Discovery of a Potent and Selective CCR4 Antagonist That Inhibits Treg Trafficking into the Tumor Microenvironment

Article abstract of DOI:10.1021/acs.jmedchem.9b00506

Recruitment of suppressive CD4⁺ FOXP3⁺ regulatory T cells (T_reg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human T_reg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, T_reg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of T_reg and increasing the population of activated effector T cells (T_eff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.

Full text of DOI:10.1021/acs.jmedchem.9b00506

Subscriber access provided by UNIV OF SOUTHERN INDIANA
Article
Discovery of a Potent and Selective CCR4 Antagonist that
Inhibits Treg Trafficking into the Tumor Microenvironment
Jeffrey James Jackson, John Michael Ketcham, Ashkaan Younai, Betty Abraham, Berenger Biannic,
Hilary Plake Beck, Minna Hue Thanh Bui, David Chian, Gene Cutler, Raymond Diokno, Dennis
X Hu, Scott Jacobson, Emily Karbarz, Paul D Kassner, Lisa Marshall, Jenny McKinnell, Cesar
Meleza, Abood Okal, Deepa Pookot, Maureen Kay Reilly, Omar Robles, Hunter Paul Shunatona,
Oezcan Talay, James Ross Walker, Angela Wadsworth, David J. Wustrow, and Mikhail Zibinsky
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00506 • Publication Date (Web): 17 Jun 2019
Downloaded from http://pubs.acs.org on June 17, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery of a Potent and Selective CCR4
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
Antagonist that Inhibits T Trafficking into the
reg
Tumor Microenvironment
†
†§
‡
Jeffrey J. Jackson , John M. Ketcham , Ashkaan Younai, Betty Abraham, Berenger Biannic ,
⊥
#
Hilary P. Beck , Minna H. T. Bui , David Chian, Gene Cutler, Raymond Diokno, Dennis X. Hu,
∥
Scott Jacobson, Emily Karbarz, Paul D. Kassner, Lisa Marshall, Jenny McKinnell , Cesar
∇
∩
≡
∥
Meleza , Abood Okal , Deepa Pookot, Maureen K. Reilly , Omar Robles, Hunter P. Shunatona ,
∆
Oezcan Talay, James R. Walker , Angela Wadsworth, David J. Wustrow*, Mikhail Zibinsky*
RAPT Therapeutics, 561 Eccles Avenue, South San Francisco, CA 94080
ABSTRACT
+
+
Recruitment of suppressive CD4 FOXP3 regulatory T cells (T ) to the tumor
reg
microenvironment (TME) has the potential to weaken the anti-tumor response in
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 126
1
2
3
4
5
6
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
patients receiving treatment with immuno-oncology (IO) agents. Human Treg express
CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and
CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis.
Preclinical data suggests that preventing the recruitment of Treg and increasing the
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
population of activated effector T cells (T ) in the TME can potentiate anti-tumor
eff
immune responses. We have developed a novel series of potent, orally bioavailable
small molecule antagonists of CCR4. From this series, several compounds exhibited
high potency in distinct functional assays in addition to good in vitro and in vivo ADME
properties. The design, synthesis, and SAR of this series and confirmation of its in vivo
activity is reported.
INTRODUCTION
CC chemokine receptor 4 (CCR4) is a seven-transmembrane G protein-coupled
receptor (GPCR) that is known to play a dominant role in the recruitment of highly
+
+
+
immunosuppressive CD4 , CD25 , and FOXP3 regulatory T cells (T ) into the tumor
reg
microenvironment (TME).1-2 Within the TME, dendritic cells and macrophages produce
ACS Paragon Plus Environment
2

