Novel Piperidinyl-Azetidines as Potent and Selective CCR4 Antagonists Elicit Antitumor Response as a Single Agent and in Combination with Checkpoint Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c00988

The C-C chemokine receptor 4 (CCR4) is broadly expressed on regulatory T cells (Treg) as well as other circulating and tissue-resident T cells. Treg can be recruited to the tumor microenvironment (TME) through the C-C chemokines CCL17 and CCL22. Treg accumulation in the TME has been shown to dampen the antitumor immune response and is thought to be an important driver in tumor immune evasion. Preclinical and clinical data suggest that reducing the Treg population in the TME can potentiate the antitumor immune response of checkpoint inhibitors. We have developed small-molecule antagonists of CCR4, featuring a novel piperidinyl-Azetidine motif, that inhibit the recruitment of Treg into the TME and elicit antitumor responses as a single agent or in combination with an immune checkpoint blockade. The discovery of these potent, selective, and orally bioavailable CCR4 antagonists, and their activity in in vitro and in vivo models, is described herein.

Full text of DOI:10.1021/acs.jmedchem.0c00988

Subscriber access provided by the University of Exeter
Article
Novel Piperidinyl-Azetidines as Potent and Selective
CCR4 Antagonists Elicit Antitumor Response as Single
Agent and in Combination with Checkpoint Inhibitors
Omar Robles, Jeffrey James Jackson, Lisa Marshall, Oezcan Talay, David Chian, Gene
Cutler, Raymond Diokno, Dennis Hu, Scott Jacobson, Emily Karbarz, Paul D Kassner, John
M Ketcham, Jenny McKinnell, Cesar Meleza, Maureen Reilly, Erin Riegler, Hunter Shunatona,
Angela Wadsworth, Ashkaan Younai, Dirk G Brockstedt, David J. Wustrow, and Mikhail Zibinsky
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00988 • Publication Date (Web): 15 Jul 2020
Downloaded from pubs.acs.org on July 15, 2020
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 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Novel Piperidinyl-Azetidines as Potent and Selective
CCR4 Antagonists Elicit Antitumor Response as
Single Agent and in Combination with Checkpoint
Inhibitors
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
Omar Robles, Jeffrey J. Jackson, Lisa Marshall, Oezcan Talay, David Chian, Gene
Cutler, Raymond Diokno, Dennis X. Hu, Scott Jacobson, Emily Karbarz, Paul D. Kassner,
John M. Ketcham, Jenny McKinnell, Cesar Meleza, Maureen K. Reilly, Erin Riegler,
Hunter P. Shunatona, Angela Wadsworth, Ashkaan Younai, Dirk G. Brockstedt, David J.
Wustrow*, and Mikhail Zibinsky*.
RAPT Therapeutics, 561 Eccles Ave, South San Francisco, CA 94080.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 135
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
ABSTRACT
The C-C chemokine receptor 4 (CCR4) is broadly expressed on regulatory T cells
Treg) as well as other circulating and tissue-resident T cells. Treg can be recruited to 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
(
tumor microenvironment (TME) through the C-C chemokines CCL17 and CCL22. Treg
accumulation in the TME has been shown to dampen anti-tumor immune response and
is thought to be an important driver in tumor immune evasion. Preclinical and clinical data
suggest that reducing the Treg population in the TME can potentiate the anti-tumor
immune response of checkpoint inhibitors. We have developed small molecule
antagonists of CCR4, featuring a novel piperidinyl-azetidine motif, that inhibit the
recruitment of Treg into the TME, and elicit anti-tumor responses as a single agent or in
combination with immune checkpoint blockade. The discovery of these potent, selective,
and orally bioavailable CCR4 antagonists, and their activity in in vitro and in vivo models,
is described herein.
INTRODUCTION
2
ACS Paragon Plus Environment

