Discovery of INCB3344, a potent, selective and orally bioavailable antagonist of human and murine CCR2

Article abstract of DOI:10.1016/j.bmcl.2010.10.020

Rational design based on a pharmacophore of CCR2 antagonists reported in the literature identified lead compound 9a with potent inhibitory activity against human CCR2 (hCCR2) but moderate activity against murine CCR2 (mCCR2). Modification on 9a led to the

Full text of DOI:10.1016/j.bmcl.2010.10.020

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7473–7478
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of INCB3344, a potent, selective and orally bioavailable antagonist
of human and murine CCR2
⇑
Chu-Biao Xue , Anlai Wang, David Meloni, Ke Zhang, Ling Kong, Hao Feng, Joseph Glenn, Taisheng Huang,
Yingxin Zhang, Ganfeng Cao, Rajan Anand, Changsheng Zheng, Michael Xia, Qi Han, D. J. Robinson,
Lou Storace, Lixin Shao, Mei Li, Carrie M. Brodmerkel, Maryanne Covington, Peggy Scherle, Sharon Diamond,
Swamy Yeleswaram, Kris Vaddi, Robert Newton, Greg Hollis, Steven Friedman, Brian Metcalf
Incyte Corporation, Experimental Station E336, Wilmington, DE 19880, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Rational design based on a pharmacophore of CCR2 antagonists reported in the literature identified lead
compound 9a with potent inhibitory activity against human CCR2 (hCCR2) but moderate activity against
murine CCR2 (mCCR2). Modification on 9a led to the discovery of a potent CCR2 antagonist 21
(INCB3344) with IC50 values of 5.1 nM (hCCR2) and 9.5 nM (mCCR2) in binding antagonism and
3.8 nM (hCCR2) and 7.8 nM (mCCR2) in antagonism of chemotaxis activity. INCB3344 exhibited >
100-fold selectivity over other homologous chemokine receptors, a free fraction of 24% in human serum
and 15% in mouse serum, and an oral bioavailability of 47% in mice, suitable as a tool compound for target
validation in rodent models.
Received 9 August 2010
Revised 1 October 2010
Accepted 5 October 2010
Available online 13 October 2010
Keywords:
Human CCR2
Murine CCR2
Chemokine
Antagonist
Ó 2010 Elsevier Ltd. All rights reserved.
The migration of leukocytes from blood vessels into inflamed
tissues is involved in the initiation of a normal, disease-fighting,
inflammatory response. The correct, controlled trafficking of these
cells is an essential feature of the immune response to infection,
but loss of control results in excessive recruitment of leukocytes
to sites of inflammation, leading to onset and progression of a vari-
ety of chronic inflammatory diseases. Excessive recruitment of
monocytes, a subset of leukocytes, and macrophages, which are
derived from monocytes, to disease sites is especially harmful as
these cells are well-characterized mediators of tissue destruction
in chronic inflammatory and autoimmune diseases such as
rheumatoid arthritis and multiple sclerosis.1,2 Accordingly, pre-
venting or reducing migration of monocytes and macrophages to
these disease sites would be expected to be beneficial in these
diseases.
The infiltration of monocytes/macrophages into sites of inflam-
mation is driven by monocyte chemoattractant protein-1 (MCP-1,
CCL2) through interaction with its specific receptor, CCR2, which
is a member of the super family of seven-transmembrane G-pro-
tein-coupled receptors (GPCRs) and is predominantly expressed
on monocytes.3 Binding of MCP-1 to CCR2 induces chemotaxis,
resulting in directed migration of monocytes/macrophages to dis-
ease sites where MCP-1 expression is elevated.4 Studies in rodent
O
H
N
CF3
N
N
H
O
1
O
O
N
HN
N
N
O
2
H
N
Cl
Cl
H
N
N
O
HO
3
Figure 1. CCR2 antagonists reported in the literature.
models by genetic deletion of either MCP-15 or CCR26–8 and use
of peptidyl CCR2 antagonists9 or anti-MCP-1 antibodies10 have
demonstrated the critical role of MCP-1/CCR2 in models of rheu-
matoid arthritis,9,10 multiple sclerosis,11–13 atherosclerosis,8,14–16
and neuropathic pain,17 and strongly suggest that CCR2 is an
attractive target for potential therapeutic intervention in these
⇑
Corresponding author. Tel.: +1 302 498 6706; fax: +1 302 425 2750.
