Discovery of N-Substituted 3-Amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic Acids as Highly Potent Third-Generation Inhibitors of Human Arginase i and II

Article abstract of DOI:10.1021/acs.jmedchem.9b00931

Recent efforts to identify new highly potent arginase inhibitors have resulted in the discovery of a novel family of (3R,4S)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid analogues with up to a 1000-fold increase in potency relative to the current standards, 2-amino-6-boronohexanoic acid (ABH) and N-hydroxy-nor-l-arginine (nor-NOHA). The lead candidate, with an N-2-amino-3-phenylpropyl substituent (NED-3238), example 43, inhibits arginase I and II with IC₅₀ values of 1.3 and 8.1 nM, respectively. Herein, we report the design, synthesis, and structure-activity relationships for this novel series of inhibitors, along with X-ray crystallographic data for selected examples bound to human arginase II.

Full text of DOI:10.1021/acs.jmedchem.9b00931

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Article
Discovery of N-substituted 3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic
acids as highly potent third generation inhibitors of human arginase I and II
Michael Christopher Van Zandt, G. Erik Jagdmann, Darren Whitehouse,
Min koo Ji, Jennifer Savoy, Olga Potapova, Alexandra Cousido-Siah,
Andre Mitschler, Eduardo I. Howard, Anna M Pyle, and Alberto D. Podjarny
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00931 • Publication Date (Web): 13 Aug 2019
Downloaded from pubs.acs.org on August 13, 2019
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TITLE PAGE
Discovery of N-substituted 3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acids as
highly potent third generation inhibitors of human arginase I and II
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Michael C. Van Zandt, * G. Erik Jagdmann, Darren L. Whitehouse, Minkoo Ji, Jennifer
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‡
++
++
Savoy, Olga Potapova, Alexandra Cousido-Siah, Andre Mitschler, Eduardo I.
#
‡
++
Howard, Anna Marie Pyle, and Alberto D. Podjarny
New England Discovery Partners, 23 Business Park Drive, Branford, CT 06405 USA
Department of Molecular, Cellular and Developmental Biology, and Department of Chemistry,
Howard Hughes Medical Institute, Yale University, 219 Prospect St, New Haven, CT 06511
Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg, 1 rue
Laurent Fries, 67404 Illkirch, France
Instituto de Fisica de Liquidos y Sistemas Biologicos, IFLYSIB - CONICET. Calle 59 Numero
7
89, 1900 La Plata, Buenos Aires, Argentina
*
To whom correspondence should be addressed. Tel: +1 203 208 2523; Fax: +1 203 208 2524. E-mail:
mvanzandt@nedp.com
†
New England Discovery Partners
‡
Yale University
+
+
Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg
#Instituto de Fisica de Liquidos y Sistemas Biologicos, IFLYSIB - CONICET. Calle 59 Numero 789, 1900 La Plata,
Buenos Aires, Argentina
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ABSTRACT
Recent efforts to identify new highly potent arginase inhibitors have resulted in the
discovery of a novel family of (3R,4S)-3-amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acid
analogs with up to a 1,000-fold increase in potency relative to the current standards, 2-amino-6-
boronohexanoic acid (ABH) and N-hydroxy-nor-l-arginine (nor-NOHA). The lead candidate, with
a N-2-amino-3-phenylpropyl substituent (NED-3238), example 43, inhibits arginase I and II with
IC50’s of 1.3 nM and 8.1 nM respectively. Herein we report the design, synthesis and structure-
activity relationships for this novel series of inhibitors, along with X-ray crystallographic data for
selected examples bound to human arginase II.
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NH2
CO H
2
N
B(OH)2
H N
2
Ph
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INTRODUCTION
Arginase is an ureohydrolase enzyme that converts L-arginine to urea and L-ornithine,
thereby playing a key role in the urea cycle and in the regulation of nitric oxide (NO) homeostasis
in mammals. There are two isoforms of the enzyme that differ in tissue specificity and
subcellular distribution. Arginase I (Arg I) is a primarily expressed in the liver, while Arginase II
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(Arg II) is expressed in other organs and in the pituitary and thyroid glands. Although these
isoforms are expressed from two different genes, Arg I and Arg II have similar enzymatic
activities and they share an almost identical molecular structure in which a binuclear manganese
cluster within the active-site plays a key role in chemical catalysis.1
It is becoming increasingly clear that the pathways for regulation of L-arginine metabolism
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5
are linked with a diversity of cardiovascular, anti-inflammatory , autoimmune, oncological, and
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infectious diseases. In many of these disorders, arginase is specifically dysregulated and its role
in the urea cycle or NO homeostasis is disrupted. Specifically, disease pathologies frequently stem
from a marked increase in arginase expression, leading to an increase in the production of
polyamines, ornithine, and proline, along with a concomitant decrease in NO production.7
Efforts to understand the specific pharmacological role that arginase plays in diverse
diseases have been limited by the availability of potent drug-like inhibitors. To date, much of the
work in this area has relied on the modestly active first-generation arginase inhibitors such as (s)-
2
-amino-6-boronohexanoic acid (ABH), (s)-(2-boronoethyl)-L-cysteine (BEC) and N-hydroxy-
nor-l-arginine (nor-NOHA) (Figure 1).
Figure 1.
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NH2
NH
NH2
NH2
S
OH
HO C
N
N
HO C
B(OH)2
HO C
B(OH)2
2
2
2
H
H
ABH
BEC
nor-NOHA
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Cl
Cl
N
HO C
F2HC
B(OH)2
2
N
H N CO H
(HO) B
2
2
2
H N
2
B(OH)2
Compound 9
HO C NH2
2
FABH
Compound 2e
Figure 1. Early arginase inhibitors
Although significant potency improvements were obtained with second-generation ,-
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9
10
disubstituted amino acid inhibitors such as FABH, Compound 9 and Compound 2e, further
optimization is needed. In our ongoing efforts to identify new, more potent and drug-like arginase
inhibitors, we envisioned a novel ring-constrained series of boronic acid-based analogs where the
critical elements of the pharmacophore would be fixed in a preferred binding orientation. Addition
of an entropically-favorable ring constraint would potentially result in a new, significantly more
potent class of arginase inhibitors. Herein we report our initial efforts to design, synthesize and
evaluate a third-generation class of boronic acid-based arginase inhibitors and the discovery of
NED-3238, which is the most potent arginase inhibitor reported to date.