Page 3 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
+
In many cancers, recruitment of CD4 FOXP3 regulatory T cells (T ) into the
reg
tumor microenvironment (TME) reduces the anti-tumor immune response and is thought
to be a key driver in tumor immune evasion.1-4 The accumulation of Treg in the TME has
been associated with poor patient prognosis in various cancers.5-10 Preclinical and clinical
data suggest that reduction of Treg populations in the TME can enhance anti-tumor
immune responses of checkpoint therapy.11-13
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
The C-C chemokine receptor 4 (CCR4) is a G protein-coupled receptor (GPCR)
highly expressed on T , in addition to other circulating and tissue-resident T cells,
reg
including T helper type 2 (Th2) cells and skin-homing T cells.14, 15 CCR4 is the receptor
of the C-C chemokines CCL17 (also known as thymus- and activation-regulated
chemokine or TARC) and CCL22 (also called macrophage-derived chemokine or MDC).
Gradients of these chemokines direct cell trafficking and homing through chemotaxis.6, 15-
17
More recently, chemokine-like factor 1 (CKLF1) has also been shown to induce
chemotaxis via CCR4.18, 19 It is recognized that CCR4 plays a key role in stimulating Treg
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 135
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
trafficking into the TME of many tumors due to high expression levels of CCL17 and
CCL22.6, 20, 21
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
Recognizing the important role of CCR4 in cell migration and homing on specific T
cell subpopulations has made this receptor an attractive target for therapeutic
intervention. Over the last decade, several small molecule CCR4 antagonists have been
reported. These antagonists can be grouped into two classes based on their proposed
binding sites.22-24 Class I antagonists (1-4a-b,25-28 Figure 1), which feature a heterocyclic
core decorated with a lipophilic substituent and an amine-containing side chain, bind
extracellularly at the allosteric antagonist binding site I. Class II antagonists (5-6a-b,29, 30
Figure 1), containing a heterocyclic core substituted with a sulfonamide side chain and a
lipophilic motif, bind intracellularly at the allosteric antagonist binding site II.
Previously reported CCR4 antagonists were developed targeting a subset of Th2
cells for the treatment of inflammation, allergic disorders, and autoimmune disease.31, 32
Among them, GSK2239633 (5,29 Figure 1), a class II antagonist developed by
GlaxoSmithKline, was evaluated in a 2011 Phase I clinical trial in healthy volunteers for
4
ACS Paragon Plus Environment

Page 5 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the treatment of asthma. Low blood exposure resulted in low target engagement and
prevented further clinical development.33 AztraZeneca has also developed a series of
CCR4 class II antagonists and have identified AZD-1678 and AZD-2098 as preclinical
candidates (6a, 6b, Figure 1),30, 34 although no clinical development has been reported.
The CCR4 receptor has also been targeted with depleting monoclonal antibodies.
Mogamulizumab (KW0761), a humanized defucosylated anti-CCR4 depleting antibody
developed by Kyowa Hakko Kirin, was approved in Japan in 2012 for the treatment of
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
+
relapsed/refractory CCR4 adult T-cell leukemia/lymphoma (ATCLL), and in 2014 for
+
15
relapsed/refractory CCR4 cutaneous T cell lymphoma (CTCL). In August of 2018, the
FDA approved mogamulizumab to treat two types of CTCL, Sézary disease and mycosis
fungoides.35 However, systemic depletion of CCR4 containing cells in healthy tissues
appears to lead to serious skin-related side effects,36, 37 including cases of Stevens-
Johnson Syndrome (SJS). This is presumably due to depletion of Treg in the skin, leading
to drastic changes in the ratio of T effector cells to Treg and thus triggering an autoimmune
response. Small-molecule reversible inhibition of CCR4-mediated cell migration and
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 135
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
homing, without depleting specific subpopulations of T cells, could offer a safer approach.
We have demonstrated in a preclinical model that Treg numbers in the skin were not
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
affected, suggesting they are neither depleted nor their migration into healthy skin is
prevented by CCR4 receptor blockade with small molecule antagonists.38
Cl
Cl
NH
NH
OMe
N
N
N
N
OMe
N
N
N
N
N
N
H
O
1
2
Bristol-Myers-Squibb (ref. 25 )
Astellas (ref. 26)
F
Cl
Cl
F
Me
NH
N
N
N
F
R
N
Me
Me
OH
N
N
N
N
O
4a, R = Me
Me 4b, R = CN
3
Daiichi Sankyo (ref. 27)
RAPT Therapeutics (ref. 28)
Me Me
OH
HN
O
Me
X
N
N
O
O
O
Cl
S
Cl
N
N
H
N
O O
S
S
N
Cl
H
OMe
6a, AZD-2098, X = H
6b, AZD-1678, X = F
AztraZeneca (ref. 30)
5
, GSK2239633
GlaxoSmithKline (ref 29)
Figure 1. Representative Class I and Class II CCR4 antagonists.
6
ACS Paragon Plus Environment