E-mail address: cxue@incyte.com (C.-B. Xue).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.020

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C.-B. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7473–7478
diseases. As a result, inhibition of CCR2 has emerged as a novel
therapeutic approach for pharmaceutical research.
O
Cbz
N
Cbz
N
H
N
Cbz
a, b
c
N
N
H
Despite the many publications on small molecule CCR2 antago-
nists,18–29 only three classes of CCR2-selective antagonists had
been disclosed at the time we initiated a CCR2 antagonist discovery
program (Fig. 1). They include Teijin compound 1,30 Roche com-
pound 231 and SmithKline Beecham compound 3,32,33 all exhibiting
moderate inhibitory activity toward human CCR2 (hCCR2). A ter-
tiary amine in the central region and two lipophilic moieties at
the ends of each molecule represent the common pharmacophore
among these inhibitors. As demonstrated by compound 2, the basic
amine is an important contributor to the binding affinity of these
molecules through a putative salt bridge interaction with an acidic
residue in transmembrane region 7 of the receptor, which is found
in most chemokine receptors.31 Since the aromatic ring on the left-
hand side and the tertiary amine are separated by one carbon in
compound 1 and by three carbons in compounds 2 and 3, we rea-
soned that separation of the aromatic ring and the tertiary amine
in 1 with a carbocycle or a saturated heterocycle should increase
the basicity of the nitrogen. This accordingly should strengthen
the salt bridge interaction with the receptor, potentially leading
to an enhancement of binding affinity if an optimal orientation of
the aromatic ring on the carbocycle or saturated heterocycle could
be defined. Based on this consideration, we designed scaffolds A, B
and C in which an OH at the 4-position of the pyrrolidine is in-
tended for further SAR exploration (Fig. 2).
O
OH
HO
NH2
CF3
6
5
4
O
H
N
d, e
R
N
N
H
O
OH
CF3
7a-9a, 7b-9b, 10
Scheme 1. Reagents and conditions: (a) mCPBA, CH2Cl2; (b) ammonium hydroxide,
70% for two steps; (c) N-(3-trifluoromethyl)benzoylglycine, BOP, Et3N, DMF, 80%;
(d) Pd/C, MeOH, 100%; (e) ketone, Na(OAc)3BH, THF.
Table 1
Compounds 7a–b, 8a–b, 9a–b and 10
O
H
N
R
N
N
H
O
OH
CF3
Compd
R
hCCR2a IC50 (nM)
MCP-1
CTX
To quickly test our design, we synthesized compounds 7a–b,
8a–b, 9a–b and 10 (Scheme 1). The synthesis started with the com-
mercially available benzyl dihydropyrrol-1-carboxylate 4. Epoxi-
7a
7b
120
>1000
>1000
dation of
4 using mCPBA followed by ring opening with
ammonium hydroxide provided trans-1-Cbz-3-hydroxy-4-amino-
pyrrolidine 5 as a racemic mixture. BOP coupling of 5 with N-(3-tri-
fluoromethylbenzoyl)glycine, which was prepared by reaction of
glycine with 3-trifluoromethylbenzoyl chloride in aqueous sodium
hydroxide and THF in one step, gave rise to compound 6. Following
removal of the Cbz group in 6 by hydrogenation, reductive amina-
tion of the resulting pyrrolidine with a substituted cyclic ketone
using sodium triacetoxyborohydride afforded the products 7a–b,
8a–b and 9a–b as a mixture of cis and trans isomers on the carbo-
cycles, with a ratio ranging from 3:1 to 5:1, and the product 10.
Separation of the cis and trans mixture in 7a–b, 8a–b and 9a–b
by flash chromatography or by reversed phase HPLC afforded the
major isomers 7a–9a and the minor isomers 7b–9b. The major iso-
mers were assigned as the cis isomers based on the employed
8a
8b
>1000
9a
9b
10
7.4
12
>1000
649
N
a
Human MCP-1 binding assay and chemotaxis assay.
O
chemistry in which the reducing agent used for reductive amina-
tion was the bulky sodium triacetoxyborohydride. This assignment
was confirmed by 2-dimensional NMR studies.