INHIBITOR DESIGN AND SYNTHESIS
1
1
9,10
Evaluation of ABH and our related second-generation series of analogs co-crystalized
with human arginase I (Arg I) and human arginase II (Arg II) led to the discovery that a constraint
can be introduced by generating a five-membered ring that connects the first carbon of the ABH
boronic acid side chain with the amino acid moiety. When the amine and the boronic acid side
chain are positioned in an anti-orientation, conformational entropy is reduced without causing
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unfavorable van der Waals interactions with active site residues (Scheme 1). In addition, atoms
within the ring can function as a scaffold for the introduction of additional substituents that can
engage in hydrogen bonds with Asp 200 and Asp 202 (Arg II), forming active-site interactions
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analogous to those observed previously9.
Scheme 1.
H N CO H
H N CO H
2 2
CO H
Ring
Constrained
2
2
New
Substituents
2
B(OH)2
Anti-configuration
B(OH)2
Target molecules
H N
2
B(OH)2
X
Reduced
Entropy
Add H-bonding
Interactions
ABH
A cyclopentane derivative was selected as an ideal model compound to test this hypothesis.
Since the three-carbon bridge would not be anticipated to have direct binding interactions with
active site amino acids, any increase in potency could be attributed to the reduced entropy resulting
from the ring constraint. This model compound was efficiently prepared using the chemistry
illustrated in Scheme 2. Diekmann cyclization of diallyl adipate followed by palladium-mediated
decarboxylative rearrangement gives the allyl ketone 3 in 56% yield (2 steps). The amino acid
moiety is formed via the Ugi reaction with t-BuNC and NH OAc in trifluoroethanol, resulting in
4
amino acid derivatives 4 and 5 as a mixture of anti- and syn-products that are easily separable by
normal phase column chromatography. The desired anti-isomer, with the acetamide opposite the
allyl sidechain, is isolated in 36% yield. Subsequent treatment with pinacolborane using
Ir Cl (COD) ethylenebis(diphenylphosphine) in dichloromethane gives exclusively the desired
2
2
2
product with the anti-Markovnikov addition to the alkene. Deprotection with a mixture of 9 N
o
hydrochloric acid and acetic acid at 130 C gives the target compound (7) in approximately 9%
overall yield from diallyl adipate. The syn-isomer (9) is prepared using the same method as
described for intermediate 5.
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Scheme 2.
O
O
O
a
b
O
O
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O
7
8%
71%
O
1
2
O
3
NHAc
CONHtBu
NHAc
CONHtBu
Bpin
NH2
c
d
e
CO H
2
B(OH)2
4: anti-isomer (36%)
5: syn-isomer (40%)
6: anti-isomer (78%)
8: syn-isomer (74%)
7: anti-isomer (55%)
9: syn-isomer (93%)
o
Reagents and conditions: (a) LiHMDS, THF, 0 C; (b) Pd(OAc) , PPh THF; (c) t-BuNC,
2
3
CF CH OH, NH Ac, room temperature, 3 days; (d) pinacolborane, Ir Cl (COD) , dppe,
3
2
4
2
2
2
o
dichloromethane; (e) 9 N HCl, AcOH, 130 C, 12 h.
The single enantiomers were also prepared via the Ugi reaction. As illustrated in Scheme
3, use of (S)--methylbenzylamine with glacial acetic acid and tert-butylisonitrile gives
diastereomers 10 and 11 which, after separation using normal phase column chromatography, are
isolated in 64% and 26% yield respectively. It is interesting to note that use of the chiral amine in
the Ugi reaction not only facilitates separation of the R and S-amino acids, but also eliminates
formation of the undesired syn-diastereomers. With diastereomers 10 and 11 separated,
hydroboration followed by cleavage of the chiral auxiliary using sodium in liquid ammonia and
hydrolysis gives the individual enantiomers 13 and 15 in 27% and 10% yield respectively from
ketone 3.
Scheme 3.
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Ac
Ac
NH2
CO2H
N
Ph
N
Ph
b
c,d
6
4%
CONHtBu
CONHtBu
B(OH)2
83%
Bpin
50%
1
0
12
13
a
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(+/-) 3
Ac
Ac
NH2
N
Ph
N
Ph
CONHtBu
Bpin
b
c,d
2
6%
CO H
2
CONHtBu
B(OH)2
6
8%
56%
11
14
15
Reagents and conditions: (a) t-BuNC, (S)-a-methylbenzylamine, AcOH, CF CH OH, RT, 3 days
3
2
(b) pinacolborane, Ir Cl (COD) , dppe, dichloromethane; (c) Na, ammonium; (d) 9 N HCl,
2
2
2
o
AcOH, 130 C, 12 h.
Based on X-ray crystallography data and screening results from our previous series, we
hypothesized that significant potency gains could be achieved by introducing a basic amine
substituent to the ring opposite the amino acid moiety to form hydrogen / ionic bonding interactions
with Asp 200 and Asp 202 (Arg II). To facilitate synthesis of these new targets, we selected a
pyrrolidine-based ring system that would provide convenient access to a versatile late-stage
intermediate that would facilitate lead optimization.
The pyrrolidine-based arginase inhibitors were synthesized using the chemistry outlined in
Scheme 4. Commercially available boc-protected pyrrolidine epoxide 16 was treated with allyl
magnesium bromide in diethylether to give alcohol 17. Subsequent oxidation with sulfur trioxide
pyridine complex gives the corresponding allyl ketone (18) in 79% yield (from epoxide). Treatment
with our standard Ugi reaction conditions gives the protected amino acid as a 10:1 mixture of anti
and syn-diastereomers that can be separated by normal phase column chromatography or
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recrystallization. The desired anti-diastereomer is resolved into its individual enantiomers using a
selective crystallization of the diastereomeric salts formed with dibenzoyl-L-tartrate [(2S,3S)-2,3-
bis(benzoyloxy)-4-(isopropylamino)-4-oxobutanoic acid] in a methanol/isopropanol mixture.