Page 3 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the cognate ligands for CCR4, CCL17 and CCL22.3-4 This chemokine gradient attracts
5
+
Treg into the TME, where they accumulate and suppress the function of CD8 effector T
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
6
cells (T ), which would otherwise act to fight the tumor. Several studies have shown
eff
that accumulation of Treg in the TME and increased levels of CCL22 can lead to poor
patient prognosis.7-11 Thus, the antagonism of CCR4 leading to the inhibition of this Treg
trafficking pathway makes CCR4 an attractive target for the development of an immuno-
oncology (IO) therapy.12
The CCR4 receptor also plays a role in the recruitment of T helper type 2 cell
(Th2) subsets for autoimmune disorders such as asthma, allergic rhinitis, and atopic
dermatitis.13,14 To probe the role of CCR4 receptor antagonists in inflammatory
disorders, several attempts have been made to design potent small molecule
antagonists of CCR4,15,16 leading to two series differing in their proposed binding
mode.17-20 Class I antagonists have been shown to bind to an extracellular portion of the
GPCR (1-3,21-24 Figure 1), while Class II antagonists (4-6,25-27 Figure 1) bind
19
intracellularly. In general, Class I antagonists contain a heteroaromatic ring with a
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 126
1
2
3
4
5
6
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
lipophilic arene, and a side chain containing a basic amine. Class II antagonists tend to
be sulfonamides that are flanked by a heteroaromatic ring and a more lipophilic arene.
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
Class I Antagonists
Cl
F
Cl
F
NH
NH
N
NH
N
OMe
OMe
F
N
Cl
N
F
N
O
N
N
N
N
N
Et2N
N
N
O
H
NH Me
Astellas (ref. 21 & 22)
Class II Antagonists
O
1
2
3
Bristol-Myers-Squibb (ref. 23)
Daiichi Sankyo (ref. 24)
NMe2
O
Me
Me
H
MeO
OH
N
O
X
N
N
OMe
O
Br
N
N
O
N
N
N
Cl
O
S
O
S
H O
S
N
Cl
S
N
Cl
H O
H O
OMe
Me
AstraZeneca (4, AZD-2098, X = H) (ref. 25)
AstraZeneca (5, AZD-1678, X = F) (ref. 25)
6
7
Ono (ref. 26)
GlaxoSmithKline (GSK2239633, ref. 27)
Figure 1. Representative CCR4 Antagonists
The most prominent of these antagonists is a Class II antagonist GSK2239633
27
(
6), developed by GlaxoSmithKline. Mentioning asthma as a possible therapeutic
indication, GSK2239633 was evaluated in a Phase I clinical trial in healthy volunteers
28
that ended in 2011. In this study, the compound suffered low target engagement and
low exposure in the blood which prevented further development. AstraZeneca has also
reported two Class II antagonists from their CCR4 program, identified as preclinical
ACS Paragon Plus Environment
4

Page 5 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
5
candidates AZD-2098 and AZD-1678 (4 and 5). While AZD-2098 was licensed to
29
Cancer Research UK as a potential therapy for kidney cancer in 2013, no further
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
development of this compound has been disclosed.
More recently, a CCR4-targeted cell-depleting monoclonal antibody,
mogamulizumab (KY-0761, Kyowa Hakko Kirin Co., Ltd.) has received approval from
the FDA to treat patients with two subtypes of relapsed Cutaneous T-Cell Lymphoma
30
(
CTCL). However, treatment has shown a risk of serious side effects such as
dermatologic toxicity, autoimmune issues, and complications from stem cell treatment
prior to mogamulizumab administration.31
While targeting CCR4 via an antibody-dependent cell mediated cytotoxicity
(
ADCC) mechanism has shown clinical efficacy, the mechanism of action is much
different compared to targeting CCR4 with a small molecule antagonist. Depletion of
Treg that serve other essential functions could cause a severe autoimmune reaction
leading to further complications.32-33 However, simply inhibiting Treg trafficking into the
TME via a small molecule antagonist may lead to tumor killing without affecting other
vital immune cell functions.
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 126
1
2
3
4
5
6
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
To this end, we have designed novel CCR4 antagonists and evaluated their
ability to inhibit Treg migration into the tumor microenvironment. Herein, we highlight the
SAR of a series of novel CCR4 antagonists that demonstrate best-in-class potency and
pharmacokinetic properties.
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
Structure-Activity Relationships. Upon reviewing the literature of known Class I
antagonists, a pharmacophore map was built to aide in the design of a new class of
17
CCR4 antagonists (Figure 2). In general, these antagonists are comprised of a
heteroarene core structure with a side chain containing a basic nitrogen on the left-hand
portion and a lipophilic arene on the northern end. The lipophilic arene and basic amine
side chain are attached to the core via a 1,3-relationship.
2
Cl
atoms
Y
X 3
Cl
1
X
X
N
atoms
3
H
O
Can be cHexyl, piperidine, piperazine, benzene, etc.
Figure 2. Pharmacophore Map of Class I CCR4 Antagonists
ACS Paragon Plus Environment
6

