Arginase inhibitor and method of using the same

Abstract

To provide pharmaceuticals for treating a respiratory inflammatory disease selected from among idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease (COPD), and chemically induced lung fibrosis.SOLUTION: A pharmaceutical comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof. [In the formula, R1 is -NHR1a; R1a is -H or -C(O)CH(R1b)NH2; and R1b is -CH3 or -CH(-CH3)2.]SELECTED DRAWING: None

Description

Background Art

[0001] Arginase is a manganese metalloenzyme that catalyzes the conversion of L-arginine to urea and L-ornithine. There are two isoforms: arginase 1 is a cytosolic enzyme mainly found in hepatocytes and plays a crucial role in removing ammonia via urea synthesis. And arginase 2 is a mitochondrial enzyme highly expressed in the kidney and is involved in the production of ornithine, which is an important precursor for polyamines and proline for cell proliferation and collagen production, respectively.

[0002] L-arginine is not an essential amino acid as it can be provided via protein turnover in healthy adults. However, increased expression and secretion of arginase decrease L-arginine levels under various physiological and pathological conditions (e.g., pregnancy, autoimmune diseases, cancer). In particular, immune cells are sensitive to the decrease in L-arginine levels. T cells, when faced with a low L-arginine microenvironment, reduce their proliferation rate and decrease the expression of CD3ζ chain, IFNγ, and lytic enzymes, impairing T cell responsiveness. Dendritic cells respond to low L-arginine conditions by reducing their antigen-presenting ability, and natural killer cells reduce both the proliferation and expression of lytic enzymes.

[0003] Tumors evade the immune system using multiple immunosuppressive mechanisms. One of these is the decrease in L-arginine via increased levels of circulating arginase, increased expression and secretion of arginase by tumor cells, and recruitment of arginase expressed and secreted by bone marrow-derived suppressor cells. Collectively, these contribute to the decrease in L-arginine in the tumor microenvironment and immunosuppressive phenotypes. Pharmacological inhibition of arginase activity has been shown to reverse low L-arginine-induced immunosuppression in animal models. Therefore, there is a need for a potent and selective arginase inhibitor, either as a single agent or in combination with therapies that reverse additional immunosuppressive mechanisms, to reverse immunosuppression and restore anti-cancer immunity in patients.

Summary of the Invention

Means for Solving the Problems

[0004] In one embodiment, disclosed is a compound of formula (I), or a pharmaceutically acceptable salt thereof:

Chemical Formula

[0005] In one embodiment, disclosed is a compound of formula (Ia), or a pharmaceutically acceptable salt thereof:

Chemical Formula

[0006] In one embodiment, disclosed is a compound of formula (Ib), or a pharmaceutically acceptable salt thereof:

Chemical Formula

[0007] In one embodiment, disclosed is a compound of formula (II), or a pharmaceutically acceptable salt thereof: [Chemical formula] In the formula R 2 is -OH or -NHR 2a ; and R 2a is -H or -C(O)CH(R 2b )NH2; and R 2b is -CH3 or -CH(CH3)2.

[0008] In one embodiment, disclosed is a compound of formula (IIa), or a pharmaceutically acceptable salt thereof: [Chemical formula] In the formula R 2 is -OH or -NHR 2a ; and R 2a is -H or -C(O)CH(R 2b )NHR 2c ; and R 2b is selected from -H, -(C1-C4)alkyl, and CH2OR 2d and R 2c is -H; or R 2b and R 2c together with the atom to which they are attached form a 5-membered heterocycle; and R 2d is -H or -CH3.

[0009] In one embodiment, disclosed is a compound of formula (IIb), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0010] In some embodiments, disclosed is a compound of formula (III), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0011] In some embodiments, disclosed is a compound of formula (IIIa), or a pharmaceutically acceptable salt thereof: [Chemical formula] In the formula R 3 is selected from -H, -(C1-C4) alkyl, and CH2OR 3a and R 3a is -H or -CH3.

[0012] In one embodiment, disclosed is a compound of formula (IIIb), or a pharmaceutically acceptable salt thereof: [Chemical formula] In the formula R 3 is selected from -H, -(C1-C4) alkyl, and -CH2OR 3a and R 3a is -H or -CH3.

[0013] In some embodiments, disclosed is a compound of formula (IV), or a pharmaceutically acceptable salt thereof: [Chemical formula] In the formula, R 4 is -OH or -NH2.

[0014] In one embodiment, disclosed is a compound of formula (IVb), or a pharmaceutically acceptable salt thereof: [Chemical formula] In the formula, R 4 is -OH or -NH2.

[0015] In some embodiments, disclosed is a compound of formula (V), or a pharmaceutically acceptable salt thereof: [Chemical formula] .

[0016] In one embodiment, disclosed is a compound of formula (Vb), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0017] In one embodiment, disclosed is a compound of formula (VI), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0018] In one embodiment, disclosed is a compound of formula (VIb), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0019] In some embodiments, disclosed is a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0020] In some embodiments, disclosed is a pharmaceutical composition comprising a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0021] In some embodiments, disclosed is a method of treating cancer, comprising a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0022] In some embodiments, disclosed is a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, for treating cancer.

[0023] In some embodiments, disclosed is the use of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any and all subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer.

[0024] In some embodiments, disclosed is a pharmaceutical composition for treating cancer, comprising a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any and all subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, disclosed is a method for treating a respiratory inflammatory disease, comprising a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any and all subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0026] In some embodiments, disclosed is a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any and all subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, for treating a respiratory inflammatory disease.

[0027] In some embodiments, disclosed is the use of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a respiratory inflammatory disease.

[0028] In some embodiments, disclosed is a pharmaceutical composition for treating a respiratory inflammatory disease, comprising a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the above-mentioned respiratory inflammatory disease is chronic obstructive pulmonary disease (COPD) or asthma.

Brief Description of the Drawings

[0030]

Figure 1

Figure 2

Figure 3

Best Mode for Carrying Out the Invention

[0031] Compound In one embodiment, disclosed is a compound of formula (I), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0032] In one embodiment, disclosed is a compound of formula (I). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (I).

[0033] In some embodiments of formula (I), R 1 is -NHR 1a and R 1a is -H.

[0034] In some embodiments of formula (I), R 1 is -NHR 1a R 1a is -C(O)CH(R 1b )NH2 and R 1b is -CH3.

[0035] In some embodiments of formula (I), R 1 is -NHR 1a R 1a is -C(O)CH(R 1b )NH2 and R 1b is -CH(CH3)2.

[0036] In one embodiment, disclosed is a compound of formula (Ia), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0037] In one embodiment, disclosed is a compound of formula (Ia). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (Ia).

[0038] In some embodiments, the compound of formula (Ia) is represented by formula (Ia1) or formula (Ia2):

Chemical formula

[0039] In some embodiments of formula (Ia), R 1a is -H.

[0040] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is -H, and R 1c is -H.

[0041] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is CH2OR 1d ; R 1c is -H, and R 1d is -H.

[0042] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is CH2OR 1d ; R 1c is -H, and R 1d is -CH3.

[0043] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is -(C1-C4)alkyl, and R 1c is -H. In some embodiments, C1-C4 alkyl is selected from methyl, ethyl, isopropyl, sec-butyl, tert-butyl, and isobutyl.

[0044] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is methyl; and R 1c is -H.

[0045] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is ethyl; and R 1c is -H.

[0046] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is isopropyl; and R 1c is -H.

[0047] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is sec-butyl; and R 1c is -H.

[0048] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is isobutyl; and R 1c is -H.

[0049] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is tert-butyl; and R 1c is -H.

[0050] In some embodiments of formula (Ia), R 1a is -C(O)CH(R 1b )NHR 1c ; and R 1b and R 1c together with the atom to which they are attached form a 5-membered ring.

[0051] In one embodiment, disclosed is a compound of formula (Ib), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0052] In one embodiment, disclosed is a compound of formula (Ib). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (Ib).

[0053] In some embodiments, the compound of formula (Ib) is represented by formula (Ib1) or formula (Ib2):

Chemical formula

[0054] In some embodiments of formula (Ib), R 1a is -H.

[0055] In some embodiments of formula (Ib), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is -H, and R 1c is -H.

[0056] In some embodiments of formula (Ib), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is CH2OR 1d ; R 1cis - H, and R 1d is - H.

[0057] In some embodiments of formula (Ib), R 1a is - C(O)CH(R 1b )NHR 1c ; R 1b is CH2OR 1d ; R 1c is - H, and R 1d is - CH3.

[0058] In some embodiments of formula (Ib), R 1a is - C(O)CH(R 1b )NHR 1c ; R 1b is -(C1 - C4)alkyl, and R 1c is - H. In some embodiments, C1 - C4 alkyl is selected from methyl, ethyl, isopropyl, sec - butyl, tert - butyl, and isobutyl.

[0059] In some embodiments of formula (Ib), R 1a is - C(O)CH(R 1b )NHR 1c ; R 1b is methyl; and R 1c is - H.

[0060] In some embodiments of formula (Ib), R 1a is - C(O)CH(R 1b )NHR 1c ; R 1b is ethyl; and R 1c is - H.

[0061] In some embodiments of formula (Ib), R 1a is - C(O)CH(R 1b )NHR 1c ; R 1b is isopropyl; and R 1c is - H.

[0062] In some embodiments of formula (Ib), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is sec-butyl; and R 1c is -H.

[0063] In some embodiments of formula (Ib), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is isobutyl; and R 1c is -H.

[0064] In some embodiments of formula (Ib), R 1a is -C(O)CH(R 1b )NHR 1c ; R 1b is tert-butyl; and R 1c is -H.

[0065] In some embodiments of formula (Ib), R 1a is -C(O)CH(R 1b )NHR 1c ; and R 1b and R 1c together with the atom to which they are attached form a 5-membered ring.

[0066] In one embodiment, disclosed is a compound of formula (II), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0067] In one embodiment, disclosed is a compound of formula (II). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (II).

[0068] In some embodiments of formula (II), R 2 is -OH.

[0069] In some embodiments of formula (II), R 2 is -NHR 2a and R 2a is -H.

[0070] In some embodiments of formula (II), R 2 is -NHR 2a R 2a is -C(O)CH(R 2b )NH2 and R 2b is -CH3.

[0071] In some embodiments of formula (II), R 2 is -NHR 2a R 2a is -C(O)CH(R 2b )NH2 and R 2b is -CH(CH3)2.

[0072] In one embodiment, disclosed is a compound of formula (IIa), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0073] In one embodiment, disclosed is a compound of formula (IIa). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (IIa).

[0074] In one embodiment of formula (IIa), R 2 is - OH.

[0075] In one embodiment of formula (IIa), R 2 is - NHR 2a ; and R 2a is - H.

[0076] In one embodiment of formula (IIa), R 2 is - NHR 2a ; and R 2a is - C(O)CH(R 2b )NHR 2c ; R 2b is H, and R 2c is H.

[0077] In one embodiment of formula (IIa), R 2 is - NHR 2a ; and R 2a is - C(O)CH(R 2b )NHR 2c ; R 2b is - CH2OR 2d ; R 2c is H, and R 2d is H.

[0078] In one embodiment of formula (IIa), R 2 is - NHR 2a ; and R 2a is - C(O)CH(R 2b )NHR 2c ; R2b is -CH2OR 2d ; R 2c is H, and R 2d is -CH3.

[0079] In some embodiments of formula (IIa), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is -(C1 - C4)alkyl, and R 2c is -H. In some embodiments, C1 - C4 alkyl is selected from methyl, ethyl, isopropyl, sec - butyl, tert - butyl, and isobutyl.

[0080] In some embodiments of formula (IIa), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is methyl; and R 2c is -H.

[0081] In some embodiments of formula (IIa), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is ethyl; and R 2c is -H.

[0082] In some embodiments of formula (IIa), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is isopropyl; and R 2c is -H.

[0083] In some embodiments of formula (IIa), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is sec-butyl; and R 2c is -H.

[0084] In some embodiments of formula (IIa), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is isobutyl; and R 2c is -H.

[0085] In some embodiments of formula (IIa), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is tert-butyl; and R 2c is -H.

[0086] In some embodiments of formula (IIa), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; and R 2b and R 2c together with the atom to which they are attached form a 5-membered ring.

[0087] In one embodiment, disclosed is a compound of formula (IIb), or a pharmaceutically acceptable salt thereof:

Chemical formula

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

[0089] In one embodiment of formula (IIb), R 2 is -OH.

[0090] In one embodiment of formula (IIb), R 2 is -NHR 2a ; and R 2a is -H.

[0091] In one embodiment of formula (IIb), R 2 is -NHR 2a ; and R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is H, and R 2c is H.

[0092] In one embodiment of formula (IIb), R 2 is -NHR 2a ; and R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is -CH2OR 2d ; R 2c is H, and R2d is H.

[0093] In one embodiment of formula (IIb), R 2 is -NHR 2a ; and R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is -CH2OR 2d ; R 2c is H, and R 2d is -CH3.

[0094] In some embodiments of formula (IIb), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is -(C1-C4)alkyl, and R 2c is -H. In some embodiments, C1-C4 alkyl is selected from methyl, ethyl, isopropyl, sec-butyl, tert-butyl, and isobutyl.

[0095] In some embodiments of formula (IIb), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is methyl; and R 2c is -H.

[0096] In some embodiments of formula (IIb), R 2 is -NHR 2a ; R 2a is -C(O)CH(R 2b )NHR 2c ; R 2b is ethyl; and R 2c is -H.

[0097] In some embodiments of formula (IIb), R 2is -NHR 2a wherein; R 2a is -C(O)CH(R 2b )NHR 2c wherein; R 2b is isopropyl; and R 2c is -H.

[0098] In some embodiments of formula (IIb), R 2 is -NHR 2a wherein; R 2a is -C(O)CH(R 2b )NHR 2c wherein; R 2b is sec - butyl; and R 2c is -H.

[0099] In some embodiments of formula (IIb), R 2 is -NHR 2a wherein; R 2a is -C(O)CH(R 2b )NHR 2c wherein; R 2b is isobutyl; and R 2c is -H.

[0100] In some embodiments of formula (IIb), R 2 is -NHR 2a wherein; R 2a is -C(O)CH(R 2b )NHR 2c wherein; R 2b is tert - butyl; and R 2c is -H.

[0101] In some embodiments of formula (IIb), R 2 is -NHR 2a wherein; R 2a is -C(O)CH(R 2b )NHR 2c wherein; and R 2b and R 2c together with the atom to which they are attached form a 5 - membered ring.

[0102] In some embodiments, disclosed is a compound of formula (III), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0103] In one embodiment, disclosed is a compound of formula (III). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (III).

[0104] In some embodiments of formula (III), R 3 is -CH3.

[0105] In some embodiments of formula (III), R 3 is -CH(CH3)2.

[0106] In some embodiments, disclosed is a compound of formula (IIIa), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0107] In one embodiment, disclosed is a compound of formula (IIIa). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (IIIa).

[0108] In one embodiment of formula (IIIa), R 3 is -H.

[0109] In one embodiment of formula (IIIa), R 3 is -CH2OR 3aand R 3a is -H.

[0110] In one embodiment of formula (IIIa), R 3 is -CH2OR 3a and R 3a is -CH3.

[0111] In one embodiment of formula (IIIa), R 3 is -(C1-C4)alkyl. In some embodiments, -(C1-C4)alkyl is selected from methyl, ethyl, isopropyl, sec-butyl, and isobutyl.

[0112] In one embodiment of formula (IIIa), R 3 is methyl.

[0113] In one embodiment of formula (IIIa), R 3 is ethyl.

[0114] In one embodiment of formula (IIIa), R 3 is isopropyl.

[0115] In one embodiment of formula (IIIa), R 3 is sec-butyl.

[0116] In one embodiment of formula (IIIa), R 3 is isobutyl.

[0117] In one embodiment of formula (IIIa), R 3 is tert-butyl.

[0118] In some embodiments, disclosed is a compound of formula (IIIb), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0119] In one embodiment, disclosed is a compound of formula (IIIb). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (IIIb).

[0120] In one embodiment of formula (IIIb), R 3 is -H.

[0121] In one embodiment of formula (IIIb), R 3 is -CH2OR 3a and R 3a is -H.

[0122] In one embodiment of formula (IIIb), R 3 is -CH2OR 3a and R 3a is -CH3.

[0123] In one embodiment of formula (IIIb), R 3 is -(C1-C4)alkyl. In some embodiments, -(C1-C4)alkyl is selected from methyl, ethyl, isopropyl, sec-butyl, and isobutyl.

[0124] In one embodiment of formula (IIIb), R 3 is methyl.

[0125] In one embodiment of formula (IIIb), R 3 is ethyl.

[0126] In one embodiment of formula (IIIb), R 3 is isopropyl.

[0127] In one embodiment of formula (IIIb), R 3 is sec-butyl.

[0128] In one embodiment of formula (IIIb), R 3 is isobutyl.

[0129] In one embodiment of formula (IIIb), R 3 is tert-butyl.

[0130] In some embodiments, disclosed is a compound of formula (IV), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0131] In one embodiment, disclosed is a compound of formula (IV). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (IV).

[0132] In some embodiments of formula (IV), R 4 is -OH.

[0133] In some embodiments of formula (IV), R 4 is -NH2.

[0134] In some embodiments, disclosed is a compound of formula (IVb), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0135] In one embodiment, disclosed is a compound of formula (IVb). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (IVb).

[0136] In some embodiments of formula (IVb), R 4 is -OH.

[0137] In some embodiments of formula (IVb), R 4 is -NH2.

[0138] In some embodiments, disclosed is a compound of formula (V), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0139] In one embodiment, disclosed is a compound of formula (V). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (V).

[0140] In some embodiments, disclosed is a compound of formula (Vb), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0141] In one embodiment, disclosed is a compound of formula (Vb). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (Vb).

[0142] In some embodiments, disclosed is a compound of formula (VI), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0143] In one embodiment, disclosed is a compound of formula (VI). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (VI).

[0144] In some embodiments of formula (VI), R 6a is -H or -CH3; and R 6b is -(C1-C3) alkyl substituted with 0 or 1 amino.

[0145] In some embodiments of formula (VI), R 6a is -H or -CH3; R 6b is -C(O)C(R 6c R 6d )NH2; R 6c is -(C1-C3) alkyl substituted with 0 or 1 amino, or -OH; and R 6d is H or -CH3.

[0146] In some embodiments, the compound of formula (VI) is represented by formula (VIa1) or formula (VIa2):

Chemical formula

[0147] In one embodiment, disclosed is a compound of formula (VIa1). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (VIa1).

[0148] In one embodiment, disclosed is a compound of formula (VIa2). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (VIa2).

[0149] In one embodiment of formula (VI), R 6b is -CH3 or -CH2CH2NH2.

[0150] In some embodiments, disclosed is a compound of formula (VIb), or a pharmaceutically acceptable salt thereof:

Chemical formula

[0151] In one embodiment, disclosed is a compound of formula (VIb). In another embodiment, disclosed is a pharmaceutically acceptable salt of a compound of formula (VIb).

[0152] In one embodiment of formula (VIb), R 6b is -CH3 or -CH2CH2NH2.

[0153] In some embodiments of formula (VIb), R 6a is -H or -CH3; and R 6bis -(C1-C3) alkyl substituted with 0 or 1 amino.

[0154] In some embodiments of formula (VIb), R 6a is -H or -CH3; R 6b is -C(O)C(R 6c R 6d )NH2; R 6c is -(C1-C3) alkyl substituted with 0 or 1 amino, or -OH; and R 6d is H or -CH3.

[0155] In some embodiments, the compound of formula (VIb) is represented by formula (VIb1) or formula (VIb2):

Chemical formula

[0156] In some embodiments, the compounds of formula (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (V), and (VI) (including their species) are converted via intramolecular cyclization to the compounds of formula (Ib), (IIb), (IIIb), (IVb), (Vb), and (VIb) (including their species), and vice versa. That is, this is an interconversion process. The compounds of formula (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (V), and (VI) (including their species), as well as the compounds of formula (Ib), (IIb), (IIIb), (IVb), (Vb), and (VIb) (including their species) are each, in part or completely, converted to others depending on conditions such as the temperature, pressure, humidity, pH, and / or composition of the medium (e.g., solvent), etc. This is illustrated by the following scheme:

Chemical formula

[0157] In some embodiments, disclosed is a compound of Table 1, or a pharmaceutically acceptable salt thereof:

[0158] [Table 1]

[0159] [Table 2]

[0160] [Table 3]

[0161] [Table 4]

[0162] [Table 5]

[0163] [Table 6]

[0164] The term "C1-C4 alkyl" includes an acyclic alkyl moiety having 1 to 4 carbon atoms. Examples of C1-C4 alkyl moieties include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

[0165] The term "pharmaceutically acceptable salts" includes acid addition salts or base addition salts that retain the biological effectiveness and properties of the compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), and those of Table 1, which are typically not biologically or otherwise undesirable. In many cases, the compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), and those of Table 1 can form acidic and / or basic salts due to the presence of basic groups and / or carboxyl groups, or groups similar thereto.