Using this procedure, the desired enantiomer 21 is obtained in 77% theoretical yield with an
enantiomeric excess of 99.7% as determined by chiral HPLC after reintroduction of the boc-group.
Subsequent hydroboration and hydrolysis gives unsubstituted pyrrolidine 23 in 68% yield (2 steps).
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Scheme 4.
NHAc
CONHtBu
NHAc
CONHtBu
OH
O
a
b
e, f
c
Boc
N
O
Boc
N
Boc N
R
d
N
Boc N
87%
91%
63%
16
17
18
19: R = Boc
0: R = H
21
2
g
86%
NH2
CO2H
NHAc
NHAc
CONHtBu
Bpin
NHAc
h
CONHtBu
Bpin
i
N
N
B(OH)2
d
CONHtBu
HN
Boc N
86%
2 steps)
Bpin
1
00%
N
N
(
26
25
24
22
h
79%
O
O
S
NH2
CO2H
NHAc
CONHtBu
Bpin
j
O
NH2
N
h
N
N
Boc
Me
CO2H
B(OH)2
HN
H2N
56%
(2 steps)
BocHN
B(OH)2
27
Me
Me
29
28
23
o
Reagents and conditions: (a) allyl magnesium bromide, diethyl ether, 0 C; (b) sulfur trioxide-
o
pyridine complex, DMSO, diisopropylethylamine, dichloromethane, 0 C; (c) NH Ac, t-BuNC,
4
CF CH OH, room temperature, 3 days; (d) TFA, dichloromethane; (e) (2S,3S)-2,3-
3
2
bis(benzoyloxy)-4-(isopropylamino)-4-oxobutanoic acid, IPA/ MeOH; (f) BOC O, aq NaHCO ,
2
3
ethyl acetate; (g) pinacolborane, Ir Cl (COD) , dppe, dichloromethane; (h) 9 N HCl, AcOH, 130
2
2
2
o
C, 12 h; (i) RCHO, Na(OAc) BH; (j) acetonitrile.
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Selective removal of the boc-group from intermediate 22 using trifluoroacetic acid gives the
corresponding amine (24) which is a highly versatile intermediate useful for a variety of alkylation,
acylation and reductive amination reactions. As an example of the reductive amination reaction,
treatment of nicotinaldehyde with sodium triacetoxyborohydride gives substituted amine 25.
Hydrolysis using our standard conditions gives 3-pyridyl analog 26 in 86% yield (2 steps) after
purification by reverse phase HPLC. When the desired aldehyde, such as N-boc-(R)-2-
aminopropanal, contains a chiral center adjacent to the aldehyde, the reductive amination reaction
results in significant epimerization. To avoid this racemization, the methyl piperidine substituent
can be introduced via alkylation chemistry using the corresponding cyclic sulfamate (27) which is
prepared from N-boc-(R)-2-aminopropan-1-ol using the general method described by Alker.12
Treatment of pyrrolidine intermediate 24 with sulfamate 27 in acetonitrile gives amine 28, the
alkylation product. Subsequent hydrolysis gives the target 2-aminopropane (29) as a single
enantiomer in 10 steps with an approximate overall yield of 9%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RESULTS AND DISCUSSION
All program compounds were tested for arginase activity using human recombinant Arg I
and Arg II using a colorimetric assay that has been previously described.13 Results from these
experiments for selected examples are listed in Table 1. As hypothesized, introduction of the five-
membered ring constraint resulted in improved potency relative to ABH, the parent compound.
Racemic analog 8, with the amine and butane boronic acid side chain in the anti-orientation has
IC s of 380 nM and 1,280 nM for Arg I and Arg II respectively. More than 100-fold loss in Arg I
5
0
potency is observed when these groups are in a syn-orientation as seen with Compound 9 (Arg I
48.0 µM; Arg II 79.2 µM). The single anti-enantiomer with the amino acid in the S-configuration
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9
(
13) is approximately 2-fold more active than the corresponding racemic compound. If a nitrogen
atom is introduced in the center of the ring constraint (23) activity is increased about 2-fold relative
to 13, the corresponding all-carbon example (23, Arg I 110 nM; Arg II 440 nM). Figure 2 illustrates
crystal structures of example 23 superimposed with ABH, each in their active conformations when
bound to Arg II. The ring constraint allows for positioning of the amino acid and boronic acid
moieties in nearly the same anti-orientation as observed with the unconstrained scaffold with less
entropic cost. Also indicated in the figure is an additional contact between the pyrrolidine nitrogen
and Asp 200, mediated by the water molecule W1 (H-bonds of 2.66 Å and 2.78 Å). This additional
contact is likely responsible for the 2-fold increase in potency of 23 relative to the cyclopentane
derivative (13).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Superposition of ABH (yellow, IC : 2,551 nM) and constrained analog 23 (PDB ID
5
0
6
Q37, cyan, IC : 440 nM) bound in the arginase II active site pocket.