Page 7 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
To assess potency of the compounds, they were first put into a calcium flux
assay. Calcium flux is a robust, high-throughput functional assay that we utilized to
rapidly triage compounds for in vitro cellular migration mediated by chemokine
receptors.34,35 Based on this pharmacophore model, a series of amides closely related
to 8 were made which were found to inhibit calcium flux in the double-digit nM range,
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
36
however these compounds suffered from low bioavailability in rats (Figure 3). Upon
surveying several different cores, a novel pyrazolopyrazine, compound 9, was
discovered that was within 5-fold potency of the parent compound 8, as a racemic
mixture. Additionally, this core increased the bioavailability in rats by more than 3-fold.
To broaden the SAR of these antagonists, various piperidinyl-containing side chains
were appended onto the left-hand side of the molecule. Replacing the amide side chain
with a piperidinyl-piperidine gave 10, which resulted in a 10-fold shift in potency. To
create more conformational restriction about the 4-piperidinyl-piperidine side chain, a
methyl group was installed in the 3-position of the piperidine to afford 11. Fortunately,
incorporation of the 3'-methyl-1,4'-bipiperidine was equipotent to the amide linkage
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 126
1
2
3
4
5
6
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
found in compound to 9. From this result, we began focusing our early stage efforts on
evaluating the SAR of these substituted piperidinyl-piperidine side chains.
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
Cl
Me
N
Cl Me
NH
N
Cl
Cl
N
N
N
N
N
N
N
N
H
H
O
O
8
9
Ca2+ flux IC50 = 54 nM
Ca2+ flux IC50 = 275 nM
F (%) = 7
F (%) = 25
Cl
Cl
Me
Me
N
N
Cl
N
N
Cl
N
N
N
N
N
N
N
N
Me
10
Ca2+ flux IC50 = 2580 nM
11
Ca2+ flux IC50 = 238 nM
Figure 3. Discovery of a Pyrazolopyrazine Core and 3'-Methyl-1,4'-Bipiperidine Side
Chain
Substitution on the C3 position of the pyrazolopyrazine core was carefully
examined while leaving the left hand 3'-methyl-1,4'-bipiperidine intact. Replacing the
C3-H substituent in 11 with more polar functional groups to give amide 12 and nitrile 13
resulted in compounds that were equipotent, but also suffered from extremely high
hepatic clearance and low AUC when dosed IV in rats. Installation of the more lipophilic
∞
Me (14) and CF (15) groups dramatically improved the IV clearance in rats, however
3
ACS Paragon Plus Environment
8

Page 9 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the C3-CF was 8-fold less potent. Consequently, our SAR focused on developing new
3
side chains to append onto the C3-methyl pyrazolopyrazine core.
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
Table 1. Evaluation of the C3 Position of the Pyrazolopyrazine Core
Cl
Me
N
N
Cl
R1
N
N
N
N
Me
Compoun
d
R1
H
Ca flux
IC50
2+
rat IV CL
rat IV
AUC∞
(hr*ng/mL)
mL/min/kg)b
(
nM)a
(
1
1
2
238
228
121.7
101.7
68
82
1
CONH
2
1
3
4
CN
Me
251
126
837
101.7
38.3
56.7
82
1
217
148
15
CF3
a
b
Assay run in the absence of serum. Dose of 0.5 mg/kg IV
Rapid evaluation of the C3-position helped to focus our efforts on confirmation of
the stereochemistry required for potent CCR4 antagonism in this series. From previous
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 126
1
2
3
4
5
6
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
literature and our extensive in-house SAR, we knew the benzylic methyl position on the
northern fragment required the R-stereochemistry. With an enantioselective route to the
C3-methyl core in hand (see experimental section for details), determination of the
stereochemistry about the piperidinyl- piperidine side chain was undertaken. Chiral
resolution of trans-3-methylpiperidin-4-yl pivalate enabled a stereoselective route for the
cis-enantiomers of 3'-methyl-1,4'-bipiperidine (see experimental section for details).
Appending these side chains onto the C3-Me-core revealed that the (R, S)-
diastereomer 16 was 19-fold more potent than the (S, R)-diastereomer 17 toward
inhibiting calcium flux (Figure 4).
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
Cl
Cl
Me
N
Me
N
N
N
Cl
Cl
Me
Me
N
N
N
N
N
N
(S)
(R)
N
(R)
N
(S)
Me
Me
1
6
17
Ca2+ flux SF IC50 = 803 nM
Ca2+ flux SF IC50 = 42 nM
Figure 4. Stereochemical Analysis
After completing the stereochemical analysis of this series, the path was cleared
to evaluate the SAR of the basic amine containing side chain. Antagonists that inhibited
ACS Paragon Plus Environment
10

