EA042598B1 - ARGINASE INHIBITORS AND METHODS OF THEIR APPLICATION - Google Patents

Description

State of the art

Arginase is a manganese-containing metalloenzyme that catalyzes the conversion of L-arginine to urea and L-ornithine. There are two isoforms: arginase 1 is a cytosolic enzyme predominantly found in hepatocytes where it plays a critical role in ammonia removal via urea synthesis, and arginase 2, a mitochondrial enzyme highly expressed in the kidney, involved in the formation of ornithine, a precursor for polyamines and prolines required for cell proliferation and collagen formation, respectively.

Although L-arginine is not an essential amino acid because it can be obtained through protein metabolism in healthy adults, increased expression and secretion of arginases results in decreased levels of L-arginine in a variety of physiological and pathological conditions (eg, pregnancy, autoimmune disease, cancer) . Immune cells are particularly sensitive to reduced levels of L-arginine. When T cells are in a microenvironment with low levels of L-arginine, they decrease their proliferation rate and reduce the expression of the CD3Z chain, IFNy, and lytic enzymes, resulting in impaired T cell reactivity. Dendritic cells respond to low L-arginine conditions by decreasing their ability to present antigens, and natural killer cells decrease both proliferation and expression of lytic enzymes.

Tumors use a variety of immunosuppressive mechanisms to evade the immune system. One of these is the reduction of L-arginine by increased levels of circulating arginase, increased expression and secretion of arginase by tumor cells, and recruitment of myeloid-derived suppressor cells expressing and secreting arginase. Together, this results in decreased levels of L-arginine in the tumor microenvironment and an immunosuppressive phenotype.

Pharmacological inhibition of arginase activity has been shown to be able to reverse L-arginine-induced immunosuppression in animal models. Therefore, there is a need for effective and selective arginase inhibitors to reverse immunosuppression and reactivate anti-cancer immunity in patients, either alone or in combination with therapies that further reverse immunosuppressive mechanisms.

Brief description of the invention

In one embodiment of the invention, a compound of formula (II) or a pharmaceutically acceptable salt thereof is disclosed:

N °L

N>- _O n

R2 \ _Z OH '-in ° ⁿ (II), where R ² is -OH or -NHR ^2a ; R ^2a is -H or -C(O)CH(R ^2b )NH2; and R ^2b is -CH3 or -CH(CH ₃ ) ₂ .

In one embodiment of the invention, a compound of formula (IIb) or a pharmaceutically acceptable salt thereof is disclosed:

Ύ NH “b vno (Ib), where R ² is -OH or -NHR ^2a ; R ^2a is -H or -C(O)CH(R ^2b )NH2; and R ^2b is -CH3 or -CH(CH ₃ ) ₂ .

In one embodiment of the invention, a compound of formula (III) or a pharmaceutically acceptable salt thereof is disclosed:

where R ³ represents -CH ₃ or -CH(CH ₃ ) ₂ .

In one embodiment of the invention, a compound of formula (IIIb) or a pharmaceutically acceptable salt thereof is disclosed:

- 1 042598

where R ³ represents -CH ₃ or -CH(CH ₃ ) ₂ .

In some embodiments, the invention discloses compounds that are:

or their pharmaceutically acceptable salts.

In one embodiment of the invention, a compound is disclosed having the following structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, pharmaceutical compositions are disclosed comprising a compound of formulas (II), (IIb), (III), (IIIb), or any of the above specific compounds, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the invention discloses the use of a compound of formula (II), (IIb), (III), (IIIb), or any of the above specific compounds, or a pharmaceutically acceptable salt thereof as a drug.

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Brief description of graphic materials

In FIG. 1 shows NMR spectra showing the conversion of compound B to compound A, where compound B was obtained in 100% d6-DMSO (labeled A), 75% D2O in d6-DMSO (labeled B), 50% D2O in d6-DMSO ( labeled C), 25% D2O in d6-DMSO (labeled E) and 100% D2O (labeled F).

In FIG. Figure 2 shows the NMR spectrum (in 0.1 M DCl in D2O) of compound B, showing how acidification leads to almost complete conversion to compound A.

In FIG. Figure 3 compares the NMR spectra of an amorphous substance in d6-DMSO (labeled B) with crystalline compound B in d6-DMSO (labeled A), showing that both substances have the same cyclic structure as crystalline compound B.

Detailed description of the invention

Connections.

In one embodiment of the invention, a compound of formula (II) or a pharmaceutically acceptable salt thereof is disclosed:

η

^he (P), where R ² represents-OH or-NHR ^2a ;

R ^2a is -H or -C(O)CH(R ^2b )NH ₂ ; And

R ^2b is -CH ₃ or -CH(CH ₃ ) ₂ .

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

In some embodiments of formula (II), R ² is —OH.

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

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.

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.

The specification discloses a compound of formula (IIa) or a pharmaceutically acceptable salt thereof:

η

° ^H (Pa), where R ² represents -OH or -NHR ^2a ; R ^2a is -H or -C(O)CH(R ^2b )NHR ^2c ;

R ^2b is selected from -H, -(C ₁ -C ₄ )alkyl and CH ₂ OR ^2d ; and R ^2c is -H; or R ^2b and R ^2c together with the atoms to which they are attached form a 5-membered heterocyclic ring; And

R ^2d is -H or -CH ₃ .

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

In one embodiment, R ² in formula (IIa) is —OH.

In one embodiment, R ² in formula (IIa) is -NHR ^2a and R ^2a is -H.

In one embodiment, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is -H, and R ^2c is -H.

In one embodiment, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is -CH ₂ OR ^2d , R ^2c is -H, and R ^2d is -H.

In one embodiment, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is -CH2OR ^2d , R ^2c is -H, and R ^2d is -CH3.

In some embodiments, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is -(C1-C4)alkyl, and R ^2c is -N. In some embodiments, -(C1-C ₄ )alkyl is selected from methyl, ethyl, isopropyl, sec-butyl, t-butyl, and isobutyl.

In some embodiments, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is methyl, and R ^2c is -H.

In some embodiments, R ² in formula (IIa) is -NHR ^2a , R ^2a is

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-C(O)CH(R ^2b )NHR ^2c , R ^2b is ethyl, and R ^2c is -H.

In some embodiments, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is isopropyl, and R ^2c is -H.

In some embodiments, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is sec-butyl, and R ^2c is -H.

In some embodiments, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is isobutyl, and R ^2c is -H.

In some embodiments, R ² in formula (IIa) is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NHR ^2c , R ^2b is t-butyl, and R ^2c is -H.

In some embodiments, R ² in formula (IIa) 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 5 -membered ring.

In one embodiment of the invention, a compound of formula (IIb) or a pharmaceutically acceptable salt thereof is disclosed:

BUT (nb), where R ² represents -OH or -NHR ^2a ;

R ^2a is -H or -C(O)CH(R ^2b )NH ₂ ;

R ^2b is -CH ₃ or -CH(CH ₃ ) ₂ .

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

In one embodiment of formula (IIb), R ² is —OH.

In one embodiment of formula (IIb), R ² is -NHR ^2a and R ^2a is -H.

In one embodiment of formula (IIb), R ² is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NH2, and R ^2b is -CH3.

In one embodiment of formula (IIb), R ² is -NHR ^2a , R ^2a is -C(O)CH(R ^2b )NH2, and R ^2b is -CH(CH3)2.

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

R ³ (III), where R ³ represents -CH ₃ or -CH(CH ₃ ) ₂ .

In one embodiment, a compound of formula (III) is disclosed. In another embodiment, a pharmaceutically acceptable soybean compound of formula (III) is disclosed.

In some embodiments of formula (III), R ³ is —CH ₃ .

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

The specification also discloses a compound of formula (IIIa) or a pharmaceutically acceptable salt thereof:

R ³ (Sha), where R ³ is selected from -H, -(C1-C ₄ )alkyl and CH ₂ OR ^3a ; And

R ^3a is -H or -CH ₃ .

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

In one embodiment, R ³ in formula (IIIa) is -H.

In one embodiment, R ³ in formula (IIIa) is -CH ₂ OR ^3a and R ^3a is -H.

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In one embodiment, R ³ in formula (IIIa) is -CH2OR ^3a and R ^3a is -CH ₃ .

In one embodiment, R ³ in formula (IIIa) is -(C1-C ₄ )alkyl. In some embodiments, -(C1-C4)alkyl is selected from methyl, ethyl, isopropyl, sec-butyl, t-butyl, and isobutyl.

In one embodiment, R ³ in formula (IIIa) is methyl.

In one embodiment, R ³ in formula (IIIa) is ethyl.

In one embodiment, R ³ in formula (IIIa) is isopropyl.

In one embodiment, R ³ in formula (IIIa) is sec-butyl.

In one embodiment, R ³ in formula (IIIa) is isobutyl.

In one embodiment, R ³ in formula (IIIa) is t-butyl.

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

ⁿ

Η ₂ Νί3)γ ^Ν γ\ _Η °°l in-±

BUT (SH), where R ³ represents -CH ₃ or -CH(CH ₃ ) ₂ .

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

In one embodiment of formula (IIIb), R ³ is -CH3.

In one embodiment of formula (IIIb), R ³ is -CH(CH ₃ ) ₂ .

The specification also discloses a compound of formula (I) or a pharmaceutically acceptable salt thereof: n °A ν .A-one

Ri \ _Z OH ^B ^he (I), where R1 is -NHR ^1a ;

R ^1a is -H or -C(O)CH(R ^1b )NH2; And

R ^1b is -CH ₃ or -CH(CH ₃ ) ₂ .

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

In some embodiments, R ¹ in formula (I) is -NHR ^1a and R ^1a is -H.

In some embodiments, R ¹ in formula (I) is -NHR ^1a , R ^1a is -C(O)CH(R ^1b )NH2, and R ^1b is -CH ₃ .

In some embodiments, R ¹ in formula (I) is -NHR ^1a , R ^1a is -C(O)CH(R ^1b )NH2, and R ^1b is -CH(CH ₃ ) ₂ .

The description also discloses a compound of formula (Ia) or a pharmaceutically acceptable salt thereof: N L .N L- ° ⁿ

W \ _z oh 'B ^0H (1a), where R ¹ is -NHR ^1a ;

R ^1a is -H or -C(O)CH(R ^1b )NHR ^1c ; And

R ^1b is selected from -H, -(C1-C ₄ )alkyl and CH2OR ^1d ; and R ^1c is -H; or

R ^1b and R ^1c together with the atom to which they are attached form a 5-membered ring; And

R ^1d is -H or -CH ₃ .

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

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In some embodiments, a compound of formula (Ia) is represented by formulas (Ia1) or (Ia2):

where R ¹ is the same as defined above.

In some embodiments, R ^1a in formula (Ia) is -H.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is -H, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is CH ₂ OR ^1d , R ^1c is -H, and R ^1d is -H.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is CH2OR ^1d , R ^1c is -H, and R ^1d is -CH3.

In some embodiments, R ^1a in formula (Ia) 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, t-butyl, and isobutyl.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is methyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is ethyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is isopropyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is sec-butyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is isobutyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ia) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is t-butyl, and R ^lc is -H.

In some embodiments, R ^1a in formula (Ia) 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.

The specification also discloses a compound of formula (Ib) or a pharmaceutically acceptable salt thereof:

^but (Ib), where R ¹ represents -NHR ^1a ;

R ^1a is -H or -C(O)CH(R ^1b )NHR ^1c ; And

R ^1b is selected from -H, -(C1-C ₄ )alkyl and CH ₂ OR ^1d ; and R ^1c is -H; or

R ^1b and R ^1c together with the atom to which they are attached form a 5-membered heterocyclic ring; And

R ^1d is -H or -CH ₃ .

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

In some embodiments, a compound of formula (Ib) is represented by formulas (Ib1) or (Ib2):

where R ¹ is the same as defined above.

In some embodiments, R ^1a in formula (Ib) is -H.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is -H, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is CH ₂ OR ^1d , R ^1c is -H, and R ^1d is -H.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is

- 6 042598 is CH2OR ^1d , R ^1c is -H, and R ^1d is -CH3.

In some embodiments, R ^1a in formula (Ib) 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, t-butyl, and isobutyl.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is methyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is ethyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is isopropyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is sec-butyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is isobutyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ib) is -C(O)CH(R ^1b )NHR ^1c , R ^1b is t-butyl, and R ^1c is -H.

In some embodiments, R ^1a in formula (Ib) 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.

The specification also discloses a compound of formula (IV) or a pharmaceutically acceptable salt thereof:

n °L

N ^he

R4 DOH ^V ~-B ^{z 0H} (IV), where R ⁴ represents -OH or -NH2.

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

In one embodiment, R ⁴ in formula (IV) is —OH. In another embodiment, R ⁴ in formula (IV) is -NH ₂ .

The specification also discloses a compound of formula (IVb) or a pharmaceutically acceptable salt thereof:

T) vHO (IVb), where R ⁴ represents -OH or -NH 2 .

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

In one embodiment, R ⁴ in formula (IVb) is —OH. In another embodiment, R ⁴ in formula (IVb) is -NH2.

The specification also discloses a compound of formula (V) or a pharmaceutically acceptable salt thereof:

n U

Ν'/ OH

ΝΗ. ' \ _Ζ ^{ΟΗ 2} ^B ^0H (V).

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

The specification also discloses a compound of formula (Vb) or a pharmaceutically acceptable salt thereof:

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

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The specification also discloses a compound of formula (VI) or a pharmaceutically acceptable salt thereof:

where R ^6a represents -H or -CH3;

R ^6b is -C(O)C(R ^6c R ^6d )NH2 poi or -OR ^6e ; and or -(C1-C ₃ )alkyl which is substituted with 0 or 1 amino group R ^6c is -(C1-C ₃ )alkyl which is substituted with 0 or 1 amino group or -OR ^6f ;

R ^6d is -H or -CH3; And

R ^6e and R ^6f are independently -H or -CH3.

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

In some embodiments, R ^6a in formula (VI) is -H or -CH3, and R ^6b is -(C1-C ₃ )alkyl which is substituted with 0 or 1 amino group.

In some embodiments, R ^6a in formula (VI) is -H or -CH3, R ^6b is -C(O)C(R ^6c R ^6d )NH2, R ^6c is -(C ₁ -C ₃ )alkyl, which is substituted with 0 or 1 amino or -OH, and R ^6d is H or -CH3.

In some embodiments, a compound of formula (VI) is represented by formulas (VIa1) or (VIa2):

\ /OH ^r6 L if \ he \ _e B \ _e in _R 6a \ _R 6a \

OH (Vial) or OH (VIa2), where R ^6a and R ^6b are the same as defined above.

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

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

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

The specification also discloses a compound of formula (VIb) or a pharmaceutically acceptable salt thereof:

^R6b

HO (vib) where R ^6a is -H or -CH3;

R ^6b is -C(O)C(R ^6c R ^6d )NH ₂ or -(C ₁ -C ₃ )alkyl which is substituted with 0 or 1 amino group or -OR ^6e ; And

R ^6c is -(C1-C ₃ )alkyl which is substituted with 0 or 1 amino group or -OR ^6f ;

R ^6d is -H or -CH ₃ ; And

R ^6e and R ^6f are independently -H or -CH3.

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

In one embodiment, R ^6b in formula (VIb) is -CH ₃ or -CH ₂ CH ₂ NH ₂ .

In some embodiments, R ^6a in formula (VIb) is -H or -CH3, and R ^6b is -(C1-C3)alkyl which is substituted with 0 or 1 amino group.

In some embodiments, R ^6a in formula (VIb) is -H or -CH3, R ^6b is -C(O)C(R ^6c R ^6d )NH=, R ^6c is -(C1-C3)alkyl which is substituted with 0 or 1 amino or -OH, and R ^6d is H or -CH ₃ .

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In some embodiments, a compound of formula (VIb) is represented by formulas (VIb1) or (VIb2):

where R ^6a and R ^6b are the same as defined above.

In some embodiments, compounds of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (V), and (VI), including species thereof, are converted to compounds formulas (Ib), (IIb), (IIIb), (IVb), (Vb) and (VIb), including their species, through intramolecular cyclization and vice versa. Those. it is a process of transformation. Compounds of formulas (I), (Ia), (II), (IIa), (III), (IIIa), (IV), (V) and (VI), including their species, and compounds of formulas (Ib) , (IIb), (IIIb), (IVb), (Vb) and (VIb), including their species, all transform into each other partially or completely depending on conditions such as temperature, pressure, humidity, pH and /or composition of the medium (e.g. solvents), etc. This is illustrated in the diagram below:

where R ¹ is the same as defined in formula (Ia) and (Ib) above. The description also discloses the compounds of table. 1 or a pharmaceutically acceptable salt thereof.