Page 7 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
We have recently reported the discovery of novel small-molecule antagonists that
inhibit the trafficking of Treg into the TME. These class I antagonists featured a novel
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
28
pyrazolopyrazine core with a unique cyclohexenyl side chain (4a-b, Figure 1). Although
4b inhibited the migration of Treg in both in vitro and in vivo models, it suffered from high
clearance in rats and also required high doses to achieve significant reduction of Treg
migration in mouse models. While 4b was a suitable tool compound for proof of concept
experiments, we wanted to advance our program by discovering a more simplified side
chain. These new antagonists should maintain potency and selectivity for CCR4 while
also encompassing improved ADME properties required for oral dosing. Our search led
us to the discovery of a novel piperidinyl-azetidine motif, which, in combination with our
pyrazolopyrazine cores, yielded highly potent, selective, and orally bioavailable
antagonists of CCR4. Herein we highlight the SAR of this novel series as well as their
activity in several in vitro and in vivo models.
RESULTS AND DISCUSSION
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 135
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
Structure–Activity Relationships. We have previously reported a pharmacophore map that
we built using the structures of published class I CCR4 antagonists (Figure 2A). This
model was further refined using our published SAR in combination with computational
studies using a known CCR4 homology model.28,39 In this model, a highly optimized right
hand phenyl group bearing two halogen substituents in positions 2 and 4 is connected to
an aromatic core (usually heterocyclic). The core is linked to an amine-containing side
chain. The size and spatial orientation of the linker critically dictates positioning of the
basic nitrogen, which is key for activity. Using this pharmacophore model, compounds
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
28
4
a-b were designed and demonstrated high potency against CCR4. We further applied
this model to the design of a simplified side chain with the goal of reducing stereochemical
complexity in our antagonists.
We turned our attention to smaller ring systems and were attracted to the simplicity
28
of symmetrically substituted four membered rings. Our previously reported SAR, as well
as other published data,25-27 suggested that the connecting atom from the side chain to
2
the core should be a nitrogen or a sp carbon in order to keep the most favorable spatial
8
ACS Paragon Plus Environment

Page 9 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
orientation. Thus, we set out to explore the nitrogen containing azetidine ring. Our refined
pharmacophore map incorporating azetidine as a linker is shown in Figure 2B.
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
Using a simple 6-4 ring system in which a piperidine ring is connected to the
azetidine, we walked the nitrogen around the piperidine ring to identify the ideal
positioning of the basic nitrogen (Figure 2C). Compound 7, with the nitrogen directly
connected to the azetidine ring, was found to be inactive in a calcium flux assay.
Increasing the distance of the basic nitrogen to the azetidinyl nitrogen, as seen in
compound 8, resulted in modest but encouraging potency with an IC of 1.37 µM. We
50
moved the basic nitrogen one atom further and tested the 3-(piperidin-3-yl)azetidine motif.
We were pleased to find compound 9 had an IC of 596 nM in the calcium flux assay as
50
a 1:1 mixture of diastereomers. Finally, we prepared the 3-(piperidin-4-yl)azetidine analog
10 that had the basic nitrogen furthest from the linker nitrogen and found that this analog
was inactive.
Next, we set out to explore the chemical space around the novel 3-(piperidin-3-
yl)azetidine side chain. This included the role substitutions on the basic nitrogen, and the
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 135
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
absolute stereochemistry of the piperidine ring, would play on potency of these
antagonists. We found that polar groups linked by one, two, or three carbons to the
piperidinyl nitrogen dramatically improved the potency of these CCR4 antagonists, as
exemplified by sulfonamide 16, which possessed an IC of 121 nM prepared as a 1:1
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
50
mixture of diastereomers from racemic amine 11 (Scheme 1). At this point, we decided
to separate the diastereomers by chiral preparative HPLC and found that antagonist 18
bearing the (R) configuration was 5-fold more potent than the corresponding diastereomer
17 with (S) configuration.
10
ACS Paragon Plus Environment