H
N
CF3
X
Ar
N
N
N
H
O
OH
Compounds were screened by binding assay to assess their
ability to inhibit MCP-1 binding to human peripheral blood
monocytes (MCP-1 assay)34 and by a functional assay to deter-
mine their ability to inhibit MCP-1 stimulated chemotaxis in hu-
man peripheral blood monocytes (CTX assay).35 As shown in
Table 1, in the cyclopentane series, the cis isomer 7a is a moder-
ate hCCR2 inhibitor with an IC50 of 120 nM in the MCP-1 binding
assay while the trans isomer 7b is inactive in the same assay (IC50
A
O
H
N
CF3
N
H
X
X
Ar
Ar
O
OH
B
O
>1
lM). In the cyclohexane series, both the cis and trans isomers
H
N
CF3
8a–b showed no inhibitory activity with IC50 values of >1
l
M
N
N
H
when the phenyl residue is at the 3-position on the cyclohexyl.
In contrast, when the phenyl residue is at the 4-position on the
cyclohexyl, the cis isomer 9a is a potent hCCR2 inhibitor with
IC50 values of 7.4 nM in the MCP-1 assay and 12 nM in the che-
motaxis assay while the trans isomer 9b is inactive (IC50
O
OH
C
X=CH, N; Ar=aryl
Figure 2. Scaffolds A, B and C.
>1 lM). By comparison, the piperidine analog 10 in which the

C.-B. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7473–7478
7475
phenyl residue at the 1-position of piperidine predominantly as-
sumes the more stable trans orientation relative to the pyrrolidine
substituent at the 4-position is a weak inhibitor (IC50 = 649 nM).
These results revealed that the geometric alignment and orienta-
tion of the phenyl ring relative to the pyrrolidine ring exert a
significant impact on the interaction of the molecule with the CCR2
receptor. The optimal geometric alignment of the phenyl ring and
the pyrrolidine ring is at 1- and 4-positions on a six-membered
carbocycle and the optimal orientation of the phenyl ring is cis
relative to the pyrrolidine ring.
tempts to directly alkylate the hydroxyl in 9a with alkyl bromide
or iodide using NaH in DMFresulted in multipleproducts. As a result,
alkylation was carried out following the protection of the amine in 5
with a Boc (Scheme 2). After removal of the Boc in the alkylated
product 11 using HCl in dioxane, the resulting amine 12 was coupled
with the glycine residue to give intermediate 13. Removal of Cbz by
hydrogenation was followed by reductive amination of the resulting
amine 14 with 4-phenylcyclohexanone to provide a mixture of cis
and trans isomers. The major cis isomers were separated by flash
chromatography to give target compounds 15–18.
One of our main goals at the early stage of the CCR2 program
was to identify a tool compound with high affinity for murine
CCR2 (mCCR2), high selectivity over other homologous chemokine
receptors and good oral bioavailability suitable for target valida-
tion in rodent models. The potent anti-hCCR2 activity of 9a
prompted us to run a screen using a murine monocytic cell line
that expresses CCR2.36 Encouragingly, 9a displayed a moderate
anti-mCCR2 activity, with IC50 values of 186 and 265 nM in
MCP-1 and chemotaxis assays, respectively.
Alkylation at the hydroxyl in 9a with ethyl (15) improved
the hCCR2 binding affinity from an IC50 of 7.4 nM to an IC50 of
0.95 nM and the chemotaxis activity from an IC50 of 12 nM to an
IC50 of 6.5 nM (Table 2). Importantly, the mCCR2 activity was more
dramatically enhanced by this modification, with 98-fold improve-
ment in binding affinity and 19-fold improvement in chemotaxis
activity. Compound 15 is now a potent mCCR2 antagonist with
IC50 values of 1.9 nM in binding affinity and 14 nM in chemotaxis
activity. Replacement of the ethyl group in 15 with longer alkyl
groups resulted in a loss in either chemotaxis activity (n-propyl
in 16) or binding affinity (n-butyl in 17 and benzyl in 18). Despite
its potent antagonistic activity against mCCR2, however, com-
pound 15 is highly protein bound, with a free fraction of 0.5% in
human serum and 2.3% in mouse serum. In addition, compound
15 exhibited a potent hERG binding activity, with an IC50 of
To improve the anti-mCCR2 activity of 9a, we first conducted SAR
investigation at the hydroxyl by introducing an alkyl group. At-
Cbz
N
Cbz
N
Cbz
N
d
c
a, b
<1
l
M in a dofetilide binding assay.