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Table 1

Example Compound Name 2 H? So ^he but''he (27?,45)-2-(4-Boronobutyl)-4hydroxypyrrolidine-2-carboxylic acid 3 n ⁰ M ^he but'<^ _in ^n he (27?,47?)-2-(4-Boronobutyl)-4hydroxypyrrolidine-2-carboxylic acid 4 n 9 n A ί ^he nX S-v' ^he he (25.45)-4-Amino-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid 5 Η? Ν 1 C OO ^he η ₂ ν' ^^ _in - ^he he (27?,45)-4-Amino-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid 6 Η? Ν L ί । ⁿ η ₂ ν C - _β - ^οη he (25',47?)-4-Amino-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid 7 n 9 C ^he nX ^ _B ' ^he he (27?,47?)-4-Amino-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid 8 n 9 A A f 7½ he o \__/ Ί _X-NH ^^v- ^on Ln ₂ ^0H (27?,47?)-4-((5)-2-Aminopropanamido)-2(4-boronobutyl)pyrrolidine-2carboxylic acid 9 n 9 / N _x A f>7 OH O \___/ Ί \ J ^-NH k ^ \ in ^ ° ^N / \ n ₂ ^0H (2Y4/?)-4-((5')-2-А mino-3 methylbutanamido)-2-(4boronobutyl)pyrrolidine-2-carboxylic acid 10 Η 0 NxJ/ o RT ^0H \ On ( )_U n ^^v^on / ΝΗ ₂ but (27?,47?)-4-((7?)-2-Amino-3methylbutanamido)-2-(4boronobutyl)pyrrolidine-2-carboxylic acid

- 10 042598

eleven ^no _{_} (27?,47?)-4-((S)-2-Amhho-3,3dimethylbutanamido)-2-(4boronobutyl)pyrrolidine-2-carboxylic acid 12 yA° ⁿ o \__/\ Vn Λ-l ⁿ λθΗ C™ Q _H (2A47?)-2-(4-Boronobutyl)-4-((X)pyrrolidine-2- carboxamido)pyrrolidine-2carboxylic acid 13 ^- ^0Η ΗΝ Co C™ νη ₂ he (27?,47?)-4-(2-Aminoacetamido)-2-(4boronobutyl)pyrrolidine-2-carboxylic acid 14 η °C he _Ν U-OI ⁰ \—/\ / ^Η 1 he νη ₂ ^Β' he (27?,47?)-4-((X)-2-Aminobutanamido)-2(4-boronobutyl)pyrrolidine-2carboxylic acid 15 Η °on ^N s/ ^ΟΗ ΗΝ ⁷ >0 Ioh η ₂ ν../ in \ ΟΗ (27?,47?)-4-((2X,35)-2-Amino-3- methylpentanamido)-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid 16 Ν V° ^H o )—/ 1 VN >/—; ^Η 1 he —( νη ₂ 9 \ ΟΗ (27?,47?)-4-[[(25)-2-Amino-4methylpentanoyl]amino]-2-(4boronobutyl)pyrrolidine-2-carboxylic acid

- 11 042598

17 . A Ά ^N X ^H <OH HO nh ₂ ? Oh (27?,47?)-4-[[(25)-2-Amino-3hydroxypropanoyl]amino]-2-(4boronobutyl)pyrrolidine-2-carboxylic acid acid 18 H°\\ NV° ^H o ;—' A Γ-C ^H \°H —0 nh ₂ ? Oh (27?,47?)-4-[[(25)-2-Amino-3methoxypropanoyl]amino]-2-(4boronobutyl)pyrrolidine-2-carboxylic acid 19 I ^H H ₂ NWy ^N 'X _NH ° Ο^κ'Χ °v— but (8)-2-Amino-U-((ZK,5K)-8-hydroxy-6oxo-7-oxa-1-aza-8- boraspiro[4.7]dodecan-3 -yl)-3 methylbutanamide 20 O nV ^on O \__/ A .\ X HO^—/ n ] ΝΗ ₂ < _v -on on (27?,47?)-4-[[(25)-2-Amino-3-hydroxy-3methylbutanoyl]amino]-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid 21 o D” \ /~Ν^ 'χ /A ⁿ ] ''' nh ₂ ^ _in - ^he OH (2R, 4R)-4-[[(25)-2-Amino-2,3d-ymethylbutanoyl]amino]-2-(4boronobutyl)pyrrolidine-2-carboxylic acid 22 About nV ^he 0 \___/ '| Vn ^z k, /—( n | ^H 2 ^N nh ₂ X ^0H OH (27?,47?)-2-(4-Boronobutyl)-4-[[(25)-2,3diaminopropanoyl]amino]pyrrolidine2-carboxylic acid

- 12 042598

23 XV ^th -Ν X H 1 x ⁿ he (27^47?)-2-(4-Boronobutyl)-4- (methylamino)pyrrolidine-2-carboxylic acid 24 NV° ^H n ] V ^H he (25.47?)-2-(4-Boronobutyl)-4- (methylamino)pyrrolidine-2-carboxylic acid 25 —Ν ^Ζ X \ Ι k _B ,OH he (27?,47?)-2-(4-Boronobutyl)-4(dimethylamino)pyrrolidine-2carboxylic acid 26 Ν V° ^H — 14 X \ ] V ^H he (25.47?)-2-(4-Boronobutyl)-4- (dimethylamino)pyrrolidine-2carboxylic acid 27 ο nF ^οη η ₂ ν-%~η 1 χ ^Η he (27?,47?)-4-(2-Aminoethylamino)-2-(4boronobutyl)pyrrolidine-2-carboxylic acid 28 Ν V° ^H H ₂ N--/^ ] k _B ,° ^H OH (25.47?)-4-(2-Aminoethylamino)-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid 29 o F \y~ N X nh ₂ L^ ^oh he (27?,47?)-4-[[(25)-2-Amino-3methylbutanoyl]methylamino]-2-(4boronobutyl)pyrrolidine-2-carboxylic acid thirty NV° ^{H 0} 3 O \ /NX / \ \ 1 νη ₂ < _β Α>η OH (25.47?)-4-[[(25)-2-Amino-3methylbutanoyl]methylamino]-2-(4boronobutyl)pyrrolidine-2-carboxylic acid

The term C1- _C4 alkyl includes acyclic alkyl moieties having 1-4 carbon atoms. Examples of C1- _C4 alkyl moieties include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl.

The expression pharmaceutically acceptable salt includes acid addition salts or when

- 13 042598 base compounds that retain the biological effectiveness and properties of the compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 and which are generally not biologically or otherwise undesirable. In many cases, compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), ( V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 are capable of forming acid and/or base salts due to the presence of basic and/or carboxyl groups or similar groups.

Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g. , gluceptate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isothionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methyl sulfate, naphthoate, napsilate, nicotinate, nitrate, octadecanoic, palmate, oleic , phosphate/hydrophosphate/dihydrophosphate, polygalacturonate, propionate, stearate, succinate, subsalicylate, sulfate/hydrosulfate, tartrate, tosylate and trifluoroacetate salts. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid acid, ethanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid, sulfosalicylic acid, and the like.

Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, ammonia and ammonium and metal salts from Groups I-XII of the Periodic Table of the Elements. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Some organic amines include isopropylamine, benzathine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

Pharmaceutically acceptable salts of compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), ( V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 can be synthesized from a basic or acid moiety using traditional chemical methods. Typically, such salts can be obtained by reacting the free acid forms of such compounds with a stoichiometric amount of a suitable base (such as hydroxide, carbonate, bicarbonate of Na ⁺ , Ca ²⁺ , Mg ²⁺ or K ⁺ or the like) or by reacting the forms the free base of such compounds with a stoichiometric amount of the appropriate acid. Such reactions are usually carried out in water or in an organic solvent or in a mixture of both. Generally, it is desirable, where applicable, to use non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. Lists 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 in Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

Any formula given herein also represents the unlabeled forms as well as the isotopically labeled forms of the compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), ( IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1. Isotopically labeled compounds are characterized by the structures depicted using the formulas given in this document, except that one or more atoms are replaced by an atom of the same element, but with a different mass number. Examples of isotopes that can be included in the compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 and ^their pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine such as ² H, ³ H, ⁿ C, 13 C, ¹⁴ C, ¹⁵ N, ³⁵ S, ³⁶ Cl and ¹²⁵ I. Isotopically labeled compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 can generally be prepared using conventional techniques known to those skilled in the art, or by methods similar to those described in the accompanying examples, using appropriate isotopically labeled reagents instead of the unlabeled reagents used previously.

Compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), in

- 14 042598 including any of their subspecies or types, and compounds from the table. 1 may have various isomeric forms. The expression optical isomer, stereoisomer, or diastereoisomer refers to any of the various stereoisomeric configurations that may exist for a given compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, and compounds from the table. 1. It is understood that a substituent may be attached at a chiral center which is a carbon atom, and thus the disclosed compounds include enantiomers, diastereomers, and racemates. The term enantiomer includes pairs of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemic mixture. The term is used to refer to a racemic mixture, if necessary. The terms diastereomers or diastereoisomers include stereoisomers that have at least two asymmetric atoms, but which are not mirror images of each other. Absolute stereochemistry is given according to the Kahn-Ingold-Prelog R-S system. If the compound is the pure enantiomer, the stereochemistry at each chiral center may be indicated as either R or S. Isolated compounds whose absolute configuration is unknown may be indicated as (+) or (-) depending on the direction (right - or left-handed), in which they rotate plane-polarized light at a wavelength D of the sodium line. Certain compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V) , (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 contain one or more asymmetric centers or axes and can thus form enantiomers, diastereomers or other stereoisomeric forms which can be defined in terms of absolute stereochemistry as (R)- or (S)-. All such possible isomers are intended to be included in the present invention, including racemic mixtures, optically pure forms, and mixtures of intermediates. Optically active (R)- and (S)-isomers can be obtained using chiral synthons or chiral reagents, or can be isolated using conventional techniques well known in the art, such as chiral HPLC.

Also disclosed herein are Intermediates 1-48 in the Examples and their salts. pharmaceutical compositions.

In some embodiments, pharmaceutical compositions are disclosed comprising a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV) , (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1 and a pharmaceutically acceptable carrier.

The expression "pharmaceutically acceptable carrier" includes compounds, materials, compositions and/or dosage forms that, based on careful medical evaluation, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problem or complication as determined by one of skill in the art. this field of technology.

The disclosed compositions may be in a form suitable for oral administration (for example, as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or tinctures), for topical application (for example, as creams, ointments, gels or aqueous or oily solutions or suspensions), for administration by inhalation (for example, as a fine powder or liquid aerosol), for administration by insufflation (for example, as a fine powder), or for parenteral administration (for example, in as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing) or as a suppository for rectal dosing.

The amount of active ingredient which is combined with one or more pharmaceutically acceptable carriers to form a unit dosage form will inevitably vary depending on the recipient being treated and the particular route of administration. For additional information on routes of administration and dosing regimens, the reader is referred to Chapter 25.3 of Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

therapeutic value.

The compounds of the present invention are useful as arginase inhibitors in therapies.

In one aspect, methods are disclosed for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), ( IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1, or a pharmaceutically acceptable salt thereof.

In one aspect, methods are disclosed for treating a respiratory inflammatory disease in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III) , (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1, or a pharmaceutically acceptable salt thereof.

In one aspect, a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV),

- 15 042598 (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1 or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.

In one aspect, a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1, or a pharmaceutically acceptable salt thereof, for use in the treatment of a respiratory inflammatory disease.

In one aspect, the use of a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb) is disclosed. , (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1 or a pharmaceutically acceptable salt in the manufacture of a medicament for the treatment of cancer.

In one aspect, the use of a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb) is disclosed. , (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1 or a pharmaceutically acceptable salt in the manufacture of a medicament for the treatment of a respiratory inflammatory disease.

In one aspect, pharmaceutical compositions are disclosed comprising a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1 or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.

In one aspect, pharmaceutical compositions are disclosed comprising a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1 or a pharmaceutically acceptable salt thereof for use in the treatment of a respiratory inflammatory disease.

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, stomach cancer, bladder cancer, melanoma, kidney cancer and ovarian cancer. In some embodiments, the cancer has metastasized. In some embodiments, cancer is associated with modulation of arginase 1 and/or arginase 2.

In some embodiments, cancer is associated with elevated plasma levels of arginase 1. In some embodiments, cancer is associated with reduced plasma levels of arginine. In some embodiments, cancer is associated with both elevated plasma levels of arginase 1 and decreased plasma levels of arginine. In some embodiments, cancers associated with elevated plasma arginase 1 levels and/or decreased plasma arginine levels 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.

In some embodiments, arginase 2 is secreted in cancer, such as acute myeloid leukemia and prostate cancer.

In some embodiments, the cancer is associated with arginase 1-positive tumor-infiltrating immune cells, e.g., lung cancer (small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), gastric cancer, bladder cancer, colorectal cancer, melanoma, squamous cell carcinoma head and neck, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer and kidney cancer.

The term respiratory inflammatory disease refers to inflammatory conditions or disorders that affect the air spaces, the pulmonary vasculature, the lung interstitial tissue, or a combination thereof. They may be isolated in the lung or may involve multiple organs. In one embodiment, the respiratory inflammatory disease is an inflammatory disease of the lung. In another embodiment, the inflammatory lung disease is non-infectious.

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

In one aspect, methods are disclosed for inhibiting arginase in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), ( IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1, or a pharmaceutically acceptable salt thereof.

In one aspect, a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1, or a pharmaceutically acceptable salt thereof for use in arginase inhibition.

- 16 042598

In one aspect, the use of a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb) is disclosed. , (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting arginase.

In one aspect, pharmaceutical compositions are disclosed comprising a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1, or a pharmaceutically acceptable salt thereof for use in arginase inhibition.

The term arginase includes manganese-containing enzymes belonging to the ureohydrolase family, which catalyze the fifth and final step in the urea cycle, converting L-arginine to Lornithine and urea. The term arginase includes two enzyme isozymes, for example, arginase 1, which functions in the urea cycle and is found predominantly in the cytoplasm of liver cells, and arginase 2, which is found in the mitochondria of several tissues in the body and is involved in the regulation of arginine/ornithine concentrations in the cell. In some embodiments, compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 or a pharmaceutically acceptable salt thereof are selective for arginase 1. In some embodiments, the compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) , (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 or a pharmaceutically acceptable salt thereof are selective for arginase 2. In some embodiments, the compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa) , (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of their subspecies or species, and compounds from the table. 1 or a pharmaceutically acceptable salt thereof inhibit both arginase 1 and arginase 2.

The expression effective amount refers to the amount of the compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb) , (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1 that elicits a biological or medical response in the subject, such as reducing or inhibiting the activity of an enzyme or protein associated with arginase or cancer, reducing the intensity of cancer symptoms, or slowing or delaying cancer progression.

In some embodiments, the expression effective amount provides an amount of a compound of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV) , (IVb), (V), (Vb), (VI), (VIb), including any of its subspecies or species, or compounds from the table. 1, which, when administered to a subject, is effective to at least partially alleviate, suppress and/or reduce the intensity of cancer, or inhibit arginase, and/or reduce or inhibit tumor growth or cancer cell proliferation in the subject.

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 is suffering from cancer. In some embodiments, the subject is in need of treatment (eg, the subject will receive a biological or medical benefit from the treatment). In some embodiments, the patient is suffering from cancer. In some embodiments, the subject is characterized by elevated plasma levels of arginase 1. In some embodiments, the subject is characterized by reduced levels of arginine. In some embodiments, the patient is characterized by both elevated levels of arginase 1 and decreased levels of arginine. In some embodiments, the subject has a cancer in which arginase 2 is secreted (eg, acute myeloid leukemia or prostate cancer). In some embodiments, the subject has arginase 1 positive tumor-infiltrating immune cells.

The terms inhibit, inhibition, or inhibitory refer to a reduction from the initial level of activity of a biological activity or process. In some embodiments, compounds of formulas (I), (Ia), (Ib), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (IVb), (V), (Vb), (VI), (VIb), including any of their subspecies or species and from Table. 1 inhibit arginase.

The terms treatment, treatment, and treatment are intended to reduce or inhibit the activity of an arginase-associated enzyme or protein in a subject, reduce the intensity of one or more symptoms of cancer, or slow or delay the progression of cancer in a subject. The terms treatment, treatment, and treatment are also intended to reduce or inhibit tumor growth or cancer cell proliferation in a subject.

- 17 042598

Examples

Aspects of the present invention may be further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain compounds and intermediates of the present invention and methods of using the compounds of the present invention. Those skilled in the art will appreciate that many modifications to both materials and methods can be practiced without departing from the scope of the present invention.

Unless otherwise specified:

(i) all syntheses were carried out at ambient temperature, i.e. in the range of 17-25°C and in an atmosphere of an inert gas such as nitrogen, unless otherwise indicated;

(ii) evaporation was performed by rotary evaporation or using Genevac equipment or a Biotage v10 in vacuo evaporator and product isolation procedures were performed after removal of residual solids by filtration;

(iii) silica gel flash chromatography purification procedures were performed on a Teledyne Isco CombiFlash® Rf or Teledyne Isco CombiFlash® Companion® automated system using pre-filled RediSep Rf Gold™ columns (20-40 µm, spherical particles), GraceResolv™ cartridges (Davisil® silica gel) or Silicycle cartridges (40-63 µm);

(iv) preparative chromatography was performed on a Gilson preparative HPLC device with a UV collection system; alternatively, preparative chromatography was performed on a Waters HPLCMS AutoPurification device with an MS and UV based collection system;

(v) Chiral prep chromatography was performed on a Gilson UV collection system (233 injector/fraction collector, 333 and 334 pumps, 155 UV detector) or a Varian Prep Star (2 x SD1 pumps, 325 UV detector, 701 fraction collector) with a pump working with a Gilson 305 injector; alternatively, chiral preparative chromatography was performed on a Waters Prep 100 SFC-MS with an MS and UV based acquisition system or a Thar MultiGram III SFC with a UV acquisition system;

(vi) output values, if present, are not necessarily the maximum achievable;

(vii) usually the structures of the final products of formula I were confirmed using nuclear magnetic resonance (NMR) spectroscopy; NMR chemical shift values were measured on a delta scale [proton magnetic resonance spectra were determined using a Bruker Avance III 600 (600 MHz), Bruker Avance 400 (400 MHz), Bruker Avance 300 (300 MHz), or Bruker DRX 500 (500 MHz) instrument] ; measurements were carried out at ambient temperature, unless otherwise indicated; the following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet of triplets; bs, wide signal;

(viii) usually the characteristics of the final products of formula I were also determined using mass spectrometry after liquid chromatography (LCMS or UPLC); UPLC was performed using a Waters UPLC equipped with a Waters SQ mass spectrometer (column temperature 40°C, UV = 220-300 nm or 190-400 nm, mass spectrometry = ESI with positive/negative ionization switching) at a flow rate of 1 ml /min, using a solvent system from 97% A + 3% B to 3% A + 97% B for 1.50 min (total assay time with equilibration back to baseline, etc. -1.70 min) where A = 0.1% formic acid or 0.05% trifluoroacetic acid in water (for acid treatment) or 0.1% ammonium hydroxide in water (for basic treatment) and B = acetonitrile. In the case of acid analysis, a Waters Acquity HSS T2 column (1.8 μm, 2.1x50 mm) was used, in the case of basic analysis, a Waters Acquity BEN C18 column (1.7 μm, 2.1x50 mm) was used. Alternatively, UPLC was performed using a Waters UPLC equipped with a Waters SQ mass spectrometer (column temperature 30°C, UV = 210-400 nm, mass spectrometer = ESI with positive/negative ionization switching), at a flow rate of 1 ml/ min, using a solvent gradient from 2 to 98% B over 1.5 min (total analysis time with equilibration back to baseline -2 min), where A = 0.1% formic acid in water and B = 0.1 % formic acid in acetonitrile (for acid treatment) or A = 0.1% ammonium hydroxide in water and B = acetonitrile (for basic treatment). In the case of acid analysis, the column used was Waters Acquity HSS T3 (1.8 µm, 2.1x30 mm), in the case of basic analysis, the column used was Waters Acquity HSS C18 (1.7 µm, 2.1x30 mm); LCMS was performed using a Waters Alliance HT (2795) system equipped with a Waters ZQ ESCi mass spectrometer and Phenomenex Gemini -NX C18 (column 5 µm, 110A, 2.1x50 mm) at a flow rate of 1.1 ml/min, from 95% A to 95% B for 4 min with a 0.5 min hold where A = 0.1% formic acid and B = 0.1% formic acid in acetonitrile (in case of acid treatment) or A = 0.1% ammonium hydroxide in water and B = acetonitrile (for the main treatment). Additionally, LCMS was performed using a Shimadzu UFLC system equipped with a Shimadzu LCMS2020 mass spectrometer and a Waters HSS C18 (1.8 µm, 2.1x50 mm) or Shim-pack XR-ODS (2.2 µm, 3.0x50 mm) column, or Phenomenex Gemini-NX C18 (3 µm, 3.0x50 mm) at a flow rate of 0.7 ml/min, (for a Waters column

- 18 042598

HSS C18), 1.0 ml/min, (for Shim-pack XR-ODS column) or 1.2 ml/min, (for Phenomenex Gemini-NX C18) 95% A to 95% B for 2.2 min with a retention of 0.6 min, where A = 0.1% formic acid or 0.05% trifluoroacetic acid in water (for acid treatment) or 0.1% ammonium hydroxide or 6.5 mM ammonium carbonate in water (for basic processing) and B = acetonitrile. The described molecular ion corresponds to [M+H]+, unless otherwise indicated; for molecules with multiple isotopes (Br, Cl, etc.), the value given is the value obtained for the isotope with the smallest mass, unless otherwise indicated;

(ix) ion exchange purification was usually carried out using an SCX-2 cartridge (Biotage);

(x) the purity of the intermediate was assessed by thin layer chromatography, mass spectrometry, LCMS, UPLC/MS, HPLC (high performance liquid chromatography) and/or NMR analysis;

(xi) used the following abbreviations:

EtOH: ethanol;

EtOAc: ethyl acetate;

LDA: lithium diisopropylamide;

MeOH: methanol;

TFA: trifluoroacetic acid;

MeCN: acetonitrile;

LCMS: liquid chromatography-mass spectrometry;

k. t. or room. temp.: room temperature;

water: water;

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).