[0166] Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and with organic acids, for example, acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorotheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methyl sulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, palmoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, subsalicylate, sulfate / hydrogen sulfate, tartrate, tosylate, and trifluoroacetate. Examples of inorganic acids from which the salts can be derived include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids from which the salts can be derived include acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid, and sulfosalicylic acid.

[0167] Pharmaceutically acceptable base addition salts can be formed with inorganic bases and with organic bases. Examples of inorganic bases from which the salts can be derived include ammonia and salts of ammonium, as well as metals from columns I - XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include salts of ammonium, potassium, sodium, calcium, and magnesium. Examples of organic bases from which the salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, etc.). Specific organic amines include isopropylamine, benzathine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0168] Pharmaceutically acceptable salts of the compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any sub - genus or species thereof), and of the compounds in Table 1 can be synthesized from the basic or acidic moieties by conventional chemical methods. Usually, such salts are prepared by reacting the free acid form of these compounds with a stoichiometric amount of the appropriate base (e.g., Na + , Ca 2+ , Mg 2+ , or K +It can be prepared by reacting a hydroxide salt, carbonate, or bicarbonate, etc., or the free base form of these compounds, with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water, in an organic solvent, or in a mixture of the two. Usually, if feasible, the use of a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desired. A list of additional suitable salts can be found, for example, in "Remington’s Pharmaceutical Sciences", 20th ed. Mack Publishing Company, Easton, Pa., (1985); Berge et al., J. Pharm. Sci., 1977, 66, 1-19, and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002)).

[0169] Also, any formula given herein is intended to represent both the unlabeled form and the isotopically labeled form for formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), and the compounds of Table 1. The isotopically labeled compounds have the structure shown by the formulas given herein, except that one or more atoms are replaced by an atom of the same element having a different mass number. Examples of isotopes that can be incorporated into formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), and the compounds of Table 1, and their pharmaceutically acceptable salts, include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, for example, 2 H, 3 H, 11 C, 13 C, 14C, 15 N, 35 S, 36 Cl, and 125 I are included. Formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), and the isotopically labeled compounds of Table 1 can usually be prepared by conventional techniques known to those skilled in the art or by processes similar to those described in the appended examples using appropriate, isotopically labeled reagents in place of the unlabeled reagents used previously.

[0170] The compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), and the compounds of Table 1 can have various isomeric forms. The terms "optical isomers", "stereoisomers", or "enantiomers" refer to any of the various stereoisomeric arrangements, which may exist for the compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), and the given compounds of Table 1. Since substituents may be attached to chiral centers of carbon atoms, it is understood that the disclosed compounds include enantiomers, enantiomers, and racemic compounds. The term "enantiomers" includes pairs of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemic mixture. The term is used to represent a racemic mixture as needed. The term "enantiomers" includes stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer, the stereochemistry of each chiral center may be specified by R or S. A resolved compound of unknown absolute configuration may be designated as (+) or (-) based on the direction (dextrorotatory or levorotatory) in which it rotates plane-polarized light at the wavelength of the sodium D line. The compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), and some of the compounds of Table 1 contain one or more asymmetric centers or axes and, thus, can give rise to enantiomers, enantiomers, or other stereoisomeric forms that can be defined as (R)- or (S)- with respect to absolute stereochemistry. The present disclosure is intended to include all such possible isomers (including racemic mixtures, optically pure forms, and intermediate mixtures).The optically active (R)-isomer and (S)-isomer may be prepared using a chiral synthon or chiral reagent, or may be resolved using conventional techniques well known in the art, such as chiral HPLC.

[0171] Also disclosed herein are intermediates 1 - 48 in the Examples, and their salts.

[0172] Pharmaceutical composition In some embodiments, disclosed is a pharmaceutical composition comprising a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), or a compound of Table 1, and a pharmaceutically acceptable carrier.

[0173] The term "pharmaceutically acceptable carrier" includes compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals, and that cause no excessive toxicity, inflammation, allergic response, or other problems and complications as determined by those of ordinary skill in the art.

[0174] The disclosed compositions may be in a form suitable for oral use (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by insufflation (e.g., as finely divided powders or liquid aerosols), for administration by inhalation (e.g., as finely divided powders), or for parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, or intramuscular dosing, or as suppositories for rectal dosing).

[0175] The amount of the active ingredient combined with one or more pharmaceutically acceptable carriers to produce a single dosage form will necessarily vary depending on the host being treated and the particular route of administration. For further information on routes of administration and dosage regimens, the reader is referred to Chapter 25.3, Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0176] Therapeutic utility The compounds are useful in therapy as arginase inhibitors.

[0177] In one aspect, disclosed is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0178] In one aspect, disclosed is a method of treating a respiratory inflammatory disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0179] In one aspect, disclosed is a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof for use in treating cancer.

[0180] In one aspect, disclosed is a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, which is used for treating a respiratory inflammatory disease.

[0181] In one aspect, disclosed is the use of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer.

[0182] In one aspect, disclosed is the use of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a respiratory inflammatory disease.

[0183] In one aspect, disclosed is a pharmaceutical composition for treating cancer, which comprises a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0184] In one aspect, disclosed is a pharmaceutical composition for treating a respiratory inflammatory disease, comprising a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0185] The term "cancer" includes, for example, renal cell carcinoma, head and neck squamous cell carcinoma, lung cancer (e.g., small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma), pancreatic cancer, colorectal cancer, breast cancer, acute myeloid leukemia (AML), prostate cancer, gastric cancer, bladder cancer, melanoma, kidney cancer, and ovarian cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is associated with the regulation of arginase 1 and / or arginase 2.

[0186] In some embodiments, the cancer is associated with an increase in plasma arginase 1 level. In some embodiments, the cancer is associated with a decrease in plasma arginine level. In some embodiments, the cancer is associated with both an increase in plasma arginase 1 level and a decrease in plasma arginine level. In some embodiments, cancers associated with an increase in plasma arginase 1 level and / or a decrease in plasma arginine level include renal cell carcinoma, head and neck squamous cell carcinoma, lung cancer (e.g., small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma), pancreatic cancer, colorectal cancer, and breast cancer.

[0187] In some embodiments, cancers, such as acute myeloid leukemia and prostate cancer, secrete arginase 2.

[0188] In some embodiments, cancers, such as lung cancer (small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC)), gastric cancer, bladder cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and kidney cancer, are associated with arginase 1-positive tumor-infiltrating immune cells.

[0189] The term "respiratory inflammatory disease" refers to an inflammatory condition or disorder that affects the airspaces, pulmonary vascular structures, lung interstitium, or combinations thereof. This can be isolated to the lungs or involve multiple organs. In one embodiment, the respiratory inflammatory disease is an inflammatory lung disease. In another embodiment, the inflammatory lung disease is non-infectious.

[0190] In some embodiments, the respiratory inflammatory disease is asthma, chronic obstructive pulmonary disease (COPD), chemically induced pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, or combinations thereof. In some embodiments, the respiratory inflammatory disease is chronic obstructive pulmonary disease (COPD) or asthma.

[0191] In one aspect, disclosed is a method of inhibiting arginase in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof.

[0192] In one aspect, disclosed is a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, for use in inhibiting arginase.

[0193] In one aspect, disclosed is the use of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenera or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting arginase.

[0194] In one aspect, disclosed is a pharmaceutical composition comprising a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), or a compound of Table 1, or a pharmaceutically acceptable salt thereof, which is used for inhibiting arginase.

[0195] The term "arginase" includes manganese-containing enzymes belonging to the ureohydrolase family that catalyze the fifth and final steps in the urea cycle that convert L-arginine to L-ornithine and urea. The term "arginase" includes two isoenzymes of the enzyme, for example, arginase 1 that functions in the urea cycle and is mainly present in the cytoplasm of the liver, and arginase 2 that is present in the mitochondria of some tissues in the body and is involved in the regulation of intracellular arginine / ornithine concentrations. In some embodiments, the compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), and the compounds of Table 1, or pharmaceutically acceptable salts thereof, are selective for arginase 1. In some embodiments, the compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), and the compounds of Table 1, or pharmaceutically acceptable salts thereof, are selective for arginase 2. In some embodiments, the compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any subgenus or species thereof), and the compounds of Table 1, or pharmaceutically acceptable salts thereof, inhibit both arginase 1 and arginase 2.

[0196] The term "effective amount" includes an amount of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any and all subgenera or species thereof), or of a compound of Table 1, which will effect in a subject a biological or medical response, such as a reduction or inhibition of arginase or an enzyme or protein activity associated with cancer, an improvement in the symptoms of cancer, or a deceleration or retardation of the progression of cancer. In some embodiments, the term "effective amount" includes an amount of a compound of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any and all subgenera or species thereof), or of a compound of Table 1, which is effective, when administered to a subject, to at least partially reduce, inhibit, and / or improve cancer in the subject, or to inhibit arginase, and / or to reduce or inhibit tumor growth or the growth of cancerous cells in the subject.

[0197] The term "subject" includes warm-blooded mammals such as primates, dogs, cats, rabbits, rats, and mice. In some embodiments, the subject is a primate such as a human. In some embodiments, the subject has cancer. In some embodiments, the subject is in need of treatment (e.g., the subject will biologically or medically benefit from treatment). In some embodiments, the patient has cancer. In some embodiments, the subject has an elevated plasma arginase 1 level. In some embodiments, the subject has a decreased arginine level. In some embodiments, the patient has an elevated plasma arginase 1 level and a decreased arginine level. In some embodiments, the subject has a cancer that secretes arginase 2 (e.g., acute myeloid leukemia or prostate cancer). In some embodiments, the subject has arginase 1-positive tumor-infiltrating immune cells.

[0198] The terms "inhibit", "inhibition", or "inhibiting" include a decrease in the baseline activity of a biological activity or process. In some embodiments, the compounds of formula (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb) (including any and all subgenera or species thereof), and Table 1 inhibit arginase.

[0199] The terms "treat", "treating", and "treatment" include reducing or inhibiting enzymatic or protein activity associated with arginase, or ameliorating one or more symptoms of cancer in a subject, or slowing or delaying the progression of cancer in a subject. Further, the terms "treat", "treating", and "treatment" include reducing or inhibiting the growth of a tumor or the proliferation of cancerous cells in a subject.

Examples

[0200] Aspects of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail the preparation of specific compounds and intermediates of the present disclosure, and methods of using the compounds of the present disclosure. It will be apparent to those skilled in the art that many modifications to both materials and methods may be practiced without departing from the spirit of the present disclosure.

[0201] Unless otherwise specified: (i) All syntheses were carried out at ambient temperature, i.e., in the range of 17 - 25 °C, and under an atmosphere of an inert gas, such as nitrogen, unless otherwise specified; (ii) Evaporation was carried out by rotary evaporation or using a Genevac instrument or a Biotage v10 vacuum evaporator, and finishing procedures were carried out after removal of residual solids by filtration; (iii) Flash chromatography purification was performed using an automated Teledyne Isco CombiFlash® Rf or Teledyne Isco CombiFlash® Companion® with packed RediSep Rf Gold™ Silica Columns (20 - 40 μm, spherical particles), GraceResolv™ Cartridges (Davisil® silica), or Silicycle cartridges (40 - 63 μm); (iv) Preparative chromatography was performed on a Gilson prep HPLC instrument with UV collection; alternatively, preparative chromatography was performed on a Waters AutoPurification HPLC - MS instrument with MS - and UV - triggered collection; (v) Chiral preparative chromatography was performed on a Gilson instrument with UV collection (233 injector / fraction collectors, 333 and 334 pumps, 155 UV detectors), or a Varian Prep Star instrument run with a Gilson 305 injection (2 x SD1 pumps, 325 UV detectors, 701 fraction collectors) pumps; alternatively, chiral preparative chromatography was performed on a Waters Prep 100 SFC - MS instrument with MS - and UV - triggered collection, or a Thar MultiGram III SFC instrument with UV collection; (vi) When present, the yields are not necessarily the maximum amounts achievable; (vii) Generally, the structure of the final product of formula I was confirmed by nuclear magnetic resonance (NMR) spectroscopy; NMR chemical shift values were measured on the delta scale [proton magnetic resonance spectra were obtained using a Bruker Avance III 600 (600 MHz), Bruker Avance 400 (400 MHz), Bruker Avance 300 (300 MHz), or Bruker DRX 500 (500 MHz) instrument]; measurements were carried out at ambient temperature unless otherwise specified; the following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublets of doublets; dt, triplet of doublets; bs, broad signal. (viii) Also, generally, the final product of formula I was characterized by mass spectrometry after liquid chromatography (LCMS or UPLC); using Waters UPLC equipped with a Waters SQ mass spectrometer (column temperature 40 °C, UV = 220 - 300 nm or 190 - 400 nm, Mass Spec = ESI by positive / negative switching), with a solvent system of 97%A + 3%B to 3%A + 97%B, at a flow rate of 1 mL / min, for 1.50 minutes (total runtime for equilibrium to return to starting conditions etc., 1.70 minutes), the UPLC was run. Here, A = 0.1% formic acid in water or 0.05% trifluoroacetic acid (for acidic studies), or 0.1% ammonium hydroxide in water (for basic studies), and B = acetonitrile. The column used for acidic analysis was Waters Acquity HSS T3 (1.8 μm, 2.1×50 mm), and the column used for basic analysis was Waters Acquity BEH C18 (1.7 μm, 2.1×50 mm). Additionally, using Waters UPLC equipped with a Waters SQ mass spectrometer (column temperature 30 °C, UV = 210 - 400 nm, Mass Spec = ESI by positive / negative switching), with a solvent gradient of 2 - 98%B at a flow rate of 1 mL / min, for 1.5 minutes (total runtime for equilibrium to return to starting conditions, 2 minutes), the UPLC was run. Here, A = 0.1% formic acid in water, and B = 0.1% formic acid in acetonitrile (for acidic studies), or A = 0.1% ammonium hydroxide in water and B = acetonitrile (for basic studies). The column used for acidic analysis was Waters Acquity HSS T3 (1.8 μm, 2.1×30 mm), and the column used for basic analysis was Waters Acquity BEH C18 (1.7 μm, 2.1×30 mm); using Waters Alliance (2795) equipped with a Waters ZQ ESCi mass spectrometer and Phenomenex Gemini-NX C18 (5 μm, 110A, 2.1×50 mm column), LCMS was run at a flow rate of 1.1 mL / min from 95%A to 95%B for 4 minutes with a 0.5-minute hold.Here, A = 0.1% formic acid in acetonitrile and B = 0.1% formic acid (for acidic studies), or A = 0.1% ammonium hydroxide in water and B = acetonitrile (for basic studies). Additionally, LCMS was performed at a flow rate of 0.7 mL / min (for Waters HSS C18 column), 1.0 mL / min (for Shim-pack XR-ODS column), or 1.2 mL / min (for Phenomenex Gemini-NX C18) over 2.2 minutes with a hold of 0.6 minutes at 95% A to 95% B using a Shimadzu UFLC equipped with a Shimadzu LCMS-2020 mass spectrometer and columns such as Waters HSS C18 (1.8 μm, 2.1×50 mm), Shim-pack XR-ODS (2.2 μm, 3.0×50 mm), or Phenomenex Gemini-NX C18 (3 μm, 3.0×50 mm). Here, A = 0.1% formic acid or 0.05% trifluoroacetic acid in water (for acidic studies), or 0.1% ammonium hydroxide or 6.5 mM ammonium carbonate in water (for basic studies), and B = acetonitrile. The reported molecular ions correspond to [M+H]+ unless otherwise specified; for molecules with multiple isotope patterns (Br, Cl, etc.), the reported values are those obtained for the lowest isotope mass unless otherwise specified. (ix) Ion exchange purification was typically performed using an SCX-2 (Biotage) cartridge. (x) The purity of the intermediate was evaluated by thin layer chromatography, mass spectrometry, LCMS, UPLC / MS, HPLC (high performance liquid chromatography), and / or NMR analysis; (xi) The following abbreviations were used: EtOH: ethanol EtOAc: ethyl acetate LDA: lithium diisopropylamide MeOH: methanol TFA: trifluoroacetic acid MeCN: acetonitrile LCMS: liquid chromatography - mass spectrometry rt or RT: room temperature aq: aqueous THF: Tetrahydrofuran KHMDS: Potassium bis(trimethylsilyl)amide DCM: Dichloromethane DMF: Dimethylformamide HATU: (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) BOC: tert-Butoxycarbonyl DTNB: 5,5’-Dithiobis(2-nitrobenzoic acid) TNB: 2-Nitro-5-thiobenzoic acid HEPES: (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)

[0202] Example 1: (R)-2-(4-Boronobutyl)pyrrolidine-2-carboxylic acid

Chem.

[0203] Intermediate 2: (3S,7aS)-7a-((E)-4-bromobuta-2-enyl)-3-(trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one A solution of LDA (2.0 M in THF / heptane / ethylbenzene, 2.05 mL, 4.09 mmol) was added dropwise to a solution of (3S,7aR)-3-(trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one (Intermediate 1, 1.00 g, 4.09 mmol) in THF (500 mL) at -78 °C under a nitrogen atmosphere. The resulting solution was stirred at -78 °C for 20 minutes. (E)-1,4-Dibromobuta-2-ene (875 mg, 4.09 mmol) was added dropwise to the reaction mixture as a solution in THF (2 mL). The reaction mixture was stirred at -78 °C for 30 minutes and then warmed to room temperature with stirring for an additional 2 hours. The reaction mixture was evaporated to dryness, and the resulting residue was diluted in EtOAc (20 mL) and washed successively with water (2 × 20 mL) and saturated brine (2 × 20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give the product (Intermediate 2, 760 mg, 49% yield). 1 1H NMR (300 MHz, CDCl3) δ 1.55 - 1.75 (1H, m), 1.85 - 2.25 (3H, m), 2.52 - 2.73 (2H, m), 3.14 - 3.32 (2H, m), 3.89 - 4.10 (2H, m), 5.01 (1H, s), 5.79 - 5.99 (2H, m); m / z (ES + ) [M + H] + = 378.

[0204] Intermediate 3: (3S,7aS)-7a-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)buta-2-enyl)-3-(trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one Pd2(dba)3 (85.0 mg, 0.0928 mmol) was added to a THF solution (30 mL) of (3S,7aS)-7a-((E)-4-bromobut-2-enyl)-3-(trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one (Intermediate 2, 700 mg, 1.85 mmol) and bis(pinacolato)diboron (942 mg, 3.71 mmol) under a nitrogen atmosphere. The resulting mixture was heated to 60 °C and stirred for 5 hours. The reaction mixture was cooled to room temperature and concentrated to dryness. The resulting residue was diluted with EtOAc (50 mL) and washed successively with water and saturated brine. The organic layer was dried over Na2SO4, filtered, and concentrated to dryness. The resulting crude material was purified by silica gel chromatography to give the product (Intermediate 3, 510 mg, 65% yield). 1 H NMR (300 MHz, CDCl3) δ 1.28 (12H, s), 1.58 - 1.80 (2H, m), 1.83 - 2.12 (3H, m) 2.42 - 2.65 (1H, m), 3.20 (1H, dd), 3.47 (1H, q), 3.71 (1H, t), 3.90 (1H, t), 4.98 (1H, s), 5.38 - 5.53 (1H, m), 5.64 - 5.83 (1H, m); m / z (ES + ) [M + H] + = 424.