To refine the protein binding and hERG activity in 15, we turned
RO
NHBoc
RO
NH2
HO
NH2
our SAR studies to the hydrophobic moiety of 4-phenylcycohexyl
by introducing a polar group such as hydroxyl to the sterically hin-
dered 4-position of the cyclohexyl. Introduction of a hydroxyl
group at the benzylic position on the cyclohexyl in 15 to give 19
led to a significant enhancement in free fraction in protein binding
and a dramatic drop in hERG binding activity (Table 3). The free
fraction was improved to 25% in 19 from 0.5% in 15 in human
serum and to 18% in 19 from 2.3% in 15 in mouse serum. The hERG
11
12
5
O
O
H
H
N
N
Cbz
HN
N
H
N
N
H
e
O
O
OR
OR
CF3
CF3
14
13
binding activity was reduced to an IC50 of 17
lM in 19 from an IC50
O
H
N
of <1 M in 15 in the dofetilide assay. Unfortunately, despite its
l
f
N
N
H
suitable profile in protein binding and hERG binding activity, com-
pound 19 is not potent enough as a tool compound for model stud-
ies in rodents. While the potent hCCR2 inhibitory activity was
retained from this modification, a fivefold loss in mCCR2 binding
affinity and 32-fold loss in mCCR2 chemotaxis activity accompa-
nied the introduction of the polar hydroxyl group, indicating that
there is no preference in polarity for hCCR2 while lipophilicity is
preferred for mCCR2 at this position.
O
O
CF3
R
15-18
Scheme 2. Reagents and conditions: (a) (Boc)2O, Et3N, THF, 90%; (b) alkyl halide,
NaH, DMF, 0 °C; (c) 4 N HCl/dioxane; (d) N-(3-trifluoromethyl)benzoylglycine, BOP,
Et3N, DMF; (e) Pd/C, MeOH; (f) 4-phenylcyclohexanone, Na(OAc)3BH, THF.
Table 2
Compounds 9a and 15–18
O
H
N
N
N
H
O
O
R
CF3
Compd
R
hCCR2a IC50 (nM)
mCCR2b IC50 (nM)
h/m PBc% free
hERGe IC50
(lM)
MCP-1
CTX
MCP-1
CTX
9a
15
16
17
18
H
Ethyl
n-Propyl
n-Butyl
Benzyl
7.4
0.95
2.9
47
12
6.5
1.8
186
1.9
1.9
54
265
14
158
2.6/NDd
<0.5/2.3
<1
<1
17
51
a
b
c
Human MCP-1 binding assay and chemotaxis assay.
Murine MCP-1 binding assay and chemotaxis assay.
% free fraction in human and mouse protein binding.
Not determined.
d
e
hERG dofetilide binding activity.

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C.-B. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7473–7478
Table 3
Compounds 15 and 19–21
HO
Ar
O
H
N
N
N
H
O
O
CF3
Compd
Ar
hCCR2a IC50 (nM)
mCCR2b IC50 (nM)
h/mPBc% free
hERGe IC50
(lM)
MCP-1
CTX
MCP-1
CTX
15
19
20
21
0.95
2.3
2.3
6.5
3
1.4
3.8
1.9
10
2.1
9.5
14
0.5/2.3
26/18
<1
17
11
13
Phenyl
4-Methylphenyl
3,4-Methylenedioxyphenyl
444
12.9
7.8
NDd/7.9
24/15
5.1
a
b
c
Human MCP-1 binding assay and chemotaxis assay.
Murine MCP-1 binding assay and chemotaxis assay.
% free fraction in human and mouse protein binding.
Not determined.
d
e
hERG dofetilide binding activity.
To improve the anti-mCCR2 activity in 19, we fine-tuned the
low yield by direct addition of 3,4-methylenedioxyphenylmagne-
sium bromide to cyclohexan-1,4-dione 25. Reductive amination
of 24a–c with intermediate 14 generated a mixture of cis and trans
isomers, with the smaller hydroxyl group (relative to aryl) trans to
the pyrrolidine moiety being the major isomer (trans:cis = ꢀ2:1).
Separation of the two isomers by flash chromatography provided
the major trans isomers 19–21. The corresponding minor cis iso-
polarity of the 4-hydroxyl-4-phenylcyclohexyl moiety by modifi-
cation at the phenyl ring. Substitution with methyl (20) at the
4-position on the phenyl in 19 improved the mCCR2 binding affin-
ity and chemotaxis activity to IC50 values of 2.1 and 12.9 nM from
IC50 values of 10 and 444 nM, respectively, but reduced the free
fraction in mouse serum by twofold (Table 3). In contrast, substitu-
tion with methylenedioxy at the 3,4-position on the phenyl in 19
afforded a potent mCCR2 inhibitor 21 (INCB3344) with retained
free fraction in mouse serum. INCB3344 exhibited IC50 values of
5.1 nM (hCCR2) and 9.5 nM (mCCR2) in binding affinity and
3.8 nM (hCCR2) and 7.8 nM (mCCR2) in chemotaxis activity. In
protein binding, it had a free fraction of 24% in human serum
and 15% in mouse serum. It showed a moderate hERG binding
mers showed IC50 values of >1 lM in the MCP-1 assay.