Example 1: (R)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

Intermediate 1: (3S,7aR)-3-(trichloromethyl)tetrahydropyrrolo[1,2-c]oxαzol-1(3H)-one.

2,2,2-trichloroethane-1,1-diol (2.155 g, 13.03 mmol) was added to a solution of D-proline (1.00 g, 8.69 mmol) in CHCl ₃ (100 ml) under nitrogen atmosphere. The reaction flask was equipped with a reverse Dean Stark trap and the reaction mixture was heated at reflux, stirred for 48 h. The reaction mixture was cooled to room temperature, diluted with DCM (100 ml) and washed successively with water (2x200 ml) and brine (2x200 ml) . The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated to give the crude product. The crude material was purified by crystallization from EtOH to give the product (Intermediate 1, 1.13 g, 53.2% yield) as a white solid.

¹ H NMR (300 MHz, CDCl3) δ 1.66-2.41 (4H, m), 3.05-3.20 (1H, m), 3.40-3.50 (1H, m), 4 .10-4.20 (1H, m), 5.18 (1H, s).

Intermediate 2: (3S,7aS)-7a-((E)-4-bromobut-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 nitrogen. The resulting solution was stirred at -78°C for 20 minutes. (E)-1,4-dibromobut-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

- 19 042598 min and then warmed to room temperature with stirring for an additional 2 h. The reaction mixture was evaporated to dryness and the resulting precipitate was diluted in EtOAc (20 ml) and washed successively with water (2x20 ml) and brine (2x20 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give the product (Intermediate 2, 760 mg, 49% yield).

¹ H 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); mass/charge (ES ⁺ ) [M+H] ⁺ =378.

Intermediate 3: (3S,7aS)-7a-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)but2-enyl)-3-( trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one.

Pd ₂ (dba) ₃ (85.0 mg, 0.0928 mmol) was added to a solution 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) in THF (30 ml) under 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 precipitate was diluted with EtOAc (50 ml) and washed successively with water and 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).

1H NMR (300 MHz, CDCl ₃ ) 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); mass/charge (ES ⁺ ) [M+H] ⁺ =424.

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% w/w, 125 mg, 0.12 mmol) was added to a solution of (3S,7aS)-7a-((E)-4-(4,4,5,5-tetramethyl1,3 in MeOH (5 ml). The reaction flask was equipped with an H ₂ balloon and 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 to dryness to give the crude product (intermediate 4, 390 mg, 78% yield) which was used without further purification. Mass/charge (ES ⁺ ) [M+H] ⁺ =426.

Example 1: (R)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

Concentrated aqueous HCl (1.00 ml, 12.0 mmol) was added to a solution of (3S,7aR)-7a-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl )-3-(trichloromethyl)tetrahydro-pyrrolo[1,2-c]oxazol-1(3H)one (intermediate 4, 300mg, 0.703mmol) and phenylboronic acid (172mg, 1.41mmol) in 1,4- dioxane (20 ml). The resulting solution was heated to 80° C. for 15 hours. The reaction mixture was cooled to room temperature and concentrated to dryness. The crude material was purified by preparative LCMS (XBridge Prep C18 OBD column, 5 µm silica gel, 19x150 mm, H2O (0.05% w/w TFA/MeCN). Pure fractions were collected and concentrated to dryness to give (R)-2- (4boronobutyl)pyrrolidine-2-carboxylic acid (Example 1, 85 mg, 37% yield) as a white solid.

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); mass/charge (ES+) [M+H] ⁺ =216.

Example 2: (2R,4S)-2-(4-boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid.

Intermediate 5 Intermediate 6

Intermediate 7 Major isomer Minor isomer

Intermediate connection 8 Connection according to example 2

Intermediate 5: (2S,4S)-2-benzyl-1-tert-butyl-4-(benzyloxy)pyrrolidine-1,2-20042598 dicarboxylate.

Dissolved (2S,4S)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (5.00 g, 21.6 mmol) in DMF (73 ml) and the solution was cooled to 0°C. Sodium hydride (60% dispersion in mineral oil) (1.81 g, 45.3 mmol) was added in portions and the suspension was stirred at 0° C. for 1 h. Benzyl bromide (12.9 ml, 108 mmol) was added and the reaction mixture was stirred during the night, while slowly warming up to room. pace. The crude reaction mixture was diluted with ethyl acetate (250 ml) and washed successively with citric acid (10% aq.) and water. The organics were dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude product was purified by silica gel chromatography (hexanes/EtOAc) to give the product as a mixture of rotamers (Intermediate 5, 5.5 g, 62% yield).

¹ H NMR (300 MHz, DMSO-d6) δ 1.28-1.40 (9H, s x2) rotamers, 2.20 (1H, dd), 2.36-2.45 (1H, m), 3 .37 (1H, dd), 3.51-3.58 (1H, m), 4.14 (1H, br s), 4.33-4.50 (3H, m), 4.94-5, 17 (2H, m), 7.25-7.32 (10H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =412.

Intermediate 6: (4S)-2-benzyl-1-tert-butyl-4-(benzyloxy)-2-(but-2enyl)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) was dissolved in THF (45 ml) and the solution was cooled to -78° C. under N 2 atmosphere. The solution was treated by adding dropwise 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 under vacuum. The crude mixture was diluted in DCM and the layers were separated. The organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give the product as a mixture of rotamers and E/Z olefins (Intermediate 6, 2.54 g, 82% yield).

1H 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); mass/charge (ES ⁺ ) [M+H] ⁺ =466.

Intermediate 8: (2R,4S)-1-(tert-butoxycarbonyl)-4-hydroxy-2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl) pyrrolidine-2-carboxylic acid.

Bis(1,5-cyclooctadiene)diiridium(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 stoppered and purged with N2. The solids were 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-2enyl)pyrrolidine-1,2-dicarboxylate (intermediate 6, 2.54 g, 5.46 mmol) was added into the reaction mixture as a solution in DCM (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 Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (4S)-2-benzyl-1-tert-butyl-4-(benzyloxy)-2-(4-(4,4,5,5-tetramethyl- 1,3,2dioxaborolan-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 [column (S,S)Whelk-O1, 21.2x250 mm, 5 µm, temperature=23°C, mobile phase=0-15% MeOH:CO2, UV detection at 220 nm, loading= 33 mg/injection, conc. = 220 ng/ml in MeOH, flow rate = 75 ml/min, outlet pressure = 100 bar] to give two diastereomers. The stereochemistry for the major isomer was assigned based on the anti product addition and for the minor isomer was assigned based on the product syn addition. The minor isomer (368 mg, 0.620 mmol) was dissolved in ethyl acetate (6.2 ml) and treated with Pd/C (10% w/w, 132 mg, 0.124 mmol). The flask was fitted with an H ₂ balloon and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and washed with methanol. The filtrate was concentrated under reduced pressure to give (2R,4S)-1-(tert-butoxycarbonyl)-4-hydroxy-2-(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl )butyl)pyrrolidine-2-carboxylic acid (intermediate 8, 228 mg, 98% yield) as a mixture of rotamers, which was used without further purification.

1H NMR (300 MHz, DMSO-d ₆ ) δ 0.57-0.74 (2H, m), 1.17 (12 H, 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 ); mass/charge (ES ⁺ ) [M+H] ⁺ =414.

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-2carboxylic acid (Intermediate 8, 175 mg, 0.423 mmol) in DCM (4 mL). The resulting solution was stirred at room temperature for 1 h and then concentrated in vacuo

- 21 042598 mom. The crude amino acid was dissolved in Et2O (3 ml) and 1 M 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 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 2M ammonia/methanol. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 to 10% and to 100% acetonitrile in water) to give (2R,4S)-2-(4-boronobutyl)-4hydroxypyrrolidine-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), 3.2-3.37 (2H, m), 4.46-4.56 (1H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =232.

Example 3: (2R,4R)-2-(4-boronobutyl)-4-hydroxyp-pyrrolidine-2-carboxylic acid.

Intermediate 9 Intermediate 10

Intermediate 11 Minor isomer Major isomer

He

Intermediate connection 12 ¹ Connection according to example 3

Intermediate 9: (2S,4R)-2-benzyl-1-tert-butyl-4-(benzyloxy)pyrrolidine-1,2-dicarboxylate.

Dissolved (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (5.00 g, 21.6 mmol) in DMF (73 ml) and the solution was cooled to 0°C. Sodium hydride (60% w/w in mineral oil, 1.81 g, 45.4 mmol) was added portionwise and the suspension was stirred at 0°C for 1 hour. Benzyl bromide (12.86 ml, 108.1 mmol) was added ) and the reaction mixture was stirred over night, while slowly warming up to room. pace. The crude reaction mixture was diluted with ethyl acetate (250 ml) and washed successively with citric acid (10% aq.) and water. The organics were dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give the product (Intermediate 9, 5.9 g, 66% yield) as a mixture of rotamers.

¹ H NMR (300 MHz, DMSO-d6) δ 1.27-1.39 (9H, s x2) rotamers, 1.95-2.08 (1H, m), 2.34-2.47 (1H, m), 3.41-3.53(2H, m), 4.17(1H, br s), 4.28(1H, q), 4.43-4.55(2H, m), 5, 06-5.22 (2H, m), 7.25-7.41 (10H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =412.

Intermediate 10: (4R)-2-benzyl-1-tert-butyl-4-(benzyloxy)-2-(but-2enyl)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) was dissolved in THF (45 ml) and the solution was cooled to -78° C. under N 2 atmosphere. The solution was treated by adding dropwise 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 under vacuum. The crude mixture was diluted in DCM and the layers were separated. The organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/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-d ₆ ) δ 1.25-1.34 (9H, s x2) rotamers, 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); mass/charge (ES ⁺ ) [M+H] ⁺ =466.

Intermediate 12: (2R,4R)-1-(tert-butoxycarbonyl)-4-hydroxy-2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl) pyrrolidine-2-carboxylic acid.

- 22 042598

Bis(1,5-cyclooctadiene)diiridium(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 stoppered and purged with N2. The solids were 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-2enyl)pyrrolidine-1,2-dicarboxylate (Intermediate 10, 1.23 g, 2.64 mmol) was added into the reaction mixture as a solution in DCM (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 Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (4R)-2-benzyl-1-tert-butyl-4-(benzyloxy)-2-(4-(4,4,5,5-tetramethyl- 1,3,2dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 11, 950 mg, 60% yield). The purified material was subjected to chiral SFC [column (S,S)Whelk-O1, 21.2x250 mm, 5 µm, temperature=23°C, mobile phase=0-15% MeOH:CO2, UV detection at 220 nm, loading= 33 mg/injection, conc. = 220 ng/ml in MeOH, flow rate = 75 ml/min, outlet pressure = 100 bar] to give two diastereomers. The stereochemistry for the major isomer was assigned based on the anti-addition of the product and for the minor isomer was assigned based on the syn-addition of the product. The main isomer (385 mg, 0.649 mmol) was dissolved in ethyl acetate (6.4 ml) and treated with Pd/C (10% w/w, 138 mg, 0.130 mmol). The flask was fitted with an H ₂ balloon and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and washed with methanol. The filtrate was concentrated under reduced pressure to give the product (intermediate 12, 249 mg, 93% yield) as a mixture of rotamers.

¹ H NMR (300 MHz, DMSO-d ₆ ) 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), 4.094.23 (1H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =414.

Example 3: (2R,4R)-2-(4-boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid. Trifluoroacetic acid (0.65 ml, 8.5 mmol) was added to a solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-hydroxy2-(4-(4,4,5,5-tetramethyl-1, 3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 12, 197 mg, 0.179 mmol) in DCM (3 mL). The resulting solution was stirred at room temperature for 1 hour and then concentrated in vacuo. The crude amino acid was dissolved in Et2O (3 ml) and 1 M 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 h. The reaction mixture was diluted with water and washed with Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn CX 60 column cc). 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 to 10% acetonitrile in water) to give (2R,4R)-2-(4-boronobutyl)-4-hydroxypyrrolidine-2-carboxylic acid (example 3, 25 mg, 25% yield) as a white solid.

Ή 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); mass/charge (ES ⁺ ) [M+H] ⁺ =232.

Example 4: (2S,4S)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

Intermediate 13 Intermediate 14

Intermediate connection 15 Intermediate Intermediate connection 16 17

Intermediate connection 18 Connection according to example 4

Intermediate 13: (2S,4S)-2-benzyl-1-tert-butyl-4-azidopyrrolidine-1,2-dicarboxylate.

- 23 042598

Methansulfonyl chloride (0.71 ml, 9.2 mmol) was added dropwise to a solution of 2-benzyl-1-(tert-butyl)-(2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (2.45 g, 7. 26 mmol) and triethylamine (1.27 ml, 9.15 mmol) in DCM (9.6 ml) at 0°C. The reaction mixture was stirred at 0° C. for 1 hour before warming to room temperature with stirring for an additional 1 hour. The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give 2-benzyl-1-(tert-butyl)-(2S,4R)-4((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (2, 9 g, 95% yield, which was used without further purification. Mass/charge (ES+) [M+H] ⁺ =400. Sodium azide (1.65 g, 25.4 mmol) was added to a solution of 2-benzyl-1-(tert-butyl)(2S,4R)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (2 .90 g, 7.26 mmol) in DMF (7.2 ml). The reaction mixture was heated to 50° C. and stirred overnight. The reaction mixture was cooled to room temperature and concentrated. The resulting precipitate was diluted with EtOAc and washed with water. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give the product (intermediate 13, 2.00 g, 80% yield) as a mixture of rotamers.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.27 and 1.40 (9H, s x2) rotamers, 1.96-2.02 (1H, m), 2.53-2.63 (1H , m), 3.24-3.29 (1H, m), 3.58-3.66 (1H, m), 4.32-4.41 (2H, m), 5.06-5.22 (2H, m), 7.33-7.39 (5H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =347.

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) was dissolved in THF (20 ml) and the solution was cooled to -78° C. under N 2 atmosphere. The solution was treated by adding dropwise 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 under vacuum. The crude mixture was diluted in DCM and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give the product (Intermediate 14, 750 mg, 65% yield) as a mixture of rotamers and E/Z olefins.

1H NMR (300 MHz, DMSO-d ₆ ) δ 1.25-1.34 (9H, s x2) rotamers, 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); mass/charge (ES ⁺ ) [M+H] ⁺ =401.

Intermediate 16: (2S,4S)-2-benzyl-1-tert-butyl-4-azido-2-(4-(4,4,5,5-tetramethyl1,3,2-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-tetramethyl1, 3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate.

Bis(1,5-cyclooctadiene)diiridium(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 stoppered and purged with N2. The solids were 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-2enyl)pyrrolidine-1,2-dicarboxylate (intermediate 14, 750 mg, 1.87 mmol) was added to the reaction mixture in solution in DCM (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 Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (4S)-2-benzyl-1-tert-butyl-4-azido-2-(4-(4,4,5,5-tetramethyl1,3 ,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 15, 678 mg, 68% yield). The purified material was subjected to chiral SFC [column (S,S)Whelk-O1, 21.2x250 mm, 5 µm, temperature=23°C, mobile phase=0-15% MeOH:CO2, UV detection at 220 nm, loading= 33 mg/injection, conc. = 220 ng/ml in MeOH, flow rate = 75 ml/min, outlet pressure = 100 bar] to give two diastereomers. The stereochemistry for the major isomer was assigned based on the anti-addition of intermediate 16 and the minor isomer based on the syn-addition of intermediate 17.

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.

1H 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); mass/charge (ES ⁺ ) [M+H] ⁺ =529.

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.

- 24 042598

Intermediate 18: (2S,4S)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl1,3,2-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-2yl)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% w/w, 128 mg, 0.120 mmol ). The flask was fitted with an H ₂ balloon and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and washed with methanol. The filtrate was concentrated under reduced pressure to give the product (intermediate 18, 190 mg, 95% yield) as a mixture of rotamers which was used without further purification.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 0.66 (2H, t), 0.88-1.03 (1H, m), 1.16 (12H, s), 1.24-1, 38 (13H, m), 1.401.56 (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); mass/charge (ES+) [M+H] ⁺ =413.

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 in vacuo. The crude amino acid was dissolved in Et ₂ O (3 ml) and 1 M 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 h. The reaction mixture was diluted with water and washed with Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn CX 60 column ss). 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 to 10% acetonitrile in water) to give (2S,4S)-4-amino-2-(4-boronobutyl)πpyrrolidine-2carboxylic acid ( Example 4, 40 mg, 37% yield as a white solid.

¹ H 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, quint); mass/charge (ES ⁺ ) [M+H] ⁺ =231.

Example 5: (2R,4S)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

Intermediate 17.

Intermediate 19.

Connection according to example 5.

Intermediate 19: (2R,4S)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl) pyrrolidine-2-carboxylic acid.

(2R,4S)-2-Benzyl-1-tert-butyl-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine- 1,2-dicarboxylate (intermediate 17, 220 mg, 0.416 mmol) was dissolved in ethyl acetate (5 ml) and methanol (5 ml) and treated with Pd/C (10% w/w, 111 mg, 0.104 mmol ). The flask was fitted with an H ₂ balloon and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and washed with methanol. The filtrate was concentrated under reduced pressure to give the product (intermediate 19, 150 mg, 87% yield) as a mixture of rotamers which was used without further purification.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 0.64-0.71 (2H, m), 1.17 (12H, s), 1.31-1.40 (15H, m), 1, 49-1.93(3H, m), 2.02-2.26(3H, m), 3.38-3.47(1H, m), 3.72-3.81(1H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =413.

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 in vacuo. The crude amino acid was dissolved in Et ₂ O (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 Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn column CX 60 ss). 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 to 10% and 0 to 100% acetonitrile in water) to give (2R,4S)-4-amino-2-(4boronobutyl)pyrrolidine -2-carboxylic acid (example 5, 33 mg, 39% yield) as a white solid

- 25 042598 substances.

1H 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), 1.952.05 (1H, m), 2.58-2.65 (1H, m), 2.87-2.95 (1H, m), 3.48-3.58 ( 3H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =231.

Example 6: (2S,4R)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

Intermediate connection 26 Connection according to example 6

Intermediate 20: (2S,4R)-1-tert-butyl-2-methyl-4-azidopyrrolidine-1,2-dicarboxylate.

Added dropwise methanesulfonyl chloride (2.86 ml, 36.7 mmol) to a solution of (2S,4S)-1-tert-butyl2-methyl-4-hydroxypyrrolidine-1,2-dicarboxylate (7.50 g, 30.6 mmol ) and triethylamine (5.11 ml, 36.7 mmol) in DCM (38 ml) at 0°C. The reaction mixture was stirred at 0° C. for 1 h before warming to room temperature with stirring for an additional 1 h. The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give 1-(tert-butyl)-2-methyl-(2S,4S)-4((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (9, 9 g, 100% yield, which was used without further purification. Mass/charge (ES ⁺ ) [M+NH4] ⁺ =341.