Page 11 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
calcium flux in the double-digit nanomolar range were also run in a physiologically more
relevant chemotaxis (CTX) assay to evaluate their ability to inhibit CCL22-mediated
migration of CCR4 expressing CEM cells. This 96-well migration platform is considered
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
35
the gold standard assay for interrogating in vitro cellular migration. Importantly, this
medium-throughput assay can be adapted for use with relevant primary immune cells
(
i.e. T ). Since the assay is run in 100% human serum (HS), the reported potencies
reg
reflect the protein binding of the compounds. Although calcium flux allowed us to triage
compounds based on their CCR4 affinity, a significant shift in CTX potency was often
observed, likely due to plasma protein binding.
Expansion or contraction of the distal piperidine ring to afford the 4-,5-,6-, and 7-
membered rings (16, 18-20) resulted in antagonists that inhibit calcium flux with similar
potencies (Table 2). Unfortunately, these compounds suffered a severe shift in potency
when evaluated in the CTX assay. While the size of the outermost ring had little effect
on the potency of these compounds, focusing on the 5-membered pyrrolidines allowed
for the installation of a variety of commercially available, enantiopure substituted
heterocycles. To this end, both enantiomers of 2-methylpyrrolidine were explored to
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 126
1
2
3
4
5
6
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
replace the simple pyrrolidine ring (18). While installation of the 2-methyl substituent did
not suppress the shift in potency between calcium flux and CTX, the (R)-2-methyl-
pyrrolidine used in 21 provided a 4-fold more potent compound than the (S)-enantiomer
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
22. To probe what effect the cLogP would have on the CTX shift, several polar
functionalities were evaluated including amides (23, 24), sulfone (25), sulfonamide (26),
nitrile (27), and carboxylic acid (28). Unfortunately, lowering the cLogP by introduction
of these polar groups had little influence on the CTX shift. Surprisingly, attenuating the
oxidation state of the carboxylic acid 28 to the prolinol derived 29 produced a compound
with a CTX IC = 151 nM. Further, replacement of the C3-Me core in 29 with a C3-CN
50
core to afford 30 resulted in one of the first compounds to exhibit an extremely low CTX
shift with a CTX IC = 58 nM.
50
Table 2. SAR of the 3,4-Substituted Piperidinyl Side Chain
Cl
Me
N
N
Cl
R1
N
N
2
R
N
(R)
N
( )n Me
ACS Paragon Plus Environment
12

Page 13 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2+
Compoun
d
R1
R2
n
Ca flux
CTX IC50
a
(nM)b
IC (nM)
5
0
16
18
19
Me
Me
Me
Me
Me
Me
Me
H
2
1
0
3
1
1
1
1
1
1
1
1
1
1
42
361
N.D.
218
195
381
N.D.
N.D.
355
N.D.
259
865
N.D.
151
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
H
H
69
58
20
H
23
21
22
23
24
Me
47
Me (S)c
C(O)NH2
172
170
37
Me CH CH C(O)NH
2
2
2
25
Me
CH CH SO Me
104
32
2
2
2
2
6
7
Me CH CH SO NH
2
2
2
2
2
Me
Me
Me
CN
CH CH CN
41
2
2
28
COOH
670
32
2
9
0
CH OH
2
3
CH OH
27
58
2
a
b
c
Assay run in absence of serum. Assay run in 100% human serum (HS). Opposite
enantiomer used.
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 126
1
2
3
4
5
6
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
Encouraged by these results, we evaluated 30 in several in vitro and in vivo
assays (Table 3). While it did not display any inhibition of CYP enzymes, when exposing
compound 30 to human and rat hepatocytes, a substantial amount remained in human
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
(74%) while rat showed a significant decrease in recovery (19%). The low in vitro
metabolic stability in rat hepatocytes correlated well with the superhepatic clearance
that was observed in vivo. In addition to extremely high clearance (114.6 mL/min/kg), 30
suffered from low bioavailability (%F = 7) and a high volume of distribution (13.4 L/kg).
Table 3. Profile of Piperidine 30
Cl
Me
N
N
Cl
CN
N
N
HO
N
N
30
Me
Assays
Compound 30
2+
Ca flux IC (nM)
27
58
50
a
CTX IC (nM)
50
b
CYP Inhibition IC (µM)
>10
74/19
50
% remaining hHep/rHep
rat in vivo PKc
ACS Paragon Plus Environment
14