Further in vitro profiling revealed that INCB3344 is a potent
antagonist towards rat and cynomolgus CCR2 as well, displaying
IC50 values of 7.3 and 16 nM in binding antagonism and 2.7 and
6.2 nM in antagonism of chemotaxis activity, respectively.
INCB3344 is a selective hCCR2 antagonist, exhibiting IC50 values
of more than 1
ers, chemokine receptors and other selected GPCRs. It is also a
selective mCCR2 antagonist, showing IC50 values of >1 M and
>3 M against murine CCR1 and murine CCR5, respectively, the
lM against a panel of >50 ion channels, transport-
activity, with an IC50 of 13 lM in the dofetilide binding assay.
Compounds 19–21 were synthesized according to Scheme 3.37
Addition of commercially available phenylmagnesium bromide or
p-tolylmagnesium bromide to hexan-1,4-dione monoethylene ke-
tal 22 gave rise to intermediate 23. Treatment of 23 with aqueous
HCl generated 4-phenyl-4-hydroxycyclohexanone 24a and 4-(p-
tolyl)-4-hydroxycyclohexanone 24b. Under this condition, the
hydroxyl group was unstable when the aryl residue was 3,4-meth-
ylenedioxyphenyl, leading to formation of a significant amount of
dehydration product. As a result, compound 24c was prepared in
l
l
two most homologous chemokine receptors to mCCR2. Other
in vitro and in vivo pharmacological properties of INCB3344 have
been characterized and reported.36,38
The pharmacokinetics of INCB3344 was assessed in both CD-1
and Balb/c mice (Table 4). When administered intravenously to
CD-1 mice, INCB3344 exhibited a high clearance and a moderate
volume of distribution, resulting in a short half life of 1 h. Despite
its high clearance, however, good oral exposure was achieved, with
an AUC at 2664 nM h at a dose of 10 mg/kg. The oral bioavailability
was 47%. By comparison, slightly better oral exposure
(AUC = 3888 nM h) was obtained when administered orally to
Balb/c mice at the same dose. This PK property, coupled with its
potent anti-mCCR2 activity and good selectivity, made this com-
pound suitable for model studies in rodents.
HO
Ar
O
O
O
O
a
O
O
23
22
25
b
HO
Ar
c
O
INCB3344 has been used as a tool compound for target valida-
tion in a mouse model of multiple sclerosis, a rat model of inflam-
matory arthritis and a mouse model of obesity, and was effective in
O
24a Ar=phenyl
24b Ar=4-methylpheny
24c Ar=3,4-methylene dioxyphenyl
d
Table 4
OH
O
Pharmacokinetic parameters of INCB3344
Ar
H
N
N
N
H
Mouse strain
CD-1
Balb/c
/10
O
Dose iv/po (mg/kg)
iv
5/10
5.1
O
CL (L/h/kg)
Vdss (L/h)
T1/2 (h)
CF3
2.8
1.0
19-21
po
Cmax (nM)
AUC (nM h)
F (%)
1886
2664
47
961
3888
Scheme 3. Reagents and conditions: (a) Phenylmagnesium bromide or p-tolyl-
magnesium bromide, THF, À78 °C; (b) 2 N HCl in water; (c) 3,4-methylenedioxy-
phenylmagnesium bromide, THF, À78 °C, 25%; (d) 14, Na(OAc)3BH, THF.

C.-B. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7473–7478
7477
30. Tarby, C. M.; Endo, N.; Moree, W. J.; Kataoka, K.; Ramirez-Weinhouse, M. M.;
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National Meeting, 1999, MEDI-082.
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R.; Jarnagin, K. J. Biol. Chem. 2000, 275, 25562.
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34. Human PBMCs were used to test compounds in a binding assay. 200,000 to
500,000 cells were incubated with 0.1–0.2 nM 125I-labeled MCP-1, with or
without unlabeled competitor (10 nM MCP-1) or various concentrations of
compounds to be tested. 125I-labeled MCP-1, were prepared by suitable
methods or purchased from commercial vendors (Perkin Elmer, Boston, MA).
lowering macrophage content in target tissues and in suppressing
development of these disease models.36,39 The results from these
studies delineated for the first time the pharmacological relevance
of inhibiting CCR2 using a small molecule antagonist in disease
models.
