10 μL of the 10X concentrated compound into the required number of wells of the preheated plate. Incubate the plate at 37°C for 2 minutes.

[0670] 9. Transfer 10 μL of test buffer only to the control well in section 2 (buffered tubulin).

[0671] 10. Transfer 100 μL of tubulin to the required number of wells (two wells should be the zero compound control, which has been treated with buffer).

[0672] 11. Immediately place the plate in a 37°C photometer and begin reading the optical density at 340 nm per minute. An increase in optical density at 340 nm is equivalent to an increase in tubulin polymerization.

[0673] The data for the compounds tested in this experiment are as follows: Figure 1-8 As shown.

[0674] Example B3

[0675] Determination of the in vitro potency of the test compound: human bladder cancer cells implanted subcutaneously in ICR SCID mice Efficacy evaluation in SW780

[0676] For in vivo animal studies, test compounds were diluted with 20 mM histidine, 5% sucrose, pH 6, and 15% DMSO. Male ICR SCID mice (Taconic Farm, Hudson, NY) were housed in standard rodent microisolation cages. Environmental control in the animal room was maintained at a temperature of 20–24 °C, relative humidity of 30%–70%, and approximately a 12-mouse light / 12-hour dark cycle. Water and food were provided freely. After 72 hours of compliance, SW780 human bladder cancer cells (2 x 10⁻⁶ cells) were suspended in a 50% complete culture mixture (Trevigen, Inc.) with PBS (Gibco). 6 (1 cell / mouse) was implanted into mice, and tumor growth rate was monitored. When the average tumor volume reached approximately 200 mm... 3 At that time, tumors were size-matched and mice were randomly assigned to treatment groups (n = 8 or 10). Tumor-bearing mice were treated intravenously for 3 weeks with the test compound or carrier at 2 or 4 mg / kg (mpk) according to the QW dosage regimen. Tumor volume was measured twice weekly using calipers.

[0677] While the compounds, uses, and methods described herein enable those skilled in the art to prepare and use the compounds, uses, and methods described herein, those skilled in the art will understand and acknowledge the existence of variations, combinations, and equivalents of the specific embodiments, methods, and examples described herein. Therefore, the compounds, uses, and methods provided herein should not be limited to the above-described embodiments, methods, or examples, but rather include all embodiments and methods within the scope and spirit of the compounds, uses, and methods provided herein.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide; (2S)-2-(dimethylamino)-N-((2S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide; (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-mercapto-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid.

2. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

3. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in a subject requiring such treatment, wherein the cancer is selected from human prostate cancer, human breast ductal carcinoma, or human colorectal adenocarcinoma, and wherein the compound is selected from the group consisting of: (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester.

4. Use of the pharmaceutical composition in the preparation of a medicament for treating cancer in a subject requiring such treatment, wherein the cancer is selected from human prostate cancer, human breast ductal carcinoma, or human colorectal adenocarcinoma, wherein the pharmaceutical composition comprises a compound selected from the group consisting of a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient: (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-4-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester.

5. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in a subject requiring such treatment, wherein the cancer is selected from human prostate cancer or human breast ductal carcinoma, and wherein the compound is selected from the group consisting of: (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide; (2S)-2-(dimethylamino)-N-((2S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-mercapto-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid.

6. Use of a pharmaceutical composition in the preparation of a medicament for treating cancer in a subject requiring such treatment, wherein the cancer is selected from human prostate cancer or human breast ductal carcinoma, wherein the pharmaceutical composition comprises a compound selected from the group consisting of a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient: (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide; (2S)-2-(dimethylamino)-N-((2S)-3-hydroxy-1-(((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)(methyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-mercapto-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolidone-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylpropionylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolid-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid; (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionate methyl ester; and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-amino-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid.

7. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating human breast ductal carcinoma in subjects requiring such treatment, wherein said compound is selected from the group consisting of: (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide; and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid.

8. Use of a pharmaceutical composition in the preparation of a medicament for treating human breast ductal carcinoma in subjects requiring such treatment, wherein the pharmaceutical composition comprises a compound selected from the group consisting of a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient: (2S,3R)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-3-hydroxy-N-((3R,4S,5S)-3-methoxy-1-((2S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((2-(piperidin-2-yl)ethyl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxohept-4-yl)-N-methylbutyramide; and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((2S,3S)-3-azido-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N-methylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionic acid.

Images