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 precipitate was diluted with EtOAc and washed with water. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give the product (Intermediate 20, 5.95 g, 72% yield) as a mixture of rotamers.

1H NMR (300 MHz, DMSO-d ₆ ) 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); mass/charge (ES ⁺ ) [M+H] ⁺ =271.

Intermediate 21: (2S,4R)-2-benzyl-1-tert-butyl-4-azidopyrrolidine-1,2-dicarboxylate.

A solution of sodium hydroxide (5.28 g, 132 mmol) in water (22 ml) was added dropwise to a solution of (2S,4R)-1-tert-butyl-2-methyl-4-azidopyrrolidine-1,2-dicarboxylate (intermediate compound 20, 5.95 g, 22.0 mmol) in THF (44 ml) and MeOH (22 ml) at 0°C. The reaction mixture was stirred overnight while warming slowly to room temperature. Volatiles were removed under vacuum and the aqueous layer was acidified to pH~3 with 5 M HCl and extracted with DCM. The combined organics were dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give (2S,4R)-4-azido-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (5.64 g, 100% yield) in as a mixture of rotamers, which was used without further purification.

1H NMR (300 MHz, DMSO-d6) 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); mass/charge (ES ^- ) [M+HCOO]-=301.

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 under vacuum and the resulting precipitate was dissolved in EtOAc and washed with water. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give the product (intermediate 21, 5.09 g, 74% yield).

- 26 042598 ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.26 and 1.39 (9H, s x2) 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); mass/charge (ES ⁺ ) [M+H] ⁺ =347.

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) was dissolved in THF (100 ml) and the solution was cooled to 78° C. under N 2 atmosphere. The solution was treated by adding dropwise 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 under vacuum. The crude mixture was diluted in DCM and the layers were separated. The organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give the product (intermediate 22, 4.6 g, 78% yield) as a mixture of rotamers and E/Z olefins.

1H NMR (300 MHz, DMSO-d ₆ ) 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); mass/charge (ES ⁺ ) [M+H] ⁺ =401.

Intermediate 24: (2S,4R)-2-benzyl-1-tert-butyl-4-azido-2-(4-(4,4,5,5-tetramethyl1,3,2-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-tetramethyl1, 3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate.

Bis(1,5-cyclooctadiene)diiridium(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 stoppered and purged with N2. The solids were 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-2enyl)pyrrolidine-1,2-dicarboxylate (intermediate 22, 4.60 g, 11.5 mmol) was added to the reaction the mixture as a solution in DCM (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 Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (4R)-2-benzyl-1-tert-butyl-4-azido-2-(4-(4,4,5,5-tetramethyl-1 ,3,2dioxaborolan-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, 21.2x250 mm, 5 µm, temperature=23°C, mobile phase=0-7% MeOH (0.2% w/w NH ₄ OH):CO ₂ . UV detection at 220 nm, load = 16.8 mg/inlet, conc = 112.5 ng/ml in MeOH, flow rate = 70 ml/min, outlet pressure = 100 bar] to give two diastereomers. for the major diastereomer, intermediate 25, was assigned based on the anti-product addition and for the minor diastereomer, intermediate 24, based on the product syn-addition.

Intermediate 24 (436 mg): (2S,4R)-2-benzyl-1-t-butyl-4-azido-2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborol 2-yl)butyl)pyrrolidine-1,2-dicarboxylate.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 0.58-0.70 (2H, m), 1.17 (12H, s), 1.25-1.40 (13 H, 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); mass/charge (ES ⁺ ) [M+H] ⁺ =529.

Intermediate 25 (1.60 g): (2R,4R)-2-benzyl-1-tert-butyl-4-azido-2-(4-(4,4,5,5tetramethyl-1,3,2- dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate.

¹ H 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); mass/charge (ES ⁺ ) [M+H] ⁺ =529.

Intermediate 26: (2S,4R)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5tetramethyl-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-2yl)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% w/w, 119 mg, 0.112 mmol). The flask was fitted with an H2 balloon and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and washed with methanol. The filtrate was concentrated under reduced pressure to give the product (intermediate 26, 275 mg, 100% yield) which 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); mass/charge (ES ⁺ ) [M+H] ⁺ =413.

- 27 042598

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 in vacuo. The crude amino acid was dissolved in Et ₂ O (2 ml) and 1 M 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 h. The reaction mixture was diluted with water and washed with Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn CX 60 column ss). 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 to 100% acetonitrile in water) to give (2S,4R)-4-amino-2-(4-boronobutyl)pyrrolidine-2carboxylic 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), 1.952.04 (1H, m), 2.58-2.64 (1H, m), 2.87-2.94 (1H, m), 3.48-3.57 (2H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =231.

Example 7: (2R,4R)-4-amino-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

Intermediate25 Intermediate!? Connection according to example 7

Intermediate 27: (2R,4R)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl) pyrrolidine-2-carboxylic acid.

(2R,4R)-2-Benzyl-1-tert-butyl-4-azido-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine- 1,2-dicarboxylate (intermediate 25, 688 mg, 1.30 mmol) was dissolved in ethyl acetate (13 mL) and methanol (4 mL) and treated with Pd/C (10% w/w, 346 mg, 0.325 mmol). The flask was fitted with an H ₂ balloon and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and washed with methanol. The filtrate was concentrated under reduced pressure to give the product (intermediate 27, 500 mg, 93% yield) which was used without further purification.

1 H NMR (300 MHz, DMSO-d ₆ ) δ 0.67 (2H, t), 0.94-1.00 (1H, m), 1.17 (12H, s), 1.22-1.38 (11H, m), 1.431.53 (1H, m), 1.85 (1H, d), 2.00-2.15 (2H, m), 3.23 (2H, dd), 3.58- 3.61 (1H, m), 3.80-3.88 (1H, m), 8.96 (2H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =413.

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 in vacuo. The crude amino acid was dissolved in Et ₂ O (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 h. The reaction mixture was diluted with water and washed with Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn column CX 60 ss). 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 to 10% to 100% acetonitrile in water) to give (2R,4R)-4-amino-2-(4-boronobumil)pyrpolidine 2- carboxylic acid (example 7, 53 mg, 34% yield) as a white solid.

1H 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), 2.102.21 (1H, m), 2.22-2.32 (1H, m), 3.07 (1H, dd), 3.46 (1H, dd), 3.71 (1H, quin.); mass/charge (ES ⁺ ) [M+H] ⁺ =231.

Example 8: (2R,4R)-4-((S)-2-aminopropanamido)-2-(4-boronotbuyl)pyrrolidine-2-carboxylic acid.

Intermediate connection!? Intermediate connection28 Connection according to example 8

Intermediate 28: (2R4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)propanamido)-2-(4-(4,4,5,5-tetramethyl-1,3 ,2-dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid.

- 28 042598

Triethylamine (0.18 ml, 1.3 mmol) and HATU (213 mg, 0.560 mmol) were added sequentially to a solution of Boc-Ala-OH (106 mg, 0.560 mmol) in DMF (2.4 ml) and the reaction mixture was stirred at room temperature for 30 min. (2R,4R)-4-Amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)butyl)pyrrolidin-2- carboxylic acid (intermediate 27, 210 mg, 0.509 mmol) was added to the reaction mixture as a solution in DMF (2.4 mL). The reaction mixture was stirred at room temperature overnight. The crude reaction mixture was concentrated and directly purified by silica gel chromatography (hexanes/EtOAc) to give the product (Intermediate 28, 236 mg, 79% yield) as a mixture of rotamers.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 0.65-0.72 (2H, m), 1.11-1.18 (18H, m), 1.26-1.37 (20H, m ), 1.63-1.73(1H, m), 2.02-2.25(2H, m), 3.09-3.20(1H, m), 3.59-3.72(1H , m), 3.83-3.94 (1H, m), 4.18-4.29 (1H, m), 6.80 (1H, br s), 7.96 (1H, s), 13 .78 (1H, brs); mass/charge (ES ⁺ ) [M+H] ⁺ =584.

Example 8: (2R, 4R)-4-((S)-2-aminopropanamido)-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-((8)-2-(tert-butoxycarbonylamino)propanamido)-2-(4- (4,4,5,5-Tetramethyl-1,3,2dioxaborolan-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 hour and then concentrated in vacuo. The crude amino acid was dissolved in Et ₂ O (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 Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn column CX 60 ss). 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 (2R,4R)-4-((S)-2-aminopropanamido)-2-(4 -boronobutyl)pyrrolidine-2-carboxylic acid (Example 8, 18 mg, 15% yield) as a white solid and a mixture of rotamers.

1H 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); mass/charge (ES ⁺ ) [M+H] ⁺ =302.

Example 9: (2R,4R)-4-((S)-2-amino-3-methylbutanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

Intermediate connection 27 Intermediate connection 29 Connection according to example 9

Intermediate 29: (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-methylbutanamido)-2-(4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid.

Triethylamine (0.21 ml, 1.5 mmol) and HATU (254 mg, 0.668 mmol) were added sequentially to a solution of Boc-Val-OH (145 mg, 0.668 mmol) in DMF (2.9 ml) and the reaction mixture was stirred at room temperature for 30 min. (2R,4R)-4-amino-1-(tert-butoxycarbonyl)-2-(4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)butyl)pyrrolidin-2- carboxylic acid (intermediate 27, 250 mg, 0.606 mmol) was added to the reaction mixture as a solution in DMF (2.9 mL). The reaction mixture was stirred at room temperature overnight. The crude reaction mixture was concentrated and directly purified by silica gel chromatography (hexanes/EtOAc) to give the product (Intermediate 29, 250 mg, 67% yield) as a mixture of rotamers.

1H NMR (300 MHz, DMSO-d ₆ ) δ 0.64-0.73 (2H, m), 0.73-0.85 (6H, m), 1.13-1.14 (1H, m) , 1.17(12H, s), 1.22-1.42(22H, m), 1.56-1.75(1H, m), 1.79-1.97(1H, m), 2 .00-2.26(2H, m), 3.08-3.24(1H, m), 3.54-3.77(2H, m), 4.12-4.36(1H, m) , 6.58 (1H, t), 7.96-8.03 (2H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =584.

Example 9: (2R,4R)-4-((S)-2-amino-3-methylbutamino)-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-((8)-2-(tert-butoxycarbonylamino)-3-methylbutanamido)-2- (4-(4,4,5,5-Tetramethyl1,3,2-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 hour and then concentrated in vacuo. The crude amino acid was dissolved in Et ₂ O (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 Et ₂ O. The aqueous layer was lyophilized and purified with ion exchange

- 29 042598 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 (2R,4R)-4((S)-2-amino-3-methylbutanamido)-2- (4-boronobutyl)-n-pyrrolidine-2-carboxylic acid (Example 9, 28 mg, 20% yield) as a white solid and mixture of rotamers.

¹ H NMR (300 MHz, D ₂ O) δ 0.66-0.76 (2H, m), 0.85 (6H, dd), 1.07-1.43 (4H, m), 1.55 -1.68(1H, m), 1.771.97(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); mass/charge (ES ⁺ ) [M+H] ⁺ =330.

Example 10: (2R,4R)-4-((R)-2-amino-3-methylbutanamido)-2-(4-boronobutyl)n-pyrrolidine-2carboxylic acid.

Intermediate Intermediate connection 30 Intermediate connection 25 connection 31

Intermediate connection 32 Connection according to example 10

Intermediate 30: (2R,4R)-2-benzyl-1-tert-butyl-4-amino-2-(4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl) butyl)pyrrolidine-1,2-dicarboxylate.

Lindlar catalyst (5% w/w, 0.275 g, 2.58 mmol) was added to a solution of (2R,4R)-2-benzyl-1-t-butyl-4-azido-2-(4-(4.4, 5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 25, 1.56 g, 2.95 mmol) in THF (25 mL). The suspension was stirred under hydrogen atmosphere (balloon, flask evacuated and backfilled with hydrogen x3) at room temperature for 8.5 hours. 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 (1-15% MeOH in DCM) to give (2R,4R)-2-benzyl-1-t-butyl-4-amino-2-(4(4,4,5, 5-Tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 30, 1.01 g, 68% yield) as a gum and as a mixture of rotamers.

Ή NMR (500 MHz, CD ₂ Cl ₂ ) δ 0.74 (2H, q), 1.20-1.22 (14H, m), 1.23-1.29 (2H, m), 1.32 (6H, s), 1.37-1.42(5H, m), 1.74-1.83(1H, m), 1.83-1.93(1H, m), 2.11-2 .19 (0.6H, m), 2.21-2.32 (1.4H, m), 3.19 (0.4H, dd), 3.28 (0.6H, dd), 3.44 -3.51(1H, m), 3.63(1H, dd), 5.07-5.20(2H, m), 7.28-7.34(1H, m), 7.34-7 .41 (4H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =503.

Intermediate 31: (2R,4R)-2-benzyl-1-tert-butyl-4-((R)-2-(tert-butoxycarbonylamino)-3-methylbutanamido)-2-(4-(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate.

N,N-diisopropylethylamine (0.235 ml, 1.34 mmol) was slowly added to a stirred solution of HATU (245 mg, 0.64 mmol) and Boc-D-Val-OH (117 mg, 0.54 mmol) in DMF (2 ml) at room temperature. The solution was stirred for 20 min and then a solution 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) in DMF (2 mL). The reaction mixture was stirred for 2.5 h, diluted with DCM (30 ml) and washed successively with water (3x25 ml) and saturated aqueous sodium chloride solution (30 ml). The organic layer was dried over MgSO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (5-65% EtOAc in hexanes) to give (2R,4R)-2-benzyl-1-tert-butyl-4-((R)-2-(Ί-tert-butoxycarbonylamino)-3- methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 31, 239 mg, 63% yield) as a colorless foam and as a mixture of rotamers.

¹ H NMR (500 MHz, CD ₂ Cl ₂ ) 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), 2.212.29(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, apparent d); mass/charge (ES ⁺ ) [M+H] ⁺ =702.

Intermediate 32: (2R,4R)-1-(tert-butoxycarbonyl)-4-((R)-2-(tert-butoxycarbonylamino)-3-methylbutanamido)-2-(4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid.

- 30 042598

Pd/C (10% w/w, 25 mg, 0.23 mmol) was added to a solution of (2R,4R)-2-benzyl-1-tert-butyl-4-((R)-2(tert- butoxycarbonylamino)-3-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate (intermediate 31, 239 mg , 0.34 mmol) in EtOAc (4 ml). The suspension was stirred under hydrogen atmosphere (balloon, flask was evacuated and backfilled with hydrogen x3) at room temperature for 2 h. 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-(t-butoxycarbonyl)-4-((R)-2-(t-butoxycarbonylamino)-3- methylbutanamido)-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-d ₆ ) 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); mass/charge (ES ⁺ ) [M+H] ⁺ =612.

Example 10: (2R,4R)-4-((R)-2-amino-3-methylbutanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid. Trifluoroacetic acid (0.37 ml, 4.8 mmol) was added dropwise to a stirred solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((R)-2-(tert-butoxycarbonylamino)-3methylbutanamido) -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) in DCM (2 ml). The reaction solution was stirred at room temperature for 22 hours and then concentrated under reduced pressure. The crude amino acid was dissolved in 1M aq. HCl (2 ml) and Et ₂ O (2 ml). Phenylboronic acid (117 mg, 0.96 mmol) was added and the clear biphasic solution was stirred at room temperature for 5 hours. The mixture was diluted with Et ₂ O (20 ml) and water (5 ml) and the layers were separated. The aqueous layer was washed with Et ₂ O and the aqueous layer was 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 in MeOH solution (20 ml). The resulting 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-3methylbutanamido)-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, apparent 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); mass/charge (ES ⁺ ) [M+H] ⁺ =330.

Example 11: (2R,4R)-4-((S)-2-amino-3,3-dimethylbutanamido)-2-(4-boronobutyl)πpyrrolidine-2carboxylic acid.

Intermediate 30 Intermediate 33

Intermediate connection 34 Connection according to example 11

Intermediate 33: (2R,4R)-2-benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanamido)-2-(4-(4,4, 5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate.

N,N-diisopropylethylamine (0.165 ml, 0.95 mmol) was slowly added to a stirred solution of HATU (158 mg, 0.42 mmol) and Boc-Tle-OH (92 mg, 0.40 mmol) in DMF (1.5 ml) at room temperature. The solution was stirred for 15 min and then a solution of (2R,4R)-2-benzyl-1-tert-butyl4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 30, 190 mg, 0.38 mmol) in DMF (1.5 mL). The reaction mixture was stirred for 3 h, diluted with EtOAc (30 ml) and washed successively with water (3x25 ml), saturated aqueous NaHCO ₃ (30 ml) and saturated aqueous sodium chloride (30 ml). The organic layer was dried over MgSO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (5-65% EtOAc in hexanes) to give (2R,4R)-2-benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)- 3,3-dimethylbutanamido)-2-(4-31 042598 (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (intermediate 33 , 261 mg, 96% yield as a white solid and as a mixture of rotamers.

1H NMR (500 MHz, DMSO-d ₆ ) δ 0.69 (2H, q), 0.87 (9H, s), 1.17 (12H, s), 1.26 (5H, s), 1, 29-1.32(1H, m), 1.34(5H, s), 1.38(9H, s), 1.72-1.85(1H, m), 1.90-2.08( 2H, m), 2.06-2.18(1H, m), 2.22-2.35(2H, m), 3.113.22(1H, m), 3.68-3.81(2H, m), 4.23-4.37(1H, m), 5.06-5.19(2H, m), 6.40(1H, t), 7.31-7.40(5H, m) , 8.11 (1H, d); mass/charge (ES ⁺ ) [M+Na] ⁺ =738.

Intermediate 34: (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanamido)-2-(4-(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid.

Pd/C (10% w/w, 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-dimethylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate (intermediate 33, 260 mg, 0.36 mmol) in EtOAc (4 ml). The suspension was stirred under hydrogen atmosphere (balloon, flask was evacuated and backfilled with hydrogen x3) at room temperature for 15 h. 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 (2-10% MeOH in DCM) to give (2R,4R)-1-(t-butoxycarbonyl)-4-((S)-2-(t-butoxycarbonylamino)-3, 3-dimethylbutanamido)-2-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (intermediate 34, 207 mg, yield 91 %) as a white solid and as a mixture of rotamers.

1 H NMR (500 MHz, DMSO-d ₆ ) 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, brs), 12.47(0.4H, br s), 12.63 (0.6H, br s); mass/charge (ES ⁺ ) [M+H] ⁺ =626.

Example 11: (2R, 4R)-4-((S)-2-amino-3,3-dimethylbutanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Trifluoroacetic acid (0.38 mL, 4.9 mmol) was added dropwise to a stirred solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3, 3-dimethylbutanamido)-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) in DCM (2 ml). 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 aq. HCl (4 ml) and Et ₂ O (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 Et ₂ O (20 ml) and water (5 ml) and the layers were separated. The aqueous layer was washed with Et ₂ O and the aqueous layer was 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 in MeOH solution (20 ml). The resulting 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-dimethylbutanamido) -2-(4boronobutyl)pyrrolidine-2-carboxylic acid (Example 11, 40 mg, 35% yield) as a white solid.