Acknowledgments
We thank Lynn Leffet, Karen Gallagher, Patricia Feldman, Bitao
Zhao, Yanlong Li, Robert Collins, Frederic Baribaud, Niu Shin and
Xiaomei Gu for their technical assistance.
The binding reactions were performed in 50–250 lL of a binding buffer
consisting of 1 M HEPES pH 7.2, and 0.1% BSA (bovine serum albumin), for
30 min at room temperature. The binding reactions were terminated by
harvesting the membranes by rapid filtration through glass fiber filters (Perkin
Elmer) which was presoaked in 0.3% polyethyleneimine or Phosphate Buffered
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buffer containing 0.5 M NaCl or PBS, then dried, and the amount of bound
radioactivity was determined by counting on a Gamma Counter (Perkin Elmer).
35. The capacity of compounds to antagonize CCR2 function was determined in a
leukocyte chemotaxis assay using human peripheral blood mononuclear cells,
in a modified Boyden Chamber (Neuro Probe). 500,000 cells in serum free
DMEM media (In Vitrogen) were incubated with or without the inhibitors and
warmed to 37 °C. The chemotaxis chamber (Neuro Probe) was also prewarmed.
400 lL of warmed 10 nM MCP-1 was added to the bottom chamber in all wells
except the negative control which had DMEM added. An 8 micron membrane
filter (Neuro Probe) was placed on top and the chamber lid was closed. Cells
were then added to the holes in the chamber lid which were associated with
the chamber wells below the filter membrane. The whole chamber was
incubated at 37 °C, 5% CO2 for 30 min. The cells were then aspirated off, the
chamber lid opened, and the filter gently removed. The top of the filter was
washed three times with PBS and the bottom was left untouched. The filter
was air dried and stained with Wright Geimsa stain (Sigma). Filters were
counted by microscopy. The negative control wells served as background and
were subtracted from all values. Antagonist potency was determined by
comparing the number of cells that migrated to the bottom chamber in wells
which contained antagonist, to the number of cells which migrated to the
bottom chamber in MCP-1 control wells.
13. Fife, B. T.; Huffnagle, G. B.; Kuziel, W. A.; Karpus, W. J. J. Exp. Med. 2000, 192,
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37. Experimental procedures for compound 21 (INCB3344):
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21. Pasternak, A.; Marino, D.; Vicario, P. P.; Ayala, J. M.; Cascierri, M. A.; Parsons,
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2007, 17, 309.
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A.; Yang, L. Bioorg. Med. Chem. Lett. 2007, 17, 3636.
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Olini, G.; Cavender, D.; Wachter, M. Bioorg. Med. Chem. Lett. 2007, 17, 4382.
26. Carter, P. H.; Brown, G. D.; Friedrich, S. R.; Cherney, R. J.; Tebben, A. J.; Lo, Y. C.;
Yang, G.; Jezak, H.; Solomon, K. A.; Scherle, P. A.; Decicco, C. P. Bioorg. Med.
Chem. Lett. 2007, 17, 5455.
27. Xia, M.; Hou, C.; Pollack, S.; Brackley, J.; DeMong, D.; Pan, M.; Singer, M.;
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Benzyl 6-Oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate. To a solution of benzyl 3-
pyrroline-1-carboxylate (30 g, 133 mmol) in CH2Cl2 (700 mL) was added
mCPBA (57.2 g, 200 mmol). The reaction mixture was stirred at room
temperature overnight and quenched with 20% NaHSO3 aqueous solution
(250 mL). The organic phase was separated, and the aqueous layer was
extracted with CH2Cl2 twice. The combined extracts were washed with
NaHCO3 and brine, dried over Na2SO4, and evaporated under reduced
pressure. Chromatography on silica gel eluting with 40% EtOAc/hexanes
provided the title compound (24 g, 83%). MS (M+H)+ 220.
Benzyl trans-3-[(tert-Butoxycarbonyl)amino]-4-hydroxypyrrolidine-1-carboxylate. To
a solution of benzyl 6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate (20.7 g,
94.4 mmol) in 80 mL of methanol was added ammonium hydroxide (80 mL).
