Design and synthesis of novel small molecule CCR2 antagonists: Evaluation of 4-aminopiperidine derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.10.060

A novel N-(2-oxo-2-(piperidin-4-ylamino)ethyl)-3-(trifluoromethyl)benzamide series of human CCR2 chemokine receptor antagonists was identified. With a pharmacophore model based on known CCR2 antagonists a new core scaffold was designed, analogues of it synthesized and structure-affinity relationship studies derived yielding a new high affinity CCR2 antagonist N-(2-((1-(4-(3-methoxyphenyl)cyclohexyl)piperidin-4-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide.

Full text of DOI:10.1016/j.bmcl.2014.10.060

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5377–5380
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel small molecule CCR2 antagonists:
Evaluation of 4-aminopiperidine derivatives
M. Vilums a, A. J. M. Zweemer a, S. Dekkers a, Y. Askar a, H. de Vries a, J. Saunders b, D. Stamos b, J. Brussee a,
L. H. Heitman a, A. P. IJzerman a,
⇑
a Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
b Vertex Pharmaceuticals Inc., San Diego, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel N-(2-oxo-2-(piperidin-4-ylamino)ethyl)-3-(trifluoromethyl)benzamide series of human CCR2
chemokine receptor antagonists was identified. With a pharmacophore model based on known CCR2
antagonists a new core scaffold was designed, analogues of it synthesized and structure–affinity relation-
ship studies derived yielding a new high affinity CCR2 antagonist N-(2-((1-(4-(3-methoxyphenyl)cyclo-
hexyl)piperidin-4-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide.
Received 28 August 2014
Revised 14 October 2014
Accepted 17 October 2014
Available online 23 October 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
CCR2
Chemokine
Antagonist
4-Aminopiperidine
The CC chemokine CCL2, through its interaction with the CCR2
protein-coupled receptor, plays an important role in the
molecule CCR2 antagonists to inhibit the chemotactic response of
CCL2.8–10 Consequently, the pharmaceutical industry has devoted
considerable efforts to the development of CCR2 antagonists to
combat these diseases. A vast number of different scaffolds used
in the design of CCR2 antagonists has been described.11,12
However, the bulk of these antagonists share the same struc-
tural motifs: a basic nitrogen atom in the center, flanked by two
aromatic rings of which one is connected to the nitrogen atom with
an amide containing linker and the other with an aliphatic linker
(Fig. 1). In some cases the latter aromatic ring is missing and only
the aliphatic group is left on one side.12 Usually, the central
G
recruitment of monocytes, natural killer cells, dendritic cells and
T-lymphocytes.1 Research on CCL2 knockout (KO) and CCR2 KO
mice suggests that inhibition of the CCL2/CCR2 axis may be
beneficial in the treatment of inflammatory diseases.2 The pair is
thought to be involved in atherosclerosis,3 insulin resistance,4
multiple sclerosis,5 neuropathic pain6 and asthma.7 Different
in vitro and in vivo models have shown the usefulness of small
Abbreviations: AcOH, acetic acid; Boc, tert-butyloxycarbonyl; CCL2, chemokine
ligand 2; CCR2, chemokine receptor 2; DCE, dichloroethane; DCM, dichlorometh-
ane; DIPEA, N,N-diisopropylethylamine; DMAP, N,N-dimethylaminopyridine;
DMSO, dimethylsulfoxide; INCB3344, N-(2-(((3S,4S)-1-(4-(benzo[d][1,3]dioxol-5-
yl)-4-hydroxycyclohexyl)-4-ethoxypyrrolidin-3-yl)amino)-2-oxoethyl)-3-(trifluo-
romethyl)benzamide; JNJ Lead, N-(2-((1-((1R,4R)-4-(3-(dimethylamino)phenyl)-4-
hydroxycyclohexyl)azetidin-3-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzam-
ide; KO, knockout; KOPh, potassium phenolate; LDA, lithium diisopropylamide; Lit-
BuO, lithium tert-butoxide; MeOH, methanol; MW, microwave; MS, molecular
sieves; Pd2(dba)3, tris(dibenzylideneacetone)dipalladium(0); Pd(dppf)Cl2, [1,10-
bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloro-
methane; Pd(OAc)2, palladium acetate; Pd(PPh3)4, tetrakis(triphenylphosphine)pal-
ladium(0); PEMB, 5-ethyl-2-methyl-pyridine borane; PPh3, triphenylphosphine;
PyBroP, bromo-tris-pyrrolidino phosphoniumhexafluorophosphate; SAR, structure–
affinity relationships; TFA, trifluoroacetic acid; THF, tetrahydrofuran; U2OS,
human bone osteosarcoma cells; XPhos, 2-dicyclohexylphosphino-20,40,60-
triisopropylbiphenyl.