1H 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, quint); mass/charge (ES ⁺ ) [M+H] ⁺ =344.

Example 12: (2R,4R)-2-(4-boronobutyl)-4-((S)-pyrrolidine-2-carboxamido)pyrrolidine-2carboxylic acid.

Intermediate 30 Intermediate 35

Intermediate connection 36 Connection according to example 12

Intermediate 35: (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-2yl)butyl)pyrrolidine-1,2-dicarboxylate.

- 32 042598

Ν,N-Diisopropylethylamine (0.182 ml, 1.04 mmol) was added slowly to a stirred solution of HATU (175 mg, 0.46 mmol) and Boc-Pro-OH (94 mg, 0.44 mmol) in DMF (1.5 ml) at room temperature. The solution was stirred for 20 min and then a solution 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) in DMF (1.5 mL). The reaction mixture was stirred for 2 h, diluted with EtOAc (30 ml) and washed successively with water (3x25 ml), saturated aqueous NaHCO ₃ and saturated aqueous sodium chloride (30 ml). The organic layer was dried over MgSO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (5-100% EtOAc in hexanes) to give (2R,4R)-2beizium-1-tert-butyl-4-((S)-1-(tert-butoxycarbonyl)pyrrolidine-2 -carboxamido)-2-(4-(4,4,5,5tetramethyl-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 rotamers.

¹ H NMR (500 MHz, CD ₂ Cl ₂ ) δ 0.68-0.79 (2H, m), 1.20 (12H, s), 1.31 (5H, s), 1.36-1, 48 (16H, m), 1.741.87 (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), 3.784.05 (1H, m), 4.49 (1H, br s), 5.10 -5.27 (2H, m), 7.10 (1H, br s), 7.30-7.42 (5H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =700.

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-2yl)butyl)pyrrolidine-2-carboxylic acid.

Pd/C (10% w/w, 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-2yl)butyl)pyrrolidine-1,2-dicarboxylate (intermediate 35, 249 mg, 0.36 mmol) in EtOAc (4 ml). The suspension was stirred under a hydrogen atmosphere (balloon, flask evacuated and backfilled with hydrogen x3) at room temperature for 5 h. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth and the filtrate concentrated under reduced pressure to give (2R,4R)-1 -(tert-butoxycarbonyl)-4-((S)-1 -(tert-butoxycarbonyl)pyrrolidine-2carboxamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane- 2-yl)butyl)pyrrolidine-2-carboxylic acid (intermediate 36, 207 mg, 87% yield) as a colorless film and as a mixture of rotamers which was used without further purification.

1H NMR (500 MHz, CD ₂ Cl ₂ ) 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, brs); mass/charge (ES ⁺ ) [M+H] ⁺ =610.

Example 12: (2R,4R)-2-(4-boronobutyl)-4-((S)-pyrrolidine-2-carboxamido)pyrrolidine-2carboxylic acid.

Trifluoroacetic acid (0.518 ml, 6.73 mmol) was added dropwise to a stirred solution 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) in DCM (2 ml). 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 aq. HCl (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 h. The mixture was diluted with Et ₂ O (20 ml) and water (5 ml) and the layers were separated. The aqueous layer was washed with Et ₂ O and the aqueous layer was 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 in MeOH solution (20 ml). The resulting 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-2carboxamido )pyrrolidine-2-carboxylic acid (example 12, 89 mg, 81% yield) as a white solid.

1H 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), 3.083.16 (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); mass/charge (ES ⁺ ) [M+H] ⁺ =328.

Example 13: (2R,4R)-4-(2-aminoacetamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

- 33 042598

Intermediate 30 Intermediate 37

Intermediate connection 38 Connection according to example 13

Intermediate 37: (2R,4R)-2-benzyl-1-tert-butyl-4-(2-(tert-butoxycarbonylamino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3 ,2-dioxaborolan-2-l)butyl)pyrrolidine 1,2-dicarboxylate.

N,N-Diisopropylethylamine (0.182 mL, 1.04 mmol) was added slowly to a stirred solution of HATU (175 mg, 0.46 mmol) and Boc-Gly-OH (77 mg, 0.44 mmol) in DMF (1.5 ml) at room temperature. The solution was stirred for 20 min and then a solution 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) in DMF (1.5 mL). The reaction solution was stirred for 2 h, diluted with EtOAc (30 ml) and washed successively with water (3x25 ml), saturated aqueous NaHCO ₃ and saturated aqueous sodium chloride (30 ml). The organic layer was dried over MgSO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (10-100% EtOAc in hexanes) to give (2R,4R)2-benzyl-1-tert-butyl-4-(2-(tert-butoxycarbonylamino)acetamido)-2-( 4-(4,4,5,5-Tetramethyl-1,3,2dioxaborolan-2-yl)butyl)pyrrolidin-1,2-dicarboxylate (Intermediate 37, 235 mg, 85% yield) as a colorless film and in as a mixture of rotamers.

¹ H NMR (500 MHz, CD2Cl2) δ 0.70-0.78 (2H, m), 1.12-1.18 (1H, m), 1.20 (12H, s), 1.25-1 .30 (1H, m), 1.33 (5H, s), 1.43 (15H, s), 1.74-1.86 (1H, m), 1.95 (0.5H, br d) , 2.03 (0.5H, br d), 2.15-2.26 (1H, m), 2.26-2.37 (1H, m), 2.40 (1H, dd), 3. 43-3.57 (3H, m), 3.57-3.64 (1H, m), 4.50 (1H, br s), 5.03 (0.5H, br s), 5.10 ( 0.5H, br s), 5.13-5.25(1H, m), 7.01(1H, dd), 7.32-7.37(1H, m), 7.36-7.41 (4H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =660.

Intermediate 38: (2R,4R)-1-(tert-butoxycarbonyl)-4-(2-(tert-butoxycarbonylamino)acetamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2 -dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid.

Pd/C (10% w/w, 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-2yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 37, 233 mg, 0.35 mmol) in EtOAc (4 ml). The suspension was stirred under a hydrogen atmosphere (balloon, flask evacuated and backfilled with hydrogen x3) at room temperature for 6 h. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth and the filtrate was concentrated to dryness to give tert-butoxycarbonyl)-4-(2-(tert-butoxycarbonylamino)acetamido)-2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidin-2- carboxylic acid (intermediate 38, 176 mg, 87% yield) as a colorless film and as a rotamer mixture which was used without further purification.

1H NMR (500 MHz, CD ₂ Cl ₂ ) 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); mass/charge (ES ⁺ ) [M+H] ⁺ =570.

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 solution of (2R,4R)-1-(t-butoxycarbonyl)-4-(2-(t-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) in DCM (2 mL). The reaction solution was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The crude amino acid was dissolved in 1 M aq. HCl (4 ml) and Et ₂ O (4 ml). Phenylboronic acid (113 mg, 0.93 mmol) was added and the clear biphasic solution was stirred at room temperature for 2 h. The mixture was diluted with Et ₂ O (20 ml) and water (5 ml) and the layers were separated. The aqueous layer was washed with Et ₂ O and the aqueous layer was 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 in MeOH solution (20 ml). received ma

- 34 042598 The material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 0 to 40% acetonitrile in water) to give (2R,4R)-4-(2-aminoacetamido)-2-(4boronobutyl)pyrrolidine- 2-carboxylic acid (example 13, 62 mg, 70% yield) as a white solid.

¹ H NMR (500 MHz, D ₂ O) δ 0.76 (2H, td), 1.15-1.25 (1H, m), 1.26-1.34 (1H, m), 1.36 -1.46(2H, m), 1.691.79(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); mass/charge (ES ⁺ ) [M+H] ⁺ =288.

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

Intermediate 30 Intermediate 39

Intermediate connection 40 Connection according to example 14

Intermediate 39: (2R,4R)-2-benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)butanamido)-2-(4-(4,4,5,5-tetramethyl -1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate.

HATU (804 mg, 2.11 mmol) was added to a solution of Boc-Abu-OH (430 mg, 2.11 mmol) in DMF (4 ml) and the reaction mixture was stirred at room temperature for 10 minutes. (2R,4R)-2-Benzyl-1tert-butyl-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidin- 1,2 dicarboxylate (intermediate 30, 885 mg, 1.76 mmol) was added to the reaction mixture as a solution in DMF (3 mL). N,N-diisopropylethylamine (0.75 ml, 4.3 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then diluted with water (15 ml) and Et ₂ O (10 ml). The layers were separated and the aqueous layer was extracted with Et ₂ O (2x10 ml). The combined organics were washed with 5% aqueous lithium chloride (10 ml), dried over MgSO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (2R,4R)-2-benzyl-1-tert-butyl-4-((S)2-(tert-butoxycarbonylamino)butanamido)-2-( 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 39, 766 mg, 63% yield) as a white solid.

¹ H NMR (500 MHz, CDCl ₃ ) δ 0.70-0.84 (5H, m), 1.09-1.19 (1H, m), 1.21 (12H, s), 1.36- 1.46 (18H, m), 1.46-1.97 (7H, m), 2.12-2.45 (2H, m), 3.45-3.60 (1H, m), 3. 68 (1H, br d), 3.78-3.94 (1H, m), 4.42-4.64 (1H, m), 4.73-5.03 (1H, m), 5.07 -5.33 (2H, m), 7.09 (1H, br d), 7.28-7.40 (5H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =688.

Intermediate 40: (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)butanamido)-2-(4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid.

(2R,4R)-2-Benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)butanamido)-2-(4-(4,4,5,5tetramethyl-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. /wt., 119 mg, 0.11 mmol). The flask was fitted with an H ₂ balloon and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and washed with EtOAc and methanol. The filtrate was concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (2R,4R)-1-(tert-butoxycarbonyl)4-((S)-2-(tert-butoxycarbonylamino)butanamido)-2-(4- (4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 40, 470 mg, 70% yield) as a white foam.

¹ H 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); mass/charge (ES ⁺ ) [M+H] ⁺ =598.

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 solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)butanamido)-2-(4-(4 ,4,5,5-tetramethyl-1,3,2

- 35 042598 dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (intermediate 40, 470 mg, 0.79 mmol) in 2 M aq. HCl (5 ml) and the reaction mixture was stirred at room temperature for 16 hours, the Reaction mixture was diluted with water (10 ml) and Et ₂ O (10 ml) and the layers were separated. The aqueous layer was washed with Et ₂ O (3x5 ml) and then lyophilized to obtain a foam. The organic layer was concentrated in vacuo. The resulting precipitate was diluted in 4 M HCl in dioxane (4 ml, 16 mmol) and the resulting solution was stirred at room temperature for 20 h. The reaction mixture was diluted with water (10 ml) and Et ₂ O (10 ml) and the layers were separated. The aqueous layer was washed with Et ₂ O (3x5 ml) and then lyophilized to obtain 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 in MeOH solution. The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18Aq, 0 to 25% acetonitrile in water) to give (2R,4R)-4-((S)-2-aminobutanamudo)-2-(4- boronobutyl)pyrroludin-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), 1.862.10 (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); mass/charge (ES ⁺ ) [M-H2O+H] ⁺ =298.

Example 15: (2R,4R)-4-((2S,3S)-2-amino-3-methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Intermediate 30 Intermediate 41

Intermediate connection 42 Connection according to example 15

Intermediate 41: (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-2yl)butyl)pyrrolidine-1,2-dicarboxylate.

HATU (804 mg, 2.11 mmol) was added to a solution of Boc-Ile-OH (489 mg, 2.11 mmol) in DMF (4 ml) and the reaction mixture 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)pyrrolidin- 1,2-dicarboxylate (intermediate 30, 885 mg, 1.76 mmol) was added to the reaction mixture as a solution in DMF (3 ml). N,N-diisopropylethylamine (0.75 ml, 4.3 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then diluted with water (15 ml) and Et ₂ O (10 ml). The layers were separated and the aqueous layer was extracted with Et ₂ O (2x10 ml). The combined organic layers were washed with 5% aqueous lithium chloride (10 ml), dried over MgSO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (2R,4R)-2-benzyl-1-t-butyl-4-((2S,3S)-2-(t-butoxycarbonylamino)-3-methylpentanamido) -2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate (Intermediate 41, 707 mg, 56% yield) as white solid.

¹ H NMR (500 MHz, CDCl ₃ ) δ 0.79 (2H, brt), 0.83-1.05 (8H, m), 1.12-1.21 (1H, m), 1.24 ( 12H, s), 1.321.61 (20H, m), 1.62-2.02 (4H, m), 2.18-2.55 (2H, m), 3.49-3.65 (1H, m), 3.65-3.78(1H, m), 3.90(1H, br s), 4.53-4.72(1H, m), 4.95(1H, br s), 5 .07-5.43(2H, m), 7.16(1H, br d), 7.30-7.44(5H, m); mass/charge (ES ⁺ )[M+H] ⁺ =716.

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-2yl)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% w/w, 105 mg, 0.10 mmol). The flask was fitted with an H ₂ balloon and the suspension was stirred overnight at room temperature. The reaction mixture was filtered through diatomaceous earth and washed with EtOAc and methanol. The filtrate was concentrated to dryness to give (2R,4R)-1-(tert

- 36 042598 butoxycarbonyl)-4-((2S,3S)-2-(tert-butoxycarbonylamino)-3-methylpentanamido)-2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan- 2-yl)butyl)pyrrolidine-2-carboxylic acid (intermediate 42, 603 mg, 98% yield), which was used without further purification.

1 H NMR (500 MHz, CDCl ₃ ) 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, brd), 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); mass/charge (ES ⁺ ) [M+H] ⁺ =626.

Example 15: (2R,4R)-4-((2S, 3S)-2-amino-3-methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Trifluoroacetic acid (1.10 mL, 14.3 mmol) was added to a solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((2S,3S)-2-(tert-butoxycarbonylamino)-3-methylpentanamido)- 2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 42, 603 mg, 0.96 mmol) in DCM ( 6 ml). The resulting solution was stirred at room temperature for 16 hours and then concentrated in vacuo. The crude amino acid was dissolved in Et ₂ O (10 ml) and re-concentrated under vacuum. These methods of re-dissolution and re-concentration were repeated twice more. The crude amino acid was then dissolved in Et ₂ O (6 ml) and 1 M aq. HCl (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 Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (Silicycle SiliaSep column SPE-R51230B-20X 5 g). The desired product was eluted from the column using 5% ammonia in MeOH solution. 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-methylpentanamido) -2-(4boronobutyl)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), 1.862.09(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); mass/charge (ES ⁺ ) [M+H] ⁺ =344.

Example 16: (2R,4R)-4-((S)-2-amino-4-methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Intermediate 30 Intermediate 43

Intermediate connection 44 Connection according to example 16

Intermediate 43: (2S,4R)-2-benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-2-(4-(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate.

HATU (247 mg, 0.65 mmol) was added to a solution of Boc-Leu-OH (125 mg, 0.54 mmol) in DCM (2 mL) and the reaction mixture was stirred at room temperature for 10 min. (2R,4R)-2-Benzyl-1tert-butyl-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidin- 1,2 dicarboxylate (intermediate 30, 272 mg, 0.54 mmol) was added to the reaction mixture as a solution in DCM (2 mL). N,N-diisopropylethylamine (0.19 ml, 1.1 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then 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 MgSO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (2R,4R)-2-benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido) -2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidin-1,2-dicarboxylate (Intermediate 43, 210 mg, 54% yield) as colorless foam and as a mixture of rotamers.

Ή NMR (500 MHz, CDCl ₃ ) 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

- 37 042598

4.75(2H, m), 5.00-5.46(2H, m), 7.08-7.22(1H, m), 7.28-7.42(5H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =716.

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-2yl)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% w/w, 100 mg, 0.094 mmol). The flask was fitted with an H ₂ balloon and the suspension was stirred at room temperature for 2 hours. The reaction mixture was filtered through diatomaceous earth and washed 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 -tetramethyl1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 44, 170 mg, 97% yield) as a white solid and as a mixture of rotamers.

¹ H NMR (500 MHz, CDCl ₃ ) 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), 3.373.74 (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); mass/charge (ES ⁺ ) [M+H] ⁺ =626.

Example 16: (2R,4R)-4-((S)-2-amino-4-methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Trifluoroacetic acid (1.00 ml, 13.0 mmol) was added to a solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-4-methylpentanamido)-2- (4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2-carboxylic acid (Intermediate 44, 170 mg, 0.27 mmol) in DCM (2 ml ). The resulting solution was stirred at room temperature for 2 hours and then concentrated in vacuo. The crude amino acid was then dissolved in Et ₂ O (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 Et ₂ O (20 ml) and the layers were separated. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 2g column) to give (2R,4R)-4-((S)-2-amino-4methylpentanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (example 16, 88 mg, 94% yield) as a white solid.

¹ H 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); mass/charge (ES ⁺ ) [M+H] ⁺ =344.

Example 17: (2R,4R)-4-((S)-2-amino-3-hydroxypropanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Intermediate 30 Intermediate 45

Intermediate connection 46 Connection according to example 17

Intermediate 45: (2R,4R)-2-benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamido)-2-(4-(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate.

N,N-Diisopropylethylamine (0.108 ml, 0.62 mmol) was added to a stirred solution of COMU (292 mg, 0.68 mmol), (2R,4R)-2-benzyl-1-tert-butyl-4-amino-2 -(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)butyl)pyrrolidin-1,2-dicarboxylate (Intermediate 30, 311 mg, 0.62 mmol) and Boc- Ser-OH (133 mg, 0.65 mmol) in DMF (5 ml) at room temperature. The reaction mixture was stirred for 3 h, diluted with water (80 ml) and EtOAc (15 ml). The phases were separated and the aqueous phase was further diluted with saturated aqueous NaHCO ₃ , then extracted with EtOAc (2x20 ml). The combined organics were washed with saturated aqueous NaCl (2x10 ml), dried over MgSO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give (2R,4R)-2-benzyl-1t-butyl-4-((S)-2-(t-butoxycarbonylamino)-3-hydroxypropanamido)-2 -(4-(4,4,5,5-tetramethyl- 38 042598

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); mass/charge (ES ⁺ ) [M+H] ⁺ =690.

Intermediate 46: (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamido)-2-(4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid.

(2R,4R)-2-Benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)-3-hydroxypropanamido)-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% w/w, 50 mg, 0.047 mmol). The flask was fitted with an H ₂ balloon and the suspension was stirred at room temperature for 3.5 hours. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, concentrated to dryness to give (2R,4R)-1-(tert-butoxycarbonyl)- 4-((S)2-(tert-Butoxycarbonylamino)-3-hydroxypropanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)butyl)pyrrolidin-2 -carboxylic acid (intermediate 46, 303 mg, 97% yield) as a white solid and as a mixture of rotamers which was used without further purification.

¹ H NMR (500 MHz, CD ₂ Cl ₂ ) 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); mass/charge (ES ⁺ ) [M+H] ⁺ =600.