Page 11 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Cl
Cl
A
Hal
Y
C
Cl
Cl
Me
Lipophilic Motif
Hal
Me
N
N
N
N
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
Me
X
Me
N
N
N
N
Heterocyclic Core
N
Me
N
N
N
Me
N
Side Chain
Basic Nitrogen Key for Activity
7
8
Ca2+ flux SF IC50 = >5 M
Ca2+ flux SF IC50 = 1.37 M
Cl
Cl
B
Cl
Cl
Me
Cl
Cl
N
N
Me
N
N
Optimized RAPT
Fragment
Me
Me
Me
N
N
N
N
EtO
O
N
N
R
N
N
N
N
N
N
Me
N
Novel Azetidine Linker
Group with Basic Nitrogen
10
Ca2+ flux SF IC50 = >5 M
9
N
Ca2+ flux SF IC50 = 596 nM
Figure 2. A. Pharmacophore map of previously reported CCR4 antagonists. B. Current
RAPT Pharmacophore model. C. Design of novel piperidinyl-azetidine series using a
pharmacophore map for class I CCR4 antagonists: topological and spatial orientation of
the basic nitrogen is crucial for maintaining potency.
Scheme 1. Synthesis of 16 and activities of the single diastereomers 17 and 18.a
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 135
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
Cl
Cl
Me
Cl
N
N
N
N
FO2S
H2NO2S
H2NO2S
NBoc
NBoc
NBoc
NH
15a
a
b
c
Me
NH
N
N
N
d
1
1
12
13
14
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
Cl
Cl
Cl
Cl
Me
Me
Me
Chiral HPLC
Separation
N
N
N
N
N
N
Me
Me
Me
O
O
O
O
N
O
O
N
N
+
e
S
S
S
N
N
2
H N
N
2
H N
H2N
N
N
H
N
H
N
N
(S)
N
(R)
16, 15%
17
Ca2+ flux SF IC50 = 455 nM
18
Ca2+ flux SF IC50 = 121 nM
Ca2+ flux SF IC50 = 84 nM
a
Reagents and conditions: (a) ethenesulfonyl fluoride, DCM. (b) MeOH, NH OH, 70℃.
4
(c) 4N HCl in dioxane, DCM. (d) (R)-6-chloro-1-(1-(2,4-dichlorophenyl)ethyl)-3-methyl-
1
H-pyrazolo[3,4-b]pyrazine, DMSO, N,N-diisopropylethylamine, 80 ℃, 4 hrs. (d)
Separation of diastereomers on chiral HPLC.
The absolute stereochemistry of compound 18 was unambiguously confirmed by
single crystal X-ray diffraction of a key intermediate (see supporting information for
details).
These findings prompted an extensive SAR campaign to improve the potency of
this series in the primary calcium flux assay, and subsequently, to evaluate the ability of
12
ACS Paragon Plus Environment