50
Substitution of the pyrrolidine nitrogen with a simple isobutyl (30) or benzyl group (31) is
tolerated but does not provide an improvement in activity. Similar results were observed for the
pyridine derivatives 26, 32, 33 though a trend is observed where potency increases for both Arg I
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9
and II as the pyridine nitrogen moves from the para- to meta- and ortho-positions. Introduction of
the 6-fluoro-substituent to the 2-pyridine results in about a 2-fold loss in activity, suggesting that
potency is decreased with reduced nitrogen basicity. This decrease is most likely due to the
electrostatic interaction with Asp 202 and/or Asp 200 strengthening with increasing nitrogen
basicity. Addition of a 4-substituted pyrazole (35 and 36) or a 5-substituted thiazole (38) did not
improve potency relative to the unsubstituted benzyl analog (31) indicating no significant hydrogen
bonding interactions from the heteroatoms. Methylimidazole example 37 is about 3-fold more
active than the benzyl analog suggesting a beneficial interaction, likely a weak electrostatic
interaction with Asp 200. A dramatic increase in potency is observed with examples containing a
basic primary or secondary amine positioned to make electrostatic contacts with Asp 202 and/or
Asp 200. Examples 29, 39-43 where a 2-aminoethyl moiety is substituted with a methyl (R-29, S-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
9), phenyl (S-40, R-41) or benzyl (R-42, S-43) moiety at the 2-position are all exceptionally potent,
with the more active R-enantiomers having Arg I IC values of 7.7 nM, 2.5 nM and 1.3 nM,
5
0
respectively. These are, by far, the most potent arginase inhibitors ever reported. Consistent with
other examples in the series, the corresponding IC values for Arg II are 2 to 4-fold less active.
5
0
Excellent potency is maintained when the basic amine is incorporated into a ring such as a
morpholine, pyrrolidine or piperidine. With a morpholine, the R-enantiomer (44; Arg I IC 10 nM,
5
0
Arg II IC 27 nM) is 2-fold more potent than the S-enantiomer (45; Arg I IC 19 nM, Arg II IC
50
5
0
50
6
5 nM). When the amine is used to form a pyrrolidine the S-enantiomer (46; Arg I IC 11 nM,
50
Arg II IC 47 nM) and R-enantiomer (47; Arg I IC 10 nM, Arg II IC 37 nM) are nearly
5
0
50
50
equipotent. In contrast, when the ring is expanded to form a piperidine, the R-enantiomer (49; Arg
I IC 2.6 nM, Arg II IC 14 nM) is approximately 10-fold more potent than the corresponding S-
5
0
50
enantiomer (48; Arg I IC 30 nM, Arg II IC 95 nM).
5
0
50
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Analysis of example 49 co-crystallized with Arg II demonstrates a hydrogen bonding
network between aspartic acid residues of the protein and the piperidine ring nitrogen (Figure 3).
The piperidine nitrogen forms a direct hydrogen bond with Asp 200 (3.11 Å) and a second
interaction with Asp 202 that is mediated by water molecule W2 (2.80 Å and 2.77 Å). It is
interesting to note that the piperidine nitrogen in example 49 displaces water molecule W1 in
example 23. Which enables a direct contact with Asp 200, and not through a water molecule. This
likely explains the large increase in potency (Arg II IC of 440 nM for 23 vs 14 nM for 49). A 2D
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
diagram of the contacts and the details of the inhibitor density in the initial difference maps are
given in the supporting information.
Figure 3.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Crystal structures of example 49 (6Q39, grey, IC : 14 nM) bound in the Arg II active
5
0
site pocket (green). Positions of ionic and hydrogen bonds (yellow) are indicated, along with bond
distances (black). Example 23 is shown in cyan (6Q37, IC : 440 nM).
5
0
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1
NH2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
CO H
2
R X
B(OH)2
Inh. IC50b
Cmpd.
R
X
Isomera
ARG I
ARG II
8
H
CH
(±-Anti
380 nM
1,280 nM
9
H
H
H
H
CH
CH
CH
N
(±-Syn
1S,2S
1R,2R
1S,2S
1S,2S
48.0 µM
210 nM
inactive
110 nM
390 nM
79.2 µM
630 nM
inactive
440 nM
1,610 nM
13
1
2
3
5
3
0
N
3
3
1
2
N
N
N
N
1S,2S
1S,2S
1S,2S
1S,2S
300 nM
600 nM
470 nM
380 nM
1,540 nM
1,860 nM
1,370 nM
1,270 nM
N
N
26
3
3
3
4
N
F
N
1S,2S
670 nM
2,160 nM
N
N
3
3
5
6
N
N
1S,2S
1S,2S
400 nM
340 nM
2,180 nM
2,230 nM
NH
N
N
3
3
7
8
N
N
N
1S,2S
1S,2S
100 nM
360 nM
330 nM
850 nM
N
S
N
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9
Me
2
3
4
4
4
9
9
0
1
2
N
N
N
N
N
1S,2S
1S,2S
1S,2S
1S,2S
1S,2S
16 nM
7.7 nM
18 nM
2.5 nM
10 nM
37 nM
16 nM
62 nM
9.5 nM
27 nM
NH2
Me
NH2
Ph
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
Ph
NH2
Ph
NH2
Ph
4
3
N
N
1S,2S
1S,2S
1.3 nM
10 nM
8.1 nM
27 nM
(
NED-3238)
NH2
O
4
4
4
5
N
H
O
N
1S,2S
19 nM
65 nM
N
H
46
N
N
N
1S,2S
1S,2S
1S,2S
11 nM
10 nM
30 nM
47 nM
37 nM
95 nM
N
H
4
4
7
8
N
H
N
H
4
9
N
1S,2S
2.6 nM
14 nM
N
H
ABH
Nor-NOHA
1,547 nM
1,360 nM
2,551 nM
1,260 nM
a
All compounds are prepared as single enantiomers with S-configuration (S), R-configuration
(R) or as racemates (±). Stereochemistry of propylboronic acid side chain relative to carboxylic
b
acid. Arginase IC50 measurements using human recombinant protein (duplicate).
Figure 4.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Superposition of the complex Arg II-Example 49 (6Q39, green protein carbons, grey
inhibitor carbons) with the Arginase I unliganded structure (PDB entry 2ZAV, colored in
cyan). Note the rotamer change of Thr 265, which opens the place for water 2604 in the Arg I
structure. This water, which is not present in the Arg II complex, makes an H-bond with Asp 200
and is well positioned to make a contact with the inhibitor Example 49 (3.0 A distance).