[0205] Intermediate 4: (3S,7aR)-7a-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)-3-(trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one Pd / C (10 wt%, 125 mg, 0.12 mmol) was added to a MeOH solution (5 mL) of (3S,7aS)-7a-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)but-2-enyl)-3-(trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one (Intermediate 3, 500 mg, 1.18 mmol). The reaction flask was equipped with a balloon of H2. The suspension was stirred at room temperature for 30 minutes. The reaction mixture was filtered through diatomaceous earth and washed with MeOH. The filtrate was concentrated and dried to obtain the crude product (Intermediate 4, 390 mg, 78% yield). This was used without further purification. m / z (ES + ) [M+H] + = 426.

[0206] Example 1: (R)-2-(4-Boronobutyl)pyrrolidine-2-carboxylic acid Aqueous concentrated HCl (1.00 mL, 12.0 mmol) was added to a 1,4-dioxane solution (20 mL) of (3S,7aR)-7a-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)-3-(trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one (Intermediate 4, 300 mg, 0.703 mmol) and phenylboronic acid (172 mg, 1.41 mmol). The resulting solution was heated at 80 °C for 15 hours. The reaction mixture was cooled to room temperature, concentrated and dried. The crude material was purified by preparative LCMS (XBridge Prep C18 OBD column, 5 μm silica, 19×150 mm, H2O (w / 0.05% TFA / MeCN)). The pure fractions were collected, concentrated and dried to obtain (R)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 1, 85 mg, 37% yield) as a white solid. 1 1H NMR (400 MHz, D2O) δ 0.63 - 0.74 (2H, m), 1.09 - 1.27 (2H, m), 1.27 - 1.37 (2H, m), 1.65 - 1.75 (1H, m), 1.77 - 2.08 (4H, m), 2.25 - 2.37 (1H, m), 3.21 - 3.37 (2H, m); m / z (ES+) [M+H] + = 216.

[0207] Example 2: (2R,4S)-2-(4-Boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid

Chemical Structure

[0208] Intermediate 6: (4S)-2-Benzyl 1-tert-butyl 4-(benzyloxy)-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylate (2S,4S)-2-Benzyl 1-tert-butyl 4-(benzyloxy)pyrrolidine-1,2-dicarboxylate (Intermediate 5, 2.75 g, 6.68 mmol) and crotyl bromide (1.03 mL, 10.0 mmol) were dissolved in THF (45 mL), and the solution was cooled to -78 °C under a N2 atmosphere. The solution was treated by dropwise addition of a solution of KHMDS (0.5 M in toluene, 20.1 mL, 10.0 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 3 hours. The crude reaction mixture was quenched with water and the volatiles were removed in vacuo. The crude mixture was diluted in DCM and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give the product (Intermediate 6, 2.54 g, 82% yield) as a mixture of rotamers and E / Z olefins. 1 H NMR (300 MHz, DMSO-d6) δ 1.20 - 1.41 (9H, s x2) rotamers, 1.54 - 1.62 (3H, m), 2.10 - 2.59 (3H, m), 2.67 - 2.97 (1H, m), 3.10 - 3.43 (1H, m), 3.50 - 3.78 (1H, m), 3.98 - 4.15 (1H, m), 4.34 - 4.49 (2H, m), 4.94 - 5.13 (2H, m), 5.18 - 5.30 (1H, m), 5.38 - 5.63 (1H, m), 7.25 - 7.36 (10H, m); m / z (ES + ) [M + H] + = 466.

[0209] Intermediate 8: (2R,4S)-1-(tert-Butoxycarbonyl)-4-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid Bis(1,5-cyclooctadiene)dirhodium(I) dichloride (0.366 g, 0.550 mmol) and bis(diphenylphosphino)methane (0.419 g, 1.09 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (31 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.74 mL, 12.0 mmol) was slowly added to the solution. The reaction mixture was stirred at room temperature for 10 minutes. (4S)-2-Benzyl 1-tert-butyl 4-(benzyloxy)-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylate (Intermediate 6, 2.54 g, 5.46 mmol) was added to the reaction mixture as a DCM solution (21 mL), and the reaction mixture was stirred overnight. The reaction mixture was diluted with DCM and quenched with water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give (4S)-2-benzyl 1-tert-butyl 4-(benzyloxy)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 7, 2.0 g, 61% yield) as a mixture of rotamers. The purified material was subjected to chiral SFC [(S,S)Whelk-O1 column, 21.2×250 mm, 5 μm, temperature = 23 °C, mobile phase = 0~15% MeOH:CO2, UV detection @220 nm, loading = 33 mg / inj, conc = 220 ng / mL in MeOH, flow rate = 75 mL / min, outlet pressure = 100 bar] to obtain two enantiomers. The stereochemistry was assigned as an anti-addition product for the major isomer and as a syn-addition product for the minor isomer. The minor isomer (368 mg, 0.620 mmol) was dissolved in ethyl acetate (6.2 mL) and treated with Pd / C (10 wt%, 132 mg, 0.124 mmol). The flask was equipped with a H2 balloon. The suspension was stirred at room temperature overnight. The reaction mixture was filtered through diatomaceous earth and rinsed with methanol.The filtrate was concentrated under reduced pressure to obtain (2R,4S)-1-(tert-butoxycarbonyl)-4-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 8, 228 mg, 98% yield) as a mixture of rotational isomers. This was used without further purification. 1 H NMR (300 MHz, DMSO-d6) δ 0.57 - 0.74 (2H, m), 1.17 (12H, s), 1.24 - 1.47 (13H, m), 1.59 - 1.78 (1H, m), 1.78 - 1.96 (1H, m), 2.01 - 2.20 (2H, m), 2.84 - 3.09 (1H, m), 3.58 - 3.73 (1H, m), 4.14 - 4.31 (1H, m), 4.98 - 5.09 (1H, m), 12.20 - 12.60 (1H, m); m / z (ES + ) [M + H] + = 414.

[0210] Example 2: (2R,4S)-2-(4-Boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.65 mL, 8.4 mmol) was added to a solution of (2R,4S)-1-(tert-butoxycarbonyl)-4-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 8, 175 mg, 0.423 mmol) in DCM (4 mL). The resulting solution was stirred at room temperature for 1 hour and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (3 mL) and 1M aq HCl (3 mL). Phenylboronic acid (103 mg, 0.847 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column using 2M ammonia / methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0-10%-100% acetonitrile in water) to give (2R,4S)-2-(4-boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid (Example 2, 33 mg, 33% yield) as a white solid. 1 H NMR (400 MHz, D2O) δ 0.67-0.78 (2H, m), 1.08-1.41 (4H, m), 1.81-2.12 (3H, m), 2.51 (1H, dd), 3.22-3.37 (2H, m), 4.46-4.56 (1H, m); m / z (ES + ) [M+H] + = 232.

[0211] Example 3: (2R,4R)-2-(4-boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid

Chemical formula

[0212] Intermediate 10: (4R)-2-Benzyl 1-tert-butyl 4-(benzyloxy)-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylate (2S,4R)-2-Benzyl 1-tert-butyl 4-(benzyloxy)pyrrolidine-1,2-dicarboxylate (Intermediate 9, 2.75 g, 6.68 mmol) and crotyl bromide (1.03 mL, 10.0 mmol) were dissolved in THF (45 mL), and the solution was cooled to -78 °C under a N2 atmosphere. The solution was treated by dropwise addition of a solution of KHMDS (0.5 M in toluene, 20.1 mL, 10.0 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 3 h. The crude reaction mixture was quenched with water and the volatiles were removed in vacuo. The crude mixture was diluted in DCM and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give the product (Intermediate 10, 1.23 g, 40% yield) as a mixture of rotamers and E / Z olefins. 1 H NMR (300 MHz, DMSO-d6) δ 1.25 - 1.34 (9H, s x2) rotamer, 1.45 - 1.63 (3H, m), 2.12 - 2.64 (2H, m), 2.64 - 3.04 (1H, m), 3.06 - 3.19 (1H, m), 3.31 - 3.45 (1H, m), 3.46 - 3.81 (1H, m), 4.03 - 4.21 (1H, m), 4.30 - 4.55 (2H, m), 4.90 - 5.16 (2H, m), 5.16 - 5.34 (1H, m), 5.38 - 5.68 (1H, m), 7.25 - 7.41 (10H, m). m / z (ES + ) [M+H] + = 466.

[0213] Intermediate 12: (2R,4R)-1-(tert-butoxycarbonyl)-4-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid Bis(1,5-cyclooctadiene)dirhodium(I) dichloride (177 mg, 0.264 mmol) and bis(diphenylphosphino)methane (203 mg, 0.527 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (15 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.84 mL, 5.8 mmol) was slowly added to the solution. The reaction mixture was stirred at room temperature for 10 minutes. (4R)-2-Benzyl 1-tert-butyl 4-(benzyloxy)-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylate (Intermediate 10, 1.23 g, 2.64 mmol) was added to the reaction mixture as a DCM solution (10 mL), and the reaction mixture was stirred overnight. The reaction mixture was diluted with DCM and quenched with water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give (4R)-2-benzyl 1-tert-butyl 4-(benzyloxy)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 11, 950 mg, 60% yield). The purified material was subjected to chiral SFC [(S,S)Whelk-O1 column, 21.2×250 mm, 5 μm, temperature = 23 °C, mobile phase = 0–15% MeOH:CO2, UV detection @ 220 nm, loading = 33 mg / inj, conc = 220 ng / mL in MeOH, flow rate = 75 mL / min, outlet pressure = 100 bar] to obtain two enantiomers. The stereochemistry was assigned as an anti-addition product for the major isomer and a syn-addition product for the minor isomer. The major isomer (385 mg, 0.649 mmol) was dissolved in ethyl acetate (6.4 mL) and treated with Pd / C (10 wt%, 138 mg, 0.130 mmol). The flask was equipped with a H2 balloon. The suspension was stirred at room temperature overnight. The reaction mixture was filtered through diatomaceous earth and rinsed with methanol.The filtrate was concentrated under reduced pressure to obtain the product (intermediate 12, 249 mg, 93% yield) as a mixture of rotamers. 1 H NMR (300 MHz, DMSO-d6) δ 0.61 - 0.73 (2H, m), 0.97 - 1.11 (1H, m), 1.12 - 1.23 (12H, m), 1.25 - 1.44 (12H, m), 1.51 - 1.71 (1H, m), 1.84 - 2.04 (2H, m), 2.05 - 2.19 (2H, m), 3.12 - 3.29 (1H, m), 3.37 - 3.59 (1H, m), 4.09 - 4.23( 1 H, m); m / z (ES + ) [M + H] + = 414.

[0214] Example 3: (2R,4R)-2-(4-Boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.65 mL, 8.5 mmol) was added to a DCM solution (3 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (intermediate 12, 197 mg, 0.179 mmol). The resulting solution was stirred at room temperature for 1 hour and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (3 mL) and 1M aq HCl (3 mL). Phenylboronic acid (102 mg, 0.837 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column using 2M ammonia / methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 - 10% acetonitrile in water) to obtain (2R,4R)-2-(4-boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid (Example 3, 25 mg, 25% yield) as a white solid. 11H NMR (300 MHz, D2O) δ 0.68 - 0.78 (2H, m), 1.13 - 1.43 (4H, m), 1.64 - 1.79 (1H, m), 1.94 - 2.14 (2H, m), 2.47 (1H, d), 3.39 (2H, m), 4.46 - 4.53 (1H, m). m / z (ES + ) [M + H] + = 232.

[0215] Example 4: (2S,4S)-4-Amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chem.

[0216] Intermediate 14: (4S)-2-benzyl 1-tert-butyl 4-azido-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylate (2S,4S)-2-Benzyl 1-tert-butyl 4-azidopyrrolidine-1,2-dicarboxylate (Intermediate 13, 1.00 g, 2.89 mmol) and crotyl bromide (0.44 mL, 4.3 mmol) were dissolved in THF (20 mL), and the solution was cooled to -78 °C under a N2 atmosphere. The solution was treated by dropwise addition of a solution of KHMDS (0.5 M in toluene, 8.66 mL, 4.33 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 3 hours. The crude reaction mixture was quenched with water, and the volatiles were removed in vacuo. The crude mixture was diluted in DCM, and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give the product (Intermediate 14, 750 mg, 65% yield) as a mixture of rotamers and E / Z olefins. 1 H NMR (300 MHz, DMSO-d6) δ 1.25 - 1.34 (9H, s x2) rotomers, 1.55 - 1.64 (3H, m), 1.99 - 2.15 (1H, m), 2.33 - 2.62 (2H, m), 2.73 - 3.10 (1H, m), 3.26 - 3.39 (1H, m), 3.52 - 3.84 (1H, m), 4.24 - 4.33 (1H, m), 5.03 - 5.21 (2H, m), 5.28 - 5.35 (1H, m), 5.49 - 5.65 (1H, m), 7.31 - 7.36 (5H, m); m / z (ES + ) [M+H] + = 401.

[0217] Intermediate 16: (2S,4S)-2-Benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate and Intermediate 17: (2R,4S)-2-Benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate Bis(1,5-cyclooctadiene)dirhodium(I) dichloride (126 mg, 0.188 mmol) and bis(diphenylphosphino)methane (144 mg, 0.375 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (10 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.60 mL, 4.1 mmol) was slowly added to the solution. The reaction mixture was stirred at room temperature for 10 minutes. (4S)-2-Benzyl 1-tert-butyl 4-azido-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylate (Intermediate 14, 750 mg, 1.87 mmol) was added to the reaction mixture as a DCM solution (8 mL), and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with DCM and quenched with water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give (4S)-2-benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 15, 678 mg, 68% yield). The purified material was subjected to chiral SFC [(S,S) Whelk-O1 column, 21.2 × 250 mm, 5 μm, temperature = 23 °C, mobile phase = 0 - 15% MeOH:CO2, UV detection @ 220 nm, loading = 33 mg / inj, conc = 220 ng / mL in MeOH, flow rate = 75 mL / min, outlet pressure = 100 bar] to obtain two enantiomers. The stereochemistry was assigned as the anti-addition product Intermediate 16 for the major isomer and the syn-addition product Intermediate 17 for the minor isomer.

[0218] Intermediate 16 (Isomer 1, 608 mg): (2S,4S)-2-Benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate. 11H NMR (500 MHz, DMSO-d6) δ 0.64 - 0.72 (2H, m), 1.04 - 1.12 (1H, m), 1.13 - 1.20 (12H, m), 1.22 - 1.39 (12H, m), 1.69 - 1.80 (1H, m), 2.01 - 2.23 (2H, m), 2.36 - 2.48 (1H, m), 3.35 - 3.42 (1H, m), 3.58 - 3.69 (1H, m), 4.33 (1H, quin), 5.05 - 5.17 (2H, m), 7.31 - 7.40 (5H, m); m / z (ES + ) [M + H] + = 529.

[0219] Intermediate 17 (isomer 2, 220 mg): (2R,4S)-2-Benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate.

[0220] Intermediate 18: (2S,4S)-4-Amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (2S,4S)-2-Benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 16, 255 mg, 0.483 mmol) was dissolved in ethyl acetate (5 mL) and methanol (5 mL), and treated with Pd / C (10 wt%, 128 mg, 0.120 mmol). The flask was equipped with a balloon of H2. The suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and rinsed with methanol. The filtrate was concentrated under reduced pressure to obtain the product (Intermediate 18, 190 mg, 95% yield) as a mixture of rotational isomers. This was used without further purification. 11H NMR (300 MHz, DMSO-d6) δ 0.66 (2H, t), 0.88 - 1.03 (1H, m), 1.16 (12H, s), 1.24 - 1.38 (13H, m), 1.40 - 1.56 (1H, m), 1.80 - 1.91 (1H, m), 2.00 - 2.15 (2H, m), 3.17 - 3.28 (1H, m), 3.58 - 3.61 (1H, m), 3.80 (1H, dd), 9.01 (2H, br s); m / z (ES+) [M + H]+ = 413.

[0221] Example 4: (2S,4S)-4-Amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.71 mL, 9.2 mmol) was added to a solution of (2S,4S)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 18, 190 mg, 0.461 mmol) in DCM (4 mL). The resulting solution was stirred at room temperature for 1 hour and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (3 mL) and 1M aq HCl (3 mL). Phenylboronic acid (112 mg, 0.919 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column using 2M ammonia / methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 - 10% acetonitrile in water) to give (2S,4S)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 4, 40 mg, 37% yield) as a white solid. 1 1H NMR (300 MHz, D2O) δ 0.73 (2H, t), 1.10 - 1.42 (4H, m), 1.69 (1H, ddd), 1.86 - 1.99 (1H, m), 2.10 - 2.30 (2H, m), 3.05 (1H, dd), 3.44 (1H, dd), 3.69 (1H, quin); m / z (ES + ) [M + H]+ =231.

[0222] Example 5: (2R,4S)-4-Amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chem.

[0223] Example 5: (2R,4S)-4-Amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.56 mL, 7.3 mmol) was added to a solution of (2R,4S)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 19, 150 mg, 0.364 mmol) in DCM (3 mL). The resulting solution was stirred at room temperature for 1 hour and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (2 mL) and 1 M aq HCl (2 mL). Phenylboronic acid (99 mg, 0.81 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column using 2 M ammonia / methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 - 10% water, 0 - 100% acetonitrile) to give (2R,4S)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 5, 33 mg, 39% yield) as a white solid. 1 H NMR (400 MHz, D2O) δ 0.72 (1H, m), 1.11 - 1.39 (3H, m), 1.46 - 1.55 (1H, m), 1.63 - 1.79 (2H, m), 1.95 - 2.05 (1H, m), 2.58 - 2.65 (1H, m), 2.87 - 2.95 (1H, m), 3.48 - 3.58 (3H, m); m / z (ES + ) [M + H] + = 231.

[0224] Example 6: (2S,4R)-4-Amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chem.

[0225] Sodium azide (5.96 g, 91.7 mmol) was added to a solution of 1-(tert-butyl) 2-methyl (2S,4S)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (9.89 g, 30.6 mmol) in DMF (30 mL). The reaction mixture was heated to 50 °C and stirred overnight. The reaction mixture was cooled to room temperature and concentrated. The resulting residue was diluted with EtOAc and washed with water. The organic layer was dried over Na2SO4, filtered, concentrated to dryness. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give the product (Intermediate 20, 5.95 g, 72% yield) as a mixture of rotamers. 1 H NMR (300 MHz, DMSO-d6) δ 1.33 and 1.40 (9H, s x2) rotamers, 2.08 - 2.22 (1H, m), 2.26 - 2.41 (1H, m), 3.41 (1H, dt), 3.48 - 3.61 (1H, m), 3.65 and 3.68 (3H, s x2) rotamers, 4.22 (1H, dd), 4.30 - 4.43 (1H, m); m / z (ES + ) [M+H] + = 271.

[0226] Intermediate 21: (2S,4R)-2-Benzyl 1-tert-butyl 4-azidopyrrolidine-1,2-dicarboxylate An aqueous solution (22 mL) of sodium hydroxide (5.28 g, 132 mmol) was added dropwise at 0 °C to a solution of (2S,4R)-1-tert-butyl 2-methyl 4-azidopyrrolidine-1,2-dicarboxylate (Intermediate 20, 5.95 g, 22.0 mmol) in THF (44 mL) and MeOH (22 mL). The reaction mixture was stirred overnight while slowly warming to room temperature. Volatiles were removed in vacuo, the aqueous layer was acidified to about pH 3 with 5M HCl and extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated to dryness to afford (2S,4R)-4-azido-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (5.64 g, 100% yield) as a mixture of rotamers. This was used without further purification. 1 H NMR (300 MHz, DMSO-d 6 ) δ 1.35 and 1.40 (9H, s x2) rotamers, 2.07 - 2.18 (1H, m), 2.26 - 2.38 (1H, m), 3.34 - 3.44 (1H, m), 3.48 - 3.63 (1H, m), 4.09 - 4.17 (1H, m), 4.30 - 4.37 (1H, m); m / z (ES - ) [M+HCOO] - = 301.