⇑
Corresponding author. Tel.: +31 71 527 4651; fax: +31 71 527 4565.
E-mail address: ijzerman@lacdr.leidenuniv.nl (A.P. IJzerman).
Figure 1. Pharmacophore of CCR2 antagonists.
http://dx.doi.org/10.1016/j.bmcl.2014.10.060
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

5378
M. Vilums et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5377–5380
O
O
H
N
CF3
CF3
N
H
N
N
H
O
N
O
N
CF3
1, IC50 < 200 nM
2, IC50 = 180 nM
Cl
O
O
N
H
N
HO
O
CF3
O
HO
N
H
H
N
N
N
CF3
O
N
H
O
O
INCB3344,
JNJ Lead,
IC50 = 5.1 nM
IC50 = 15 nM
Figure 2. Known CCR2 antagonists.
O
O
NH2
H
H
N
N
CF3
CF3
c
c
a, b
a, b
N
H
N
H
N
Boc
Boc
HN
O
O
N
O
R
3
5
7-11, 13-20
O
N
H
N
H
N
NH
N
CF3
CF3
N
O
R
O
H
H2N
N
H
4
6
21
0
Scheme 1. Reagents and conditions: (a) PyBroP, DIPEA, DMAP, N-(3-(trifluoromethyl)benzoyl)glycine, DCM, MS 4 ÅA, room temperature; (b) dry 3 M HCl in MeOH, room
temperature, yield in two steps: 57–89%; (c) corresponding aldehyde or ketone, Na(AcO)3BH, AcOH, DCE, room temperature, (2–64%).
O
O
O
a
b
O
O
O
O
N
O
c
,
d
NH
22
23
Ts
24
O
e
R
O
O
d
,
c
O
f
33-39
O
Tf
O
R
g
O
h
25
27-32
O
O
B
O
26
Scheme 2. Reagents and conditions: (a) p-toluenesulfonhydrazide, dioxane, 130 °C, 45 min, MW, (65%); b) Pd2(dba)3, XPhos, Lit-BuO 1.0 M in hexanes, dioxane, 110 °C, 2 h,
MW, (24%); (c) Pd/C 10% wt, Pd(OAc)2, MeOH, H2, room temperature, 4–12 h, (85–99%); (d) FeCl3ꢀ6H2O, acetone, DCM, 5–12 h, room temperature, (42–99%); (e) (i)1.3 equiv
LDA, THF, under N2, ꢁ78 °C ? ꢁ25 °C, 2 h, cool down to ꢁ78 °C; (ii) N-phenyl-bis(trifluoromethanesulfonimide), ꢁ78 °C ? room temperature, 24 h, (80%); (f) 3,4-
(methylenedioxy)phenylboronic acid, KF, Pd(dppf)Cl2, room temperature overnight, (58%); (g) PdCl2, PPh3, bis(pinacolato)diboron, KOPh, toluene, under N2, 4 h at 50 °C, 24 h
at room temperature, (59%); (h) Pd(PPh3)4, corresponding arylhalogen, Na2CO3 2 M in H2O, dioxane, under N2, 80 °C, 5 h, MW, (80–99%).
nitrogen is part of an aliphatic heterocycle (e.g., piperidine, 113
pyrrolidine, 214 and INCB334415 or azetidine, JNJ Lead16) (Fig. 2).