The reaction mixture was stirred at 60 °C overnight and concentrated under
reduced pressure to give an oil residue (22.3 g, 94.4 mmol) which was used
directly for the next reaction. MS: (M+H)+ = 237.
To a solution of the above crude product in THF (200 mL) was added di-tert-
butyldicarbonate (26.8 g, 123 mmol) and triethylamine (17.1 mL, 123 mmol)
at 0 °C. The reaction mixture was stirred at room temperature overnight. After
addition of EtOAc and water, the organic phase was separated, and the aqueous
layer was extracted with EtOAc. The combined extracts were washed with
NaHCO3 and brine, dried over Na2SO4, and evaporated under reduced pressure.
Chromatography on silica gel eluting with 70% EtOAc/hexanes provided the
title compound (27.3 g, 86%). MS: (M+H)+ = 337. 1H NMR (400 MHz, CD3OD): d
ppm 7.34 (m, 5H), 5.10 (s, 2H), 4.10 (m, 1H), 3.86 (m, 1H), 3.70 (m, 1H), 3.58
(m, 1H), 3.36 (m, 2H), 1.40 (s, 9H).
Benzyl trans-3-ethoxy-4-[(tert-butoxycarbonyl)amino]pyrrolidine-1-carboxylate.
To a solution of benzyl 3-[(tert-butoxycarbonyl)amino]-4-hydroxypyrrolidine-1-
carboxylate (26 g, 77 mmol) in THF (120 mL) was added NaH (5 g, 211 mmol) at
0 °C. The reaction mixture was stirred at 0 °C for 1 h and iodoethane (17.9 g,
115 mmol) was added. After being stirred at room temperature overnight,
water and EtOAc were added. The organic phase was separated, and the
aqueous layer was extracted with EtOAc twice. The combined extracts were
washed with brine, dried over Na2SO4, and evaporated under reduced pressure.
Chromatography on silica gel eluting with 25% EtOAc/hexanes provided the
title compound (19.6 g, 70%). MS: (M+H)+ = 365.1. 1H NMR (400 MHz, CD3OD):
d ppm 7.35 (m, 5H), 5.12 (s, 2H), 4.00 (m, 1H), 3.82 (m, 1H), 3.60 (m, 4H), 3.40
(m, 2H), 1.42 (s, 9H), 1.18 (t, J = 6.8 Hz, 3H).
28. Yang, L.; Butora, G.; Jiao, R. X.; Pasternak, A.; Zhou, C.; Parsons, W. H.; Mills, S.
G.; Vicario, P. P.; Ayala, J. M.; Cascieri, M. A.; MacCoss, M. J. Med. Chem. 2007, 50,
2609.
29. Xia, M.; Hou, C.; DeMong, D. E.; Pollack, S. R.; Pan, M.; Brackley, J. A.; Jain, N.;
Gerchak, C.; Singer, M.; Malaviya, R.; Matheis, M.; Olini, G.; Cavender, D.;
Wachter, M. J. Med. Chem. 2007, 50, 5561.

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C.-B. Xue et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7473–7478
Benzyl trans-3-ethoxy-4-[({[3-(trifluoromethyl)benzoyl]amino}acetyl)amino]
trans-N-(2-{1-[4-(Benzo[d][1,3]dioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxypyrr-
olidin-3-ylamino}-2-oxoethyl)-3-(trifluoromethyl)benzamide (21, INCB3344). To
a solution of benzyl trans-3-(ethoxy)-4-[({[3-(trifluoromethyl) benzoyl]amino}
acetyl)amino]pyrrolidine-1-carboxylate (4.9 g, 10 mmol) in 50 mL of MeOH
was added 10% Pd on carbon (250 mg) under nitrogen. The reaction mixture
was stirred at room temperature under hydrogen overnight and filtered
through Celite. The filtrate was concentrated under reduced pressure to give
trans-N-[2-(4-ethoxypyrrolidin-3-ylamino)-2-oxoethyl]-3-
pyrrolidine-1-carboxylate. To a solution of benzyl 3-ethoxy-4-[(tert-butoxy-
carbonyl)amino]pyrrolidine-1-carboxylate (18.2 g, 50 mmol) in THF (100 mL)
was added a solution of 4 N HCl in dioxane (250 mL). After being stirred at
room temperature for 2 h, the solution was concentrated under reduced
pressure. The residue was dissolved in saturated NaHCO3 solution. The
resulting solution was extracted with EtOAc twice. The combined extracts
were washed with brine, dried over Na2SO4, and evaporated under reduced
pressure to give benzyl trans-3-ethoxy-4-aminopyrrolidine-1-carboxylate as
an oil. MS: (M+H)+ = 265.1.