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9
As shown in Table 1, compounds in this series are generally 3~5 times more potent against Arg I
than Arg II, while nor-NOHA is roughly equally potent with both enzymes. In an attempt to better
understand this, the structure of the complex Arg II-Example 49 was superimposed with the
nonliganded structure of Arg I (pdb entry 2ZAV, see figure 4). The superposition showed a change
in the rotamer of Thr 265 in Arg II (Thr 246 in ARG I) which opens a space near the inhibitor
occupied by water molecule 2604 in Arg I. This water molecule is well positioned to make a close
contact with the inhibitor, which could explain the increase in potency. On the other hand, in the
structure of Arg I with nor-NOHA (pdb entry 1HQH), Thr 246 makes an H-bond with nor-NOHA,
fixing it in the same rotamer as that observed for Arg II, in agreement with the observed potency
values.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSIONS
In conclusion, a novel series of highly potent 3-amino-4-(3-boronopropyl) pyrrolidine-3-
carboxylic acid-based inhibitors of arginase have been identified. Their increase in potency relative
to the parent ABH template results from at least two structural changes in the scaffold: introduction
of a ring constraint for reduction of conformational entropy, and incorporation of a basic amine side
chain that forms additional ionic interactions with aspartic acids in the active site. For our lead
compound, example 43 (NED-3238), the Arg I potency enhancements for these modifications are
approximately 14-fold and 84-fold respectively, and when these values are combined, they
represent a 1,190-fold enhancement relative to ABH. In addition to being exquisitely potent,
compounds in this series also have physical-chemical properties consistent with drug-like
structures.14 It is hoped that this exciting new class of inhibitors will not only provide a path to
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
important new therapeutics, but will also facilitate our understanding of arginine metabolism and
the role played by arginase in health and disease.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Chemistry. General methods. Melting points were determined in open capillary
tubes on a Thomas-Hoover apparatus and are uncorrected. Proton nuclear magnetic
1
resonance ( H NMR) spectra were determined on Agilent DD2 400 MHz, 500 MHz or 600
MHz NMR spectrometers. Chemical shifts are reported in parts per million (ppm)
downfield from tetramethylsilane (internal standard) with coupling constants in hertz (Hz).
Multiplicity is indicated by the following abbreviations: singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), broad (br). Mass spectra were recorded using an Advion
Expression CMS mass spectrometer coupled with an Agilent 1260 analytical HPLC.
Elemental analyses (C,H,N) were performed by Atlantic Microlab, Inc. (Norcross, Georgia)
and are within ±4% of theory unless otherwise noted. Intermediate products from all
reactions (non-polar compounds) were purified by either flash column chromatography or
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medium pressure liquid chromatography using a Biotage Isolera One with SNAP cartridge
unless otherwise indicated. All final products were purified on a Gilson 215 robotic liquid
handler equipped with a Gilson ELSD detector and Phenomenex Synergi 4  Polar-RF
preparative column (250 mm x 21.2 mm) or a Shimadzu Prominence LC-20AP preparative
HPLC equipped with a Shimadzu ELSD-LT II using a Phenomenex Synergi 10  Polar-RF
preparative column (250 mm x 50 mm). Analytical purity for all final compounds was
determined to be > 99% by HPLC. This analysis was completed using an Agilent Eclipse
XDB-Phenyl analytical HPLC column (3.5 m; 4.6 x 150 mm) with a evaporative light
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
scattering detector.
Thin-layer chromatography using glass-backed silica plates
containing a fluorescent indicator (0.25 mm, Whatman, Merck) was used to monitor
reactions. Chromatograms were visualized using ultraviolet illumination, exposure to
iodine vapors, or by dipping in an aqueous potassium permanganate solution. All starting
materials were used without further purification. All reactions were carried out under an
atmosphere of dried nitrogen or argon. Compound names are derived from the structures
using ChemDraw Ultra 14.0.
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1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General Procedures for the Synthesis of the 1-Amino-2-(3-boronopropyl)cyclopentane-1-
carboxylic acids and 3-Amino-4-(3-boronopropyl)pyrrolidine-3-carboxylic acids. The 1-
amino-2-(3-boronopropyl)cyclopentane-1-carboxylic acids in Tables 1 were prepared
using the methods illustrated in Schemes 1 and 2 which are detailed below. The 3-amino-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-(3-boronopropyl)pyrrolidine-3-carboxylic acids were prepared using the method
described in Scheme 4. Details for compounds 23, 26 and 43 are provided below.
Analytical data for the remaining compounds are provided in the supplementary material.
O
O
2
O
Preparation of anti-1-amino-2-(3-boronopropyl)cyclopentane-1-carboxylic acid (8).
Step 1: synthesis of allyl 2-oxocyclopentanecarboxylate (2). A stirred solution of diallyl adipate
o
(
4.53 g, 20 mmol) in anhydrous tetrahydrofuran (100 mL) was cooled to 0 C and treated with
lithium bis(trimethylsilyl)amide (40 mL, 1.0 N in THF, 40 mmol). After the addition was complete,
o
the solution was warmed to room temperature, stirred for an additional 2 h, re-cooled to 0 C and
quenched with acetic acid (2.53 mL, 44 mmol) in a dropwise manner. The turbid mixture was
warmed to room temperature and filtered and concentrated. Purification by flash column
chromatography (silica gel, dichloromethane) afforded allyl 2-oxocyclopentanecarboxylate, 2 (2.62
1
g, 78%) as a colorless oil. H NMR (CDCl , 600 MHz)  5.90 (ddt, J = 16.5 Hz, J = 10.9 Hz, J
3
1
2
3
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=
5.7 Hz, 1 H), 5.33 (dd, J = 17.2 Hz, J = 1.9 Hz, 1 H), 5.23 (d, J = 10.4 Hz, 1 H), 4.63 (t, J =
1
2
6
.1 Hz, 1 H), 3.17 (t, J = 9.0 Hz, 1 H), 2.34 – 2.27 (m, 4 H), 2.16 – 2.09 (m, 1 H), 1.91 – 1.82 (m,
1
3
1 H); C NMR (600 MHz, D O): δ 212.26, 169.15, 131.82, 118.59, 65.97, 54.83, 38.17, 27.51,
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
2
1.07.; MS (CI): m/z for C H O : expected 168.1; found 169.1 (M+H) . Anal. Calcd for C H O :
9
12
3
9
12
3
C 64.27; H, 7.19. Found C, 63.89; H, 7.05.