In this Letter, we describe our efforts towards the identification
of a new class of CCR2 receptor antagonists. Using the structural
knowledge of CCR2 antagonists as in Figure 2 we generated hybrid
scaffolds based on a piperidine ring. We explored different linkers
between the basic nitrogen and aromatic groups as well as
different substituents on the aromatic group. All compounds were
evaluated in a 125I-CCL2 displacement assay on a human bone
osteosarcoma (U2OS) cell membrane preparation expressing
CCR2 as described previously by our group.17
The synthetic methods to arrive at these compounds are
depicted in Schemes 1–3. The commercially available N-Boc-
protected piperidineamines 3 and 4 were used in a peptide-
coupling reaction with N-(3-(trifluoromethyl)benzoyl)glycine
under bromo-tris-pyrrolidino phosphoniumhexafluorophosphate

M. Vilums et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5377–5380
5379
O
O
H
N
H
N
a
b
CF3
CF3
N
H
N
H
N
O
HN
O
40
5
O
H
N
CF3
N
N
N
H
N
N
O
12
Scheme 3. Reagents and conditions: (a) propargyl bromide, K2CO3, acetone, reflux overnight, (99%); (b) iodobenzene, proline, Na2CO3, NaN3, ascorbic acid, CuSO4ꢀ5H2O,
DMSO/H2O 3:1, 80 °C, 48 h, (4%).
however, to introduce other substituents we decided to transform
the triflate into boronic ester 26 with bis(pinacolato)diboron. This
allowed us to use a wider range of arylhalogens as coupling part-
ners in the Suzuki-coupling to eventually generate the desired
intermediates with good overall yields. Subsequently, reduction
of the double bond and removal of the acetal protecting group
yielded the desired ketones 33–39, which were used in reductive
amination reactions to yield the final compounds.
Table 1
CCR2 affinities of compounds 7–12
O
H
N
CF3
N
H
N
O
R
7 - 12
R
Nr.
Ki, (nM) SEM (n = 3)a
To explore the influence of a methylenetriazole group as a lin-
ker between the piperidine and phenyl moieties we used click
chemistry. First, we alkylated the piperidine of compound 5 with
propargyl bromide to generate compound 40, which was used in
a further reaction with sodium azide and iodobenzene in the pres-
ence of proline, ascorbic acid and CuSO4 as described by Feldman
et al.20
7
0%
Cl
8
9
6%
24%
As mentioned before we combined the different scaffolds from
two known CCR2 antagonists (compound 1 of Epix Delaware;13
compound 2 from Tejin14) to generate a hybrid scaffold by trans-
fecting the N-(3-(trifluoromethyl)benzoyl)glycine part onto the
piperidine ring. We argued that the expansion of the central ring
to piperidine (compared to INCB3344 and JNJ Lead) might have a
minor effect only on the configuration of the molecule. However,
the 4-chlorobenzyl group (compound 7) which had yielded good
affinity in combination with the pyrrolidine scaffold14 (compound
2), provided no affinity in the case of piperidine (Table 1). Extend-
ing the linker to propyl (compound 8) had a minor effect on the
affinity and the rigidification of the linker into tetrahydronaphtha-
lene (compound 9) yielded negligible improvement (displacement
10
11
12
74
9
10%
12%
N
N
N
a
at 1 lM concentration of 6% and 24%, respectively). However, sep-
Human CCR2 binding affinity in [125I]CCL2 assay or % displ. at 1
l
M of [125I]CCL2
aration of the rings into a 4-phenylcyclohexyl group (compound
10) resulted in a boost of affinity (Ki = 74 nM). To explore the cor-
rect location of the phenyl ring we moved it to the 3 position on the
cyclohexane ring (compound 11), which resulted in a complete
loss of affinity. In addition, the cyclohexane’s exchange to methyl-
enetriazole as a linker (compound 12) did not yield any affinity
either. Apparently, the distance, 3D orientation and lipophilicity
provided by the cyclohexane moiety is just right for the binding
of these molecules to the CCR2 receptor and any deviation from
it results in complete loss of affinity. This could also be the reason
why the 4-aryl-cyclohexane motif is used in so many pyrroli-
dine21,22 and azetidine16,23 derivatives (e.g., INCB3344, JNJ Lead,
see Fig. 2). We continued the SAR studies with different substitu-
ents on the phenyl ring of the 4-phenyl-cyclohexyl group. Intro-
duction of a methyl group on different positions indicated that
substitution on the 2 and 4 positions (compounds 13 and 15)
decreased the affinity (Table 2). The 3 position can tolerate substi-
tution, albeit with a slight decrease in affinity (14, Ki = 270 nM).
