Discovery and optimization of pyrazole amides as antagonists of CCR1

Article abstract of DOI:10.1016/j.bmcl.2018.11.015

A HTS screen for CCR1 antagonists afforded a novel sub-micromolar hit 5 containing a pyrazole core. In this report the design, optimization, and SAR of novel CCR1 antagonists based on a pyrazole core motif is presented. Optimization led to the advanced ca

Full text of DOI:10.1016/j.bmcl.2018.11.015

Accepted Manuscript
Discovery and Optimization of Pyrazole Amides as Antagonists of CCR1
Christian Harcken, Christopher Sarko, Can Mao, John Lord, Brian Raudenbush,
Hossein Razavi, Pingrong Liu, Alan Swinamer, Darren Disalvo, Thomas Lee,
Siqi Lin, Alison Kukulka, Heather Grbic, Mita Patel, Monica Patel, Kim
Fletcher, David Joseph, Della White, Laura Amodeo, Karen Berg, Maryanne
Brown, David S. Thomson
PII:
S0960-894X(18)30880-1
https://doi.org/10.1016/j.bmcl.2018.11.015
BMCL 26126
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 September 2018
4 November 2018
8 November 2018
Please cite this article as: Harcken, C., Sarko, C., Mao, C., Lord, J., Raudenbush, B., Razavi, H., Liu, P., Swinamer,
A., Disalvo, D., Lee, T., Lin, S., Kukulka, A., Grbic, H., Patel, M., Patel, M., Fletcher, K., Joseph, D., White, D.,
Amodeo, L., Berg, K., Brown, M., Thomson, D.S., Discovery and Optimization of Pyrazole Amides as Antagonists
of CCR1, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.015
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Discovery and Optimization of Pyrazole Amides as
Antagonists of CCR1
Christian Harcken a,*, Christopher Sarko a,‡, Can Mao a,‡, John Lord a,‡, Brian Raudenbush
a,‡, Hossein Razavia,‡, Pingrong Liu a, Alan Swinamer a, Darren Disalvo a,‡, Thomas Lee
a,‡, Siqi Lin b, Alison Kukulka b,‡, Heather Grbic c, Mita Patel c,‡, Monica Patel c,‡, Kim
Fletcher c,‡, David Joseph c, Della White d, Laura Amodeo d, Karen Berg d,‡, Maryanne
Brown d,‡, and David S. Thomson a,‡
a Medicinal Chemistry Department, b Compound Profiling Department, c Drug Discovery Support
Department, and d Immunology & Respiratory Disease Research Department, Boehringer Ingelheim
Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877-0368, USA
‡Former employee
 To whom correspondence should be addressed. Tel: (203) 778-7924, Fax: (203)
791-6410, E-mail: christian.harcken@boehringer-ingelheim.com
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Key words: Chemokine Receptors; CCR1; Pyrazole; HTS hit; Development candidate
Abstract—A HTS screen for CCR1 antagonists afforded a novel sub-micromolar hit 5 containing a
pyrazole core. In this report the design, optimization, and SAR of novel CCR1 antagonists based on a
pyrazole core motif is presented. Optimization led to the advanced candidate compounds (S)-16q and (S)-
16r with 250-fold improved CCR1 potency, excellent off-target selectivity and attractive drug-like
properties.
The selective accumulation and activation of leukocytes in inflamed tissues contributes to
the pathogenesis of inflammatory and autoimmune diseases. It has been suggested that
chemokines and their receptors, which belong to a family of seven transmembrane G-
protein coupled receptors (GPCRs), may be involved in this process. Chemokine
Receptor 1 (CCR1) is a receptor for at least three chemokines (MIP-1, MCP-3, and
RANTES).1-4 Despite set-backs in the use of CCR1 antagonists in autoimmune disease
and inflammation, pharmacological intervention to disease via CCR1 remains of strong
1