Page 15 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Cl (mL/min/kg)
114.6
Vss (L/kg)
13.4
2.1
23.2
7
T (h)
1/2
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
PO AUC0-∞ (hr*ng/mL)
F (%)
a
b
Assay run in 100% human serum (HS). Enzymes tested: 1A2, 2C9, 2C19, 2D6, 3A4.
cDose of 0.5 mg/kg IV or 2 mg/kg PO
To address these issues, we hypothesized that by replacing the piperidine
nitrogen attached to the core in compound 30 with a carbon, the shift in cLogP may
bring about more favorable pharmacokinetics for these antagonists. Up to this point,
none of the nitrogen-linked antagonists displayed acceptable pharmacokinetic
properties, therefore, we integrated the carbon-carbon link similar to our earlier
antagonists 8 and 9. The requisite carbon-carbon bond to achieve this goal brought
about a significant synthetic challenge, and thus we envisioned first replacing the 3-Me-
piperidine ring with a 3-Me-cyclohexene ring (vide infra).
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 126
1
2
3
4
5
6
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
Given the properties of prolinol-piperidine 30, the cyclohexenyl compound 31
was also evaluated in several in vitro and in vivo studies (Table 4). Replacement of the
anilinic nitrogen connection to the core with a styrenyl bond gave a compound that was
equipotent in the CTX assay, no inhibition of CYP enzymes, and similar in vitro hepatic
stability in human and rat hepatocytes, 81% and 32% remaining, respectively.
Interestingly, the in vivo pharmacokinetic properties of 31 were far superior to that of
compound 30, displaying a longer terminal half-life (T ) and a 4-fold higher
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
bioavailability (%F = 28).
Table 4. Profile of Cyclohexene 31
Cl
Me
N
N
Cl
CN
N
N
HO
N
31
Me
Assays
Compound
31
2+
Ca flux IC (nM)
40
70
50
a
CTX IC (nM)
50
b
CYP Inhibition IC (µM)
>10
50
ACS Paragon Plus Environment
16

Page 17 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
%
remaining hHep/rHep
81/32
rat in vivo PKc
Cl (mL/min/kg)
Vss (L/kg)
T (h)
74.7
9.8
4.1
125
28
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
1/2
PO AUC0-∞ (hr*ng/mL)
F (%)
a
b
Assay run in 100% human serum (HS). Enzymes tested: 1A2, 2C9, 2C19, 2D6, 3A4.
cDose of 0.5 mg/kg IV or 2 mg/kg PO
Encouraged by the in vivo pharmacokinetics of compound 31, the SAR of these
cyclohexenyl antagonists was further assessed (Table 5). To begin, the C3-position of
1
the core (R ) was again interrogated. Replacing the C3-nitrile in 31 with a C3-methyl
substituent (32) resulted in an almost 3-fold drop in potency within the CTX assay and
little change in the in vitro hepatic stability. Given these data, the C3-nitrile was
2
conserved throughout to focus our efforts on the R substituents on the pyrrolidine ring.
Building steric hindrance around the primary alcohol in 31 to give the tertiary alcohol in
3
3 resulted in a significant loss of potency in the CTX assay. Replacement of prolinol
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 126
1
2
3
4
5
6
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
with prolinamine (34) or prolinamide (35) increased the stability of these compounds
toward human and rat hepatocytes. However, the increase in stability was accompanied
by a significant decrease in potency in the CTX assay. Interestingly, installation of a 2-
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
(R)-Me-pyrrolidine (36) resulted in a compound that was equipotent to 31 and displayed
2
higher in vitro hepatic stability in rats. Replacement of the R methyl with a more
lipophilic trifluoromethyl substituent (37), rendered the compound completely inactive.
Table 5. Primary SAR of Cyclohexenyl Antagonists
Cl
Me
N
N
Cl
R1
N
N
R2
N
Examples 31-37
Me
Example R1
R2
Ca flux
2+
CTX IC50
(nM)b
% remaining
hHep/rHep
a
IC (nM)
50
31
32
33
CN
Me
CH OH
40
36
70
81/32
99/35
N.D.
2
CH OH
179
2
CN C(Me) OH
384
1200
2
ACS Paragon Plus Environment
18