Example 17: (2R,4R)-4-((S)-2-amino-3-hydroxypropanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Trifluoroacetic acid (0.771 ml, 10.01 mmol) was added dropwise to a stirred solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3hydroxypropanamido)-2 -(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2carboxylic acid (Intermediate 46, 300 mg, 0.50 mmol) in DCM (4 ml) at room temperature. After 1.5 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (4 ml, 4.00 mmol) and Et ₂ O (4 ml). Phenylboronic acid (183 mg, 1.50 mmol) was added and the clear biphasic solution was stirred at room temperature for 3 hours. The mixture was diluted with Et ₂ O (20 ml) and water (5 ml) and the layers were separated. The aqueous layer was washed with Et ₂ O, the layers were separated and the aqueous layer was lyophilized. The resulting solid was dissolved in MeOH (3 ml) and subjected to ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column using 5% ammonia in MeOH solution (20 ml). The resulting material was further purified by reverse phase chromatography (RediSep Rf

Gold® C18, 0 to 30% acetonitrile in water) to give (2R,4R)-4-((S)-2-amino-3hydroxypropanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (example 17.94 mg, 59% yield as a white solid.

¹ H NMR (500 MHz, D ₂ O) 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); mass/charge (ES ⁺ ) [M+H] ⁺ =318.

Example 18: (2R,4R)-4-((S)-2-amino-3-methoxypropanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Intermediate 30 Intermediate 47

Intermediate connection 48 Connection according to example 18

Intermediate 47: (2R,4R)-2-benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)-3-methoxypropanamido)-2-(4-(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-1,2-dicarboxylate.

- 39 042598

Ν,N-Diisopropylethylamine (0.082 ml, 0.47 mmol) was added to a stirred solution of COMU (220 mg, 0.51 mmol), (2R,4R)-2-benzyl-1-tert-butyl-4-amino-2 -(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)butyl)pyrrolidin-1,2-dicarboxylate (Intermediate 30, 235 mg, 0.47 mmol) and N- Boc-O-methyl-L-serine (108 mg, 0.49 mmol) in DMF (3 ml) at room temperature. The reaction mixture was stirred for 2 h, then diluted with water (60 ml) and DCM (15 ml). The phases were separated and the aqueous phase was extracted with DCM (2x20 ml). The combined organics were washed with saturated aqueous NaHCO ₃ (30 ml), saturated aqueous NaCl (2x10 ml), dried over MgSO ₄ , filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (hexanes/EtOAc) to give (2R,4R)-2-benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)-3-methoxypropanamido) -2-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidin-1,2-dicarboxylate (Intermediate 47, 145 mg, 44% yield) in as a colorless dry film and as a mixture of rotamers.

¹ H NMR (500 MHz, CDCl ₃ ) δ 0.78 (2H, t), 1.18 (1H, br dd), 1.22-1.26 (13H, m), 1.33 (6H, s ), 1.44 (5H, br s), 1.46 (9H, s), 1.57-1.74 (1H, m), 1.74-1.85 (1H, m), 1.94 (0.4H, d), 2.01(0.6H, d), 2.18-2.27(0.6H, m), 2.322.47(1.4H, m), 3.32(3H , s), 3.39-3.46 (1H, m), 3.49-3.55 (0.4H, m), 3.60 (1H, dd), 3.65-3.76 (1 .6H, m), 4.15(1H, br d), 4.51-4.64(1H, m), 5.06-5.22(2H, m), 5.23-5.34( 1H, m), 7.32-7.39 (5H, m); mass/charge (ES ⁺ ) [M+H] ⁺ =704.

Intermediate 48: (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3-methoxypropanamido)-2-(4-(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-2carboxylic acid.

(2R,4R)-2-Benzyl-1-tert-butyl-4-((S)-2-(tert-butoxycarbonylamino)-3-methoxypropanamido)-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% w/w, 22 mg, 0.021 mmol). The flask was fitted with an H ₂ balloon and the suspension was stirred at room temperature for 4 hours. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth, concentrated to dryness to give (2R,4R)-1-(tert-butoxycarbonyl)-4- ((S)-2(tert-butoxycarbonylamino)-3-methoxypropanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl)pyrrolidine-2-carboxylic acid (48 intermediate, 126 mg, 100% yield) as a white solid and as a mixture of rotamers which was used without further purification.

1 H NMR (500 MHz, CD ₂ Cl ₂ ) 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), 3.383.47 (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, brs); mass/charge (ES ⁺ ) [M+H] ⁺ =614.

Example 18: (2R,4R)-4-((S)-2-amino-3-methoxypropanamido)-2-(4-boronobutyl)pyrrolidine-2carboxylic acid.

Trifluoroacetic acid (0.25 ml, 3.26 mmol) was added dropwise to a stirred solution of (2R,4R)-1-(tert-butoxycarbonyl)-4-((S)-2-(tert-butoxycarbonylamino)-3- methoxypropanamido)-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 ) in DCM (2 ml) at ambient temperature. After 1 h, the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (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 Et ₂ O (20 ml) and water (5 ml) and the layers were separated. The aqueous layer was washed with Et ₂ O, the layers were separated and the aqueous layer was lyophilized. The resulting solid was dissolved in MeOH (3 ml) and subjected to ion exchange chromatography (PoraPak Rxn CX 20 cc column). The desired product was eluted from the column using 5% ammonia in MeOH solution (20 ml). The resulting material was further purified by reverse phase chromatography (RediSep Rf Gold® C18, 0 to 20% acetonitrile in water). Fractions containing product were lyophilized and the resulting material was again subjected to reverse phase flash chromatography (RediSep Rf Gold® C18, 0 to 2% acetonitrile in water). Fractions containing product were lyophilized and the resulting material was again repurified by reverse phase flash chromatography (RediSep Rf Gold® C18, 0 to 5% acetonitrile in water) to give (2R,4R)-4-((S) -2-Amino-3-methoxypropanamido)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 18, 18 mg, 35% yield) as a white solid.

1H 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), 1.721.81 (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); mass/charge (ES ⁺ ) [M+H] ⁺ =332.

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.

- 40 042598

Compound B (Example 19) was obtained by intramolecular cyclization of the compound of Example 9 (Compound A) via an interconversion process. In addition, interconversions between Compound A (Example 9) and Compound B were observed under various conditions. For example, compound B was converted to compound A in the presence of water, and such conversion is proportional to the concentration of water in the solvent. This is shown in FIG. 1 showing NMR spectra of compound B obtained in 100% d6-DMSO (labeled A), 75% D2O in d6-DMSO (labeled B), 50% D2O in d6-DMSO (labeled C), 25% D2O in d6-DMSO (labeled E) and 100% D2O (labeled F). In d6-DMSO, Compound B predominates, with a proportional increase in Compound A with increasing D2O concentration. The proportion of compound A relative to compound B in 100% D ₂ O reaches about 90%. In addition, compound B was converted to compound A under acidic conditions. In FIG. 2 NMR spectra of compound B in 0.1 M DCl (in D2O) demonstrate that acidification provides almost complete conversion of compound A. In addition, it was determined that the crystalline compound B and the amorphous compound B have the same structural form. In FIG. 3 NMR spectra (obtained in d6-DMSO) show that both crystalline compound B and amorphous compound B have the same ring structure.

Example 20: (2R,4R)-4-[[(2S)-2-amino-3-hydroxy-3-methylbutanoyl]amino]-2-(4boronobutyl)pyrrolidine-2-carboxylic acid.

Intermediate 52: 2-benzyl-1-(tert-butyl)-(2R,4R)-4-((S)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanamido)-2-( 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)butyl)pyrrolidine-1,2-dicarboxylate.

Added N,N-diisopropylethylamine (0.13 ml, 0.76 mmol) to a solution of (S)-N-alpha-tert-butyloxycarbonyl-3,3-dimethylserine (106 mg, 0.454 mmol) and HATU (0.173 g, 0.454 mmol) in MF (2.6 ml) at 0°C and the reaction mixture was stirred for 15 min. A solution of (2R,4R)-2-benzyl1-tert-butyl-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl) was added pyrrolidine-1,2-dicarboxylate (intermediate 30, 190 mg, 0.38 mmol) in DMF (1 ml) and the reaction mixture was stirred for 2 h while slowly warming to room temperature. The reaction mixture was diluted with EtOAc (40 ml) and washed with saturated aqueous NH4Cl (2x20 ml), saturated aqueous NaHCO ₃ (2x20 ml) and brine (20 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (0-50% EtOAc in hexanes) to give 2-benzyl 1-(tert-butyl)-(2R,4R)-4-((S)-2-((tert-butoxycarbonyl) amino)-3-hydroxy-3-methylbutanamido)-2-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl)pyrrolidine-1,2-dicarboxylate (intermediate compound 52, 223 mg, 82% yield as a white foam and as a mixture of rotamers.

1H NMR (500 MHz, MeOH-d ₄ ) δ 0.70-0.83 (m, 2H) 1.14-1.27 (m, 19 H) 1.31 (s, 6 H) 1.36- 1.42 (m, 5H) 1.44 (s, 10H) 1.81-1.93 (m, 1H) 2.02-2.14 (m, 1H) 2.14-2.30 ( m, 1H) 2.36-2.49 (m, 1H) 3.39-3.48 (m, 1H) 3.73-3.83 (m, 1H) 3.87-3.97 (m, 1H) 4.43-4.53 (m, 1H) 5.09-5.25 (m, 2 H) 7.28-7.45 (m, 5 H); mass/charge (ES ⁺ ) [M+H] ⁺ =718.

Example 20: (2R,4R)-4-[[(2S)-2-amino-3-hydroxy-3-methylbutanoyl]amino]-2-(4boronobutyl)pyrrolidine-2-carboxylic acid.

Pd/C (10% w/w, 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-methylbutanamido)-2-(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)butyl)pyrrolidin-1,2 -dicarboxylate (intermediate 52, 220 mg, 0.31 mmol) in EtOAc (3 mL). The flask was equipped with an H ₂ balloon and the suspension was stirred at room temperature for 3 hours. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth and concentrated to dryness. The white solid was dissolved in DCM (1 ml) and trifluorouk

- 41 042598 acetic acid (0.50 ml, 6.5 mmol) and the reaction mixture was stirred at room temperature for 2 h. The solution was concentrated and the resulting precipitate was dissolved in Et ₂ O (2 ml) and 1 M aq. HCl (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 Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn column CX 20cc). The desired product was eluted from the column using 5% ammonia in MeOH (20 ml) to give (2R,4R)-4-[[(2S)-2-amino-3-hydroxy-3-methyl-butanoyl]amino]-2 -(4boronobutyl)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), 4.474.55(1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =346.

Example 21: (2R,4R)-4-[((2S)-2-amino-2,3-dimethylbutanoyl]amino]-2-(4-boronobutyl)pyrrolidine2-carboxylic acid.

Intermediate Intermediate Connection according to example 21 connection 30 connection 53

Intermediate 53: 2-benzyl-1-tert-butyl-(2R,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-2,3-dimethylbutanoyl]amino]-2-[4 -(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl]pyrrolidine-1,2-dicarboxylate.

N,N-diisopropylethylamine (0.14 ml, 0.82 mmol) was added to a solution of (B)-2-((tert-butoxycarbonyl)amino)-2,3-dimethylbutanoic acid (0.113 g, 0.489 mmol) and HATU (0.186 g , 0.489 mmol) in DMF (3 ml) at 0°C and the reaction mixture was stirred for 15 min. A solution of (2R,4R)-2-benzyl-1-tert-butyl-4-amino-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl) was added pyrrolidine-1,2-dicarboxylate (intermediate 30, 205 mg, 0.408 mmol) in DMF (1 ml) and the reaction mixture was stirred for 3 h while slowly warming to room temperature. The reaction mixture was diluted with EtOAc (50 ml) and washed with saturated aqueous NH ₄ Cl (2x25 ml), saturated aqueous NaHCO ₃ (2x25 ml) and brine (20 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (0-50% EtOAc in hexanes) to give 2-benzyl-1-tert-butyl-(2R,4R)-4-[[(2.5)-2-(tert- butoxycarbonylamino)-2,3-dimethylbutanoyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidin-1,2dicarboxylate (intermediate 53, 211 mg, 72% yield as a white foam and as a mixture of rotamers.

Ή NMR (500 MHz, MeOH-d4) δ 0.72-0.79 (2H, m), 0.80-0.93 (6H, m), 1.17-1.25 (13H, m), 1.31(9H, d), 1.36-1.47(16H, m), 1.82-1.92(2H, m), 1.97-2.09(1H, m), 2, 15-2.30(1H, m), 2.34-2.51(1H, m), 3.61-3.75(1H, m), 4.42-4.56(1H, m), 5.08-5.31 (2H, m), 7.29-7.46 (5H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =716.

Example 21: (2R,4R)-4-[[(2S)-2-amino-2,3-dimethylbutanoyl]amino]-2-(4-boronobutyl)pyrrolidine2-carboxylic acid.

Pd/C (10% w/w, 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-1,3,2dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (intermediate 53, 210 mg, 0.29 mmol) in EtOAc (4 mL). The flask was fitted with an H2 balloon and the suspension was stirred at room temperature for 3 hours. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth and concentrated to dryness. The white solid was dissolved in DCM (1 ml) and trifluoroacetic acid (0.50 ml, 6.5 mmol) and the reaction mixture was stirred at room temperature for 2 h. The solution was concentrated and the resulting residue was dissolved in Et ₂ O (2 ml) and 1 M aq. HCl (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 Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn column CX 20cc). 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-dimethylbutanoyl]amino]-2-( 4boronobutyl)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

- 42 042598 (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) ; mass/charge: (ES ⁺ ) [M+H] ⁺ =344.

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

Intermediate connection 30 Intermediate Connection according to example 22 connection 54

Intermediate 54: 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-2yl)butyl]pyrrolidine-1,2-dicarboxylate.

N,N-diisopropylethylamine (0.14 ml, 0.82 mmol) was added to a solution of Boc-Dap(Boc)-OH-DCHA (0.238 g, 0.489 mmol) and HATU (0.186 g, 0.489 mmol) in DMF (3 ml ) at 0°C and the reaction mixture was stirred for 15 min. A solution of (2R,4R)-2-benzyl-1-tert-butyl-4-amino-2-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl) was added pyrrolidine-1,2-dicarboxylate (intermediate 30, 205 mg, 0.408 mmol) in DMF (1 ml) and the reaction mixture was stirred for 16 h while slowly warming to room temperature. The reaction mixture was diluted with EtOAc (50 ml) and washed with saturated aqueous NH ₄ Cl (2x25 ml), saturated aqueous NaHCO ₃ (2x25 ml) and brine (20 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (0-50% EtOAc in hexanes) to give 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-2yl)butyl]pyrrolidine-1,2-dicarboxylate (intermediate 54, 214 mg, yield 66%) as a white foam and as a mixture of rotamers.

¹ H NMR (500 MHz, MeOH-d4) δ 0.76 (2H, q), 1.19-1.26 (14H, m), 1.27-1.34 (6H, m), 1.37 -1.52(24H, m), 1.76-1.93(1H, m), 2.06-2.31(2H, m), 2.50(1H, s), 3.19-3 .27 (2H, m), 3.66-3.84 (1H, m), 4.00-4.13 (1H, m), 4.35-4.50 (1H, m), 5.04 -5.25 (2H, m), 7.26-7.50 (5H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =789.

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

Pd/C (10% w/w, 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-1,3,2dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (intermediate 54, 210 mg, 0.27 mmol) in EtOAc (4 ml). The flask was fitted with an H ₂ balloon and the suspension was stirred at room temperature for 3 hours. The reaction mixture was diluted with MeOH, filtered through diatomaceous earth and concentrated to dryness. The white solid was dissolved in DCM (1 ml) and trifluoroacetic acid (0.50 ml, 6.5 mmol) and the reaction mixture was stirred at room temperature for 2 h. The solution was concentrated and the resulting residue was dissolved in Et ₂ O (2 ml) and 1 M aq. HCl (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 Et ₂ O. The aqueous layer was lyophilized and purified using ion exchange chromatography (PoraPak Rxn column CX 20cc). The desired product was eluted from the column using 5% ammonia in MeOH (20 ml) to give (2R,4R)-2-(4-boronobutyl)-4-[[(2S)-2,3diaminopropanoyl]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), 2.002.13 (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), 4.414.52 (1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =317.

Example 23: (2R,4R)-2-(4-boronobutyl)-4-(methylamino)pyrrolidine-2-carboxylic acid.

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Intermediate 55 Intermediate 56 oN

B'°

Intermediate ό connection 59

Intermediate 57

Intermediate 58

Intermediate connection 62 connection

intermediate

Connection according to example 23

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 solution of 1-(tert-butyl)-2-methyl-(2S,4R)-4-aminopyrrolidine-1,2-dicarboxylateα (4 .50 g, 13.5 mmol) and triethylamine (5.63 ml, 40.4 mmol) in DCM (57 ml) and the reaction mixture was stirred at room temperature overnight under N 2 atmosphere. The crude reaction mixture was diluted with DCM (200 ml) and washed successively with 0.5 M HCl (aq.), saturated sodium bicarbonate solution and saline. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (DCM/MeOH) to give 1-tert-bugyl-2-methyl-(2S,4R)-4-(tert-bugoxycarαrbonylamino)pyrrolidine-1,2-dikαrboxylateα (intermediate 55, 3.66 g, 79% yield as a white solid.

¹ H NMR (500 MHz, DMSO-d ₆ ) 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =345.

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 in portions to a solution of 1-tert-butyl-2-methyl-(2S,4R)-4-(tert-butoxycarbonylamino)pyrrolidine-1, 2-dicarboxylate (intermediate 55, 3.66 g, 10.7 mmol) in DMF (35 mL). After the addition, the reaction mixture was stirred for 10 min, then methyl iodide (0.715 ml, 11.4 mmol) was added and the reaction mixture was stirred for an additional 3 h. 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 Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/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.

1H 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =359.

- 44 042598

Intermediate 57: 2-benzyl-1-tert-butyl-(2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate.

A solution of sodium hydroxide (2.10 g, 52.4 mmol) in water (11 ml) was added to a solution of 1-tert-butyl-2methyl-(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 warming slowly to room temperature. The volatiles were removed under reduced pressure and the aqueous layer was acidified to pH~3 with 5M HCl (aq) and extracted with DCM. The combined organics were dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give the crude carboxylic acid as a white solid which was used without further purification.

Benzyl bromide (1.24 ml, 10.5 mmol) was added to a solution of 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 solids were washed with EtOAc. The filtrate was concentrated and purified by silica gel chromatography (hexanes/EtOAc) to give 2-benzyl-1-t-butyl-(2S,4R)-4-[t-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (intermediate compound 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =435.

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) was dissolved in THF (25 ml) and the solution was cooled to -78° C. under N 2 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 mixture 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 and concentrated to dryness. The crude material was purified by silica gel purification (hexanes/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 diastereomers, E/Z olefin isomers and rotamers.

1H 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), 5.015.27(2H, m), 5.28-5.47(1H, m), 5.49-5.72 (1H, m), 7.25-7.42 (5H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =489.