interest. For example, recent publications discussed CCR1 antagonism for the potential
treatment of systemic fungal infection or in oncology.5,6
A number of publications have disclosed ligands as antagonists of CCR1 and several
have shown promising results in animal models of disease.7-11 However, several early
clinical candidates have failed to show efficacy (Figure 1):12 BX-471 (1) failed to show
efficacy in a Phase II clinical trial in patients with relapsing remitting multiple sclerosis
(MS).13-15 CP-481715 (2) failed to show efficacy in Rheumatoid Arthritis (RA) patients
after 6 weeks of treatment. Compound 1 and 2 may not have achieved sufficient
inhibition of the receptor since their PK profile led to high human doses. The Millennium
compound MLN-3897 (3) was also stopped in the clinic after lack of efficacy in a Phase
II clinical trial for RA.16-18 It is presumed that compound 3 could not be dosed
sufficiently high due to safety limitations. In 2012, ChemoCentryx reported positive
proof of clinical concept for an oral CCR1 antagonist CCX354 (4; dose 200 mg, once
daily) in a 12-week Phase II trial in patients (n = 160) with RA (ACR20 response at 12
weeks = 56%).19-22 In contrast, in 2014, results from a 12-week Phase II trial in patients
(n=123) with RA with BMS-817399 (5) did not show statistically significant differences
in disease scores (ACR20, DAS28) or synovitis MRI read-outs despite achieving
excellent coverage of the receptor.23 Additional compound classes have been described
and have advanced into clinical trials.24,25
2

Cl
OH
F
O
N
O
N
O
O
N
Cl
NH
N
N
NH2
N
OH
H
H2N
O
OH
N
O
BX-471 (1)
F
CP-481715 (2)
OH
MLN-3897 (3)
N
Cl
O
N
OH
N
N
N
H
O
N
N
N
N
N
OH
H
H
O
BMS-817399 (5)
CCX-354 (4)
O
Cl
Figure 1: Clinical CCR1 antagonists
A CCR1 calcium flux assay high-throughput screening (HTS) campaign using the
Boehringer Ingelheim compound collection was performed.26 Several promising hit
clusters were identified from this screen and subsequent ADME profiling yielded three
compound classes of interest. Of these, the pyrazole class of compounds represented by 6
was prioritized as it was amenable to rapid structure-activity relationship (SAR)
expansion via high-throughput synthesis (Figure 2). The compound showed sub-
micromolar CCR1 potency (CCR1 calcium flux IC50 500 nM), and showed some
functional activity in a THP-1 chemotaxis assay (IC50 1200 nM).27 However, the
compound was extremely lipophilic with a calculated logP of 5.8. This translated into
very low aqueous solubility (<1 µg/mL).
3

Benzylic Amine
Substitution
CF3
NH
N
O
Pyrazole Substitution
and/or Replacement
N
Cl
Cl
Aryl Substitution
6
hCCR1 IC50 = 500 nM
Figure 2. Pyrazole starting hit and SAR evaluation plan
The pyrazole portion of the molecules was generally prepared through cyclization of an
alkyl β-oxoacetate 7 substituted with R2 and dimethylformamide dimethylacetal (8) in the
presence of acid (Scheme 1). The resulting intermediate was then reacted with a
hydrazine salt 9 bearing R1, in the presence of base to provide the pyrazole ester 10. The
ester was hydrolyzed to provide the corresponding carboxylic acid which is coupled with
the desired benzylic amine 11 using standard coupling conditions to provide the desired
amides 12.
Alternatively the desired β-oxoacetate 7 substituted with R2 was heated with
dimethylformamide dimethylacetal (8), followed by the addition of hydrazine hydrate (9;
R1 = H) to provide the pyrazole ester 10 (R1 = H). Conversion to the acid was then
followed by coupling with the benzyl amine 11 as described above. Coupling of the
resulting pyrazole 12a (R1 = H) with an iodoarene in the presence of CuI, base and a
suitable diamine ligand, provided the desired amide 12b (R1 = aryl).
The synthesis of specific substituted benzyl amines 11, and the post-coupling
modification of some amide compounds 12 (e.g. to install sulfonamide substituents) has
been described elsewhere.28,29 Chiral amines were prepared using Ellman’s sulfinamide
chemistry from the corresponding bromopyridine.30,31 The asymmetric synthesis of
enantiopure chiral benzylamines has been described elsewhere.32
4