Page 19 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
3
3
4
5
6
7
CN
CN
CN
CN
CH NH
229
54
638
1380
44
100/100
97/71
86/72
N.D.
2
2
C(O)NH2
Me
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
34
3
CF3
>5000
N.D.
a
b
Assay run in absence of serum. Assay run in 100% human serum (HS).
Compounds 31 and 36 were the most potent analogs in their ability to inhibit
CCL22-mediated CTX of CCR4 expressing CEM cells in 100% human serum. These
compounds were further assessed for their ability to inhibit CTX in mouse induced Treg
(
miT ) and for their PK characteristics in mice. Although the in vivo clearance in mice
reg
was lower for 36 (1.17 vs 12.0 mL/min/kg), leading to a substantially longer T1/2 (30
hours vs 7.6, Table 6), the superior potency, synthetic availability, and lower lipophilicity
of compound 31 ultimately led us to advance this compound into an in vivo mouse
model to determine its ability to suppress Treg trafficking into the tumor
microenvironment.
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 126
1
2
3
4
5
6
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
Table 6. Comparison of Compounds 31 and 36 in iTreg CTX and Mouse In Vivo PK
Cl
Cl
Me
N
Me
N
N
N
Cl
Cl
CN
CN
N
N
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
N
N
HO
Me
N
31
N
36
Me
Me
Example cLogP/tPSA
miTreg
mouse in vivo PK:
CTX IC50
Cl / Vss / T1/2
/
(nM)
%F
3
1
4.5/87.6
5.6/67.4
165
325
12.0 / 5.7 / 7.6 /
26
36
1.17 / 4.9 / 30.3 /
27
In Vivo Pharmacology in Mouse. Various murine tumor models were examined to
assess their levels of CCR4 ligand (CCL17 and CCL22) expression, and it was found
that the pancreatic ductal adenocarcinoma (Pan02) model displayed a high expression
37
of these ligands. Compound 31 was dosed QD for 6 days at 10 and 30 mg/kg in mice
ACS Paragon Plus Environment
20

Page 21 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
bearing Pan02 tumors (Figure 5A). Mouse Treg were generated in vitro from naive CD4+
+
+
+
38,39
T cells isolated from Foxp3 GFP transgenic mice (GFP miT )
as described in the
reg
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
+
supporting information. These GFP miT were injected into the animals 24 h after the
reg
+
first dose. Inhibition of the migration of GFP miT into tumor and other tissues was
reg
compared to vehicle treated animals (Figure 5B). A significant dose-dependent
+
decrease in the number of GFP miT was found in the tumor tissue when compared
reg
to the vehicle group. The 30 mg/kg dose showed nearly complete suppression of Treg
migration into the tumor. Plasma levels at 24-hr post dose for 31 showed that 30 mg/kg
QD covered the approximate IC of the miT CTX. Importantly, compound 31 was
90
reg
able to selectively inhibit Treg trafficking into the tumor without affecting the migration of
these cells into the spleen (Figure 5), further demonstrating that these antagonists may
not disrupt the functions of Treg in other healthy tissues.40
A)
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 126
1
2
3
4
5
6
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
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
B)
Figure 5. Treatment with Compound 31 selectively reduces Treg trafficking into the
tumor. A) Schematic outline of treatment and tumor model that is used in this study. B)
+
Number of GFP T in tumor (left) and spleen (right). Plasma concentration of compound 31 is
reg
ACS Paragon Plus Environment
22