[0227] Benzyl bromide (2.83 mL, 23.8 mmol) was added dropwise to a solution of (2S,4R)-4-azido-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (5.19 g, 19.9 mmol) and triethylamine (3.46 mL, 24.8 mmol) in DMF (60 mL) and the reaction mixture was stirred overnight at room temperature. Volatiles were removed in vacuo, the resulting residue was dissolved in EtOAc and washed with water. The organic layer was dried over Na2SO4, filtered, concentrated to dryness. The crude material was purified by silica gel chromatography (hexane / EtOAc) to afford the product (Intermediate 21, 5.09 g, 74% yield). 11H NMR (300 MHz, DMSO-d6) δ 1.26 and 1.39 (9H, s x 2) rotamers, 2.11 - 2.23 (1H, m), 2.31 - 2.43 (1H, m), 3.43 (1H, ddd), 3.50 - 3.59 (1H, m), 4.25 - 4.40 (2H, m), 5.07 - 5.22 (2H, m), 7.31 - 7.40 (5H, m); m / z (ES + ) [M+H] + = 347.

[0228] Intermediate 22: (4R)-2-Benzyl 1-tert-butyl 4-azido-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylate (2S,4R)-2-Benzyl 1-tert-butyl 4-azidopyrrolidine-1,2-dicarboxylate (Intermediate 21, 5.09 g, 14.7 mmol) and crotyl bromide (2.27 mL, 22.0 mmol) were dissolved in THF (100 mL), and the solution was cooled to -78 °C under a N2 atmosphere. The solution was treated by dropwise addition of a solution of KHMDS (0.5 M in toluene, 44.1 mL, 22.0 mmol). The reaction mixture was slowly warmed to room temperature and stirred for 3 hours. The crude reaction mixture was quenched with water and the volatiles were removed in vacuo. The crude mixture was diluted with DCM and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give the product (Intermediate 22, 4.6 g, 78% yield) as a mixture of rotamers and E / Z olefins. 1 1H NMR (300 MHz, DMSO-d6) δ 1.26 - 1.43 (9H, m), 1.59 - 1.66 (3H, m), 2.07 - 2.17 (1H, m), 2.32 - 2.48 (2H, m), 2.57 - 3.12 (2H, m), 3.35 - 3.82 (1H, m), 4.20 - 4.38 (1H, m), 5.02 - 5.22 (2H, m), 5.24 - 5.41 (1H, m), 5.46 - 5.68 (1H, m), 7.28 - 7.42 (5H, m); m / z (ES + ) [M+H] + = 401.

[0229] Intermediate 24: (2S,4R)-2-benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate and Intermediate 25: (2R,4R)-2-benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate Bis(1,5-cyclooctadiene)dirhodium(I) dichloride (772 mg, 1.15 mmol) and bis(diphenylphosphino)methane (883 mg, 2.30 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (66 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3.67 mL, 25.3 mmol) was slowly added to the solution. The reaction mixture was stirred at room temperature for 10 minutes. (4R)-2-Benzyl 1-tert-butyl 4-azido-2-(but-2-enyl)pyrrolidine-1,2-dicarboxylate (Intermediate 22, 4.60 g, 11.5 mmol) was added to the reaction mixture as a DCM solution (44 mL), and the reaction mixture was stirred overnight. The reaction mixture was diluted with DCM and quenched with water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give (4R)-2-benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 23, 2.7 g, 44% yield). The purified material was subjected to chiral SFC (Chiralpak IG column, 21.2×250 mm, 5 μm, temperature = 23 °C, mobile phase = 0 - 7% MeOH (w / 0.2% NH4OH):CO2, UV detection @ 220 nm, loading = 16.8 mg / inj, conc = 112.5 ng / mL in MeOH, flow rate = 70 mL / min, outlet pressure = 100 bar] to obtain two enantiomers. The stereochemistry was assigned as the anti-addition product for the major enantiomer Intermediate 25, and the syn-addition product for the minor enantiomer Intermediate 24.

[0230] Intermediate 24 (436 mg): (2S,4R)-2-Benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate. 11H NMR (400 MHz, DMSO-d6) δ 0.58 - 0.70 (2H, m), 1.17 (12H, s), 1.25 - 1.40 (13H, m), 1.74 - 1.83 (1H, s), 2.00 - 2.11 (2H, m), 2.38 - 2.47 (1H, m), 3.07 - 3.16 (1H, m), 3.81 (1H, m), 4.29 - 4.34 (1H, m), 5.04 - 5.17 (2H, m), 7.34 - 7.39 (m, 5H); m / z (ES + ) [M + H] + = 529.

[0231] Intermediate 25 (1.60 g): (2R,4R)-2-Benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate. 1 1H NMR (400 MHz, DMSO-d6) δ 0.56 - 0.73 (2H, m), 0.98 - 1.13 (1H, m), 1.17 (12H, s), 1.26 - 1.37 (13H, m), 1.66 - 1.79 (1H, m), 2.01 - 2.22 (2H, m), 2.34 - 2.47 (1H, m), 3.60 (1H, br dd), 4.29 - 4.35 (1H, m), 5.04 - 5.18 (2H, m), 7.31 - 7.40 (5H, m); m / z (ES + ) [M + H] + = 529.

[0232] Intermediate 26: (2S,4R)-4-Amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (2S,4R)-2-Benzyl 1-tert-butyl 4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 24, 236 mg, 0.447 mmol) was dissolved in ethyl acetate (4.5 mL) and treated with Pd / C (10 wt%, 119 mg, 0.112 mmol). The flask was equipped with a balloon of H2. The suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and rinsed with methanol. The filtrate was concentrated under reduced pressure to give the product (Intermediate 26, 275 mg, 100% yield). This was used without further purification. 1 H NMR (300 MHz, DMSO-d6) δ 0.64 - 0.71 (2H, M), 1.17 (12H, s), 1.27 - 1.40 (15H, m), 1.57 - 1.82 (4H, m), 1.98 - 2.08 (3H, m), 3.70 - 3.78 (1H, m); m / z (ES + ) [M + H] + = 413.

[0233] Example 6: (2S,4R)-4-Amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.69 mL, 8.9 mmol) was added to a solution of (2S,4R)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 26, 184 mg, 0.446 mmol) in DCM (4 mL). The resulting solution was stirred at room temperature for 1 hour and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (2 mL) and 1M aq HCl (2 mL). Phenylboronic acid (109 mg, 0.894 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column using 2M ammonia / methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 - 100% acetonitrile in water) to give (2S,4R)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 6, 38 mg, 37% yield) as a white solid. 1 H NMR (400 MHz, D2O) δ 0.72 (2H, td), 1.09 - 1.19 (1H, m), 1.22 - 1.39 (3H, m), 1.65 - 1.76 (2H, m), 1.95 - 2.04 (1H, m), 2.58 - 2.64 (1H, m), 2.87 - 2.94 (1H, m), 3.48 - 3.57 (2H, m); m / z (ES + ) [M+H] + = 231.

[0234] Example 7: (2R,4R)-4-Amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical formula

[0235] Example 7: (2R,4R)-4-Amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (1.02 mL, 13.3 mmol) was added to a solution of (2R,4R)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 27, 275 mg, 0.667 mmol) in DCM (4 mL). The resulting solution was stirred at room temperature for 1 hour and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (2 mL) and 1 M aq HCl (2 mL). Phenylboronic acid (163 mg, 1.34 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column using 2 M ammonia / methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 - 10% - 100% acetonitrile in water) to give (2R,4R)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 7, 53 mg, 34% yield) as a white solid. 1 H NMR (400 MHz, D2O) δ 0.76 (2H, dt), 1.10 - 1.46 (4H, m), 1.62 - 1.71 (1H, m), 1.84 - 1.96 (1H, m), 2.10 - 2.21 (1H, m), 2.22 - 2.32 (1H, m), 3.07 (1H, dd), 3.46 (1H, dd), 3.71 (1H, quin); m / z (ES + ) [M + H] + = 231.

[0236] Example 8: (2R,4R)-4-((S)-2-aminopropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical Structure

[0237] Example 8: (2R,4R)-4((S)-2-Aminopropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.62 mL, 8.1 mmol) was added to a solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)propanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 28, 236 mg, 0.404 mmol) in DCM (4 mL). The resulting solution was stirred at room temperature for 1 h and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (2 mL) and 1 M aq HCl (2 mL). Phenylboronic acid (99 mg, 0.81 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 h. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column with 2 M ammonia / methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 - 10% acetonitrile in water) to give (2R,4R)-4((S)-2-aminopropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 8, 18 mg, 15% yield) as a white solid and a mixture of rotamers. 1 H NMR (500 MHz, D2O) δ 0.69 (2H, dt), 1.05 - 1.14 (1H, m), 1.21 (3H, d), 1.23 - 1.35 (3H, m), 1.65 (1H, dt), 1.91 - 1.96 (1H, m), 2.17 (1H, dd), 2.35 (1H, dd), 3.26 (1H, dd), 3.46 - 3.57 (2H, m), 4.29 - 4.34 (1H, m); m / z (ES + ) [M + H] + = 302.

[0238] Example 9: (2R,4R)-4-((S)-2-amino-3-methylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chem.

[0239] Example 9: (2R,4R)-4-((S)-2-Amino-3-methylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.63 mL, 8.2 mmol) was added to a solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-methylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 29, 250 mg, 0.409 mmol) in DCM (4 mL). The resulting solution was stirred at room temperature for 1 h and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (2 mL) and 1 M aq HCl (2 mL). Phenylboronic acid (99 mg, 0.81 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 h. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column using 2 M ammonia / methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0-10% acetonitrile in water) to give (2R,4R)-4((S)-2-amino-3-methylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 9, 28 mg, 20% yield) as a white solid and a mixture of rotamers. 1 H NMR (300 MHz, D2O) δ 0.66 - 0.76 (2H, m), 0.85 (6H, dd), 1.07 - 1.43 (4H, m), 1.55 - 1.68 (1H, m), 1.77 - 1.97 (2H, m), 2.13 - 2.33 (2H, m), 3.07 (1H, d), 3.08 - 3.16 (1H, m), 3.37 - 3.48 (1H, m), 4.27 - 4.40 (1H, m); m / z (ES + ) [M + H] + = 330.

[0240] Example 10: (2R,4R)-4-((S)-2-Amino-3-methylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical Structure

[0241] Intermediate 31: (2R,4R)-2-Benzyl 1-tert-butyl 4-((R)-2-(tert-butoxycarbonylamino)-3-methylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate N,N-Diisopropylethylamine (0.235 mL, 1.34 mmol) was slowly added to a stirred DMF solution (2 mL) of HATU (245 mg, 0.64 mmol) and Boc-D-Val-OH (117 mg, 0.54 mmol) at room temperature. After the solution was stirred for 20 minutes, a DMF solution (2 mL) of (2R,4R)-2-benzyl 1-tert-butyl 4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 30, 270 mg, 0.54 mmol) was added. The reaction mixture was stirred for 2.5 hours, diluted with DCM (30 mL), and washed successively with water (3 × 25 mL) and saturated aqueous sodium chloride (30 mL). The organic layer was dried over MgSO4, filtered, concentrated to dryness. The crude material was purified by silica gel chromatography (5 - 65% EtOAc in hexane) to give (2R,4R)-2-benzyl 1-tert-butyl 4-((R)-2-(tert-butoxycarbonylamino)-3-methylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 31, 239 mg, 63% yield) as a colorless foam and as a mixture of rotamers. 11H NMR (500 MHz, CD2Cl2) δ 0.74 - 0.81 (2H, m), 0.84 (3H, d), 0.87 - 0.94 (3H, m), 1.24 (12H, s), 1.26 - 1.34 (2H, m), 1.37 (5H, s), 1.40 - 1.43 (2H, m), 1.45 (4H, s), 1.46 (9H, s), 1.78 - 1.89 (1H, m), 1.95 - 2.07 (2H, m), 2.21 - 2.29 (0.6H, m), 2.31 - 2.46 (1.4H, m), 3.51 - 3.60 (1.5H, m), 3.65 (0.5H, br d), 3.72 (1H, br dd), 4.49 - 4.58 (1H, m), 5.01 (1H, br d), 5.19 - 5.29 (2H, m), 6.93 - 7.09 (1H, m), 7.36 - 7.40 (1H, m), 7.43 (4H, app d); m / z (ES + ) [M + H] + = 702.

[0242] Intermediate 32: (2R,4R)-1-(tert-Butoxycarbonyl)-4-((R)-2-(tert-Butoxycarbonylamino)-3-methylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 25 mg, 0.23 mmol) was added to a solution of (2R,4R)-2-benzyl 1-tert-butyl 4-((R)-2-(tert-butoxycarbonylamino)-3-methylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 31, 239 mg, 0.34 mmol) in EtOAc (4 mL). The suspension was stirred at room temperature for 2 h under a hydrogen atmosphere (balloon, the flask was evacuated and refilled with hydrogen × 3). The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, and the filtrate was concentrated to dryness. The crude material was purified by silica gel chromatography (2 - 15% MeOH in DCM) to give (2R,4R)-1-(tert-butoxycarbonyl)-4-((R)-2-(tert-butoxycarbonylamino)-3-methylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 32, 196 mg, 94% yield) as a white solid and as a mixture of rotamers. 1 H NMR (500 MHz, DMSO-d6) δ 0.63 - 0.71 (2H, m), 0.75 - 0.82 (6H, m), 1.15 (12H, s), 1.21 - 1.30 (2H, m), 1.32 (6H, s), 1.36 (13H, br s), 1.61 - 1.72 (1H, m), 1.81 - 1.90 (1H, m), 1.92 - 2.05 (2H, m), 2.05 - 2.13 (0.6H, m), 2.13 - 2.28 (1.4H, m), 3.03 - 3.14 (1H, m), 3.62 (0.6H, t), 3.66 (1.4H, t), 4.18 - 4.29 (1H, m), 6.59 (1H, d), 7.99 (1H, br s), 12.48 (0.4H, br s), 12.65 (0.6H, br s); m / z (ES + ) [M + H] + = 612.

[0243] Example 10: (2R,4R)-4-((R)-2-Amino-3-methylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.37 mL, 4.8 mmol) was added dropwise to a stirred DCM solution (2 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-((R)-2-(tert-butoxycarbonylamino)-3-methylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 32, 195 mg, 0.32 mmol). The reaction solution was stirred at room temperature for 22 h and then concentrated under reduced pressure. The crude amino acid was dissolved in 1M HCl aq (2 mL) and Et2O (2 mL). Phenylboronic acid (117 mg, 0.96 mmol) was added and the clear biphasic solution was stirred at room temperature for 5 h. The mixture was diluted with Et2O (20 mL) and water (5 mL) and the layers were separated. The aqueous layer was washed with Et2O and freeze-dried. The resulting solid was dissolved in MeOH (3 mL) and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column with 5% ammonia solution in MeOH (20 mL). The obtained material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 0-80% acetonitrile in water) to give (2R,4R)-4-((R)-2-amino-3-methylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 10, 46 mg, 44% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.73-0.80 (2H, m), 0.90 (6H, app t), 1.13-1.25 (1H, m), 1.26-1.35 (1H, m), 1.40 (2H, quin), 1.68-1.80 (1H, m), 1.85-1.96 (1H, m), 2.00 (1H, td), 2.29 (1H, dd), 2.37-2.45 (1H, m), 3.18 (1H, d), 3.28 (1H, dd), 3.59 (1H, dd), 4.36-4.49 (1H, m); m / z (ES + ) [M+H] + = 330.

[0244] Example 11: (2R,4R)-4-((S)-2-Amino-3,3-dimethylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chem.

[0245] Intermediate 34: (2R,4R)-1-(tert-Butoxycarbonyl)-4-((S)-2-(tert-Butoxycarbonylamino)-3,3-dimethylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 25 mg, 0.23 mmol) was added to a solution of (2R,4R)-2-benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 33, 260 mg, 0.36 mmol) in EtOAc (4 mL). The suspension was stirred at room temperature for 15 h under a hydrogen atmosphere (balloon, flask evacuated and refilled with hydrogen × 3). The reaction mixture was diluted with MeOH, filtered through celite, and the filtrate was concentrated to dryness. The crude material was purified by silica gel chromatography (2 - 10% MeOH in DCM) to give (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 34, 207 mg, 91% yield) as a white solid and as a mixture of rotamers. 1 H NMR (500 MHz, DMSO-d6) δ 0.63 - 0.72 (2H, m), 0.86 (9H, s), 1.09 - 1.20 (14H, m), 1.21 - 1.30 (2H, m), 1.33 (5H, s), 1.35 - 1.38 (13H, m), 1.61 - 1.73 (1H, m), 1.89 - 2.11 (2H, m), 2.14 - 2.27 (1H, m), 3.06 - 3.14 (1H, m), 3.59 - 3.72 (1H, m), 3.72 - 3.80 (1H, m), 4.20 - 4.30 (1H, m), 6.35 (1H, d), 8.08 (1H, br s), 12.47 (0.4H, br s), 12.63 (0.6H, br s); m / z (ES + ) [M + H] + = 626.

[0246] Example 11: (2R,4R)-4-((S)-2-Amino-3,3-dimethylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.38 mL, 4.9 mmol) was added dropwise to a stirred DCM solution (2 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 34, 206 mg, 0.33 mmol). The reaction mixture was stirred at room temperature for 15 hours and then concentrated under reduced pressure. The crude amino acid was dissolved in 1M HCl aq (4 mL) and Et2O (4 mL). Phenylboronic acid (120 mg, 0.99 mmol) was added and the clear biphasic solution was stirred at room temperature for 3 hours. The mixture was diluted with Et2O (20 mL) and water (5 mL) and the layers were separated. The aqueous layer was washed with Et2O and lyophilized. The resulting solid was dissolved in MeOH (3 mL) and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column using 5% ammonia solution in MeOH (20 mL). The obtained material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 2 - 50% acetonitrile in water) to give (2R,4R)-4-((S)-2-amino-3,3-dimethylbutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 11, 40 mg, 35% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.72 (2H, td), 0.89 (9H, s), 1.10 - 1.21 (1H, m), 1.22 - 1.30 (1H, m), 1.35 (2H, quin), 1.64 - 1.75 (1H, m), 1.90 - 2.02 (1H, m), 2.22 - 2.34 (2H, m), 3.04 (1H, s), 3.22 (1H, dd), 3.56 (1H, dd), 4.41 (1H, quin); m / z (ES + ) [M + H] + = 344.

[0247] Example 12: (2R,4R)-2-(4-Boronobutyl)-4((S)-pyrrolidine-2-carboxamido)pyrrolidine-2-carboxylic acid [Chemical formula] Intermediate 35: (2R,4R)-2-Benzyl 1-tert-butyl 4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate N,N-Diisopropylethylamine (0.182 mL, 1.04 mmol) was slowly added to a stirred DMF solution (1.5 mL) of HATU (175 mg, 0.46 mmol) and Boc-Pro-OH (94 mg, 0.44 mmol) at room temperature. After the solution was stirred for 20 minutes, a DMF solution (1.5 mL) of (2R,4R)-2-benzyl 1-tert-butyl 4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 30, 210 mg, 0.42 mmol) was added. The reaction mixture was stirred for 2 hours, diluted with EtOAc (30 mL), and washed successively with water (3 × 25 mL), saturated aqueous NaHCO3, and saturated aqueous sodium chloride (30 mL). The organic layer was dried over MgSO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (5 - 100% EtOAc in hexane) to give (2R,4R)-2-benzyl 1-tert-butyl 4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 35, 249 mg, 85% yield) as a colorless film and as a mixture of rotational isomers. 11H NMR (500 MHz, CD2Cl2) δ 0.68 - 0.79 (2H, m), 1.20 (12H, s), 1.31 (5H, s), 1.36 - 1.48 (16H, m), 1.74 - 1.87 (3H, m), 1.89 - 2.10 (3H, m), 2.13 - 2.46 (2H, m), 3.27 - 3.40 (1H, m), 3.44 (2H, br s), 3.50 - 3.64 (2H, m), 3.78 - 4.05 (1H, m), 4.49 (1H, br s), 5.10 - 5.27 (2H, m), 7.10 (1H, br s), 7.30 - 7.42 (5H, m); m / z (ES + ) [M + H] + = 700.