O
O
H
O
O
O
O
N
N
R1
a
NH2
R2
O
+
+
H
R2
N
N
R1
7
10
9
8
R3
b, c
NH
N
12a: R1 = H
12b: R1 = Aryl
O
d
R3
R2
N
H2N
11
R1
Scheme 1. Reagents and conditions: (a) TsOH, microwave; NEt3, MeCN (b) HCl, dioxane (c) PyBop, Et3N (d) substituted
iodobenzene, K2HPO4, trans-N,N’-dimethylcyclohexane-1,2-diamine, toluene or DMF
Initial SAR efforts were focused on defining the minimum pharmacophore and
improving the CCR1 potency of the screening hit 6 (Figure 2). Table 1 shows activity
data for a number of N-1 substituted pyrazoles. The tolerance to variation of the N-1-
substituent was extremely limited: A number of small alkyl groups for R1 were prepared
(data not shown) which were not active at the highest concentrations tested in the CCR1
calcium flux assay (CCR1 IC50 > 3000 nM). Removing the halogen atoms from the N-
phenyl substituent such as in the unsubstituted phenyl analogue 13a, led to a complete
loss of CCR1 activity. Removing one halogen from 6 resulted in compounds with
retained potency, such as in the para-fluorophenyl and para-chlorophenyl analogues 13b
and 13c (CCR1 IC50 790 and 600 nM, respectively). Mono-substitution in meta-position
was not tolerated: The meta-chlorophenyl compound 13d was inactive. Ortho-
substitution was not tolerated either: The ortho-chlorophenyl analogue 13e was also
inactive. Compounds 13f-h represent three examples of variation of the type of
substituent: Neither a meta-tolyl group in 13f, nor a meta-(trifluoromethyl)phenyl as in
13g was tolerated, while the para-nitrophenyl compound 13h retained some activity
(CCR1 IC50 960 nM). Replacing the phenyl group with a benzyl group as in 13i was also
not tolerated. No improvements in potency or physical chemical properties could be
achieved through modification of the N-1 substitution. Since the original 3,4-
5

dichlorophenyl compounds such as 6 showed consistently very low aqueous solubility,
the slightly less lipophilic but equally active para-chlorophenyl substituted compound
13c was selected as a starting point for further SAR.
Table 1. N-1 substituted pyrazole SAR
CF3
NH
O
N
N
R1
CCR1 Ca2+ flux
Compound
R1
IC50 (nM)
3,4-dichlorophenyl
phenyl
500
6
>3000
790
13a
13b
13c
13d
13e
13f
13g
13h
13i
4-fluorophenyl
4-chlorophenyl
3-chlorophenyl
2-chlorophenyl
3-methylphenyl
3-(trifluoromethyl)phenyl
4-nitrophenyl
600
>3000
>3000
>3000
>3000
960
benzyl
>3000
Further optimization of compound 13c began with an evaluation of pyrazole substitution
and pyrazole replacements (Figure 2). Table 2 shows that the SAR in this moiety of the
molecule was also extremely limited. Removal of the 5-methyl group as in 14a resulted
in loss of all activity. Replacement of the methyl group with trifluoromethyl as in 14b,
methoxymethyl as in 14d, amino as in 14e, or N,N-dimethylamino as in 14f was not
tolerated. Interestingly, the polar hydroxymethyl substituent in 14c was tolerated (CCR1
IC50 1200 nM), however this group introduced a metabolically labile benzylic oxidation
site that resulted in significantly lower metabolic stability (human liver microsome
(HLM) predicted clearance 80% of hepatic bloodflow (%Qh)). Introduction of an
additional substituent in the 3-position in 14g was not tolerated (CCR1 IC50 > 3000 nM).
Table 2. 3- and 5-substituted pyrazole SAR
6