Page 23 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
measured 24 hours after last treatment (bottom). For statistical analysis, the one-way ANOVA
was used.
Pharmacokinetic Profiling of 31. Results from the Treg migration study were
encouraging and led to further profiling the in vivo pharmacokinetics of compound 31 in
rat, dog, and cyno (Table 7). Additionally, the predicted hepatic clearance of 31 was
found across species by monitoring the in vitro metabolic stability of 31 in a time-course
assay with hepatocytes. The in vivo CL of all species were within 2-fold of their
predicted hepatic CL, demonstrating good in vivo/in vitro correlation. When comparing
the in vivo hepatic clearance across species, 31 suffered from very high clearance in
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
rats while displaying lower clearance in dog and cynomolgus monkeys.
Table 7. PK Profile of 31 Across Speciesa
ratb dogb cynob
human
7.2
Pred. Hepatic CL
(mL/min/kg)
45.0 18.3
12.5
in vivo CL (mL/min/kg) 74.7 10.7
11.3
9.9
---
---
IV Terminal Half Life
(hr)
2.2 16.4
F (%)
28
75
30
---
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 126
1
2
3
4
5
6
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
a
b
Three animals per group were used in each study. Dose of 0.5 mg/kg IV or 2
mg/kg PO
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
Synthetic Chemistry. Synthetic efforts began by devising similar routes for
piperidinyl amides 8 and 9. Coupling of pyrazinyl chlorides 38 and 39 to tert-butyl 4-
(
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate via
a Pd-catalyzed Suzuki coupling in the presence of aqueous base yielded the desired C-
C bond formation (Scheme 1). Removal of the Boc-protecting group followed by a
HATU coupling resulted in the installation of the basic piperidyl left hand piece. Finally,
treatment with 4N HCl in dioxane revealed the basic amine to furnish compounds 8 and
9.
Scheme 1. Synthesis of Compounds 8 and 9a
Cl Me
Cl Me
NH
NH
a, b, c, d
Cl
N
N
Cl
N
N
N
Cl
N
H
3
8
O
8
Cl
Me
Cl
Me
N
N
N
Cl
a, b, c, d
Cl
N
N
N
N
N
N
Cl
N
H
O
3
9
9
ACS Paragon Plus Environment
24

Page 25 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
aReagents and conditions: (a) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
3
,6-dihydropyridine-1(2H)-carboxylate, 1M Na CO (aq.), Pd(dppf)Cl , THF, 80-100 °C;
2
3
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
(b) 4N HCl (in dioxane), MeOH, rt; (c) HATU, DIPEA, DMF, rt; (d) 4N HCl (in dioxane),
MeOH, rt.
As a representative example of the piperidyl-pyrrolidine series, the synthesis of
compound 30 is shown (Scheme 2). The core structure was prepared by first heating
hydrazine 40 (see experimental section for synthesis) with glyoxylate 41 in THF to form
a mixture of hydrazones 42 and 43. Treating these hydrazones with NaH in THF
initiated an intramolecular S Ar to form the pyrazolo portion of the core structure in 44.
N
Conversion of ester 44 to the final nitrile core 46 proceeded cleanly upon stirring with
NH OH to form 45, followed by dehydration with Burgess reagent. Reductive amination
4
of prolinol 47 with ketone 48 followed by treatment with TFA to remove the Boc-
protecting group provided the requisite amino side chain 49 as a mixture of
stereoisomers. Completion of compound 30 was achieved via simple S Ar of 49 with
N
the chloropyrazine core 46 under thermal conditions to afford the desired antagonist 30
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 126
1
2
3
4
5
6
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
after chiral separation. Stereochemical confirmation for these antagonists was
confirmed via an enantioselective synthesis of a similar analogue (see experimental
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
section for details).
Scheme 2. Synthesis of Compound 30a
Me Cl
Cl
N
N
Cl
HN
N
Cl
Cl
Me
N
Cl Me
Cl
N
N
Cl
O
O
CO2Et
+
42
NH2
HCl
a
b
N
N
+
Cl
OEt
H
OEt
Cl
N
Cl
Cl
Me Cl
N
O
N
N
Cl
44
40
41
N
N
H
CO2Et
Cl
43
Cl
Cl
Me
Me
c
Cl
N
N
d
Cl
N
N
NH2
Cl
N
CN
Me
N
N
N
O
N
N
N
Cl
Cl
Cl
45
46
g
CN
N
N
HO
OH
NH
OH
N
NBoc
NH
N
30
e, f
+
Me
O
Me
Me
47
48
49
aReagents and conditions: (a) THF, 80 °C; (b) NaH, THF, 0 °C to rt, 50% yield over two
steps; (c) NH OH (29% in water), dioxane, rt, 95% yield; (d) Burgess Reagent, CH Cl ,
4
2
2
rt, 88%; (e) Na(OAc) BH, AcOH, 1,2-DCE, rt; (f) TFA, CH Cl , 40% over two steps (g)
3
2
2
DIPEA, DMF, 100 °C, 10%.
ACS Paragon Plus Environment
26