(trifluoromethyl)benzamide (3.5 g, 97%). MS (M+H)+ 360.1.
To a solution of 4-(benzo[d][1,3]dioxol-5-yl)-4-hydroxycyclohexanone (281 mg,
1.2 mmol) and trans-N-[2-(4-ethoxypyrrolidin-3-ylamino)-2-oxoethyl]-3-
(trifluoromethyl)benzamide (360 mg, 1 mmol) in THF (10 mL) was added
Na(OAc)3BH (424 mg, 2 mmol). The mixture was stirred at room temperature
To
a solution of benzyl trans-3-ethoxy-4-aminopyrrolidine-1-carboxylate
(9.5 g, 36 mmol) in DMF (100 mL) in an ice bath was added N-methylmor
pholine (12 g, 105 mmol), BOP (19 g, 44 mmol) and N-[(3-trifluoromethyl)
benzoyl]glycine (10 g, 39 mmol). After being stirred at room temperature
overnight, the solution was diluted with EtOAc. The resulting solution was
washed with NaHCO3 and brine, dried (MgSO4) and concentrated.
Chromatography on silica gel eluting with 50% EtOAc-hexanes provided the
title compound (14.2 g, 80%). MS: (M+H)+ = 494.1. 1H NMR (400 MHz, CD3OD):
d ppm 8.20 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 8 Hz, 1H), 7.68 (t, J = 8 Hz,
1H), 7.35 (m, 5H), 5.12 (s, 2H), 4.35 (m, 1H), 4.02 (s, 2H), 3.95 (m, 1H), 3.60 (m,
4H), 3.42 (m, 2H), 1.17 (t, J = 6.8 Hz, 3H).
overnight and poured into
a brine solution. The resulting solution was
extracted with EtOAc twice. The combined extracts were washed with
NaHCO3 and brine, dried over MgSO4 and concentrated. Purification on silica
gel eluting with 5% MeOH/CH2Cl2 gave 230 mg of the fast moving isomer 21
with 99% purity (trans isomer, MS: (M+H)+ = 578.2) and 120 mg of the slow
moving isomer (cis isomer, MS: (M+H)+ = 578.2). 1H NMR of 21 (400 MHz,
CD3OD): d ppm 8.20 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 8 Hz, 1H), 7.67
(t, J = 8 Hz, 1H), 7.02 (s, 1H), 6.99 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 5.90
(s, 2H), 4.25 (m, 1H), 4.02 (s, 2H), 3.85 (m, 1H), 3.62 (m, 1H), 3.45 (m, 1H), 3.02
(m, 1H), 2.96 (m, 1H), 2.50 (m, 2H), 2.22 (m, 3H), 1.94 (m, 2H), 1.52 (m, 4H),
1.18 (t, J = 6.8 Hz, 3H).
4-(Benzo[d][1,3]dioxol-5-yl)-4-hydroxycyclohexanone. To
a solution of 1,4-
cyclohexanedione (11.2 g, 100 mmol) in THF (200 mL) cooled at À78 °C was
added a 1 M solution of 3,4-(methylenedioxy)phenylmagnesium bromide in
toluene/THF (50:50) (50 mL, 50 mmol). The solution was allowed to warm to
room temperature. After being stirred at room temperature for 3 h, the
reaction was quenched with a solution of NH4Cl. The resulting solution was
extracted with EtOAc three times. The combined extracts were washed with
brine, dried over MgSO4 and concentrated. Purification on silica gel eluting
with 50% EtOAc/hexanes yielded 2.35 g (20%) of the title compound. MS:
(M+HÀH2O)+ = 217.1.
38. Shin, N.; Baribaud, F.; Wand, K.; Yang, G.; Wynn, R.; Covington, M. B.; Feldman,
P.; Gallagher, K. B.; Leffet, L. M.; Lo, Y. Y.; Wang, A.; Xue, C.-B.; Newton, R. C.;
Scherle, P. A. Biochem. Biophys. Res. Commun. 2009, 387, 251.
39. Weisberg, S. P.; Hunter, D.; Huber, R.; Lemieux, J.; Slaymaker, S.; Vaddi, K.;
Charo, I.; Leibel, R. L.; Ferrante, A. W., Jr. J. Clin. Invest. 2006, 116, 1457.