CF3
R4
N
NH
O
R2
N
Cl
CCR1 Ca2+ flux
IC50 (nM)
Compound
R2
R4
Me
H
H
H
600
13c
14aa
14b
14c
14d
14e
14f
>3000
>3000
1200
CF3
H
CH2OH
CH2OMe
NH2
H
H
>3000
>3000
>3000
>3000
H
N(Me)2
Me
H
14ga
Me
a) contains N-(3,4-dichlorophenyl) substituent on pyrazole
Next, the entire pyrazole ring was replaced with other scaffolds. The 1,2,3-triazole 15a
was 4-fold less active (CCR1 IC50 2400 nM) than the pyrazole reference compound 13c.
Interestingly, the pyrrol 15b was slightly more potent (CCR1 IC50 350 nM) than the
pyrazole 13c. However, this moiety was not pursued further since it introduced potential
reactive sites. None of the synthesized thiophene 15c, oxazole 15d, thiazole 15e, 1,2,4-
triazole 15f and imidazole isomers 15g-h retained any CCR1 activity.
Table 3. Pyrazole replacement SAR
CF3
NH
O
X
Cl
CCR1 Ca2+ flux
IC50 (nM)
Compound
X
7

600
2400
350
13c
15a
15b
15c
N
N
N
N
N
N
1900
S
>3000
>3000
>3000
>3000
>3000
15d
15e
15f
O
S
N
N
N
N
15g
15h
N
N
N
N
SAR exploration in the benzylamine moiety of the series was more fruitful (Figure 2).
Table 4 shows selected examples. More than 500 compounds with various benzylamine
moieties were explored, initially in directed libraries, and ultimately as single point
analogs and combination molecules. Simple substitution with small and/or polar
substituents on the benzylamine was not tolerated, such as in the 3-hydroxybenzyl
compound 16a or the 3-aminobenzyl compound 16b. However, the high polar-surface
area methylsulfonyl group was tolerated: The 3-methylsulfonyl compound 16c retained
activity with a modest loss of potency (CCR1 IC50 2100 nM). While the same group was
not tolerated in the 2-position in 16d, the group could alternatively be placed in the 4-
8

position as in 16e (CCR1 IC50 1700 nM). Related polar groups were explored and were
generally not tolerated e.g. amide 16g (CCR1 IC50 >3000 nM). Solely the sulfonamide
16f retained activity (CCR1 IC50 420 nM). Follow-up analogues revealed that the
sulfonamide compounds in this series suffered from low solubility and the majority of
SAR focused on the sulfones. Heteroaryl replacement to the benzyl group were not
tolerated, all three pyridyl isomers 16h-j were inactive. The combination of sulfonyl
substitution and pyridyl replacement as in 16k was equally inactive.
A break-through occurred when additional branching in the benzylic position was
explored. The methylbenzyl analogue 16l showed significantly improved potency (CCR1
IC50 28 nM) that could be further improved by increasing the size of the branching
substituent to ethyl in 16m (CCR1 IC50 13 nM) and n-propyl in 16n (CCR1 IC50 10 nM).
These compounds were initially prepared using a slightly different substitution pattern on
the benzyl ring (2-fluoro-3-(trifluoromethyl)phenyl) which increases potency by 2-3 fold
over the 3-(trifluoromethyl)benzyl as in 13c, however, the potency increase due to the
added benzylic substituent was 10-20-fold. 16l-n were prepared as single enantiomers
from commercially available amines. It was later demonstrated that all activity resides in
the (S)-isomer for chiral compounds in this series.30
In these compounds, polar modifications to improve physical chemical properties that
were previously not tolerated, were not only tolerated, but led to further improvement in
potency: The combination of heteroaryl replacement (pyridines), large polar substitution
(alkylsulfonyl) and small alkyl substitution in the benzyl position (propyl) resulted in
compounds with further increased potency and improved physical chemical properties:
The pyridylsulfone 16o showed acceptable potency (CCR1 IC50 47 nM) without the use
of overly lipophilic substituents (calculated logP 3.9). Moving the sulfonyl group to the
preferred 3-position resulted in the identification of 16p with a CCR1 IC50 of 15 nM.
Additionally, moving the pyridyl nitrogen atom to the 4-position, resulted in the first
single-digit nanomolar CCR1 antagonist from this series: 16q showed a CCR1 IC50 of 3
nM. The N-methyl sulfonamide analogue 16r was equipotent (CCR1 IC50 of 4 nM).
Table 4. Benzylamide SAR
9