Changing the methyl to methoxy resulted in a regain of the affinity
(compound 16, Ki = 66 nM) pointing to a possible H-bond forma-
tion in the receptor binding pocket. However, insertion of two
binding.
(PyBroP) conditions.18 Subsequent removal of the Boc-protecting
group with dry HCl in methanol produced the free amines 5 and
6. These amines were used in reductive amination reactions with
different aldehydes and ketones to yield the desired products 7–
11, 13–21 (Scheme 1).
For the synthesis of the desired ketones we first used a synthetic
route via hydrazone intermediates (Scheme 2) under conditions
described by Barluenga et al.19 Commercially available ketone 22
was reacted with tosylhydrazide to generate hydrazone 23, which
was used subsequently in a Pd-catalyzed cross-coupling reaction
to generate 24 with moderate yield. However, attempts to use this
method with other substituents on the phenyl ring resulted in very
poor yields or no product at all. Another synthetic route was there-
fore chosen to yield the desired ketones. The acetal protected cyclo-
hexanone 22 was deprotonated with lithium diisopropylamide
(LDA) and reacted with N-phenyl-bis(trifluoromethanesulfoni-
mide) to generate triflate 25. This compound was used directly in
a Suzuki-coupling with 3,4-methylenedioxy⁄⁄⁄phenylboronic acid,

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M. Vilums et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5377–5380
Table 2
CCR2 affinities of compounds 13-21
O
H
N
CF3
N
H
H
N
N
O
O
N
CF3
N
H
R
O
13-20
21
Nr.
R
Ki, (nM) SEM (n = 3)a
13
14
15
16
17
18
19
20
21
2-Me
3-Me
4-Me
3-OMe
3,5-Di-OMe
2,6-Di-OMe
4-OH
26%
270 20
38%
66 12
41%
42%
139 35
90 18
31%
3,4-OCH2O–
—
a
Human CCR2 binding affinity in [125I]CCL2 assay or % displ. at 1 M of [125I]CCL2 binding.
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and 18) yielded a decrease in affinity (displacement of 41% and
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compared to 10. Combining substituents of 16 and 19 into a 3,4-
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In conclusion, we have synthesized a novel series of N-(2-oxo-2-
(piperidin-4-ylamino)ethyl)-3-(trifluoromethyl)benzamide deriva-
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identified as good hits for CCR2 antagonism and might be consid-
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Acknowledgments
This study was financially supported by the Dutch Top Institute
Pharma, project number D1-301. We thank Dr. Julien Louvel and
Jacobus van Veldhoven for their input in analytical data analysis.
We acknowledge Prof. Dr. Martine Smit (Vrije Universiteit, Amster-
dam, The Netherlands) for helpful comments and suggestions.
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Supplementary data
Supplementary data (experimental procedures for the synthesis
of the compounds) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2014.10.060.
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