Page 13 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
these antagonists to inhibit the migration of cells expressing CCR4 in the presence of a
CCL22 gradient. We have previously reported a 96-well medium-throughput chemotaxis
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
(
CTX) assay using CCR4-expressing CCRF-CEM cells.28 Antagonists that inhibited
calcium flux in the double-digit nanomolar range were also run in a physiologically more
relevant CTX assay. This platform is considered the gold standard assay for interrogating
in vitro cellular migration. Since the assay is run in 100% human serum (HS), the reported
potencies 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. Importantly, this platform can be
extended to other relevant cell types, including primary immune cells (Treg and Th2 cells).
The results are summarized in Table 1. As mentioned above, sulfonamide 18 was
highly potent in the calcium flux assay, however this compound suffered a large shift in
the CTX assay. Reversing the terminal sulfonamide was tolerated and antagonists (19
and 20) displayed increased potency in the primary calcium flux assay (29 and 31 nM,
respectively) with only a 4-5-fold shift in the cell migration assay. 2-carbon linked sulfone
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 135
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
2
1 was equipotent to sulfonamide 18 and suffered a similar CTX shift. While the 3-carbon
linked sulfone 22 was 3-fold more potent in the calcium flux assay (22 nM), it had a 10-
fold shift in the CTX assay. Replacing the sulfonamide functional group for a 3-carbon
linked carboxylic acid 23 displayed similar inhibition in the calcium flux and similar shift in
the CTX assay, as compared to the parent sulfonamide. The 2-carbon linked carboxylic
acid 24 had a similar potency in the calcium flux assay but was 2-fold more potent in the
CTX assay. Interestingly, replacing the C3-methyl in the pyrazolopyrazine core for a C3-
cyano pyrazolopyrazine core yielded carboxylic acid 25 that was 2-fold more potent in the
calcium flux assay than the C3-methyl analog, but with similar CTX potencies. Decreasing
the length of the linker by one carbon was tolerated and provided antagonist 26 that
displayed increased activity in the primary calcium flux assay (36 nM) but also showed a
large shift in the CTX assay. Terminal amides were also active CCR4 antagonists as seen
in amide 27, which was 42 nM in the calcium flux assay and 242 nM in the cell migration
assay. Reversed amides 28 and 29 were found to be marginally more potent CCR4
antagonists. Amide 28 had an IC of 6 nM in the calcium flux assay and 40 nM in 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
50
14
ACS Paragon Plus Environment

Page 15 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CTX assay, while the more lipophilic amide 29 was slightly less potent. Methyl carbamate
30 was also highly potent in the calcium flux assay (23 nM) but suffered a 5-fold shift 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
the chemotaxis assay. Introduction of a small heterocycle was also tolerated. Imidazole
31, connected by a 1-carbon linker attached to the C3-cyano pyrazolopyrazine core,
possessed a calcium flux potency of 21 nM and CTX potency of 97 nM.
Table 1. SAR of the Piperidinyl Side Chain
Cl
Cl
Me
N
N
R2
N
N
N
R1
H
N
Ca2+
CTX
IC50
flux
cmpd
R1
R2
IC50
nM)a
nM)b
(
(
O
O
S
H2N
1
8
9
84
29
717
136
Me
H
Me
N
S
1
O
O
Me
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 135
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
Me
H
N
Me
S
2
0
Me
31
115
O
O
O
O
S
21
22
23
24
25
Me
Me
Me
Me
Me
CN
77
22
69
91
47
575
259
559
370
409
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
Me
S
O
O
HO
O
O
O
HO
HO
HO
26
27
28
O
CN
Me
Me
36
42
6
775
247
40
O
H2N
H
Me
N
O
Me
H
N
Me
2
9
0
Me
Me
18
23
59
O
H
N
O
Me
3
133
O
H
N
3
1
2
CN
CN
21
97
N
NC
3
1470
N.D.
16
ACS Paragon Plus Environment

Page 17 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
NC
3
3
3
3
3
3
3
3
4
5
6
7
8
9
CN
Me
28
505
370
37
523
N.D.
1500
109
74
F3C
F3C
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
CN
HO
HO
Me
Me
24
HO
HO
HO
HO
HO
CN
22
8
50
36
CONH2
40
CO H
>5000
25
N.D.
446
379
2
41
CONHMe
CONMe2
42
9
a
b
Assay ran in absence of serum. Assay ran in 100% human serum. N.D. Not
determined.
Terminal nitriles produced less potent CCR4 antagonists. Antagonist 32, bearing
a terminal nitrile connected to the piperidine through a one carbon linker, was 1470 nM in
the calcium flux assay. Extending the linker by one carbon improved the potency of analog
3
3, but it had a large shift in the chemotaxis assay. Similarly, introducing a terminal
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 135
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
trifluoromethyl with a 2-carbon tether was detrimental for potency, as compound 34 was
505 nM in the calcium flux assay and its analog 35 on the C3-cyano pyrazolopyrazine
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
core had similar activity. Simple alcohols were also studied and yielded highly active
CCR4 antagonists. The 3-carbon linked alcohol 36 was moderately less potent than its
2
-carbon analog 37. Antagonist 37 showed an IC of 24 nM in the inhibition of calcium
50
flux and 74 nM in the chemotaxis assay. Replacing the C3-methyl for a C3-cyano on the
core yielded compound 38 that was highly potent in both CCR4 functional assays. In the
primary calcium flux assay, compound 38 had an IC of 22 nM and 50 nM in the CTX
50
cell migration assay. We then explored other substitutions at the C3-position of the
pyrazolopyrazine core. Placing terminal amides at the C3-position was productive and
generated potent CCR4 antagonist 39. Compound 39, bearing an unsubstituted amide at
the C3 position, displayed an IC 0f 8 nM in the calcium flux assay and 36 nM in the
50
chemotaxis assay. The C3-carboxylic acid substituted pyrazolopyrazine 40 was found to
be inactive. Monomethylated amide 41 was 3-fold less potent than the unsubstituted
analog 39 and had a large shift in the migration assay. Finally, adding a second methyl
18
ACS Paragon Plus Environment