R3
NH
O
N
N
Cl
CCR1 Ca2+ flux
IC50 (nM)
Compound
R3
600
13c
CF3
OH
>3000
>3000
2100
16a
16b
16c
NH2
S
O O
>3000
1700
420
16d
16e
16f
S O
O
O O
S
O O
S
N
H
NH2
>3000
>3000
16g
16h
O
N
10

N
N
>3000
>3000
16i
16j
O O
S
16ka
>3000
N
28
13
10
16l
16m
16n
CF3
F
CF3
F
CF3
F
O O
S
47
16o
N
N
15
3.0
4.0
16p
16q
S
O O
N
S
O O
N
16r
S
O O
a) contains N-(3,4-dichlorophenyl) substituent on pyrazole
16q and 16r were initially synthesized in racemic form. The compounds were
subsequently resolved into pure enantiomers and the active (S)-isomers were extensively
profiled as potential development candidates.
Additional data for the two compounds (S)-16q and (S)-16r are shown in Table 5. The
CCR1 potency of 2.0 and 2.9 nM IC50, respectively, was approximately 250-fold more
potent than the original screening hit 6. This translated into functional activity in a THP-1
11

cell chemotaxis assay with 6.9 and 12 nM IC50, respectively. Compounds of the series
were minimally cross-reactive with mouse or rat CCR1 (mouse CCR1 binding IC50
>3000 nM and 600 nM, respectively) and, therefore, were not feasible to be tested in any
in vivo PoP models. (S)-16q was tested for general receptor selectivity and showed no
significant inhibition in a panel of 68 pharmacologically relevant receptors at the tested
concentration of 10 µM, with the possible exception of 55% inhibition of melatonin MT1
receptor at this concentration. The compounds (S)-16q and (S)-16r showed moderate
inhibition of the hERG channel (22 and 9.6 M IC50, respectively, in an IonWorks
automated patch-clamp hERG study).
The drug-like properties of the (S)-16q and (S)-16r were attractive: The compounds had
MW < 500 g/mole and were moderately polar with calculated logP values of 3.9 and 3.3,
respectively. The simultaneous optimization of potency and properties had resulted in
only a 19 and 38 g/mole increase in MW, while reducing the logP by over 2 units. The
compounds showed good metabolic stability in vitro with <11% and 14% Qh predicted
clearance from HLM. No significant inhibition of Cytochrome P450 enzymes was
detected. The compounds showed moderate solubility in a kinetic solubility screen at pH
7. No crystalline forms allowing determination of thermodynamic solubility were
identified.
The compounds showed acceptable PK properties, for example (S)-16q showed low
clearance of 10%Qh, VSS 1.3 L/kg, and excellent bioavailability of 87% in a rat PK study
(Sprague-Dawley rat, po at 10 mg/kg in 0.5% methyl cellulose/0.015% tween 80 in
water; iv at 1 mg/kg in 70% PEG400 in water).
Table 5. Profile of advanced compounds
HT
Solubility
pH 7
(g/mL)
CCR1
CCR1
HLMb
CL
CYP3A4
IC50
(M)
hERG
IC50
(M)c
Compound
Ca2+ flux Chemotaxis
MW clogP
IC50 (nM)
IC50 (nM)a (%Qh)
2.9
2.0
6.9
12
<11
14
29
64
43
22
447
462
3.9
3.3
(S)-16q
(S)-16r
>30
9.6
aCCR1 chemotaxis in THP-1 cells27
bHuman liver microsomes (HLM)
cIonWorks Quattro automated patch clamp system
Compounds like (S)-16q and (S)-16r offered an attractive potent, selective CCR1
antagonist profile with excellent drug-like properties. The compounds were among the
12