[0248] Intermediate 36: (2R,4R)-1-(tert-Butoxycarbonyl)-4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 25 mg, 0.23 mmol) was added to a solution of (2R,4R)-2-benzyl 1-tert-butyl 4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 35, 249 mg, 0.36 mmol) in EtOAc (4 mL). The suspension was stirred at room temperature for 5 h under a hydrogen atmosphere (balloon, the flask was evacuated and refilled with hydrogen × 3). The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 36, 207 mg, 87% yield) as a colorless film and as a mixture of rotational isomers. This was used without further purification. 11H NMR (500 MHz, CD2Cl2) δ 0.66 - 0.83 (2H, m), 1.21 (12H, s), 1.34 - 1.51 (21H, m), 1.63 - 1.97 (4H, m), 2.06 (1H, m), 2.11 - 2.29 (2H, m), 2.33 - 2.67 (1H, m), 3.24 - 3.52 (3H, m), 3.53 - 3.67 (1H, m), 4.15 - 4.34 (1H, m), 4.47 - 4.74 (1H, m), 6.76 - 7.23 (1H, m), 7.17 - 7.69 (1H, m), 9.74 (1H, br s); m / z (ES + ) [M + H] + = 610.

[0249] Example 12: (2R,4R)-2-(4-Boronobutyl)-4((S)-pyrrolidine-2-carboxamide)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.518 mL, 6.73 mmol) was added dropwise to a stirred DCM solution (2 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 36, 205 mg, 0.34 mmol). The reaction mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The crude amino acid was dissolved in 1M HCl aq (4 mL) and Et2O (4 mL). Phenylboronic acid (123 mg, 1.01 mmol) was added and the clear biphasic solution was stirred at room temperature for 2 hours. The mixture was diluted with Et2O (20 mL) and water (5 mL) and the layers were separated. The aqueous layer was washed with Et2O and lyophilized. The resulting solid was dissolved in MeOH (3 mL) and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column using 5% ammonia solution in MeOH (20 mL). The obtained material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 0 - 50% acetonitrile in water) to give (2R,4R)-2-(4-boronobutyl)-4-((S)-pyrrolidine-2-carboxamido)pyrrolidine-2-carboxylic acid (Example 12, 89 mg, 81% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.70 - 0.79 (2H, m), 1.11 - 1.23 (1H, m), 1.24 - 1.33 (1H, m), 1.34 - 1.42 (2H, m), 1.60 - 1.71 (1H, m), 1.81 - 1.91 (3H, m), 1.91 - 1.99 (1H, m), 2.18 (1H, dd), 2.22 - 2.29 (1H, m), 2.40 (1H, dd), 3.08 - 3.16 (1H, m), 3.16 - 3.22 (1H, m), 3.25 (1H, dd), 3.48 (1H, dd), 3.99 (1H, dd), 4.29 - 4.38 (1H, m); m / z (ES + ) [M + H] + = 328.

[0250] Example 13: (2R,4R)-4-(2-Aminoacetamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical formula

[0251] Intermediate 38: (2R,4R)-1-(tert-Butoxycarbonyl)-4-(2-(tert-butoxycarbonylamino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 25 mg, 0.23 mmol) was added to a solution of (2R,4R)-2-benzyl 1-tert-butyl 4-(2-(tert-butoxycarbonylamino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 37, 233 mg, 0.35 mmol) in EtOAc (4 mL). The suspension was stirred at room temperature for 6 h under a hydrogen atmosphere (balloon, the flask was evacuated and refilled with hydrogen three times). The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, and the filtrate was concentrated to dryness to give (2R,4R)-1-(tert-butoxycarbonyl)-4-(2-(tert-butoxycarbonylamino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 38, 176 mg, 87% yield) as a colorless film and as a mixture of rotamers. This was used without further purification. 1 H NMR (500 MHz, CD2Cl2) δ 0.65 - 0.80 (2H, m), 1.11 - 1.18 (1H, m), 1.18 - 1.23 (12H, m), 1.25 - 1.32 (1H, m), 1.36 - 1.45 (13H, m), 1.47 (7H, s), 1.70 - 1.83 (0.4H, m), 1.84 - 1.95 (0.6H, m), 2.06 - 2.27 (2H, m), 2.33 - 2.47 (0.4H, m), 2.63 (0.6H, br d), 3.44 - 3.62 (2H, m), 3.63 - 3.82 (2H, m), 4.28 (0.6H, br s), 4.36 - 4.60 (0.4H, m), 5.26 (0.6H, br s), 5.58 - 5.90 (0.3H, m), 6.83 (0.6H, br s), 6.97 - 7.44 (0.4H, m); m / z (ES + ) [M + H] + = 570.

[0252] Example 13: (2R,4R)-4-(2-Aminoacetamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.476 mL, 6.18 mmol) was added dropwise to a stirred DCM solution (2 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-(2-(tert-butoxycarbonylamino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 38, 176 mg, 0.31 mmol). The reaction solution was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The crude amino acid was dissolved in 1M HCl aq (4 mL) and Et2O (4 mL). Phenylboronic acid (113 mg, 0.93 mmol) was added and the clear biphasic solution was stirred at room temperature for 2 hours. The mixture was diluted with Et2O (20 mL) and water (5 mL) and the layers were separated. The aqueous layer was washed with Et2O and lyophilized. The resulting solid was dissolved in MeOH (3 mL) and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column using 5% ammonia solution in MeOH (20 mL). The obtained material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 0 - 40% acetonitrile in water) to give (2R,4R)-4-(2-aminoacetamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 13, 62 mg, 70% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.76 (2H, td), 1.15 - 1.25 (1H, m), 1.26 - 1.34 (1H, m), 1.36 - 1.46 (2H, m), 1.69 - 1.79 (1H, m), 2.00 (1H, ddd), 2.27 (1H, dd), 2.44 (1H, dd), 3.33 (1H, dd), 3.42 (2H, s), 3.59 (1H, dd), 4.36 - 4.45 (1H, m); m / z (ES + ) [M + H] + = 288.

[0253] Example 14: (2R,4R)-4-((S)-2-aminobutanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical Structure

[0254] Intermediate 40: (2R,4R)-1-(tert-Butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)butanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (2R,4R)-2-Benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)butanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 39, 766 mg, 1.11 mmol) was dissolved in EtOAc (11 mL) and treated with Pd / C (10 wt%, 119 mg, 0.11 mmol). The flask was equipped with a balloon of H2. The suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and rinsed with EtOAc and methanol. The filtrate was concentrated to dryness. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)butanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 40, 470 mg, 70% yield) as a white foam. 11H NMR (500 MHz, CDCl3) δ 0.67 - 0.82 (2H, m), 0.89 (3H, br t), 1.11 - 1.28 (14H, m), 1.37 - 1.51 (20H, m), 1.53 - 1.65 (1H, m), 1.65 - 1.94 (2H, m), 2.02 - 2.12 (1H, m), 2.13 - 2.31 (1H, m), 2.70 (1H, br d), 3.40 - 3.62 (2H, m), 3.88 - 4.04 (1H, m), 4.26 (1H, br s), 5.01 (1H, br s), 6.73 (1H, br d); m / z (ES + ) [M + H] + = 598.

[0255] Example 14: (2R,4R)-4-((S)-2-Aminobutanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Phenylboronic acid (192 mg, 1.57 mmol) was added to a 2M HCl aq solution (5 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)butanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 40, 470 mg, 0.79 mmol), and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water (10 mL) and Et2O (10 mL), and the layers were separated. The aqueous layer was washed with Et2O (3×5 mL) and then lyophilized to give a foam. The organic layer was concentrated under vacuum. The resulting residue was diluted in 4M HCl (4 mL, 16 mmol) in dioxane, and the resulting solution was stirred at room temperature for 20 h. The reaction mixture was diluted with water (10 mL) and Et2O (10 mL), and the layers were separated. The aqueous layer was washed with Et2O (3×5 mL) and then lyophilized to give a foam. The foams from these two operations were combined, and the resulting crude amino acid was purified by ion exchange chromatography (Silicycle SiliaSep SPE-R51230B-20X 5 g column). The desired product was eluted from the column using 5% ammonia solution in MeOH. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0-25% acetonitrile in water) to give (2R,4R)-4-((S)-2-aminobutanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 14, 96 mg, 39% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.55 - 0.82 (2H, m), 0.88 (3H, t), 1.12 - 1.48 (4H, m), 1.56 - 1.81 (3H, m), 1.86 - 2.10 (1H, m), 2.12 - 2.53 (2H, m), 3.15 - 3.37 (1H, m), 3.41 - 3.53 (1H, m), 3.62 (1H, dd), 4.35 - 4.52 (1H, m); m / z (ES + ) [M - H2O + H] + = 298.

[0256] Example 15: (2R,4R)-4-((2S,3S)-2-Amino-3-methylpentanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical formula

[0257] Intermediate 42: (2R,4R)-1-(tert-Butoxycarbonyl)-4-((2S,3S)-2-(tert-butoxycarbonylamino)-3-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (2R,4R)-2-Benzyl 1-tert-butyl 4-((2S,3S)-2-(tert-butoxycarbonylamino)-3-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 41, 707 mg, 0.99 mmol) was dissolved in EtOAc (10 mL) and treated with Pd / C (10 wt%, 105 mg, 0.10 mmol). The flask was equipped with a balloon of H2. The suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and rinsed with EtOAc and methanol. The filtrate was concentrated to dryness to give (2R,4R)-1-(tert-butoxycarbonyl)-4-((2S,3S)-2-(tert-butoxycarbonylamino)-3-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 42, 603 mg, 98% yield). This was used without further purification. 11H NMR (500 MHz, CDCl3) δ 0.68 - 0.80 (2H, m), 0.83 - 0.93 (6H, m), 1.05 - 1.14 (1H, m), 1.21 (12H, s), 1.28 - 1.36 (1H, m), 1.37 - 1.55 (22H, m), 1.71 - 1.96 (2H, m), 2.18 - 2.31 (1H, m), 2.72 (1H, br d), 3.42 - 3.51 (2H, m), 3.52 - 3.63 (1H, m), 3.86 - 4.04 (1H, m), 4.16 - 4.34 (1H, m), 4.98 (1H, br d), 6.69 (1H, br s); m / z (ES + ) [M + H] + = 626.

[0258] Example 15: (2R,4R)-4-((2S,3S)-2-Amino-3-methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (1.10 mL, 14.3 mmol) was added to a DCM solution (6 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-((2S,3S)-2-(tert-butoxycarbonylamino)-3-methylpentanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 42, 603 mg, 0.96 mmol). The resulting solution was stirred at room temperature for 16 h and then concentrated under vacuum. The crude amino acid was dissolved in Et2O (10 mL) and reconcentrated under vacuum. This dissolution and reconcentration process was repeated two more times. Next, the crude amino acid was dissolved in Et2O (6 mL) and 1 M HCl aq (6 mL). Phenylboronic acid (235 mg, 1.93 mmol) was added and the clear biphasic solution was stirred at room temperature for 3 h. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (Silicycle SiliaSep SPE-R51230B-20X 5 g column). The desired product was eluted from the column using 5% ammonia solution in MeOH. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 - 25% acetonitrile in water) to give (2R,4R)-4-((2S,3S)-2-amino-3-methylpentanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 15, 136 mg, 41% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.55 - 0.81 (2H, m), 0.89 (6H, dd), 1.07 - 1.52 (6H, m), 1.63 - 1.83 (2H, m), 1.86 - 2.09 (1H, m), 2.13 - 2.51 (2H, m), 3.11 - 3.40 (2H, m), 3.45 - 3.67 (1H, m), 4.39 - 4.54 (1H, m); m / z (ES + ) [M + H] + = 344.

[0259] Example 16: (2R,4R)-4-((S)-2-Amino-4-methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid [Chemical formula] Intermediate 43: (2R,4R)-2-Benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate HATU (247 mg, 0.65 mmol) was added to a DCM solution (2 mL) of Boc-Leu-OH (125 mg, 0.54 mmol), and the reaction solution was stirred at room temperature for 10 minutes. (2R,4R)-2-Benzyl 1-tert-butyl 4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 30, 272 mg, 0.54 mmol) was added to the reaction mixture as a DCM solution (2 mL). N,N-Diisopropylethylamine (0.19 mL, 1.1 mmol) was added, and the reaction solution was stirred at room temperature for 1 hour. Next, the reaction solution was diluted with DCM (20 mL) and washed successively with water (25 mL) and saturated aqueous sodium chloride (30 mL). The organic layer was dried over MgSO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to obtain (2R,4R)-2-Benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 43, 210 mg, 54% yield) as a colorless foam and as a mixture of rotational isomers. 11H NMR (500 MHz, CDCl3) δ 0.77 (2H, t), 0.91 (6H, d), 1.06 - 1.19 (1H, m), 1.19 - 1.24 (12H, m), 1.31 - 1.51 (20H, m), 1.50 - 1.63 (2H, m), 1.73 - 2.02 (2H, m), 2.17 - 2.55 (2H, m), 3.35 - 3.75 (2H, m), 3.84 - 4.06 (1H, m), 4.35 - 4.75 (2H, m), 5.00 - 5.46 (2H, m), 7.08 - 7.22 (1H, m), 7.28 - 7.42 (5H, m); m / z (ES + ) [M + H] + = 716.

[0260] Intermediate 44: (2R,4R)-1-(tert-Butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (2R,4R)-2-Benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 43, 201 mg, 0.28 mmol) was dissolved in EtOAc (4 mL) and treated with Pd / C (10 wt%, 100 mg, 0.094 mmol). The flask was equipped with a balloon of H2. The suspension was stirred at room temperature for 2 h. The reaction mixture was filtered through diatomaceous earth and rinsed with EtOAc and methanol. The filtrate was concentrated to dryness to give (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 44, 170 mg, 97% yield) as a white solid and as a mixture of rotamers. 11H NMR (500 MHz, CDCl3) δ 0.78 (2H, t), 0.93 (6H, d), 1.15 - 1.25 (12H, m), 1.25 - 1.31 (2H, m), 1.39 - 1.51 (19H, m), 1.55 - 1.72 (2H, m), 1.73 - 1.89 (1H, m), 2.01 - 2.11 (1H, m), 2.18 - 2.36 (1H, m), 2.47 - 2.83 (1H, m), 3.37 - 3.74 (2H, m), 3.96 - 4.10 (1H, m), 4.17 - 4.32 (1H, m), 4.82 - 5.31 (1H, m), 6.62 - 7.12 (1H, m); m / z (ES + ) [M + H] + = 626.

[0261] Example 16: (2R,4R)-4-((S)-2-Amino-4-methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (1.00 mL, 13.0 mmol) was added to a DCM solution (2 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 44, 170 mg, 0.27 mmol). The resulting solution was stirred at room temperature for 2 hours and then concentrated under vacuum. Next, the crude amino acid was dissolved in Et2O (5 mL) and water (4 mL). Phenylboronic acid (66 mg, 0.54 mmol) was added and the clear biphasic solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (5 mL) and Et2O (20 mL) and the layers were separated. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 2 g column) to give (2R,4R)-4-((S)-2-amino-4-methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 16, 88 mg, 94% yield) as a white solid. 11H NMR (500 MHz, D2O) δ 0.46 - 0.68 (2H, m), 0.73 - 0.82 (6H, m), 1.03 - 1.13 (1H, m), 1.13 - 1.23 (1H, m), 1.23 - 1.42 (4H, m), 1.42 - 1.52 (1H, m), 1.54 - 1.66 (1H, m), 1.76 - 1.93 (1H, m), 2.07 - 2.19 (1H, m), 2.29 (1H, dd), 3.13 (1H, q), 3.34 (1H, t), 3.48 (1H, q), 4.23 - 4.40 (1H, m); m / z (ES + ) [M + H] + = 344.

[0262] Example 17: (2R,4R)-4-((S)-2-Amino-3-hydroxypropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid [Chemical Structure] Intermediate 45: (2R,4R)-2-Benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate N,N-Diisopropylethylamine (0.108 mL, 0.62 mmol) was added to a stirred DMF solution (5 mL) of COMU (292 mg, 0.68 mmol), (2R,4R)-2-benzyl 1-tert-butyl 4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 30, 311 mg, 0.62 mmol), and Boc-Ser-OH (133 mg, 0.65 mmol) at room temperature. The reaction mixture was stirred for 3 h and diluted with water (80 mL) and EtOAc (15 mL). The phases were separated and the aqueous phase was further diluted with saturated aqueous NaHCO3 and then extracted with EtOAc (2 × 20 mL). The combined organics were washed with saturated aqueous NaCl (2 × 10 mL), dried over MgSO4, filtered, concentrated to dryness. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give (2R,4R)-2-benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 45, 358 mg, 84% yield) as a colorless dry film and as a mixture of rotamers. 1 H NMR (500 MHz, CDCl3) δ 0.76 (2H, t), 1.16 (1H, m), 1.21 (12H, s), 1.31 (6H, s), 1.35 - 1.42 (5H, m), 1.43 (11H, s), 1.73 - 1.87 (1H, m), 1.87 - 2.02 (2H, m), 2.14 - 2.24 (1H, m), 2.29 - 2.41 (1H, m), 3.43 - 3.52 (0.4H, m), 3.52 - 3.61 (2H, m), 3.66 (0.6H, d), 3.79 - 3.92 (1H, m), 3.92 - 4.04 (1H, m), 4.51 (1H, br s), 5.06 - 5.26 (2H, m), 5.36 (1H, br s), 7.30 - 7.40 (5H, m); m / z (ES + ) [M + H] + = 690.

[0263] Intermediate 46: (2R,4R)-1-(tert-Butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (2R,4R)-2-Benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 45, 358 mg, 0.52 mmol) was dissolved in EtOAc (4 mL) and treated with Pd / C (10 wt%, 50 mg, 0.047 mmol). The flask was equipped with a balloon of H2. The suspension was stirred at room temperature for 3.5 h. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, concentrated and dried to give (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 46, 303 mg, 97% yield) as a white solid and as a mixture of rotamers. This was used without further purification. 1 H NMR (500 MHz, CD2Cl2) δ 0.67 - 0.91 (2H, m), 1.25 (14H, s), 1.41 - 1.47 (3H, m), 1.48 (9H, s), 1.53 (10H, s), 1.82 - 1.90 (2H, m), 2.06 - 2.14 (1H, m), 2.24 - 2.33 (1H, m), 2.76 - 2.91 (1H, m), 3.48 - 3.54 (1H, m), 3.59 (1H, dd), 3.70 (1H, dd), 3.93 (1H, d), 4.03 - 4.17 (1H, m), 4.29 (1H, d), 6.79 - 6.98 (1H, m); m / z (ES + ) [M + H] + = 600.

[0264] Example 17: (2R,4R)-4-((S)-2-Amino-3-hydroxypropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.771 mL, 10.01 mmol) was added dropwise to a stirred DCM solution (4 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 46, 300 mg, 0.50 mmol) at room temperature. After 1.5 h, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1M HCl aq (4 mL, 4.00 mmol) and Et2O (4 mL). Phenylboronic acid (183 mg, 1.50 mmol) was added and the clear biphasic solution was stirred at room temperature for 3 h. The mixture was diluted with Et2O (20 mL) and water (5 mL) and the layers were separated. The aqueous layer was washed with Et2O, the layers were separated and the aqueous layer was lyophilized. The resulting solid was dissolved in MeOH (3 mL) and applied to an ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column with 5% ammonia solution in MeOH (20 mL). The obtained material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 0 - 30% acetonitrile in water) to give (2R,4R)-4-((S)-2-amino-3-hydroxypropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 17, 94 mg, 59% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.77 (2H, td), 1.16 - 1.26 (1H, m), 1.26 - 1.35 (1H, m), 1.35 - 1.45 (2H, m), 1.76 (1H, ddd), 2.02 (1H, ddd), 2.29 (1H, dd), 2.46 (1H, dd), 3.35 (1H, dd), 3.48 (1H, t), 3.62 (1H, dd), 3.66 - 3.77 (2H, m), 4.40 - 4.50 (1H, m); m / z (ES + ) [M + H] + = 318.