O
3
Step 2: synthesis of 2-allylcyclopentanone (3). While under a nitrogen atmosphere, a
solution of palladium(II) acetate (51 mg, 0.23 mmol) and triphenylphosphine (0.24 g, 0.9 mmol) in
o
anhydrous THF (20 mL) was heated to 65 C and treated with a second solution of allyl 2-
oxocyclopentanecarboxylate (2, 2.52 g, 15 mmol) in anhydrous THF (rapid gas evolution upon
o
addition). After 45 minutes at 65 C, the reaction mixture was cooled and concentrated.
Purification by flash column chromatography (silica gel, dichloromethane) afforded 2-
1
allylcyclopentanone (1.32 g, 71%) as a colorless oil. H NMR (CDCl , 600 MHz)  5.76 (td, J =
3
1
16.8 Hz, J = 7.2 Hz, 1 H), 5.06 (d, J = 17.0 Hz, 1 H), 5.01 (d, J = 10.1 Hz, 1 H), 2.53-2.48 (m, 1
2
H), 2.31 (dd, J = 18.9 Hz, J = 8.6 Hz, 1 H), 2.22 – 1.97 (m, 5 H), 1.83-1.73 (m, 1 H), 1.62 – 1.52
1
2
1
3
(m, 1 H); C NMR (600 MHz, CDCl ): δ 220.72, 136.05, 116.52, 48.73, 38.31, 34.02, 29.10, 20.78.
3
NHAc
CONHtBu
NHAc
CONHtBu
4
5
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Step 3: synthesis of anti-1-acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide (4)
and syn-1-acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide (5). A solution of 2-
allylcyclopentanone, 3 (0.993 g, 8 mmol) and ammonium acetate (1.54 g, 20 mmol) in 2,2,2-
trifluoroethanol (2.5 mL) was treated with tert-butyl isocyanide (1.81 mL, 16 mmol). After stirring
at room temperature for 4 days, the solution was concentrated. Purification by flash column
chromatography (silica gel, 60% ethyl acetate in heptane) afforded first, the anti-isomer, 4 (0.771g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
6%), then after increasing the polarity of the eluent (80% ethyl acetate in hexanes) the later
1
migrating syn-isomer, 5 (0.851g, 40%), both obtained as white solids. Anti-isomer (4): H NMR
(
600 MHz, CDCl ): δ 6.47 (brs, 1 H), 6.21 (brs, 1 H), 5.75-5.65 (m, 1 H), 4.99 (d, J = 18.4 Hz, 1
3
H), 4.97 (d, J = 11.0 Hz, 1 H), 2.46 (dtd, J = 11.5 Hz, J = 7.4 Hz, J = 4.3 Hz, 1H), 2.40 (dt, J
1
1
2
3
=
2
14.1 Hz, J = 8.8 Hz, 1 H), 2.25-2.18 (m, 1 H), 2.02-1.98 (m, 1 H), 1.97 (s, 3 H), 1.91-1.80 (m,
2
1
3
H), 1.79-1.72 (m, 2 H), 1.51-1.45 (m, 1 H), 1.32 (s, 9 H); C NMR (600 MHz, CDCl ): δ 171.47,
3
170.44, 137.15, 116.20, 70.85, 51.36, 46.66, 34.97, 34.38, 29.45, 28.81, 24.28, 21.56.; MS (CI):
m/z for C H N O : expected 266.2; found 289.1 (M+Na), 166.1 (M-Boc+1), 265.1 (M-1); Anal.
1
5
26
2
2
Calcd for C H N O : C 67.63; H, 9.84; N, 10.52. Found C, 67.57; H, 9.95; N, 10.26. Syn-isomer
1
5
26
2
2
1
(
5): H NMR (600 MHz, CDCl ): δ 6.80 (brs, 1 H), 5.97 (brs, 1 H), 5.77 (ddt, J = 17.1 Hz, J =
3
1
2
1
2
1
0.0 Hz, J = 6.9 Hz, 1 H), 5.04 (dd, J = 17.1 Hz, J = 1.6 Hz, 1 H), 4.99 (d, J = 10.1 Hz, 1 H),
3 1 2
.48 (tdd, J = 9.2 Hz, J = 7.6 Hz, J = 4.6 Hz, 1 H), 2.31-2.15 (m, 3 H), 1.99 (s, 3 H) , 1.91-
1
2
3
1
3
.81 (m, 2 H), 1.70-1.59 (m, 2 H ), 1.51-1.42 (m, 1 H), 1.30 (s, 9 H); C NMR (600 MHz, CDCl ):
3
δ 172.50, 170.79, 137.03, 116.29, 69.90, 51.03, 46.38, 34.50, 34.31, 29.10, 28.71, 24.18, 21.58.;
MS (CI): m/z for C H N O : expected 266.2; found 289.1 (M+Na), 166.1 (M-Boc+1), 265.1 (M-
1
5
26
2
2
1
); Anal. Calcd for C H N O : C 67.63; H, 9.84; N, 10.52. Found C, 67.80; H, 9.99; N, 10.35.