Page 19 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
group on the terminal amide 42 restored potency in the calcium flux assay (9 nM) but this
substituted amide suffered from a large shift in the chemotaxis assay.
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
We then evaluated the in vitro metabolic stability and pharmacokinetic properties
of several of these potent CCR4 antagonists in human and rat hepatocytes as well as rat
in vivo PK (Table 2). Sulfonamide 19 was moderately stable in a human hepatocyte assay
with 85% remaining, while in rat hepatocytes only 53% remained after incubating for 60
minutes. The lower rat in vitro metabolic stability of 19 was also reflected in a rat in vivo
PK study, which showed medium clearance and low bioavailability. The reverse amide
28 displayed good stability in both rat and human hepatocytes. In rat PK, 28 had relatively
low clearance (11 mL/min/kg), however the bioavailability of this antagonist was only
13%. The in vitro metabolic stability of the 2-carbon linked alcohol 37 was also assessed
and revealed that this antagonist was stable in human hepatocytes (87% remaining) and
moderately stable in the rat hepatocyte assay (62% remaining). Consistent with these
findings, the rat in vivo PK of compound 37 was remarkably improved, with moderate
clearance (32.3 mL/min/kg) and 61% bioavailability.
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 135
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 2. In Vitro Metabolic Stability and Rat in Vivo PK of Selected CCR4 Antagonist
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
Rat
Hepatocyt
Rat IV CL
mL/min/kg)b
a
cmpd es % rem
(
%Fc
11
13
61
49
0
r/h
1
9
53/85
41.6
11
28
68/94
62/87
73/91
95/95
37
32.3
47.6
190.8
38
39
a
b
c
% remaining after incubating for 60 min . Dose of 0.5 mg/kg IV. Dose of 2 mg/kg PO.
Compound 38 had slightly improved metabolic stability in the in vitro rat and human
hepatocyte assay as compared to its analog 37. Compound 38 possessed medium
clearance (47.6 mL/min/kg) in a rat in vivo PK experiment and was 49% bioavailable upon
oral dosing. Amide 39 was remarkably stable in both in human and rat hepatocytes (95%
20
ACS Paragon Plus Environment