[0265] Example 18: (2R,4R)-4-((S)-2-Amino-3-methoxypropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical formula

[0266] Intermediate 48: (2R,4R)-1-(tert-Butoxycarbonyl)-4-((S)-2-(tert-Butoxycarbonylamino)-3-methoxypropanamide)-2-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (2R,4R)-2-Benzyl 1-tert-butyl 4-((S)-2-(tert-butoxycarbonylamino)-3-methoxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 47, 145 mg, 0.21 mmol) was dissolved in EtOAc (2 mL) and treated with Pd / C (10 wt%, 22 mg, 0.021 mmol). The flask was equipped with a balloon of H2. The suspension was stirred at room temperature for 4 hours. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, concentrated and dried to give (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-methoxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 48, 126 mg, 100% yield) as a white solid and as a mixture of rotational isomers. This was used without further purification. 1 H NMR (500 MHz, CD2Cl2) δ 0.65 - 0.80 (2H, m), 1.14 - 1.29 (14H, m), 1.35 - 1.43 (5H, m), 1.44 (7H, s), 1.46 - 1.60 (8H, m), 1.77 - 1.95 (1H, m), 2.03 - 2.14 (1H, m), 2.14 - 2.26 (1H, m), 2.68 (1H, br d), 3.33 (3H, s), 3.38 - 3.47 (1H, m), 3.47 - 3.60 (2H, m), 3.65 - 3.76 (1H, m), 4.02 - 4.15 (1H, m), 4.24 (1H, br s), 5.38 (1H, br s), 7.06 (1H, br s); m / z (ES + ) [M + H] + = 614.

[0267] Example 18: (2R,4R)-4-((S)-2-Amino-3-methoxypropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.25 mL, 3.26 mmol) was added dropwise to a stirred DCM solution (2 mL) of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-methoxypropanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 48, 100 mg, 0.16 mmol) at ambient temperature. After 1 hour, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1M HCl aq (2 mL, 2.00 mmol) and Et2O (2 mL). Phenylboronic acid (60 mg, 0.49 mmol) was added and the clear biphasic solution was stirred at room temperature for 3 hours. The mixture was diluted with Et2O (20 mL) and water (5 mL) and the layers were separated. The aqueous layer was washed with Et2O, the layers were separated, and the aqueous layer was lyophilized. The resulting solid was dissolved in MeOH (3 mL) and applied to an ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column using 5% ammonia solution in MeOH (20 mL). The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 0 - 20% acetonitrile in water). The product fractions were lyophilized and the resulting material was again applied to reverse phase flash chromatography (RediSep Rf Gold® C18, 0 - 2% acetonitrile in water). The product fractions were lyophilized and the resulting material was again purified by reverse phase flash chromatography (RediSep Rf Gold® C18, 0 - 5% acetonitrile in water) to give (2R,4R)-4-((S)-2-amino-3-methoxypropanamide)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 18, 18 mg, 35% yield) as a white solid. 11H NMR (500 MHz, D2O) δ 0.77 (2H, td), 1.14 - 1.27 (1H, m), 1.27 - 1.35 (1H, m), 1.35 - 1.45 (2H, m), 1.72 - 1.81 (1H, m), 2.02 (1H, ddd), 2.30 (1H, dd), 2.41 (1H, dd), 3.31 - 3.38 (4H, m), 3.55 - 3.60 (3H, m), 3.63 (1H, dd), 4.42 - 4.51 (1H, m); m / z (ES + ) [M + H] + = 332.

[0268] Example 19: (S)-2-Amino-N-((3R,5R)-8-hydroxy-6-oxo-7-oxa-1-aza-8-boraspiro[4.7]dodecan-3-yl)-3-methylbutanamide - Compound B

Chemical Structure

[0269] Example 20: (2R,4R)-4-[[(2S)-2-Amino-3-hydroxy-3-methyl-butanoyl]amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical Structure

[0270] Example 20: (2R,4R)-4-[[(2S)-2-Amino-3-hydroxy-3-methyl-butanoyl]amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 100 mg, 0.09 mmol) was added to a solution of 2-benzyl 1-(tert-butyl) (2R,4R)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanamide)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 52, 220 mg, 0.31 mmol) in EtOAc (3 mL). The flask was equipped with a balloon of H2. The suspension was stirred at room temperature for 3 h. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, concentrated and dried. The white solid was dissolved in DCM (1 mL) and trifluoroacetic acid (0.50 mL, 6.5 mmol), and the reaction solution was stirred at room temperature for 2 h. The solution was concentrated, and the resulting residue was dissolved in Et2O (2 mL) and 1 M HCl aq (2 mL). Phenylboronic acid (100 mg, 0.82 mmol) was added, and the clear biphasic solution was stirred at room temperature for 2 h. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column with 5% ammonia in MeOH (20 mL) to give (2R,4R)-4-[[(2S)-2-amino-3-hydroxy-3-methyl-butanoyl]amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 20, 92 mg, 87% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.70 (2H, t), 1.20 (4H, s), 1.28 (4H, s), 1.31 - 1.40 (2H, m), 1.77 - 1.88 (1H, m), 2.04 - 2.14 (1H, m), 2.40 - 2.47 (1H, m), 2.48 - 2.54 (1H, m), 3.40 (1H, dd), 3.74 (1H, s), 3.75 - 3.80 (1H, m), 4.47 - 4.55 (1H, m); m / z: (ES + ) [M + H] +=346.

[0271] Example 21: (2R,4R)-4-[[(2S)-2-Amino-2,3-dimethyl-butanoyl]amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical formula

[0272] Example 21: (2R,4R)-4-[[(2S)-2-Amino-2,3-dimethyl-butanoyl]amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 90 mg, 0.08 mmol) was added to a solution of 2-benzyl 1-tert-butyl (2R,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-2,3-dimethyl-butanoyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 53, 210 mg, 0.29 mmol) in EtOAc (4 mL). The flask was equipped with a balloon of H2. The suspension was stirred at room temperature for 3 h. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, concentrated, and dried. The white solid was dissolved in DCM (1 mL) and trifluoroacetic acid (0.50 mL, 6.5 mmol), and the reaction solution was stirred at room temperature for 2 h. The solution was concentrated, and the resulting residue was dissolved in Et2O (2 mL) and 1M HCl aq (2 mL). Phenylboronic acid (100 mg, 0.82 mmol) was added, and the clear biphasic solution was stirred at room temperature for 2 h. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 mL) to give (2R,4R)-4-[[(2S)-2-amino-2,3-dimethyl-butanoyl]amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 21, 90 mg, 89% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.74 - 0.78 (2H, m), 0.79 (3H, d), 0.89 (3H, d), 1.17 - 1.27 (4H, m), 1.28 - 1.35 (1H, m), 1.36 - 1.47 (2H, m), 1.70 - 1.81 (1H, m), 1.93 - 2.07 (2H, m), 2.27 (1H, dd), 2.43 (1H, dd), 3.27 - 3.39 (1H, m), 3.60 (1H, dd), 4.39 - 4.48 (1H, m); m / z: (ES + ) [M + H] + = 344.

[0273] Example 22: (2R,4R)-2-(4-Boronobutyl)-4-[[(2S)-2,3-diaminopropanoyl]amino]pyrrolidine-2-carboxylic acid

Chem.

[0274] Example 22: (2R,4R)-2-(4-Boronobutyl)-4-[[(2S)-2,3-diaminopropanoyl]amino]pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 57 mg, 0.053 mmol) was added to a solution of 2-benzyl 1-tert-butyl (2R,4R)-4-[[(2S)-2,3-bis(tert-butoxycarbonylamino)propanoyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 54, 210 mg, 0.27 mmol) in EtOAc (4 mL). The flask was equipped with a balloon of H2. The suspension was stirred at room temperature for 3 h. The reaction mixture was diluted with MeOH, filtered through celite, concentrated, and dried. The white solid was dissolved in DCM (1 mL) and trifluoroacetic acid (0.50 mL, 6.5 mmol), and the reaction solution was stirred at room temperature for 2 h. The solution was concentrated, and the resulting residue was dissolved in Et2O (2 mL) and 1M HCl aq (2 mL). Phenylboronic acid (100 mg, 0.82 mmol) was added, and the clear biphasic solution was stirred at room temperature for 3 h. The reaction mixture was diluted with water and washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column with 5% ammonia in MeOH (20 mL) to give (2R,4R)-2-(4-boronobutyl)-4-[[(2S)-2,3-diaminopropanoyl]amino]pyrrolidine-2-carboxylic acid (Example 22, 74 mg, 88% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.71 (2H, t), 1.11 - 1.24 (1H, m), 1.26 - 1.41 (3H, m), 1.75 - 1.89 (1H, m), 2.00 - 2.13 (1H, m), 2.31 - 2.48 (1H, m), 2.51 - 2.67 (1H, m), 3.39 - 3.53 (3H, m), 3.68 - 3.80 (1H, m), 4.27 (1H, t), 4.41 - 4.52 (1H, m); m / z: (ES + ) [M + H] + = 317.

[0275] Example 23: (2R,4R)-2-(4-boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid [Chem.] Intermediate 55: 1-tert-butyl 2-methyl (2S,4R)-4-(tert-butoxycarbonylamino)pyrrolidine-1,2-dicarboxylate Di-tert-butyl dicarbonate (4.41 g, 20.2 mmol) was added to a DCM solution (57 mL) of 1-(tert-butyl) 2-methyl (2S,4R)-4-aminopyrrolidine-1,2-dicarboxylate oxalate (4.50 g, 13.5 mmol) and triethylamine (5.63 mL, 40.4 mmol), and the reaction mixture was stirred overnight at room temperature under a N2 atmosphere. The crude reaction mixture was diluted with DCM (200 mL) and washed successively with 0.5 M HCl(aq), saturated sodium bicarbonate, and brine. The organic layer was dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (DCM / MeOH) to give 1-tert-butyl 2-methyl (2S,4R)-4-(tert-butoxycarbonylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 55, 3.66 g, 79% yield) as a white solid. 1 H NMR(500MHz,DMSO-d6)δ 1.29-1.42(18H,m),1.96-2.17(2H,m),3.12-3.17(1H,m),3.45-3.56(1H,m),3.62-3.69(3H,m),3.96-4.06(1H,m),4.24-4.34(1H,m),7.17-7.26(1H,m);m / z:(ES + )[M+H] + =345.

[0276] Intermediate 56: 1-tert-butyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate Sodium hydride (60% dispersion in mineral oil) (0.491 g, 12.3 mmol) was added portionwise to a DMF solution (35 mL) of 1-tert-butyl 2-methyl (2S,4R)-4-(tert-butoxycarbonylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 55, 3.66 g, 10.7 mmol). After the addition, the reaction mixture was stirred for 10 minutes, then methyl iodide (0.715 mL, 11.4 mmol) was added and the reaction mixture was stirred for an additional 3 hours. The reaction mixture was cooled to 0 °C and quenched with water. The mixture was diluted with EtOAc (200 mL) and the layers were separated. The organic layer was washed successively with water and brine, dried over Na2SO4, filtered, concentrated and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give 1-tert-butyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 56, 3.13 g, 82% yield) as a colorless oil. 1 H NMR (500 MHz, DMSO-d6) δ 1.28 - 1.41 (18H, m), 1.87 - 2.02 (1H, m), 2.29 - 2.44 (1H, m), 2.69 (3H, s), 3.13 - 3.25 (1H, m), 3.44 - 3.57 (1H, m), 3.60 - 3.68 (3H, m), 4.23 - 4.32 (1H, m), 4.62 (1H, br s); m / z: (ES + )[M+H] + = 359.

[0277] Intermediate 57: 2-Benzyl 1-tert-butyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate An aqueous solution (11 mL) of sodium hydroxide (2.10 g, 52.4 mmol) was added to a solution of 1-tert-butyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 56, 3.13 g, 8.73 mmol) in THF (22 mL) and MeOH (11 mL) at 0 °C. The reaction mixture was stirred for 3 hours while slowly warming to room temperature. Volatiles were removed under reduced pressure, and the aqueous layer was acidified to a pH of approximately 3 with 5 M HCl(aq) and extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated to dryness, and the crude carboxylic acid was obtained as a white solid. This was used without further purification.

[0278] Benzyl bromide (1.24 mL, 10.5 mmol) was added to a solution of the crude carboxylic acid, sodium iodide (1.737 g, 11.59 mmol), and K2CO3 (3.01 g, 21.8 mmol) in DMF (28 mL), and the reaction mixture was stirred at room temperature for 17 hours. The reaction mixture was filtered, and the solid was rinsed with EtOAc. The filtrate was concentrated and purified by silica gel chromatography (hexane / EtOAc) to give 2-benzyl 1-tert-butyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 57, 3.05 g, 81% yield) as a colorless oil. 1 H NMR (500 MHz, DMSO-d6) δ 1.20 - 1.42 (18H, m), 1.91 - 2.07 (1H, m), 2.33 - 2.46 (1H, m), 2.69 (3H, s), 3.12 - 3.26 (1H, m), 3.44 - 3.57 (1H, m), 4.20 - 4.38 (1H, m), 4.66 (1H, br s), 5.07 - 5.20 (2H, m), 7.25 - 7.41 (5H, m); m / z: (ES + )[M + H] + = 435.

[0279] Intermediate 58: 2-benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate 2-Benzyl 1-tert-butyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 57, 3.05 g, 7.02 mmol) and crotyl bromide (1.08 mL, 10.5 mmol) were dissolved in THF (25 mL), and the solution was cooled to -78 °C under a N2 atmosphere. A solution of KHMDS (0.5 M in toluene, 21.0 mL, 10.5 mmol) was added dropwise to the reaction mixture, and the reaction solution was stirred for 17 hours while slowly warming to room temperature. The crude reaction mixture was quenched with water, and the volatile substances were removed under reduced pressure. The crude mixture was diluted in DCM, and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel purification (hexane / EtOAc) to give 2-benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 58, 1.26 g, 37% yield) as a yellow oil and as a mixture of enantiomers, E / Z olefin isomers, and rotational isomers. 1 H NMR (500 MHz, DMSO-d6) δ 1.23 - 1.44 (18H, m), 1.51 - 1.70 (3H, m), 2.00 - 2.26 (2H, m), 2.38 - 2.47 (1H, m), 2.57 - 2.68 (3H, m), 2.71 - 3.00 (1H, m), 3.00 - 3.25 (1H, m), 3.40 - 3.74 (1H, m), 4.54 - 4.80 (1H, m), 5.01 - 5.27 (2H, m), 5.28 - 5.47 (1H, m), 5.49 - 5.72 (1H, m), 7.25 - 7.42 (5H, m); m / z: (ES + )[M + H] + = 489.

[0280] Intermediate 59: 2-Benzyl 1-tert-butyl (4R)-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate Bis(1,5-cyclooctadiene)dirhodium(I) dichloride (269 mg, 0.400 mmol) and bis(diphenylphosphino)methane (308 mg, 0.801 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (11 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.28 mL, 8.82 mmol) was slowly added to the solution. The reaction mixture was stirred at room temperature for 10 minutes. 2-Benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 58, 1.96 g, 4.01 mmol) was added to the reaction mixture as a DCM solution (7.5 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was cooled to 0 °C and carefully quenched with MeOH and water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated, and dried. The resulting residue was purified by flash silica chromatography (hexane / EtOAc) to give 2-benzyl 1-tert-butyl (4R)-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 59, 2.5 g, 100% yield) as a yellow oil and as a mixture of enantiomers and rotamers. The purified material was subjected to chiral SFC [(S,S)Whelk-O1 column, 30 mm × 250 mm, 5 μm, temperature = 20 °C, mobile phase = 0–30% MeOH:CO2, UV detection @ 220 nm, loading = 31 mg / inj, conc = 125 mg / mL in MeOH, flow rate = 75 mL / min, outlet pressure = 100 bar] to give two enantiomers. The stereochemistry of each enantiomer was assigned retroactively based on the enzyme potencies of Examples 23 and 24 to be consistent with the other exemplified compounds.

[0281] Intermediate 60 (Isomer 2, 637 mg): 2-Benzyl 1-tert-butyl (2R,4R)-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate. 1 H NMR (500 MHz, DMSO-d6) δ 0.58 - 0.76 (2H, m), 1.15 (12H, d), 1.21 - 1.47 (22H, m), 1.69 - 1.82 (1H, m), 1.94 - 2.23 (3H, m), 2.56 - 2.62 (3H, m), 3.19 - 3.28 (1H, m), 3.45 - 3.57 (1H, m), 4.58 - 4.79 (1H, m), 5.01 - 5.26 (2H, m), 7.26 - 7.41 (5H, m).

[0282] Intermediate 61 (Isomer 1, 860 mg): 2-Benzyl 1-tert-butyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate. 1 H NMR (500 MHz, DMSO-d6) δ 0.62 - 0.71 (2H, m), 1.15 (12H, s), 1.20 - 1.42 (22H, m), 1.67 - 1.84 (1H, m), 1.92 - 2.32 (3H, m), 2.69 (3H, s), 3.04 - 3.15 (1H, m), 3.57 - 3.71 (1H, m), 4.52 - 4.73 (1H, m), 4.98 - 5.25 (2H, m), 7.24 - 7.40 (5H, m).

[0283] Intermediate 62: (2R,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 165 mg, 0.155 mmol) was added to a solution of 2-benzyl 1-tert-butyl (2R,4R)-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 60, 637 mg, 1.03 mmol) in EtOAc (7 mL). The flask was equipped with a balloon of H2. The suspension was stirred overnight at room temperature. The reaction mixture was diluted with MeOH and filtered through diatomaceous earth. The filtrate was concentrated to dryness and purified by silica gel chromatography (hexane / EtOAc) to give (2R,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 62, 350 mg, 64% yield) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 0.63 - 0.73 (2H, m), 1.14 - 1.19 (12H, m), 1.22 - 1.42 (21H, m), 1.63 - 1.77 (1H, m), 1.99 - 2.18 (3H, m), 2.63 - 2.67 (3H, m), 3.21 - 3.27 (2H, m), 3.41 - 3.55 (1H, m), 4.57 - 4.80 (1H, m), 12.32 - 12.75 (1H, m); m / z: (ES + )[M + H] + = 527.

[0284] Example 23: (2R,4R)-2-(4-Boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.51 mL, 6.7 mmol) was added dropwise to a stirred solution of (2R,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 62, 350 mg, 0.66 mmol) in DCM (4 mL) at room temperature. After 1 hour, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1M HCl aq (5 mL) and Et2O (5 mL). Phenylboronic acid (162 mg, 1.33 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The mixture was diluted with Et2O and water and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column with 5% ammonia in MeOH (60 mL) to give (2R,4R)-2-(4-boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid (Example 23, 140 mg, 86% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.72 - 0.82 (2H, m), 1.09 - 1.43 (4H, m), 1.62 - 1.77 (1H, m), 1.83 - 1.95 (1H, m), 2.23 (2H, d), 2.46 (3H, s), 3.02 - 3.11 (1H, m), 3.37 - 3.49 (1H, m), 3.49 - 3.61 (1H, m); m / z: (ES + )[M + H] + = 245.

[0285] Example 24: (2S,4R)-2-(4-boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid

Chemical formula

[0286] Example 24: (2S,4R)-2-(4-Boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (1.23 mL, 16.0 mmol) was added dropwise to a stirred DCM solution (8.5 mL) of (2S,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 63, 520 mg, 0.80 mmol) at room temperature. After 1 hour, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1M HCl aq (5 mL) and Et2O (5 mL). Phenylboronic acid (196 mg, 1.60 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The mixture was diluted with Et2O and water and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column with 5% ammonia in MeOH (60 mL) to give (2S,4R)-2-(4-boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid (Example 24, 163 mg, 83% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.70 - 0.90 (2H, m), 1.10 - 1.46 (4H, m), 1.65 - 1.77 (2H, m), 1.96 - 2.07 (1H, m), 2.43 (3H, s), 2.68 - 2.77 (1H, m), 2.96 - 3.10 (1H, m), 3.37 - 3.50 (1H, m), 3.50 - 3.60 (1H, m); m / z: (ES + )[M + H] + = 245.