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)diiridium(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 stoppered and purged with N2. The solids were 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) was added , 4.01 mmol) into the reaction mixture as a solution in DCM (7.5 ml) and the reaction mixture was stirred for 16 hours at room temperature. 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 Na ₂ SO ₄ , filtered and concentrated to dryness. The resulting residue was purified by silica gel flash chromatography (hexanes/EtOAc) to give 2benzyl-1-tert-butyl-(4R)-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4.4 ,5,5-Tetramethyl-1,3,2dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 59, 2.5 g, 100% yield) as a yellow oil and as a mixture of diastereomers and rotamers. The purified material was subjected to chiral SFC [column (S,S)Whelk-O1, 30 mm x 250 mm, 5 µm, temperature = 20°C, mobile phase = 0-30% MeOH:CO2, UV detection at 220 nm, load = 31 mg/input., conc. = 125 mg/ml in MeOH, flow rate = 75 ml/min, outlet pressure = 100 bar] to give two diastereomers. The stereochemistry of each diastereomer was assigned retrospectively based on the enzyme activity of Example 23 and Example 24 to match other exemplary compounds.

Intermediate 60 (isomer 2, 637 mg): 2-benzyl-1-t-butyl-(2R,4R)-4-[t-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5 -tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine 1,2-dicarboxylate.

1H 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),

- 45 042598

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).

Intermediate 61 (isomer 1, 860 mg): 2-benzyl-1-t-butyl-(2S,4R)-4-[t-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-d ₆ ) 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).

Intermediate 62: (2R,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane- 2-yl)butyl]pyrrolidine-2carboxylic acid.

Pd/C (10% w/w, 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-2yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 60, 637 mg, 1.03 mmol) in EtOAc (7 ml). The flask was fitted with an H ₂ balloon and 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 (hexanes/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-d ₆ ) 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), 12.3212.75(1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =527.

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-1,3,2dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 62, 350 mg, 0.66 mmol) in DCM (4 ml) at room temperature. After 1 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (5 ml) and Et ₂ O (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 Et ₂ O and water and the layers were separated. The aqueous layer was washed with Et ₂ O. 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 (2R,4R)-2-(4-boronobutyl)-4(methylamino)pyrrolidine-2-carboxylic acid (Example 23, 140 mg, yield 86%) 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =245.

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

Intermediate 63: (2S,4R)-1-tert-butokCicarbonyl-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)butyl]pyrrolidine-2carboxylic acid.

Pd/C (10% w/w, 223 mg, 0.209 mmol) was added to a solution of 2-benzyl-1-t-butyl-(2S,4R)-4[t-butoxycarbonyl(methyl)amino]-2- [4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 61, 860 mg, 1.39 mmol) in EtOAc (9 .3 ml). The flask was fitted with an H2 balloon and 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 (hexanes/EtOAc) to give (2S,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4,4 ,5,5Tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 63, 520 mg, 71% yield) as a white solid.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 0.60-0.75 (2H, m), 1.16 (12H, s), 1.28-1.45 (22H, m), 1, 56-1.74 (1H, m),

- 46 042598

1.84-1.95(1H, m), 2.01-2.22(2H, m), 2.69(3H, s), 3.01-3.17(1H, m), 3, 56-3.70(1H, m), 4.52-4.73(1H, m), 12.35-12.77(1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =527.

Example 24: (2S,4R)-2-(4-boronobutyl)-4-(methylamuno)nu-rrrolidine-2-carboxylic acid.

Trifluoroacetic acid (1.23 ml, 16.0 mmol) was added dropwise to a stirred solution of (2S,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl(methyl)amino]-2-[4-(4 ,4,5,5-Tetramethyl-1,3,2dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 63, 520 mg, 0.80 mmol) in DCM (8.5 ml) at room temperature temperature. After 1 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (5 ml) and Et ₂ O (5 ml). Phenylboronic acid (196 mg, 1.60 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 h. The mixture was diluted with Et ₂ O and water and the layers were separated. The aqueous layer was washed with Et ₂ O. 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)-2-(4-boronobutyl)-4(methylamino)pyrrolidine-2-carboxylic acid (Example 24, 163 mg, yield 83%) 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =245.

Example 25: (2R,4R)-2-(4-boronobutyl)-4-(dimethylamino)pyrrolidine-2-carboxylic acid.

Intermediate 64: 2-benzyl-1-tert-butyl-(2R,4R)-4-aminopyrrolidine-1,2-dicarboxylate.

Triphenylphosphine (7.87 g, 30.0 mmol) and water (0.54 ml, 30.0 mmol) were added to a solution of 2-benzyl-1(tert-butyl)-(2R,4R)-4-azidopyrrolidine-1 ,2-dicarboxylate (5.20 g, 15.0 mmol) in THF (68 ml) at room temperature. The reaction mixture was heated to 60° C. and stirred for 6 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc and washed successively with water (2x100 ml) and saturated aqueous sodium chloride solution (100 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (DCM/MeOH) to give 2-benzyl-1-tert-butyl-(2R,4R)-4aminopyrrolidine-1,2-dicarboxylate (intermediate 64, 3.2 g, 67% yield). %) as a colorless oil.

1 H NMR (500 MHz, DMSO-d ₆ ) δ 1.20-1.49 (9H, m), 1.53-1.64 (1H, m), 1.65-1.79 (2H, m) , 2.26-2.40(1H, m), 2.85-3.01(1H, m), 3.31-3.42(1H, m), 3.45-3.56(1H, m), 4.13-4.24(1H, m), 4.99-5.25(2H, m), 7.36(5H, s); mass/charge: (ES ⁺ ) [M+H] ⁺ =321.

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

- 47 042598 butyl-(2R,4R)-4-aminopyrrolidine-1,2-dicarboxylate (intermediate 64, 3.20 g, 9.99 mmol) and formaldehyde (37% w/w in H ₂ O, 4.46 ml, 59.9 mmol) in MeOH (79 ml). After the addition, the reaction mixture was stirred at room temperature for 17 hours. The volatiles were removed under reduced pressure and the resulting precipitate was diluted with DCM. Solids were removed by filtration and the filtrate was concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc with NH ₄ OH) 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-d ₆ ) δ 1.24 (9H, s), 1.53-1.67 (1H, m), 2.10 (6H, s), 2.40-2, 48 (1H, m), 2.532.73 (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); mass/charge: (ES ⁺ )[M+H] ⁺ =350.

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) was dissolved in THF (18 ml) and the solution was cooled to -78° C. under N 2 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 mixture 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 Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel purification (hexanes/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 diastereomers, E/Z olefin isomers and rotamers.

L NMR (500 MHz, DMSO-d ₆ ) 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =403.

Intermediate 67: 2-benzyl-1-tert-butyl-(4R)-4-(dimethylamino)-2-[4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl) butyl]pyrrolidine-1,2-dicarboxylate.

Bis(1,5-cyclooctadiene)diiridium(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 stoppered and purged with N ₂ . The solids were 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 solution in DCM (6.7 ml) and the reaction mixture was stirred for 16 h at room temperature. 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 and concentrated to dryness. The resulting residue was purified by silica gel flash chromatography (hexanes/EtOAc) to give 2benzyl-1-tert-butyl-(4R)-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl -1,3,2-dioxaborolan-2yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 67, 1.5 g, 79% yield) as a yellow oil and as a mixture of diastereomers and rotamers. The purified material was subjected to chiral SFC [column (S,S)Whelk-O1, 30 mm x 250 mm, 5 μm, temperature = 20°C, mobile phase = 0-15% MeOH:CO ₂ , UV detection at 220 nm, load = 32 mg/injection, conc. = 80 mg/ml in MeOH, flow rate = 120 ml/min, outlet pressure = 100 bar] to give two diastereomers. The stereochemistry of each diastereomer was assigned retrospectively based on the enzyme activity of Example 25 and Example 26 to match other exemplary compounds.

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-d ₆ ) 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).

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).

Intermediate 70: (2R,4R)-1-tert-butoxycarbonyl-4-(dimethylamino)-2-[4-(4,4,5,5

- 48 042598 tetramethyl-],3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid.

Pd/C (10% w/w, 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 fitted with an H ₂ balloon and 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-(dimethylamino)-2-[4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl ]pyrrolidine-2-carboxylic acid (intermediate 70, 150 mg, 96% yield) as a white solid, which was used without further purification.

¹ H NMR (500 MHz, DMSO-d ₆ ) 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =441.

Example 25: (2R, 4R)-2-(4-boronobutyl)-4-(dimethylamino)-n-pyrrolidine-2-carboxylic acid.

Trifluoroacetic acid (0.26 ml, 3.4 mmol) was added dropwise to a stirred solution of (2R,4R)-1-tert-butoxycarbonyl-4-(dimethylamino)-2-[4-(4,4,5,5 -tetramethyl-1,3,2-dioxaborolan-2yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 70, 150 mg, 0.34 mmol) in DCM (2 ml) at room temperature. After 1 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (5 ml) and Et ₂ O (5 ml). Phenylboronic acid (83 mg, 0.68 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 h. The mixture was diluted with Et ₂ O and water and the layers were separated. The aqueous layer was washed with Et ₂ O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 ml) to give (2R,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); mass/charge: (ES ⁺ ) [M+H] ⁺ =259.

Example 26: (2S,4R)-2-(4-boronobutyl)-4-(dimethylamino)pyrroludin-2-carboxylic acid.

Intermediate connection 69 Intermediate connection according to example 26 connection 71

Intermediate 71: (2S,4R)-1-tert-butoxycarbonyl-4-(dimethylamino)-2-[4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl] pyrrolidine-2-carboxylic acid.

Pd/C (10% w/w, 148 mg, 0.139 mmol) was added to a solution of 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 (Intermediate 69, 490 mg, 0.93 mmol) in EtOAc (6.7 mL). The flask was fitted with an H ₂ balloon and 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 (2S,4R)-1-tert-butoxycarbonyl-4-(dimethylamino)-2-[4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)butyl ]pyrrolidine-2-carboxylic acid (intermediate 71, 405 mg, 99% yield) as a white solid, which was used without further purification.

ΊI NMR (500 MHz, DMSO-d ₆ ) δ 0.52-0.71 (2H, m), 0.83-1.06 (1H, m), 1.16 (12H, s), 1.32 (12H, s), 1.531.67 (1H, m), 1.75-1.88 (1H, m), 2.01-2.13 (7H, m), 2.54-2.69 (1H , m), 2.79-2.95(1H, m), 3.66-3.83(1H, m), 7.24-7.46(1H, m), 11.86-12.88 (1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =441.

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-2yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 71, 405 mg, 0.919 mmol) in DCM (5.1 ml) at room temperature. After 1 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (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 Et ₂ O. 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% am-49 042598 myac 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =259.

Example 27: (2R,4R)-4-(2-aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

Intermediate 79

Intermediate 74

Connection according to example 27

1-tert-butyl-2-methyl-(2S,4R)-4-[2-(tert Intermediate

intermediate

intermediate connection

72:

butoxycarbonylamino)ethylamino]pyrrolidine-1,2-dicarboxylate.

Acetic acid (42 µl, 0.73 mmol) was added to a solution of 1-(tert-butyl)-2-methyl-(2S,4R)aminopyrrolidine-1,2-dicarboxylate (7.48 g, 30.6 mmol) and tert-butyl-(2-oxoethyl)carbamate (4.64 g, 29.2 mmol) in MeOH (194 ml) and the reaction mixture was stirred at room temperature for 3 hours. The solution was cooled to 0°C and sodium triacetoxyborohydride was added in portions (9.27 g, 43.7 mmol). The reaction mixture was stirred overnight while warming slowly to room temperature.

The reaction mixture was concentrated under reduced pressure, and the resulting residue was diluted with DCM, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (DCM/EtOAc/MeOH) to give

1-tert-Butyl-2-methyl-(2S,4R)-4-[2-(tert-butoxycarbonylamino)ethylamino]pyrrolidine-1,2-dicarboxylate (Intermediate 72, 4.77 g, 42% yield) as colorless oils.

1 H NMR (500 MHz, DMSO-d6) δ 1.25-1.48 (20H, m), 1.84-2.07 (3H, m), 2.88-2.98 (2H, m), 3.02-3.17(1H, m), 3.17-3.27(1H, m), 3.40-3.51(1H, m), 3.64(3H, s), 4, 14-4.24 (1H, m), 6.63-6.74 (1H, m); mass/charge: (ES ⁺ )[M+H] ⁺ =388.

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 solution 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) in DCM (53 ml) at room temperature and reaction the mixture was stirred for 17 hours. The reaction mixture was diluted with DCM and washed successively with water and saturated aqueous sodium chloride. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (hexanes/EtOAc) to give 1-tert-butyl-2methyl-(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), 6.796.94(1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =488.

Intermediate 74: 2-benzyl-1-tert-butyl-(2S,4R)-4-tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2-dicarboxylate.

A solution of sodium hydroxide (2.09 g, 52.3 mmol) in water (11 ml) was added to a solution of 1-tert-butyl-2methyl-(2S,4R)-4-[tert-butoxycarbonyl-[2-(tert- butoxycarbonylamino)ethyl]amino]pyrrolidine-1,2dicarboxylate (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. The volatiles were removed under reduced pressure and the aqueous layer was acidified to pH~3 with 5M HCl (aq) and extracted with DCM. The combined organics were dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give the crude carboxylic acid as a white solid which was used without further purification.

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 mixture was stirred at room temperature for 17 hours. The reaction mixture was filtered and the solids were washed with EtOAc. The filtrate was concentrated and purified by silica gel chromatography (hexanes/EtOAc) to give 2-benzyl-1-t-butyl-(2S,4R)-4-[t-butoxycarbonyl-[2(t-butoxycarbonylamino)ethyl]amino] pyrrolidine-1,2-dicarboxylate (intermediate 74, 3.91 g, 80% yield) as a white solid.

¹ H NMR (500 MHz, DMSO-d ₆ ) 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), 7.257.41(5H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =564.

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 N 2 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 mixture 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 and concentrated to dryness. The crude material was purified by silica gel purification (hexanes/EtOAc) to give 2benzyl-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 diastereomers, E/Z olefin isomers and rotamers.

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), 5.005.20(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); mass/charge: (ES ⁺ ) [M+H] ⁺ =618.

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-2yl)butyl]pyrrolidine-1,2-dicarboxylate.

Bis(1,5-cyclooctadiene)diiridium(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 stoppered and purged with N2. The solids were 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 compound 75, 2.04 g, 3.30 mmol) was added to the reaction mixture as a solution in DCM (6.1 ml) and the reaction mixture was stirred for 16 h at room temperature. 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 Na ₂ SO ₄ , filtered

- 51 042598 and concentrated to dryness. The resulting residue was purified by silica gel flash chromatography (hexanes/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-2yl)butyl]pyrrolidine-1,2-dicarboxylate (Intermediate 76, 1.14 g, 46% yield) as yellow oil and as a mixture of diastereomers and rotamers. The purified material was subjected to chiral SFC [(S,S)Whelk-O1 column, 30x250 mm, 5 µm, temperature=20°C, mobile phase=0-20% IPA with 0.2% NH ₄ OH:CO ₂ , UV detection at 220 nm, load = 18 mg/inlet, conc. = 46 mg/ml in MeOH, flow rate = 120 ml/min, outlet pressure = 100 bar] to give two diastereomers. The stereochemistry of each diastereomer was assigned retrospectively based on the enzyme activity of Example 27 and Example 28 to match other exemplary compounds.

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-1,3,2dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate.

¹ H NMR (500 MHz, DMSO-d ₆ ) 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).

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-1,3,2dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate.

1H 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).

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-2yl)butyl]pyrrolidine-2-carboxylic acid.

Pd/C (10% w/w, 99 mg, 0.093 mmol) was added to a solution of 2-benzyl-1-t-butyl-(2R,4R)-4-[t-butoxycarbonyl-[2-(t-butoxycarbonylamino) ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)butyl]pyrrolidin-1,2-dicarboxylate (Intermediate 77, 347 mg, 0.465 mmol ) in EtOAc (2.3 ml). The flask was fitted with an H ₂ balloon and 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-(4,4,5,5tetramethyl-1 ,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 79, 287 mg, 94% yield) as a white solid which was used without further purification.

1H 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), 6.846.93 (1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =656.

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 solution 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) in DCM ( 3.7 ml) at room temperature. After 1 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1M aq. HCl (5 ml) and Et ₂ O (5 ml). Phenylboronic acid (107 mg, 0.878 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 h. The mixture was diluted with Et ₂ O and water and the layers were separated. The aqueous layer was washed with Et ₂ O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 ml) to give (2R,4R)-4-(2aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid (Example 27, 106 mg, 89% yield as a white solid.

1H NMR (500 MHz, 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), 3.283.43 (1H, m), 3.43-3.51 (1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =274.

Example 28: (2S,4R)-4-(2-aminoethylamino)-2-(4-boronobutyl)pyrrolidine-2-carboxylic acid.

- 52 042598

Intermediate 80: (2S,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[4-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2yl)butyl]pyrrolidine-2-carboxylic acid.

Pd/C (10% w/w, 137 mg, 0.129 mmol) was added to a solution of 2-benzyl-1-t-butyl-(2S,4R)-4[t-butoxycarbonyl-[2-(t-butoxycarbonylamino )ethyl]amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)butyl]pyrrolidin-1,2-dicarboxylate (Intermediate 78, 480 mg, 0 .64 mmol) in EtOAc (7.2 ml). The flask was fitted with an H ₂ balloon and 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 (hexanes/EtOAc) to give (2S,4R)-1-tert-butoxycarbonyl-4-[tert-butoxycarbonyl-[2-(tert-butoxycarbonylamino)ethyl]amino]-2-[ 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)butyl]pyrrolidine-2-carboxylic acid (80 intermediate, 170 mg, 40% yield) as a white solid.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 0.58-0.73 (2H, m), 1.15 (12H, s), 1.37 (31H, br d), 1.58-1 .74 (2H, m), 2.18-2.33 (2H, m), 2.92-3.16 (6H, m), 3.59-3.73 (1H, m), 4.31 -4.50 (1H, m), 6.84-6.95 (1H, m); mass/charge: (ES+) [M+H] ⁺ =656.

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 solution 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) in DCM (5 ml) at room temperature. After 1 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (5 ml) and Et ₂ O (5 ml). Phenylboronic acid (63 mg, 0.52 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 h. The mixture was diluted with Et ₂ O and water and the layers were separated. The aqueous layer was washed with Et ₂ O. The aqueous layer was lyophilized and purified by ion exchange chromatography (PoraPak Rxn CX 20cc column). The desired product was eluted from the column using 5% ammonia in MeOH (20 ml) to give (2S,4R)-4-(2aminoethylamino)-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); mass/charge: (ES ⁺ ) [M+H] ⁺ =274.

Example 29: (2R,4R)-4-[[(2S)-2-amino-3-methylbutanoyl]methylamino]-2-(4-boronobutyl)pyrrolidine2-carboxylic acid.

- 53 042598 n ₂

Intermediate 90

Intermediate 89 in- ^he he

Connection according to example 29

Intermediate 81

Intermediate 84

Intermediate 87

Intermediate 82

Intermediate 85

Intermediate 88

Intermediate 83

Intermediate 81: 1-benzyl-2-methyl-(2S,4R)-4-(tert-butoxycarbonylamino)pyrrolidine-1,2-dicarboxylate.