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NHAc
CONHtBu
Bpin
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Step 4: synthesis of anti-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)propyl)cyclopentanecarboxamide (6). While under nitrogen, a stirred
solution of anti-1-acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide, 4 (0.599 g, 2.25 mmol)
in anhydrous methylene chloride (9 mL) was treated with Ir Cl (COD) (45 mg, 0.07 mmol) and
2
2
2
DiPhos (54 mg, 0.136 mmol) and stirred at room temperature for 30 min. pinacolborane (0.65 mL,
4.48 mmol) was added dropwise and stirring was continued at room temperature for 20 h. The
reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (40 mL, then 2 x
1
5 mL), and the combined organic solution was washed with saturated aqueous sodium chloride
(30 mL), dried over MgSO , and concentrated. Purification by flash column chromatography (silica
4
gel, 40-50% ethyl acetate in heptane) afforded anti-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentanecarboxamide, 6 (0.695g, 78%) as a white
1
solid. H NMR (600 MHz, CDCl ): δ 6.22 (brs, 1 H), 6.13 (brs, 1 H), 2.44-2.30 (m, 2 H), 2.07-
3
1
9
1
.99 (m, 1 H), 1.98 (s, 3 H) , 1.97-1.91 (m, 1 H), 1.83-1.74 (m, 2 H ), 1.51-1.36 (m, 3 H), 1.30 (s,
1
3
H), 1.22 (s, 12 H), 1.15-1.06 (m, 1 H), 0.82-0.67 (m, 2 H ); C NMR (600 MHz, CDCl ): δ
3
71.78, 170.13, 83.06, 70.81, 51.34, 47.50, 34.47, 33.13, 29.96, 28.83, 24.94, 24.32, 22.99, 21.97,
11.53.; MS m/z for C H BN O : expected 394.3; found 417.2 (M+Na), 395.3 (M+1), 393.2 (M-
2
1
39
2
4
1
); Anal. Calcd for C H BN O : C 63.96; H, 9.97; N, 7.10. Found C, 64.06; H, 10.09; N, 7.22.
21 39 2 4
NH2
CO H
2
B(OH)2
7
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9
1
1
1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
Step 5: synthesis of anti-1-amino-2-(3-boronopropyl)cyclopentanecarboxylic acid (7).
A solution of anti-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)propyl)cyclopentanecarboxamide, 6 (400 mg, 1.01 mmol) in concentrated hydrochloric acid (1
mL), acetic acid (1 mL) and water (2 mL) in a pressure bottle was stirred for 30 min at 60°C, then
capped and stirred for 18 h at 130 °C, cooled to room temperature, and uncapped. The solution was
diluted with water (20 mL), extracted with toluene (20 mL) and concentrated. Purification by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
preparative HPLC afforded the desired product (55 mg, 55%) as an off-white foam. H NMR (600
MHz, D O): δ 2.40 (dt, J = 14.3 Hz, J = 8.5 Hz, 1 H), 2.15-2.04 (m, 2 H), 1.97-1.85 (m, 2 H),
2
1
2
1
.83-1.71 (m, 1 H), 1.54-1.41 (m, 3 H ), 1.36-1.25 (m, 1 H), 1.24-1.13 (m, 1 H), 0.76 (ddd, J =
1
15.1 Hz, J = 8.8 Hz, J = 6.2 Hz, 1 H), 0.70 (ddd, J = 15.1 Hz, J = 9.1 Hz, J = 6.1 Hz, 1 H); 13C
2
3
1
2
3
NMR (600 MHz, D O): δ 173.78, 68.07, 49.21, 34.84, 31.66, 30.62, 22.64, 22.42, 14.02.; MS (CI):
2
m/z for C H BNO : expected 215.1; found 198.1 (M-H O+1).
9
18
4
2
NHAc
CONHtBu
Bpin
8
Preparation of syn-1-amino-2-(3-boronopropyl)cyclopentane-1-carboxylic acid (9).
Step 1:
syn-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)propyl)cyclopentanecarboxamide (8). While under nitrogen, a stirred solution of syn-1-
acetamido-2-allyl-N-tert-butylcyclopentanecarboxamide, 5 (0.285 g, 1.07 mmol) in anhydrous
methylene chloride (5.5 mL) was treated with Ir Cl (COD) (22 mg, 0.032 mmol) and 1,2-
2
2
2
bis(diphenylphosphino)ethane (26 mg, 0.06 mmol). After stirring at room temperature for 30 min,
o
the solution was cooled to -10 C (ice/ethanol bath) and treated with pinacolborane (0.23 mL, 1.61
mmol) dropwise. After the addition was complete, the bath was allowed to warm to room
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temperature with stirring for 20 h. The reaction mixture was poured into water (20 mL) and
extracted with ethyl acetate (40 mL, then 2 x 15 mL), and the combined organic solution was
washed with saturated aqueous sodium chloride (30 mL), dried over MgSO , and concentrated.
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Purification by flash column chromatography (silica gel, 40-50% ethyl acetate in heptane) afforded
syn-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)propyl)cyclopentanecarboxamide, 7 (0.313g, 74%) as a white solid. 1H NMR (600 MHz,
CDCl ): δ 6.71 (bs, 1 H), 5.71 (bs, 1 H), 2.39-2.31 (m, 2 H), 2.13 (dt, J = 13.6 Hz, J = 7.4 Hz, 1
3
1
2
H), 2.01 (s, 3 H) , 1.95 (tt, J = 13.6 Hz, J = 7.4 Hz, 1 H), 1.68-1.60 (m, 2 H), 1.52-1.43 (m, 2
1
2
1
3
H), 1.42-1.32 (m, 2 H), 1.30 (s, 9 H) , 1.22 (s, 12 H), 1.13-1.02 (m, 1 H), 0.76 (m, 2 H); C NMR
(600 MHz, CDCl ): δ 172.49, 170.75, 83.08, 70.02, 50.99, 47.56, 34.35, 32.50, 29.67, 28.74, 24.94,
3
2
3
4.34, 22.97, 21.90, 11.72.; MS (CI): m/z for C H BN O : expected 394.3; found 417.2 (M+Na),
21 39 2 4
95.3 (M+1), 393.2 (M-1).