[0287] Example 25: (2R,4R)-2-(4-Boronobutyl)-4-(dimethylamino)pyrrolidine-2-carboxylic acid

Chemical formula

[0288] Intermediate 65: 2-benzyl 1-tert-butyl (2R,4R)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate Sodium triacetoxyborohydride (6.35 g, 29.9 mmol) was added portionwise to a solution of 2-benzyl 1-tert-butyl (2R,4R)-4-aminopyrrolidine-1,2-dicarboxylate (Intermediate 64, 3.20 g, 9.99 mmol) and formaldehyde (37 wt% in H2O, 4.46 mL, 59.9 mmol) in MeOH (79 mL). After the addition, the reaction mixture was stirred at room temperature for 17 h. Volatiles were removed under reduced pressure and the resulting residue was diluted with DCM. The solid was removed by filtration and the filtrate was concentrated to dryness. The crude material was purified by silica gel chromatography (hexane / EtOAc (with NH4OH)) to give 2-benzyl 1-tert-butyl (2R,4R)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 65, 3.00 g, 86% yield). 1 H NMR (500 MHz, DMSO-d6) δ 1.24 (9H, s), 1.53 - 1.67 (1H, m), 2.10 (6H, s), 2.40 - 2.48 (1H, m), 2.53 - 2.73 (1H, m), 2.83 - 3.06 (1H, m), 3.59 - 3.69 (1H, m), 4.15 - 4.29 (1H, m), 5.02 - 5.21 (2H, m), 7.26 - 7.42 (5H, m); m / z: (ES + ) [M + H] + = 350.

[0289] Intermediate 66: 2-benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate 2-Benzyl 1-tert-butyl (2R,4R)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 65, 3.00 g, 8.61 mmol) and crotyl bromide (1.33 mL, 12.9 mmol) were dissolved in THF (18 mL), and the solution was cooled to -78 °C under a N2 atmosphere. A solution of KHMDS (0.5 M in toluene, 25.8 mL, 12.9 mmol) was added dropwise to the reaction mixture, and the reaction solution was stirred for 17 hours while slowly warming to room temperature. The crude reaction mixture was quenched with water, and the volatiles were removed under reduced pressure. The crude mixture was diluted in DCM, and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel purification (hexane / EtOAc) to give 2-benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 66, 1.55 g, 45% yield) as a yellow oil and as a mixture of enantiomers, E / Z olefin isomers, and rotational isomers. 1 H NMR(500MHz,DMSO-d6)δ 1.21-1.44(9H,m),1.51-1.70(3H,m),1.79-1.96(1H,m),2.05(6H,s),2.08-2.24(1H,m),2.31-2.43(1H,m),2.54-2.74(1H,m),2.75-3.08(2H,m),3.56-3.86(1H,m),5.00-5.23(2H,m),5.23-5.42(1H,m),5.43-5.73(1H,m),7.23-7.41(5H,m);m / z:(ES + )[M+H] + =403.

[0290] Intermediate 67: 2-Benzyl 1-tert-butyl (4R)-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate Bis(1,5-cyclooctadiene)dirhodium(I) dichloride (240 mg, 0.36 mmol) and bis(diphenylphosphino)methane (275 mg, 0.715 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (10 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.14 mL, 7.87 mmol) was slowly added to the solution. The reaction mixture was stirred at room temperature for 10 minutes. 2-Benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 66, 1.44 g, 3.58 mmol) was added to the reaction mixture as a DCM solution (6.7 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was cooled to 0 °C and carefully quenched with MeOH and water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated, and dried. The resulting residue was purified by flash silica chromatography (hexane / EtOAc) to give 2-benzyl 1-tert-butyl (4R)-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 67, 1.5 g, 79% yield) as a yellow oil and as a mixture of enantiomers and rotamers. The purified material was subjected to chiral SFC [(S,S)Whelk-O1 column, 30 mm × 250 mm, 5 μm, temperature = 20 °C, mobile phase = 0–15% MeOH:CO2, UV detection @ 220 nm, loading = 32 mg / inj, conc = 80 mg / mL in MeOH, flow rate = 120 mL / min, outlet pressure = 100 bar] to give two enantiomers. The stereochemistry of each enantiomer was assigned retrospectively based on the enzyme potencies of Examples 25 and 26 to be consistent with the other exemplified compounds.

[0291] Intermediate 68 (Isomer 2, 190 mg): 2-Benzyl 1-tert-butyl (2R,4R)-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate. 1 H NMR (500 MHz, DMSO-d6) δ 0.64 - 0.71 (2H, m), 0.97 - 1.12 (1H, m), 1.13 - 1.17 (12H, m), 1.24 (13H, s), 1.57 - 1.81 (1H, m), 1.81 - 1.99 (1H, m), 2.06 (6H, s), 2.09 - 2.21 (1H, m), 2.55 - 2.74 (1H, m), 3.02 - 3.12 (1H, m), 3.59 - 3.70 (1H, m), 5.00 - 5.20 (2H, m), 7.27 - 7.41 (5H, m).

[0292] Intermediate 69 (Isomer 1, 491 mg): 2-Benzyl 1-tert-butyl (2S,4R)-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate. 1 H NMR (500 MHz, DMSO-d6) δ 0.52 - 0.72 (2H, m), 0.93 - 1.12 (1H, m), 1.12 - 1.20 (12H, m), 1.20 - 1.39 (13H, m), 1.59 - 1.74 (1H, m), 1.77 - 1.93 (1H, m), 2.06 (6H, s), 2.08 - 2.15 (1H, m), 2.53 - 2.65 (1H, m), 2.78 - 2.96 (1H, m), 3.67 - 3.82 (1H, m), 5.00 - 5.21 (2H, m), 7.27 - 7.42 (5H, m).

[0293] Intermediate 70: (2R,4R)-1-tert-butoxycarbonyl-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 57 mg, 0.054 mmol) was added to a solution of 2-benzyl 1-tert-butyl (2R,4R)-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 68, 189 mg, 0.356 mmol) in EtOAc (2.4 mL). The flask was equipped with a balloon of H2. The suspension was stirred overnight at room temperature. The reaction mixture was diluted with MeOH and filtered through celite. The filtrate was concentrated to dryness to give (2R,4R)-1-tert-butoxycarbonyl-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 70, 150 mg, 96% yield) as a white solid. This was used without further purification. 1 H NMR (500 MHz, DMSO-d6) δ 0.53 - 0.70 (2H, m), 1.11 - 1.20 (12H, m), 1.21 - 1.41 (14H, m), 1.56 - 1.71 (1H, m), 1.92 - 2.06 (2H, m), 2.20 (7H, s), 2.69 - 2.78 (1H, m), 3.37 - 3.51 (1H, m), 3.67 - 4.21 (1H, m); m / z: (ES + ) [M + H] + = 441.

[0294] Example 25: (2R,4R)-2-(4-Boronobutyl)-4-(dimethylamino)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.26 mL, 3.4 mmol) was added dropwise to a stirred DCM solution (2 mL) of (2R,4R)-1-tert-butoxycarbonyl-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 70, 150 mg, 0.34 mmol) at room temperature. After 1 hour, the solution was concentrated under reduced pressure, and the resulting residue was dissolved in 1M HCl aq (5 mL) and Et2O (5 mL). Phenylboronic acid (83 mg, 0.68 mmol) was added, and the clear biphasic solution was stirred at room temperature for 1 hour. The mixture was diluted with Et2O and water, and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column with 5% ammonia in MeOH (20 mL) to give (2R,4R)-2-(4-boronobutyl)-4-(dimethylamino)pyrrolidine-2-carboxylic acid (Example 25, 73 mg, 83% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.62 - 0.73 (2H, m), 1.14 - 1.36 (4H, m), 1.79 - 1.90 (1H, m), 1.99 - 2.10 (1H, m), 2.57 - 2.73 (2H, m), 2.82 - 2.90 (6H, m), 3.48 - 3.56 (1H, m), 3.89 - 3.98 (1H, m), 4.12 - 4.20 (1H, m); m / z: (ES + )[M + H] + = 259.

[0295] Example 26: (2S,4R)-2-(4-boronobutyl)-4-(dimethylamino)pyrrolidine-2-carboxylic acid

Chemical formula

[0296] Example 26: (2S,4R)-2-(4-Boronobutyl)-4-(dimethylamino)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (1.0 mL, 13 mmol) was added dropwise to a stirred solution of (2S,4R)-1-tert-butoxycarbonyl-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 71, 405 mg, 0.919 mmol) in DCM (5.1 mL) at room temperature. After 1 hour, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1 M HCl aq (5 mL) and Et2O (5 mL). Phenylboronic acid (224 mg, 1.84 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The mixture was diluted with Et2O and water and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column with 5% ammonia in MeOH (60 mL) to give (2S,4R)-2-(4-boronobutyl)-4-(dimethylamino)pyrrolidine-2-carboxylic acid (Example 26, 206 mg, 87% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.71 - 0.89 (2H, m), 1.12 - 1.44 (4H, m), 1.70 - 1.82 (2H, m), 1.97 - 2.11 (1H, m), 2.29 (6H, s), 2.62 - 2.71 (1H, m), 2.95 - 3.10 (2H, m), 3.61 - 3.68 (1H, m); m / z: (ES + )[M + H] + = 259.

[0297] Example 27: (2R,4R)-4-(2-aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical Structure

[0298] Intermediate 73: 1-tert-butyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate Di-tert-butyl dicarbonate (4.29 mL, 18.5 mmol) was added to a DCM solution (53 mL) of 1-tert-butyl 2-methyl (2S,4R)-4-[2-(tert-butoxycarbonylamino)ethylamino]pyrrolidine-1,2-dicarboxylate (Intermediate 72, 4.77 g, 12.3 mmol) and N,N-diisopropylethylamine (4.30 mL, 24.6 mmol) at room temperature, and the reaction mixture was stirred for 17 h. The reaction mixture was diluted with DCM and washed successively with water and saturated aqueous sodium chloride. The organic layer was dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel chromatography (hexane / EtOAc) to give 1-tert-butyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate (Intermediate 73, 4.25 mg, 71% yield). 1 H NMR (500 MHz, DMSO-d6) δ 1.21 - 1.27 (1H, m), 1.27 - 1.42 (27H, m), 1.99 - 2.07 (1H, m), 2.33 - 2.46 (1H, m), 2.93 - 3.14 (3H, m), 3.17 - 3.24 (1H, m), 3.48 - 3.61 (1H, m), 3.61 - 3.69 (3H, m), 4.20 - 4.44 (2H, m), 6.79 - 6.94 (1H, m); m / z: (ES + ) [M + H] + = 488.

[0299] Intermediate 74: 2-Benzyl 1-tert-butyl (2S,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate An aqueous solution (11 mL) of sodium hydroxide (2.09 g, 52.3 mmol) was added to a solution of 1-tert-butyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate (Intermediate 73, 4.25 g, 8.72 mmol) in THF (22 mL) and MeOH (11 mL) at 0 °C. The reaction mixture was stirred for 6 hours while slowly warming to room temperature. Volatiles were removed under reduced pressure, and the aqueous layer was acidified to about pH 3 with 5M HCl(aq) and extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated to dryness, and the crude carboxylic acid was obtained as a white solid. This was used without further purification.

[0300] Benzyl bromide (1.24 mL, 10.5 mmol) was added to a solution of the crude carboxylic acid, sodium iodide (1.96 g, 13.1 mmol), and K2CO3 (3.62 g, 26.2 mmol) in DMF (28 mL), and the reaction solution was stirred at room temperature for 17 hours. The reaction mixture was filtered, and the solid was rinsed with EtOAc. The filtrate was concentrated and purified by silica gel chromatography (hexane / EtOAc) to give 2-benzyl 1-tert-butyl (2S,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate (Intermediate 74, 3.91 g, 80% yield) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ 1.20 - 1.47 (27H, m), 2.00 - 2.13 (1H, m), 2.36 - 2.45 (1H, m), 2.93 - 3.01 (2H, m), 3.02 - 3.26 (3H, m), 3.48 - 3.64 (1H, m), 4.26 - 4.43 (2H, m), 5.05 - 5.22 (2H, m), 6.78 - 6.93 (1H, m), 7.25 - 7.41 (5H, m); m / z: (ES + ) [M + H] + = 564.

[0301] Intermediate 75: 2-Benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate 2-Benzyl 1-tert-butyl (2S,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate (Intermediate 74, 3.91 g, 6.94 mmol) and crotyl bromide (0.93 mL, 9.0 mmol) were dissolved in THF (10.6 mL), and the solution was cooled to -78 °C under a N2 atmosphere. A solution of KHMDS (0.5 M in toluene, 34.7 mL, 17.3 mmol) was added dropwise to the reaction mixture, and the reaction solution was stirred for 17 hours while slowly warming to room temperature. The crude reaction mixture was quenched with water, and the volatile substances were removed under reduced pressure. The crude mixture was diluted in DCM, and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel purification (hexane / EtOAc) to give 2-benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate (Intermediate 75, 1.34 g, 31% yield) as a yellow oil and as a mixture of enantiomers, E / Z olefin isomers, and rotational isomers. 1 H NMR (500 MHz, DMSO-d6) δ 1.21 - 1.45 (27H, m), 1.53 - 1.71 (3H, m), 1.91 - 2.32 (2H, m), 2.37 - 2.47 (1H, m), 2.69 - 2.86 (1H, m), 2.86 - 3.11 (4H, m), 3.46 - 3.58 (1H, m), 3.58 - 3.77 (1H, m), 4.29 - 4.57 (1H, m), 5.00 - 5.20 (2H, m), 5.28 - 5.46 (1H, m), 5.46 - 5.71 (1H, m), 6.83 - 7.00 (1H, m), 7.19 - 7.43 (5H, m); m / z: (ES + )[M + H] + = 618.

[0302] Intermediate 76: 2-Benzyl 1-tert-butyl (4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate Bis(1,5-cyclooctadiene)dirhodium(I) dichloride (222 mg, 0.331 mmol) and bis(diphenylphosphino)methane (254 mg, 0.661 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (9 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.05 mL, 7.26 mmol) was slowly added to the solution. The reaction mixture was stirred at room temperature for 10 minutes. 2-Benzyl 1-tert-butyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate (Intermediate 75, 2.04 g, 3.30 mmol) was added to the reaction mixture as a DCM solution (6.1 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was cooled to 0 °C and carefully quenched with MeOH and water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organics were dried over Na2SO4, filtered, concentrated, and dried. The resulting residue was purified by flash silica chromatography (hexane / EtOAc) to give 2-benzyl 1-tert-butyl (4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 76, 1.14 g, 46% yield) as a yellow oil and as a mixture of enantiomers and rotamers. The purified material was subjected to chiral SFC [(S,S)Whelk-O1 column, 30 mm × 250 mm, 5 μm, temperature = 20 °C, mobile phase = 0–20% IPA (with 0.2% NH4OH):CO2, UV detection @ 220 nm, loading = 18 mg / inj, conc = 46 mg / mL in MeOH, flow rate = 120 mL / min, outlet pressure = 100 bar] to give two enantiomers. The stereochemistry of each enantiomer was assigned retroactively based on the enzyme potencies of Examples 27 and 28 to be consistent with the other exemplified compounds.

[0303] Intermediate 77 (Isomer 2, 347 mg): 2-Benzyl 1-tert-butyl (2R,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate. 1 H NMR (500 MHz, DMSO-d6) δ 0.60 - 0.76 (2H, m), 1.15 (12H, s), 1.21 - 1.52 (31H, m), 1.66 - 1.82 (1H, m), 1.96 - 2.19 (2H, m), 2.19 - 2.33 (1H, m), 2.83 - 3.10 (4H, m), 3.48 - 3.66 (1H, m), 4.34 - 4.53 (1H, m), 5.00 - 5.23 (2H, m), 6.83 - 6.93 (1H, m), 7.25 - 7.43 (5H, m).

[0304] Intermediate 78 (Isomer 1, 540 mg): 2-Benzyl 1-tert-butyl (2S,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate. 1 H NMR (500 MHz, DMSO-d6) δ 0.61 - 0.76 (2H, m), 1.15 (12H, s), 1.21 - 1.47 (31H, m), 1.61 - 1.80 (1H, m), 1.93 - 2.24 (2H, m), 2.26 - 2.43 (1H, m), 2.89 - 3.13 (5H, m), 3.60 - 3.81 (1H, m), 4.33 - 4.49 (1H, m), 5.00 - 5.20 (2H, m), 6.86 - 6.94 (1H, m), 7.27 - 7.39 (5H, m).

[0305] Intermediate 79: (2R,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 99 mg, 0.093 mmol) was added to a solution of 2-benzyl 1-tert-butyl (2R,4R)-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 77, 347 mg, 0.465 mmol) in EtOAc (2.3 mL). The flask was equipped with a balloon of H2. The suspension was stirred overnight at room temperature. The reaction mixture was diluted with MeOH and filtered through diatomaceous earth. The filtrate was concentrated to dryness to give (2R,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 79, 287 mg, 94% yield) as a white solid. This was used without further purification. 1 H NMR (500 MHz, DMSO-d6) δ 0.65 - 0.74 (2H, m), 1.14 - 1.21 (12H, m), 1.31 - 1.43 (31H, m), 1.66 - 1.76 (1H, m), 2.00 - 2.27 (3H, m), 2.96 - 3.12 (5H, m), 3.37 - 3.42 (1H, m), 3.49 - 3.61 (1H, m), 4.34 - 4.49 (1H, m), 6.84 - 6.93 (1H, m); m / z: (ES + ) [M + H] + = 656.

[0306] Example 27: (2R,4R)-4-(2-Aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.67 mL, 8.8 mmol) was added dropwise to a stirred DCM solution (3.7 mL) of (2R,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 79, 287 mg, 0.438 mmol) at room temperature. After 1 hour, the solution was concentrated under reduced pressure, and the resulting residue was dissolved in 1M HCl aq (5 mL) and Et2O (5 mL). Phenylboronic acid (107 mg, 0.878 mmol) was added, and the clear biphasic solution was stirred at room temperature for 1 hour. The mixture was diluted with Et2O and water, and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 mL) to give (2R,4R)-4-(2-aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 27, 106 mg, 89% yield) as a white solid. 1 H NMR(500MHz,D2O)δ 0.63-0.77(2H,m),1.13-1.23(1H,m),1.37(3H,br d),1.63-1.76(1H,m),1.92-2.02(1H,m),2.06-2.14(1H,m),2.24-2.33(1H,m),2.78(2H,s),2.96(2H,s),3.08-3.19(1H,m),3.28-3.43(1H,m),3.43-3.51(1H,m);m / z:(ES + )[M+H] + =274.

[0307] Example 28: (2S,4R)-4-(2-Aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical formula

[0308] Example 28: (2S,4R)-4-(2-Aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (1.00 mL, 13.0 mmol) was added dropwise to a stirred DCM solution (5 mL) of (2S,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 80, 170 mg, 0.26 mmol) at room temperature. After 1 hour, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1M HCl aq (5 mL) and Et2O (5 mL). Phenylboronic acid (63 mg, 0.52 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The mixture was diluted with Et2O and water and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column with 5% ammonia in MeOH (20 mL) to give (2S,4R)-4-(2-aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 28, 67 mg, 95% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.65 - 0.77 (2H, m), 1.11 - 1.24 (1H, m), 1.25 - 1.34 (1H, m), 1.34 - 1.42 (2H, m), 1.62 - 1.79 (2H, m), 1.97 - 2.06 (1H, m), 2.61 - 2.70 (1H, m), 2.72 - 2.84 (2H, m), 2.93 (3H, s), 3.30 - 3.41 (1H, m), 3.47 - 3.55 (1H, m); m / z: (ES + ) [M + H] + = 274.

[0309] Example 29: (2R,4R)-4-[[(2S)-2-amino-3-methyl-butanoyl]-methyl-amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical Structure

[0310] Intermediate 82: 1-Benzyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate Sodium hydroxide (60% dispersion in mineral oil) (0.423 g, 12.3 mmol) was added portionwise at 0 °C to a solution of 1-benzyl 2-methyl (2S,4R)-4-(tert-butoxycarbonylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 81, 3.20 g, 8.46 mmol) in DMF (33 mL). The reaction mixture was stirred at 0 °C for 1 hour and then warmed to room temperature with stirring for an additional 1 hour. Methyl iodide (0.63 mL, 10.2 mmol) was added and the reaction solution was stirred at room temperature for 16 hours. The reaction mixture was cooled to 0 °C and quenched with water. Volatiles were removed under reduced pressure and the suspension was diluted with DCM (200 mL). The layers were separated and the organic layer was washed successively with water (4 × 50 mL) and saturated aqueous sodium chloride (2 × 50 mL). The organic layer was dried over Na2SO4, filtered, concentrated to dryness, and 1-benzyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 82, 2.33 g, 70% yield) was obtained as a colorless oil. This was used without further purification. 1 H NMR (500 MHz, DMSO-d6) δ 1.25 - 1.47 (9H, m), 1.89 - 2.08 (1H, m), 2.31 - 2.46 (1H, m), 2.69 (3H, d), 3.22 - 3.39 (2H, m), 3.65 (4H, s), 4.22 - 4.49 (1H, m), 4.87 - 5.17 (2H, m), 7.18 - 7.46 (5H, m); m / z: (ES + )[M + H] + = 393.