Page 27 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis of the cyclohexenyl CCR4 antagonists created a significant challenge
and is represented in the synthesis of compound 31 (Scheme 3). Gilman addition of
methyl cuprate to the α, β-unsaturated ketone 50 was accomplished to form 51.
Deprotonation of 51 with LDA and treatment with phenyl triflimide gave triflate 52 as a
mixture of olefin regio-isomers, which underwent a palladium catalyzed formation of the
Bpin-53. Separation of the desired olefin isomer followed by Suzuki-Miyaura coupling
with chloride 46 gave ketal 54, followed by treatment with TFA to unmask ketone 55.
Formation of the final analogue was completed via a simple reductive amination with
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
(R)-prolinol (47) and ketone 55, followed by chiral separation to afford compound 31.
Scheme 3. Synthesis of Cyclohexenyl CCR4 Antagonist 31a
Me Me
Me Me
O
O
OTf
O
O
B
a
b
c
Me
Me
O
O
O
O
O
O
Me
O
O
50
51
52
53
Cl
Me
N
N
Cl
Cl
OH
Cl
Cl
Me
N
Me
N
Me
CN
47
NH
N
N
N
N
N
N
Cl
Cl
Cl
N
46
Cl
CN
e
CN
CN
N
N
N
d
N
f
OH
N
N
O
54
O
55
31
O
Me
Me
Me
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 126
1
2
3
4
5
6
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
a
Reagents and conditions: (a) MeLi, CuCN, TMSCl, Et O:THF, -40 °C; (b) LDA, then
2
PhNTf , THF, -78 °C, 53% over two steps; (c) Bpin , Pd(dppf)Cl , KOAc, KBr, dioxane,
2
2
2
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
9
5 °C; (d) Pd(dppf)Cl , Na CO , THF:H O, 90 °C; (e) TFA, CH Cl , rt; (f) NaBH CN,
2
2
3
2
2
2
3
AcOH, 1,2-DCE, rt.
For larger scale synthesis of 31, a more selective route was needed that avoided
chiral separations in the final step (Scheme 4). Reductive amination between ketone 56
and (R)-a-methylbenzylamine (57) set the stereocenters on the cyclohexane ring in
>10:1 selectivity, with the desired isomer being easily isolated by silica gel
chromatography. Subsequent protecting group manipulation and formation of the vinyl
triflate proceeded in >30:1 regioselectivity of olefin 61. Suzuki coupling with Bpin-62
(obtained from 46) provided compound 63 in good yield. Removal of the Boc group and
bisalkylation with 65 afforded the desired protected prolinol 66, which after a final TBS-
deprotection yielded compound 31 as a single isomer.
nd
a
Scheme 4. 2 Generation Synthesis of Cyclohexenyl CCR4 Antagonist 31
ACS Paragon Plus Environment
28

Page 29 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Me
Ph
Me
O
NHBoc
NHBoc
O
NHBoc
OTf
H N
HN
Ph
Me
2
Me
Me
Me
c, d
e
f
57
Me
a, b
O
O
O
O
O
O
56
58
59
60
61
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
Cl
Me
OTBS
Cl
Cl
Cl
N
Cl
N
Me
N
Me
N
65
Me
CN
OMs
N
N
N
N
N
62
Cl
Cl
Cl
N
CN
h
CN
OMs
CN
j
BPin
N
N
N
3
1
i
g
N
N
OTBS
N
N
BocHN
H2N
63
64
66
Me
Me
Me
a
Reagents and conditions: (a) benzene, 95 °C, quant. yield; (b) NaBH(OAc)3,
CH Cl :AcOH (10:1), rt, 65%; (c) Pd(OH) , H , MeOH, rt, 97%; (d) Boc O, Et N, CH Cl ,
2
2
2
2
2
3
2
2
rt, 98%; (e) TsOH, THF:H O, rt, 70%; (f) LiHMDS, PhNTf , THF, -78 °C, 97%; (g)
2
2
Pd(PPh ) , Na CO , toluene:EtOH:H O, 90 °C, 76%; (h) 4 N HCl in dioxane, CH Cl , rt,
3
4
2
3
2
2
2
quant. yield; (i) DIPEA, MeCN, rt, 75%; (j) 4 N HCl in dioxane, CH Cl , rt, 82%.
2
2
CONCLUSIONS
A novel, orally bioavailable cyclohexenyl series of CCR4 antagonists has been
discovered that is potent and selective (see Supporting Information for data) against
CCR4. Compound 31 exhibits good in vitro and in vivo ADME properties, but
importantly, it inhibits Treg trafficking into the tumor microenvironment without
ACS Paragon Plus Environment
29