NH2
CO H
2
B(OH)2
9
Step 2: syn-1-amino-2-(3-boronopropyl)cyclopentanecarboxylic acid (9) (syn-isomer,
racemic)
A stirred mixture of syn-1-acetamido-N-tert-butyl-2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)propyl)cyclopentanecarboxamide 8 (0.627 g, 1.59 mmol) in 6 N HCl (15 mL) was heated to 90
oC for 20 h, cooled to room temperature, and diluted with water (15 mL). The mixture was extracted
with dichloromethane (2 x 15 mL) and concentrated in vacuo. The resulting residue was dissolved
in methanol (5 mL) and diluted to a volume of 40 mL with ether and stirred for 1 hour, to remove
solid tertbutylamine hydrochloride by filtration. The resulting filtrate, was concentrated, dissolved
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in saturated aqueous ammonium hydroxide (15 mL), and re-concentrated (3 x). The resulting solid
white residue was triturated using acetonitrile and dried to afford the target compound syn-1-amino-
1
2-(3-boronopropyl) cyclopentanecarboxylic acid 9 (0.397g, 93%) as a white powder. H NMR (600
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, D O): δ 2.40 (dt, J = 14.4 Hz, J = 8.5 Hz, 1 H), 2.08 (d, J = 7.1 Hz, J = 2.3 Hz, 2 H),
2
1
2
1
2
1
.96-1.84 (m, 2 H), 1.83-1.70 (m, 1 H), 1.54-1.42 (m, 3 H ), 1.36-1.25 (m, 1 H), 1.23-1.14 (m, 1
H), 0.77 (ddd, J = 15.1 Hz, J = 8.7 Hz, J = 6.2 Hz, 1 H), 0.70 (ddd, J = 15.3 Hz, J = 9.2
1
2
3
1
2
1
3
Hz, J = 6.2 Hz, 1 H); C NMR (600 MHz, D O): δ 173.82, 68.08, 49.21, 34.85, 31.67, 30.63,
3
2
+
2
2.65, 22.43, 14.02; MS (+CI): m/z for C H BNO : expected 215.1; found 215.3 (M+H) . Anal.
9 18 4
Calcd for C H BClNO : C 42.98; H, 7.61; N, 5.57. Found C, 42.69; H, 7.43; N, 5.60.
9
19
4
Ac
Ac
N
Ph
N
Ph
CONHtBu
CONHtBu
10
11
Preparation of (1S,2S)-1-amino-2-(3-boronopropyl)cyclopentane carboxylic acid (13).
Step 1: synthesis of (1S,2R)-2-allyl-N-(tert-butyl)-1-(N-((S)-1-phenylethyl)acetamido)
cyclopentanecarboxamide (10) and (1R,2S)-2-allyl-N-(tert-butyl)-1-(N-((S)-1-
phenylethyl)acetamido) cyclopentanecarboxamide (11). A stirred solution of 2-(propene-3-
yl)cyclopentanone, 3 (0.745 g, 6.0 mmol), (S)--methylbenzylamine (3.1 mL, 24 mmol) and glacial
acetic acid (1.38 mL, 24 mmol) in methanol (5 mL) was treated with tert-butylisonitrile (2.04 mL,
o
1
8 mmol) and warmed to 60 C for 2 days. After cooling to room temperature, the mixture was
concentrated, diluted with dichloromethane (60 mL) and washed successively with water (50 mL)
and saturated aqueous sodium chloride. The combined organic solution was dried over MgSO4,
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8
9
filtered and concentrated. Purification via column chromatography (silica gel, 5-30% ethyl acetate
in heptane) afforded anti-diastereomer 10 (1.42 g, 3.83 mmol, 64%) and syn-diastereomer, 11 (0.57
1
g, 1.54 mmol, 26%), both as pale-yellow viscous oils. Compound 10: H NMR (600 MHz, CDCl ):
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
δ 7.53 (d, J = 7.5 Hz, 2 H), 7.37 (t, J = 7.6 Hz, 2 H), 7.25 (m, 1 H), 6.33 (brs, 1 H), 5.74 (ddt, J =
1
1
7.1 Hz, J = 10.8 Hz, J = 7.1 Hz, 1 H), 5.01-4.91 (m, 2 H), 4.91-4.74 (m, 1 H), 3.00-2.89 (m, 1
2 3
H), 2.55-2.42 (m, 2 H), 1.99-1.86 (m, 1 H), 1.75 (d, J = 7.0 Hz, 3 H), 1.85-1.56 (m, 7 H), 1.35 (s, 9
1
3
H); C NMR (600 MHz, CDCl ): δ 174.13, 171.89, 142.98, 137.10, 128.71, 127.03, 126.63,
3
1
16.12, 53.78, 51.36, 46.25, 36.08, 29.78, 29.65, 29.52, 28.74, 25.94, 21.94, 19.81.; MS (CI): m/z
1
for C H N O : expected 370.3; found 393.8 (M+Na), 371.1 (M + 1). Compound 11: H NMR
23 34
2
2
(600 MHz, CDCl ): δ 7.36 (t, J = 7.8 Hz, 2 H), 7.30 (d, J = 7.6 Hz, 2 H), 7.26 (m, 1 H), 7.41-7.20
3
(m, 5 H), 6.00 (brs, 1 H), 5.72 (brs, 1 H), 5.10-4.79 (m, 3 H), 3.15-2.96 (m, 1 H), 2.55-2.34 (m, 1
1
3
H), 2.34-2.18 (m, 1 H), 2.09-1.74 (m, 9 H), 1.71-1.53 (m, 3 H), 1.37 (s, 9 H); C NMR (600 MHz,
CDCl ): δ 173.23, 172.11, 143.06, 136.91, 128.78, 126.91, 125.80, 116.12, 53.53, 51.46, 47.44,
3
3
3
7.15, 36.26, 30.27, 29.78, 28.72, 25.42, 22.45, 20.08.; MS (CI): m/z for C H N O : expected
23 34 2 2
70.3; found 393.2 (M+Na).
Ac
N
Ph
CONHtBu
Bpin
12
Step 2: synthesis of (1S,2S)-N-(tert-butyl)-1-(N-((S)-1-phenylethyl)acetamido)-2-(3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)cyclopentanecarboxamide (12). While
under nitrogen, (1S,2R)-2-allyl-N-(tert-butyl)-1-(N-((S)-1-phenylethyl)acetamido)
cyclopentanecarboxamide, 10 (1.00 g, 2.7 mmol) in anhydrous methylene chloride (14 mL) was
29
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