[0311] Intermediate 83: Dibenzyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate An aqueous solution (7.5 mL) of sodium hydroxide (1.425 g, 35.62 mmol) was added to a solution of 1-benzyl 2-methyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 82, 2.33 g, 5.94 mmol) in THF (15 mL) and MeOH (7.5 mL) at 0 °C. The reaction mixture was stirred for 6 hours while slowly warming to room temperature. Volatiles were removed under reduced pressure, and the aqueous layer was acidified to a pH of approximately 3 with 5M HCl(aq) and extracted with DCM (4 x 50 mL). The combined organics were dried over Na2SO4, filtered, concentrated to dryness, and the crude carboxylic acid was obtained as a white solid. This was used without further purification.

[0312] Benzyl bromide (0.66 mL, 5.6 mmol) was added to a solution of the crude carboxylic acid, sodium iodide (1.11 g, 7.38 mmol), and K2CO3 (1.92 g, 13.9 mmol) in DMF (28 mL), and the reaction was stirred at room temperature for 17 hours. The reaction mixture was filtered, and the solid was rinsed with EtOAc. The filtrate was concentrated and purified by silica gel chromatography (hexane / EtOAc) to give dibenzyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 83, 2.45 g, 94% yield) as a pale yellow oil. 1 H NMR (500 MHz, DMSO-d6) δ 1.28 - 1.45 (9H, m), 1.98 (1H, s), 2.30 - 2.46 (1H, m), 2.64 - 2.71 (3H, m), 3.30 (1H, s), 3.51 - 3.76 (1H, m), 4.48 (1H, d), 4.53 - 4.75 (1H, m), 4.88 - 5.21 (4H, m), 7.18 - 7.42 (10H, m); m / z: (ES + )[M + H] + = 469.

[0313] Intermediate 84: Dibenzyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate Dibenzyl (2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 83, 2.45 g, 5.23 mmol) and crotyl bromide (0.81 mL, 7.8 mmol) were dissolved in THF (18 mL), and the solution was cooled to -78 °C under a N2 atmosphere. A solution of KHMDS (0.5 M in toluene, 15.7 mL, 7.85 mmol) was added dropwise to the reaction mixture, and the reaction solution was stirred for 17 hours while slowly warming to room temperature. The crude reaction mixture was quenched with water, and the volatile substances were removed under reduced pressure. The crude mixture was diluted in DCM, and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated, and dried. The crude material was purified by silica gel purification (hexane / EtOAc) to give dibenzyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 84, 2.10 g, 86% yield) as a yellow oil and as a mixture of enantiomers, E / Z olefin isomers, and rotational isomers. 1 H NMR(500MHz,DMSO-d6)δ 1.31-1.40(9H,m),1.46-1.68(3H,m),1.99-2.31(2H,m),2.40-2.47(1H,m),2.55-2.68(3H,m),2.69-3.00(1H,m),3.30(1H,s),3.51-3.77(1H,m),4.55-4.74(1H,m),4.79-5.21(4H,m),5.26-5.41(1H,m),5.42-5.71(1H,m),7.16-7.42(10H,m);m / z:(ES + )[M+H] + =523.

[0314] Intermediate 85: Dibenzyl (4R)-2-(but-2-enyl)-4-(methylamino)pyrrolidine-1,2-dicarboxylate Trifluoroacetic acid (3.84 mL, 50.2 mmol) was added dropwise to a DCM solution (32 mL) of dibenzyl (4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (Intermediate 84, 2.10 g, 4.02 mmol), and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and dried to obtain the TFA salt of dibenzyl (4R)-2-(but-2-enyl)-4-(methylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 85, 2.55 g, 100% yield) as a colorless oil. This was used without purification. 1 H NMR (500 MHz, DMSO-d6) δ 1.45 - 1.74 (3H, m), 2.14 - 2.33 (1H, m), 2.34 - 2.46 (1H, m), 2.57 - 2.65 (3H, m), 2.66 - 3.07 (1H, m), 3.15 - 3.56 (1H, m), 3.66 - 3.77 (1H, m), 3.81 - 4.13 (1H, m), 4.79 - 5.22 (4H, m), 5.26 - 5.73 (2H, m), 7.20 - 7.41 (10H, m), 8.62 - 8.87 (2H, m); m / z: (ES + )[M+H] + = 423.

[0315] Intermediate 86: Dibenzyl (4R)-2-(but-2-enyl)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]pyrrolidine-1,2-dicarboxylate N,N-Diisopropylethylamine (0.70 mL, 4.0 mmol) was added to a DMF solution (15 mL) of HATU (1.52, 4.02 mmol) and Boc-Val-OH (873 mg, 4.02 mmol), and the reaction mixture was stirred at room temperature for 30 minutes. A solution of dibenzyl (4R)-2-(but-2-enyl)-4-(methylamino)pyrrolidine-1,2-dicarboxylate TFA salt (Intermediate 85, 2.16 g, 4.02 mmol) in DMF (15 mL) and N,N-diisopropylethylamine (0.70 mL, 4.0 mmol) was added, and the reaction mixture was stirred at room temperature for an additional 17 hours. The reaction mixture was concentrated and purified directly by silica gel chromatography (hexane / EtOAc) to obtain dibenzyl (4R)-2-(but-2-enyl)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]pyrrolidine-1,2-dicarboxylate (Intermediate 86, 1.97 g, 79% yield) as a mixture of rotamers. 1 H NMR (500 MHz, DMSO-d6) δ 1 H NMR (500 MHz, DMSO-d6) 0.61 - 0.92 (6H, m), 1.35 (9H, br s), 1.62 (3H, br d), 1.79 - 1.92 (1H, m), 2.03 - 2.33 (2H, m), 2.57 - 2.76 (2H, m), 2.76 - 3.00 (3H, m), 3.30 (1H, s), 3.49 - 3.77 (1H, m), 4.04 - 4.16 (1H, m), 4.78 - 5.20 (5H, m), 5.26 - 5.72 (2H, m), 6.72 - 7.03 (1H, m), 7.17 - 7.38 (10H, m); m / z: (ES + ) [M + H] + = 622.

[0316] Intermediate 87: Dibenzyl (4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate Bis(1,5-cyclooctadiene)dirhodium(I) dichloride (213 mg, 0.317 mmol) and bis(diphenylphosphino)methane (244 mg, 0.635 mmol) were added to an oven-dried round-bottom flask. The flask was sealed and purged with N2. The solid was dissolved in DCM (8.9 mL), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.01 mL, 6.97 mmol) was slowly added to the solution. The reaction mixture was stirred at room temperature for 10 minutes. Dibenzyl (4R)-2-(but-2-enyl)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]pyrrolidine-1,2-dicarboxylate (Intermediate 86, 1.97 g, 3.17 mmol) was added to the reaction mixture as a DCM solution (5.9 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was cooled to 0 °C and carefully quenched with MeOH and water. The layers were separated, and the aqueous layer was extracted with DCM. The combined organic matter was dried over Na2SO4, filtered, concentrated, and dried. The resulting residue was purified by flash silica chromatography (hexane / EtOAc) to give dibenzyl (4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 87, 1.5 g, 63% yield) as a yellow oil and as a mixture of diastereoisomers and rotational isomers. The purified material was subjected to chiral SFC [(S,S)Whelk-O1 column, 30 mm × 250 mm, 5 μm, temperature = 20 °C, mobile phase = 0 - 30% MeOH:CO2, UV detection @ 220 nm, loading = 31 mg / inj, conc = 125 mg / mL in MeOH, flow rate = 75 mL / min, outlet pressure = 100 bar] to obtain two diastereoisomers. The stereochemistry of each diastereoisomer was assigned retrospectively based on the enzyme potencies of Examples 29 and 30 to be consistent with the other exemplified compounds.

[0317] Intermediate 88 (Isomer 2, 280 mg): Dibenzyl (2R,4R)-4-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate. 1 H NMR (500 MHz, DMSO-d6) δ 0.51 - 0.70 (2H, m), 0.72 - 0.93 (6H, m), 1.14 (12H, s), 1.20 - 1.52 (13H, m), 1.67 - 2.31 (5H, m), 2.54 - 2.90 (3H, m), 3.33 - 3.48 (1H, m), 3.49 - 3.85 (1H, m), 4.01 - 4.18 (1H, m), 4.71 - 5.23 (5H, m), 6.71 - 7.07 (1H, m), 7.19 - 7.33 (10H, m).

[0318] Intermediate 89 (Isomer 1, 590 mg): Dibenzyl (2S,4R)-4-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate. 1 H NMR (500 MHz, DMSO-d6) δ 0.49 - 0.72 (2H, m), 0.73 - 0.90 (6H, m), 1.15 (12H, s), 1.34 (13H, br s), 1.59 - 2.34 (5H, m), 2.94 (3H, br s), 3.30 (1H, s), 3.63 - 3.76 (1H, m), 3.96 - 4.17 (1H, m), 4.75 - 5.18 (5H, m), 6.71 - 7.05 (1H, m), 7.17 - 7.41 (10H, m).

[0319] Intermediate 90: (2R,4R)-4-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid Pd / C (10 wt%, 99 mg, 0.093 mmol) was added to a solution of dibenzyl (2R,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 88, 280 mg, 0.37 mmol) in EtOAc (1.8 mL). The flask was equipped with a balloon of H2. The suspension was stirred at room temperature for 4 h. The reaction mixture was diluted with MeOH and filtered through diatomaceous earth. The filtrate was concentrated to dryness to give (2R,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 90, 180 mg, 92% yield) as a white solid. This was used without further purification. 1 H NMR (500 MHz, DMSO-d6) δ 0.59 - 0.69 (2H, m), 0.74 - 0.87 (6H, m), 1.16 (12H, s), 1.22 - 1.30 (3H, m), 1.35 (9H, s), 1.49 - 1.65 (1H, m), 1.65 - 1.82 (1H, m), 1.82 - 1.95 (2H, m), 2.15 - 2.26 (1H, m), 2.63 - 2.91 (3H, m), 2.92 - 3.21 (3H, m), 4.05 - 4.22 (1H, m), 4.80 - 5.16 (1H, m), 6.76 (1H, s), 7.56 - 8.05 (1H, m); m / z: (ES + ) [M + H] + = 526.

[0320] Example 29: (2R,4R)-4-[[(2S)-2-Amino-3-methyl-butanoyl]-methyl-amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (0.53 mL, 6.9 mmol) was added dropwise to a stirred solution of (2R,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 90, 180 mg, 0.34 mmol) in DCM (10 mL) at room temperature. After 1 hour, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1M HCl aq (5 mL) and Et2O (5 mL). Phenylboronic acid (84 mg, 0.69 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The mixture was diluted with Et2O and water and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60 cc column). The desired product was eluted from the column with 5% ammonia in MeOH (60 mL) to give (2R,4R)-4-[[(2S)-2-amino-3-methyl-butanoyl]-methyl-amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 29, 98 mg, 86% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.72 - 0.83 (2H, m), 0.85 - 1.01 (6H, m), 1.10 - 1.45 (4H, m), 1.61 - 1.84 (1H, m), 1.85 - 2.03 (2H, m), 2.19 - 2.34 (1H, m), 2.34 - 2.49 (1H, m), 3.00 (3H, s), 3.16 - 3.51 (2H, m), 3.76 - 3.96 (1H, m), 4.95 - 5.10 (1H, m); m / z: (ES + ) [M + H] + = 344.

[0321] Example 30: (2S,4R)-4-[[(2S)-2-amino-3-methyl-butanoyl]-methyl-amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid

Chemical Structure

[0322] Example 30: (2S,4R)-4-[[(2S)-2-Amino-3-methyl-butanoyl]-methyl-amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid Trifluoroacetic acid (1.17 mL, 15.2 mmol) was added dropwise to a stirred DCM solution (10 mL) of (2S,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]-methyl-amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 91, 397 mg, 0.755 mmol) at room temperature. After 1 hour, the solution was concentrated under reduced pressure and the resulting residue was dissolved in 1M HCl aq (5 mL) and Et2O (5 mL). Phenylboronic acid (186 mg, 1.52 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 hour. The mixture was diluted with Et2O and water and the layers were separated. The aqueous layer was washed with Et2O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 60cc column). The desired product was eluted from the column using 5% ammonia in MeOH (60 mL) to give (2S,4R)-4-[[(2S)-2-amino-3-methyl-butanoyl]-methyl-amino]-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 30, 163 mg, 92% yield) as a white solid. 1 H NMR (500 MHz, D2O) δ 0.68 - 0.78 (2H, m), 0.83 - 0.94 (6H, m), 1.10 - 1.41 (4H, m), 1.66 - 2.13 (4H, m), 2.49 - 2.68 (1H, m), 3.02 (3H, s), 3.22 - 3.30 (1H, m), 3.43 - 3.60 (1H, m), 3.70 - 3.88 (1H, m), 4.53 - 4.66 (1H, m); m / z: (ES + )[M + H] + = 344.

[0323] Example 31: Biological Activity of Examples 1 - 30 Using recombinant arginase 1 or arginase 2 produced from Escherichia coli (E. coli), the inhibitory effects of Examples 1 to 30 on human arginase 1 activity and arginase 2 activity were quantified by measuring the formation of thiol groups from thioarginine. The thiol groups were detected with the Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB reacts with thiols to give mixed disulfides and 2-nitro-5-thiobenzoic acid (TNB), which is quantified by the absorbance of the anion (TNB 2- ) at 412 nm.

[0324] The assay was performed in clear 384-well plates (Greiner cat no: 781101). The plates were sealed immediately after using an Echo acoustic dispenser and centrifuged to dispense various concentrations of Examples 1 to 30 in 300 nL of DMSO, and the plates were assayed. Two premixes were prepared from reagents thawed immediately prior to addition to the assay plates. Premix 1 contained human arginase 1 or human arginase 2 with a final concentration of 5 nM and 0.5 mM DTNB in assay buffer, 45 mM HEPES pH 7.5, brij 35, 0.045% (w / v), and 100 μM MnCl2. Premix 2 contained freshly thawed 0.5 mM thioarginine in assay buffer. 15 microliters of Premix 1 was dispensed, the plates containing Examples 1 to 30 were centrifuged, incubated at room temperature for 30 minutes, then 15 microliters of Premix 2 was added and assayed.

[0325] The assay plate was centrifuged and then the absorbance at 412 nm was read using a Pherastar multimode plate reader to collect data at time point 0 (T0). The plate was incubated at room temperature for 60 minutes and then read again to collect data at time point 1 (T1). The data was derived by subtracting the A412 signal measured at T0 (time point 0) from the A412 signal measured at T1 (time point 1). The data was converted to % effect using the following formula:

[0326] Compound % effect = 100 * [(X - minimum amount) / (maximum amount - minimum amount)], where X represents the value normalized for the compound based on the minimum amount (vehicle) and maximum amount (reference compound) inhibition controls.

[0327] The concentration that inhibits the activity by 50% (i.e., IC 50 ) for Examples 1 to 30 was calculated by plotting the % effect against the test compound concentration and fitting the data using the Genedata Screener Smart fit algorithm. The results of these assays are shown in Table 2:

[0328]

Table 7

[0329] Example 32: Bioavailability Study Examples 8, 9, and 13 - 20 are in the prodrug form of Example 7. The following pharmacokinetic studies were performed to demonstrate the bioavailability of Example 8 relative to Example 7. Example 8 was formulated in 0.9 w / v% saline pH 4 (adjusted with 1 M HCl) for IV dosing. The formulation was dosed at 2 mg / kg each by femoral catheter to two male rats (170 - 250 g). Jugular vein catheter continuous blood samples were taken at 0.033, 0.083, 0.167, 0.5, 1, 2, 4, 8, and 24 hours post - dosing. For PO dosing, Example 8 was formulated in deionized water pH 4 (adjusted with 1 M HCl) and dosed at 5 mg / kg each by oral gavage to two male rats (170 - 250 g). Continuous blood samples were taken at 0.25, 0.5, 1, 1.5, 2, 3, 4, 8, and 24 hours post - dosing by jugular vein catheter. Plasma samples were generated from the blood using low - speed centrifugation. A single set of calibration standards containing Example 7 and Example 8 was prepared by spiking blank plasma. Samples and standards were extracted by precipitation with 2 volumes of acetonitrile followed by centrifugation. Using the results obtained, for both Example 7 and Example 8, Cl (mL / min / kg), Vdss (L / kg), Cmax (μM), AUC (μMh), tmax (h), and %F were determined. The absolute bioavailability was determined by comparing the PO - dose - normalized AUC of Example 7 when dosed as Example 8 to the dose - normalized IV AUC of Example 7 when dosed as Example 7. Bioavailability was calculated using the measured non - nominal doses as necessary. Similarly, the same procedure was repeated for Example 9 and Examples 13 - 20. The results are shown in Tables 3 - 12. These results indicate that bioavailability can be increased by incorporating specific amino acid moieties as prodrugs.

[0330]

Table 8

[0331]

Table 9

[0332]

Table 10

[0333]

Table 11

[0334]

Table 12

[0335]

Table 13

[0336]

Table 14

[0337]

Table 15

[0338]

Table 16

[0339]

Table 17

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Claims

1. Formula (I): 【Chemical 1】 (I) [wherein, R １ is -NHR １ａ ; R １ａ is -H or -C(O)CH(R １ｂ )NH ２ and R １ｂ is -CH ３ or -CH(CH ３ ) ２ is]] A medicament for treating a respiratory inflammatory disease selected from idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, and chemically induced pulmonary fibrosis, comprising a compound represented by or a pharmaceutically acceptable salt thereof.

2. Formula (II): [Chemical 2] (II) [wherein, R ２ is —OH or —NHR ２ａ and; R ２ａ is -H or -C(O)CH(R ２ｂ )NH ２ and; and R ２ｂ is -CH ３ or -CH(CH ３ ) ２ is]] A medicament for treating a respiratory inflammatory disease selected from idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, and chemically induced pulmonary fibrosis, comprising a compound represented by or a pharmaceutically acceptable salt thereof.

3. The medicament according to claim 1 or 2, wherein the compound or its pharmaceutically acceptable salt is a compound represented by formula (III): 【Chemical Formula 3】 (III) [wherein, R ３ is -CH ３ or -CH(CH ３ ), ２ respectively] or a pharmaceutically acceptable salt thereof.

4. Formula (Ib): 【Chemical Formula 4】 (Ib) [wherein, R １ is -NHR １ａ and R １ａ is -H or -C(O)CH(R １ｂ )NH ２ and; and R １ｂ is -CH ３ or -CH(CH ３ ) ２ is]] A medicament for treating a respiratory inflammatory disease selected from idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, and chemically induced pulmonary fibrosis, comprising a compound represented by or a pharmaceutically acceptable salt thereof.

5. Formula (IIb): 【Chemical Formula 5】 (IIb) [wherein, R ２ is —OH or —NHR ２ａ ; R ２ａ is -H or -C(O)CH(R ２ｂ )NH ２ and; and R ２ｂ is -CH ３ or -CH(CH ３ ) ２ is]] A medicament for treating a respiratory inflammatory disease selected from idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, and chemically induced pulmonary fibrosis, comprising a compound represented by or a pharmaceutically acceptable salt thereof.

6. The medicament according to claim 4 or 5, wherein the compound or its pharmaceutically acceptable salt is a compound represented by formula (IIIb): [Chemical Formula 6] (IIIb) [wherein, R ３ is -CH ３ or -CH(CH ３ ) ２ ​ or a pharmaceutically acceptable salt thereof.

7. 【Fig. 7】 A medicament for treating a respiratory inflammatory disease selected from idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, and chemically induced pulmonary fibrosis, comprising a compound selected from or a pharmaceutically acceptable salt thereof.

8. The medicament according to claim 4, wherein the compound or its pharmaceutically acceptable salt is the following: 【Chemical 8】 or a pharmaceutically acceptable salt thereof.

9. The medicament according to any one of claims 1 to 8, wherein the respiratory inflammatory disease is idiopathic pulmonary fibrosis.

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