Di-tert-butyl dicarbonate (6.27 g, 28.8 mmol) was added to a solution of 1-benzyl-2-methyl-(2S,4R)-4aminopyrrolidine-1,2-dicarboxylate (5.00 g, 18.0 mmol ) and triethylamine (5.00 ml, 35.9 mmol) in DCM (78 ml) and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with DCM and washed successively with 0.5 M (aq. ) HCl, saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel chromatography (DCM/MeOH) to give 1-benzyl-2-methyl-(2S,4R)-4-(tert-butoxycarbonylamino)pyrrolidine-1,2-dicarboxylate (Intermediate 81, 5.4 g , yield 79%) as a white solid.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 1.31-1.42 (9H, m), 1.99-2.23 (2H, m), 3.22-3.28 (1H, m ), 3.52-3.65(4H, m), 3.96-4.10(1H, m), 4.30-4.48(1H, m), 4.92-5.13(2H , m), 7.18-7.41 (5H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =379.

Intermediate 82: 1-benzyl-2-methyl-(2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate.

Sodium hydride (60% dispersion in mineral oil) (0.423 g, 12.3 mmol) was added portionwise 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) at 0°C. The reaction mixture was stirred at 0°C for 1 h before warming to room temperature while stirring for an additional 1 h. Methyl iodide (0.63 ml, 10.2 mmol) was added and the reaction mixture was stirred at room temperature for 16 h 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 (4x50 ml) and saturated aqueous sodium chloride solution (2x50 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give 1-benzyl-2-methyl-(2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (intermediate 82, 2.33 g, 70% yield as a colorless oil which 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =393.

Intermediate 83: dibenzyl-(2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate.

A solution of sodium hydroxide (1.425 g, 35.62 mmol) in water (7.5 ml) was added to a solution of 1-benzyl-2methyl-(2S,4R)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1, 2-dicarboxylate (intermediate

- 54 042598 compound 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 pH~3 with 5 M HCl (aq.) and was extracted with DCM (4x50 ml). The combined organics were dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give the crude carboxylic acid as a white solid which was used without further purification.

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 K ₂ CO ₃ (1.92 g, 13.9 mmol) in DMF (28 ml) and the reaction mixture was stirred at room temperature for 17 hours, the Reaction mixture was filtered and the solids washed with EtOAc. The filtrate was concentrated and purified by silica gel chromatography (hexanes/EtOAc) to give dibenzyl-(2S,4R)-4-[t-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-d ₆ ) 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =469.

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) was dissolved in THF (18 ml) and the solution was cooled to -78° C. under N 2 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 mixture 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 Na ₂ SO ₄ , filtered and concentrated to dryness. The crude material was purified by silica gel purification (hexanes/EtOAc) to give dibenzyl-(4R)-2-(but-2-enyl)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2-dicarboxylate (intermediate compound 84, 2.10 g, 86% yield as a yellow oil and as a mixture of diastereomers, E/Z olefin isomers and rotamers.

¹ H NMR (500 MHz, DMSO-d ₆ ) 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =523.

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 solution of dibenzyl-(4R)-2(but-2-enyl)-4-[tert-butoxycarbonyl(methyl)amino]pyrrolidine-1,2- dicarboxylate (intermediate 84, 2.10 g, 4.02 mmol) in DCM (32 ml) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated to dryness to give the dibenzyl(-4R)-2- TFA salt (but-2-enyl)-4-(methylamino)pyrrolidine-1,2-dicarboxylate (intermediate 85, 2.55 g, 100% yield) as a colorless oil which was used without purification.

1H NMR (500 MHz, DMSO-d ₆ ) 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =423.

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 solution of HATU (1.52, 4.02 mmol) and Boc-Val-OH (873 mg, 4.02 mmol) in DMF (15 ml ) and the reaction mixture was stirred at room temperature for 30 min. 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) and the reaction mixture was stirred at room temperature for an additional 17 hours. The reaction mixture was concentrated and directly purified by silica gel chromatography (hexanes/EtOAc) to give 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.

1H 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =622.

Intermediate 87: dibenzyl-(4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]methylamino]-2-[4-(4,4,5,5-tetramethyl -1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-1,2-dicarboxylate.

Bis(1,5-cyclooctadiene)diiridium(I) dichloride (213 mg, 0.317 mmol) and bis(diphenylphosphino)methane

- 55 042598 (244 mg, 0.635 mmol) was added to an oven dried round bottom flask. The flask was stoppered and purged with N ₂ . The solids were 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 solution in DCM (5.9 ml) and the reaction mixture was stirred for 16 hours at room temperature. 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 Na ₂ SO ₄ , filtered and concentrated to dryness. The resulting residue was purified by silica gel flash chromatography (hexanes/EtOAc) to give dibenzyl-(4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]methyl-amino]-2- [4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidin-1,2dicarboxylate (Intermediate 87, 1.5 g, 63% yield) as yellow oils and as a mixture of diastereomers and rotamers. The purified material was subjected to chiral SFC [column (S,S)Whelk-O1, 30x250 mm, 5 µm, temperature=20°C, mobile phase=0-30% MeOH:CO ₂ , UV detection at 220 nm, loading=31 mg/input., conc. = 125 mg/ml in MeOH, flow rate = 75 ml/min, outlet pressure = 100 bar] to give two diastereomers. The stereochemistry of each diastereomer was assigned retrospectively based on the enzyme activity of Example 29 and Example 30 to match other exemplary compounds.

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-dioxaborolan2-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).

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-dioxaborolan2-yl)butyl]pyrrolidine-1,2-dicarboxylate.

1 H NMR (500 MHz, DMSO-d ₆ ) 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), 6.717.05(1H, m), 7.17-7.41(10H, m).

Intermediate 90: (2R,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]methylamino]-2-[4-(4,4,5,5-tetramethyl -1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2carboxylic acid.

Pd/C (10% w/w, 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-dioxaborolan2-yl)butyl]pyrrolidin-1,2-dicarboxylate (Intermediate 88, 280 mg, 0.37 mmol) in EtOAc (1.8 ml). The flask was fitted with an H2 balloon and the suspension was stirred at room temperature for 4 hours. 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)-3methyl-butanoyl]methyl-amino]-2-[4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2carboxylic acid (Intermediate 90, 180 mg, 92% yield) as a white solid which was used without further purification.

1H 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =526.

Example 29: (2R,4R)-4-[[(2S)-2-amino-3-methylbutanoyl]methylamino]-2-(4-boronobutyl)pyrrolidine2-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-(4,4,5,5tetramethyl-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 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (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 Et ₂ O. 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 (2R,4R)-4-[[(2S)-2-amino-3-methylbutanoyl]methyl-amino]-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid (example 29, 98 mg, 86% yield) as a white solid.

- 56 042598

1H 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =344.

Example 30: (2S,4R)-4-[[(2S)-2-amino-3-methylbutanoyl]methylamino]-2-(4-boronobutyl)pyrrolidine2-carboxylic acid.

Intermediate Intermediate Connection by connection 89 connection 91 example 30

Intermediate 91: (2S,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]methyl-amino]-2-[4-(4,4,5,5-tetramethyl -1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2carboxylic acid.

Pd/C (10% w/w, 209 mg, 0.196 mmol) was added to a solution of dibenzyl-(2S,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]methyl -amino]-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)butyl]pyrrolidin-1,2-dicarboxylate (intermediate 89, 590 mg, 0.79 mmol) in EtOAc (4 ml). The flask was fitted with an H2 balloon and 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 (2S,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3methyl-butanoyl]methyl-amino]-2-[4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2carboxylic acid (Intermediate 91, 397 mg, 96% yield) as a white solid which was used without further purification.

1 H NMR (500 MHz, DMSO-d ₆ ) δ 0.58-0.67 (2H, m), 0.73-0.87 (6H, m), 1.16 (12H, s), 1.22 -1.31(3H, m), 1.35(9H, s), 1.51-1.67(1H, m), 1.68-1.95(3H, m), 2.28-2 .37 (2H, m), 2.95 (4H, s), 4.05-4.20 (1H, m), 4.664.79 (1H, m), 6.65-6.90 (1H, m ), 7.85-8.15 (1H, m); mass/charge: (ES ⁺ ) [M+H] ⁺ =526.

Example 30: (2S,4R)-4-[[(2S)-2-amino-3-methylbutanoyl]methylamino]-2-(4-boronobutyl)pyrrolidine2-carboxylic acid.

Trifluoroacetic acid (1.17 mL, 15.2 mmol) was added dropwise to a stirred solution of (2S,4R)-4-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]methyl-amino ]-2-[4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)butyl]pyrrolidine-2-carboxylic acid (Intermediate 91, 397 mg, 0.755 mmol) in DCM ( 10 ml) at room temperature. After 1 h the solution was concentrated under reduced pressure and the resulting precipitate was dissolved in 1 M aq. HCl (5 ml) and Et ₂ O (5 ml). Phenylboronic acid (186 mg, 1.52 mmol) was added and the clear biphasic solution was stirred at room temperature for 1 h. The mixture was diluted with Et ₂ O and water and the layers were separated. The aqueous layer was washed with Et ₂ O. 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-methylbutanoyl]methyl-amino]-2-(4- boronobutyl)pyrrolidine-2-carboxylic acid (Example 30, 163 mg, 92% yield) as a white solid.

1H 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); mass/charge: (ES ⁺ ) [M+H] ⁺ =344.

Example 31: biological activity of the compounds of examples 1-30.

The inhibitory effects of the compounds of Examples 1-30 on human arginase 1 and arginase 2 activity were quantified by measuring thiol group formation from thioarginine using recombinant arginase 1 or arginase 2 derived from E. coli. The thiol group was determined using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB reacts with thiol to give a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB) which is quantified by anion absorbance (TNB ² -) at 412 nm.

The assays were performed in clean 384-well plates (Greiner, Cat # 781101). Various concentrations of the compounds of Examples 1-30 in 300 nl DMSO were dispensed into assay plates using an Echo sonic pipette followed by immediate capping of the plate and centrifugation.

Two premixes were prepared from reagents thawed just prior to addition to assay plates. The first premix contains human arginase 1 or human arginase 2 at a final concentration of 5 nM and 0.5 mM DTNB in assay buffer, 45 mM HEPES

- 57 042598 pH 7.5, brij 35, 0.045% (w/v) and 100 μM MnCl ₂ . The second premix contains freshly thawed 0.5 mM thioarginine in assay buffer. Fifteen microliters of the first premix were dispensed into assay plates containing the compounds of Examples 1-30, centrifuged and incubated for 30 minutes at room temperature before adding fifteen microliters of the second premix.

The assay plates were centrifuged prior to reading absorbance at 412 nm in a Pherastar multi-mode data collection plate reader at time 0 (T0). Plates were incubated at room temperature for 60 minutes before rereading for data collection at time 1 (T1). The data is obtained by subtracting the signal A412 measured at T0 (time 0) from that measured at T1 (time 1). Data were converted to % effect values using the equation:

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

The concentration of the compounds of Examples 1-30 that inhibited activity by 50% (ie IC ₅₀ ) was calculated by plotting % effect versus test compound concentration and fitting the data using the Smart fit algorithm in Genedata Screener. The results of such analyzes are in table. 2.

table 2

Example Enzyme arginase 1 person IC50 (µM) Enzyme arginase 2 human ICso (μM) 1 14.69 22.98 2 26.57 21.44 3 4.55 10.40 4 41.92 74.07 5 6.41 16.63 6 3.96 10.41 7 0.01 0.02 8 0.61 0.56 9 0.32 0.33 10 3.77 4.77 eleven 0.21 0.20 12 0.70 0.85 13 0.43 0.63 14 0.26 0.31 15 0.31 0.26 16 0.31 0.31 17 0.20 0.23 18 0.32 0.38 19 0.22 0.28 20 0.34 0.52 21 0.48 0.68 22 0.93 1.32 23 1.14 2.12 24 <0.003 0.01 25 10.80 24.55 26 0.06 0.15 27 1.43 3.32 28 0.09 0.19 29 5.27 9.28 thirty 1.35 2.42

Example 32 Bioavailability Studies.

Examples 8, 9 and 13-20 are prodrugs of the compounds of Example 7. The following pharmacokinetic study was performed to demonstrate the bioavailability of the compounds of Example 7 and Example 8. The compound of Example 8 was formulated at 0.9% w/v. saline pH 4 (adjust using 1 M HCl) for IV dosing. The formulation was administered at a dose of 2 mg/kg via a femoral catheter to each of two male rats (170-250 g). Serial blood samples were taken from the jugular vein catheter at 0.033, 0.083, 0.167, 0.5, 1, 2, 4, 8, and 24 hours after injection.

- 58 042598 doses. During PO dosing, Example 8 was formulated in pH 4 deionized water (adjusted with 1M HCl) and dosed at 5 mg/kg by gavage to each of two male rats (170-250 g). Serial blood samples were taken with a jugular vein catheter at 0.25, 0.5, 1, 1.5, 2, 3, 4, 8 and 24 hours post-dose. Plasma samples were obtained from blood using low speed centrifugation. A separate set of calibration standards containing the compounds of example 7 and example 8 was obtained by adding control blood plasma. Samples and calibration standards were extracted by precipitation with two volumes of acetonitrile followed by centrifugation. The results obtained were used to determine Cl (ml/min/kg), Vdss (l/kg), Cmax (μM), AUC (μM/h), tmax (h) and %F for both the compound of Example 7 and the compound as in Example 8. Absolute bioavailability was determined by comparing the AUC, normalized to the PO dose of the compound of Example 7, when dosing as in Example 8, relative to the AUC normalized to the IV dose of the compound of Example 7, when dosing as in Example 7. If necessary, measured and non-nominal doses were used to calculate bioavailability. Similarly, the same procedure was repeated for the compounds of examples 9 and 13-20. The results are shown in Tables 3-12. These results indicate that bioavailability can be enhanced by including certain amino acid fragments as prodrugs.

Table 3

Example 8 Example 7 C1 (ml/min./kg) N.V. ^# 8.90* Vdss (l/kg) N.V. ^# 0.26* Stach RO (µM) N.V. ^# 3.60 ^# AUC RO (µM.h.) N.V. ^# 10.40 ^# Ttah (h) N.V. ^# 1.00 ^# %F N.V. ^# 33.00 ^#

# observed value if dosing as a prodrug.

* observed value if dosing is done as a payload. NV - no final value.

Table 4

Example 9 Example 7 C1 (ml/min./kg) 33.30 ^# 8.90* Vdss (l/kg) 0.20 ^# 0.26* Stach RO (µM) 0.60 ^# 5.70 ^# AUC RO (µM.h.) 0.24 ^# 16.80 ^# Ttah (h) 0.25 ^# 1.00 ^# %F 3.20 ^# 59.00 ^#

# observed value if dosing as a prodrug.

* observed value if dosing is done as a payload. NV - no final value.

Table 5

Example 13 Example 7 C1 (ml/min./kg) 17.90 ^# 8.90* Vdss (l/kg) 0.20 ^# 0.26* Stach RO (µM) 0.46 ^# 3.30 ^# AUC RO (µM.h.) 0.62 ^# 13.30 ^# Ttah (h) 0.50 ^# 1.25 ^# %F 3.80 ^# 40.70 ^#

# observed value if dosing as a prodrug.

* observed value if dosing is done as a payload. NV - no final value.

- 59 042598

Table 6

Example 14 Example 7 C1 (ml/min./kg) N.V. ^# 8.90* Vdss (l/kg) N.V. ^# 0.26* Stach RO (µM) N.V. ^# 3.80 ^# AUC RO (µM.h.) N.V. ^# 8.90 ^# Ttah (h) N.V. ^# 1.00 ^# %F N.V. ^# 29.90 ^#

# observed value if dosing as a prodrug.

* observed value if dosing is done as a payload. NV - no final value.

Table 7

Example 15 Example 7 C1 (ml/min./kg) 104.00 ^# 8.90* Vdss (l/kg) 0.46 ^# 0.26* Stach RO (µM) N.V. ^# 5.70 ^# AUC RO (µM.h.) N.V. ^# 15.60 ^# Ttah (h) N.V. ^# 1.00 ^# %F N.V. ^# 57.10 ^#

# observed value if dosing as a prodrug. NV - no final value.

Table 8

Example 16 Example 7 C1 (ml/min./kg) 585.00 ^# 8.90* Vdss (l/kg) 7.41 ^# 0.26* Stach RO (µM) N.V. ^# 1.60 ^# AUC RO (µM.h.) N.V. ^# 5.80 ^# Ttah (h) N.V. ^# 1.00 ^# %F N.V. ^# 17.00 ^#

# observed value if dosing as a prodrug.

* observed value if dosing is done as a payload. NV - no final value.

Table 9

Example 17 Example 7 C1 (ml/min./kg) 40.50 ^# 8.90* Vdss (l/kg) 0.36 ^# 0.26* Stach RO (µM) 0.43 ^# 4.90 ^# AUC RO (µM.h.) 0.38 ^# 18.30 ^# Ttah (h) 0.38 ^# 1.50 ^# %F 5.80 ^# 61.90 ^#

# observed value if dosing as a prodrug.

* observed value if dosing is done as a payload. NV - no final value.

-

Claims (6)

Table 10 Example 18 Example 7 C1 (ml/min./kg) 317.00 ^# 8.90 * Vdss (l/kg) 0.64 ^# 0.26 * Stach RO (µM) NV ^# 4.10 ^# AUC PO (µM.h.) NV ^# 13.10 ^# Tmax (p.) NV ^# 1.50 ^# %F NV ^# 45.70 ^# # Observable value if dosing as a prodrug. * observed value if dosing is done as a payload. NV - no final value. Table 11 Example 19 Example 7 C1 (ml/min./kg) 36.80 ^# 8.90 * Vdss (l/kg) 0.20 ^# 0.26 * Stach RO (µM) 0.33 ^# 5.70 ^# AUC PO (μM.h.) 0.21 ^# 16.80 ^# Tmax (h) 0.25 ^# 1.00 ^# %F 2.50 ^# 59.00 ^# # Observable value if dosing as a prodrug. * observed value if dosing is done as a payload. NV - no final value. Table 12 Example 20 Example 7 C1 (ml/min/kg) 8.90 ^# 8.90 * Vdss (l/kg) 0.29 ^# 0.26 * Stach RO (µM) 1.80 ^# 2.20 ^# AUC PO (μM.h.) 6.90 ^# 12.20 ^# Tmax (h) 1.00 ^# 3.50 ^# %F 25.50 ^# 59.00 ^# # Observable value if dosing as a prodrug. * observed value if dosing is done as a payload. NV - no final value. CLAIM 1. The compound of formula (II) or its pharmaceutically acceptable salt: R ^2a is -H or -C(O)CH(R ^2b )NH ₂ ; R ^2b is -CH ₃ or -CH(CH ₃ ) ₂ . 2. A compound of formula (III) or a pharmaceutically acceptable salt thereof: - 61 042598 _H2N R3 ABOUT He is in' OH (ΙΠ), where R ³ represents -CH ₃ or -CH(CH ₃ ) ₂ . 3. A compound of formula (IIb) or a pharmaceutically acceptable salt thereof: ^R2 BUT (IIb), where R ² represents -OH or -NHR ^2a ; R ^2a is -H or -C(O)CH(R ^2b )NH ₂ ; R ^2b is -CH ₃ or -CH(CH ₃ ) ₂ . 4. A compound of formula (IIIb) or a pharmaceutically acceptable salt thereof: ^R3 .. h ₂ n where R ³ represents -CH ₃ or -CH(CH ₃ ) ₂ . 5. Connection selected from: or a pharmaceutically acceptable salt thereof. 6. Connection having the following structure: -