Page 21 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
and 95% remaining, correspondingly) but had extremely high clearance in a rat in vivo
PK study (190.8 mL/min/kg) and was not bioavailable.
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
Compounds 37 and 38 were evaluated for inhibition of the five most common
isoforms of cytochrome P450 and found that both possessed clean CYP450 profiles
(Table 3). Furthermore, compounds 37 and 38 showed no activity in a CYP450 induction
assay. However, in a CYP450 Time-Dependent Inhibition assay (CYP450 TDI) for the
3
A4 isoform, there was a 2.6-fold shift in its IC when compound 37 was incubated in the
50
presence NADPH as compared to when it was incubated in the absence of the cofactor.
Compound 38 did not display a significant shift in its IC when it was incubated in the
50
presence NADPH as compared to incubation in the absence of the cofactor. We
speculate that metabolism at the C3-methyl in the pyrazolopyrazine core might be
responsible for the CYP450 TDI.
Table 3. In Vitro CYP Profile of Compounds 37 and 38
Assay
Cmpd
Cmpd
38
37
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 135
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
CYP450 Inhibition
IC ) for isoforms 1A2,
(
50
>
40 µM > 40 µM
2C9, 2C19, 2D6 and
3A4 a
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
CYP Induction a
Negative Negative
CYP450 TDI IC shift
50
2
.6-fold
1.2-fold
for isoform 3A4a
aSee supporting information for details.
Based on these results, we further profiled compound 38 in several in vitro and in
vivo experiments. A mouse in vivo PK study revealed that compound 38 had low
clearance (10.2 mL/min/kg), medium volume of distribution (5.2 L/kg), a half-life of 6.9 h,
and good bioavailability (%F = 29) that led to high exposure after oral dosing (Table 4).
When CCL17 was used as a chemoattractant instead of CCL22 the CTX IC value
5
0
was 64 nM. Compound 38 was also evaluated for its ability to inhibit the CCR4-mediated
migration of the more physiologically relevant induced mouse and human regulatory T
cells (iT ) in an in vitro chemotaxis assay (Table 4). Compound 38 inhibited the
reg
migration of mouse iTreg cells with an IC of 39 nM, while the IC in human iT cells
50
50
reg
was 33 nM.
22
ACS Paragon Plus Environment

Page 23 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 4. Mouse PK, in Vitro iTreg Chemotaxis and Selectivity Profile of Antagonist 38
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
Assay
Result
mouse in vivo PK
Cl (mL/min/kg)a
Vss (L/kg)a
10.2
5.2
a
T (hr)
6.9
1
/2
PO AUC0-last (ng *hr
751
mL)b
/
F (%)b
29
CTX IC (nM)
50 / 64c
5
0
miTreg CTX IC50
(nM)d
3
9
3
hiTreg CTX IC50
3
nM)e
(
a
b
c
Dose of 0.5 mg/kg IV. Dose of 2 mg/kg PO CCL17 was used as a chemoattractant
d
e
Assay run in 100% mouse serum. Assay run in 100% human serum.
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 135
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
120
00
1
8
6
4
2
0
0
0
0
0
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
-20
0
1
2
3
4
5
6
7
8
9
1
1
1
2
3
4
5
6
7
1
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
C
C
C
C
C
C
C
C
C
L
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
K
3
X
X
X
X
X
X
X
C
M
X
C
C
C
C
C
C
C
C
C
Chemokine Receptor
Figure 4. Selectivity of CCR4 antagonist 38 at 500 nM against a panel of human
chemokine receptors in a beta arrestin recruitment assay.
The selectivity of CCR4 antagonist 38 against other prevalent chemokine
receptors was also assessed and it was found that compound 38 was highly selective for
CCR4, as it did not inhibit other chemokine receptors from the panel at a concentration
of 500 nM (Figure 4).
In Vivo Treg Migration Studies in Mouse. Having established that antagonist 38 potently
and selectively inhibited the migration of Treg cells in in vitro models, we then evaluated
the ability of 38 to inhibit the migration of Treg into the TME using a previously reported in
24
ACS Paragon Plus Environment

Page 25 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
vivo murine pancreatic ductal adenocarcinoma model (Pan02).40,41 Compound 38
+
significantly inhibited the migration of adoptively transferred GFP iT to the TME in a
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
dose-dependent manner, as compared to the control group (Figure 5).42 Importantly,
treatment with compound 38 did not show any significant reduction in the number of GFP+
or endogenous Treg in the spleen, nor other healthy tissues, including the skin and lung.38
B
5000
000
3000
*
4
*
*
2
1
000
000
0
* p = 0.05 vs Vehicle
* p < 0.01 vs Vehicle
*
le
kg
kg
kg
/
hic
/
/
g
g
g
e
m
m
m
0
V
3
0
1
3
Figure 5. Treatment with Compound 38 reduces Treg migration into the tumor. (A)
Schematic outline of treatment and tumor model that is used in this study. (B) Number of
+
GFP T in tumor. For statistical analysis, the two-tailed t-test was used.
reg
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 135
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
Anti-tumor efficacy of compound 38 in a Pan02-OVA and CT26 tumor models as
a single agent and in combination with checkpoint inhibitors. After confirming that CCR4
antagonist 38 effectively inhibited the migration of regulatory T cells into the TME in an in
vivo model, we sought to explore the extent of anti-tumor efficacy that could be achieved
through CCR4 antagonism. To that end, we used a Pan02-OVA tumor model (see
supporting information), to examine the anti-tumor efficacy of antagonist 38 as a single
agent and in combination with an anti-CTLA-4 checkpoint inhibitor with therapeutic dosing
of both agents (Figure 6). Antagonist 38 significantly reduced the tumor growth as
compared to the vehicle control group and was comparable to the group that received the
anti-CTLA-4 antibody. Importantly, when CCR4 antagonist 38 was combined with anti-
CTLA-4, a further reduction on the tumor growth was observed.
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
26
ACS Paragon Plus Environment

Page 27 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Median Tumor Volume
5
4
3
00
00
00
Vehicle
Compound 38
Anti-CTLA-4
Combo
***
***
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
*
*
200
100
0
*** p < 0.001
** p < 0.01
0
10
20
30
40
Days post-inoculation
Figure 6. Anti-tumor efficacy of compound 38 in a Pan02-OVA model with 50 mg/kg PO
QD dosing regimen. See supporting information for details. For statistical analysis, the two-
tailed t-test was used.
Next, to evaluate synergy with another widely used checkpoint inhibitor, an anti-
PD-L1 antibody, we designed a therapeutic combination study in the CT26 mouse model
(
see supporting information). In this study (Figure 7) we observed significant tumor growth
inhibition in animals who received combination treatment compared to the single agent
treatment groups. This suggests that inhibiting Treg migration may enhance an immune-
mediated anti-tumor response by removing the inhibitory effect of Treg in TME.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 135
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
Median tumor volume
1
500
Vehicle
Compound 38
Anti-PD-L1
Combo
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
1000
*
5
00
0
****
*
p < 0.05
*
*** p < 0.00001
0
10
20
30
Days post innoculation
Figure 7. Anti-tumor efficacy of compound 38 in a CT26 model with 50 mg/kg PO QD
dosing regimen. See supporting information for details. For statistical analysis, the two-tailed
t-test was used.
Pharmacokinetic profiling of compound 38 in dog and cynomolgus monkey. Considering
the promising in vitro and in vivo pharmacology profile of antagonist 38, we sought to fully
profile its pharmacokinetic properties in higher species (see Supporting Information). In
higher species the in vivo clearance of compound 38 was 3-fold lower than predicted by
the in vitro assay. In dog, compound 38 had low clearance (7.3 mL/min/kg), a half-life of
12.7 hr, and was 44% bioavailable. Similarly, in cynomolgus monkey, compound 38 had
28
ACS Paragon Plus Environment

Page 29 of 135
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
low clearance (3.7 mL/min/kg), a long terminal half-life (10.7 hr), and good bioavailability
(%F = 41).
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
Synthetic Chemistry. CCR4 antagonists 9 and 16-42 were synthesized through a
straightforward sequence starting with introduction of an alkyl group to amine 11 (racemic
or enantioenriched), followed by deprotection of the Boc group, and completed by
nucleophilic aromatic substitution with an appropriately substituted pyrazolopyrazine
28
intermediate 15a-d. In general, introduction of an alkyl group was achieved via reductive
amination, alkylation with an alkyl halide, or Michael addition (General Procedures A-C in
Experimental Section).
Compounds 7-10 were made via nucleophilic aromatic substitution reaction
between intermediate 15a and the corresponding amines, which were either commercially
available or accessed via reductive amination as described in Scheme 2.
Compounds 9, 19, 20, 22, 24, 27, 28-31, 36, 37 and 39 were synthesized using racemic
amine 11 and diastereomers were separated by chiral HPLC or SFC. Chiral resolution of racemic
1
1 was accomplished by fractional crystallization with R-mandelic acid. X-ray crystallography
29
ACS Paragon Plus Environment