best CCR1 antagonist available in the field with regard to their balance of physical-
chemical properties and potency and selectivity. Ultimately, the compounds were not
advanced into further pre-clinical testing (higher species PK, general safety
pharmacology, exploratory toxicology studies), since at that time a series with
significantly further improved potency was identified. The indazole series of CCR1
antagonists was designed through an innovative scaffold hopping approach using the
pyrazole series as a starting point. The approach is described in a subsequent publication.
Competing Interest Statement: The authors have no competing interests to declare.
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Phenylpiperazin-1-yl)-2-(1H-pyrazol-1-yl)ethanones as novel CCR1 antagonists. Biorg.
Med. Chem. Lett. 2013, 23, 1228-1231.
(22) Tak, P. P.; Balanescu, A.; Tseluyko, V.; Bojin, S.; Drescher, E.; Dairaghi, D.; Miao,
S.; Marchesin, V.; Jaen, J.; Schall, T. J.; Bekker, P. Chemokine receptor CCR1 antagonist
CCX354-C treatment for rheumatoid arthritis: CARAT-2, a randomized, placebo
controlled clinical trial. Ann. Rheum. Dis. 2012, 72, 337-344.
(23) Santalla, J. B., III; Gardner, D. S.; Duncia, J. V.; Wu, H.; Dhar, M.; Cavallaro, C.;
Tebben, A. J.; Carter, P. H.; Barrish, J. C.; Yarde, M.; Briceno, S. W.; Cvijic, M. E.;
Grafstrom, R. R.; Liu, R.; Patel, S. R.; Watson, A. J.; Yang, G.; Rose, A. V.; Vickery, R.
D.; Caceres-Cortes, J.; Caporuscio, C.; Camac, D. M.; Khan, J. A.; An, Y.; Foster, W. R.;
Davies, P.; Hynes, J., Jr. Discovery of the CCR1 Antagonist, BMS-817399, for the
Treatment of Rheumatoid Arthritis. J. Med. Chem. 2014, 57, 7550-7564.
(24) Hossain, N.; Mensonides-Harsema, M.; Cooper, M. E.; Eriksson, T.; Ivanova, S.;
Bergström, L. Structure activity relationships of fused bicyclic and urea derivatives of
15

spirocyclic compounds as potent CCR1 antagonists. Bioorg. Med. Chem. Lett. 2014, 24,
108-112.
(25) Trummer, D.; Walzer, A.; Groettrup-Wolfers, E.; Schmitz, H. Efficacy, safety and
tolerability of the CCR1 antagonist BAY 86-5047 for the treatment of endometriosis-
associated pelvic pain: a randomized controlled trial. Acta Obstet. Gynecol. Scand. 2017,
96, 694-701.
(26) Compounds are assessed for the ability to block the interaction of CCR1 and its
ligand in a functional cellular assay measuring calcium flux in response to MIP-1α in
CCR1-transfected cells. Published CCR1 reference compounds gave the following IC50
values in this assay: BX-471 (1): 1.3 nM; CP-481715 (2): 59 nM; MLN-3897 (3): 0.8
nM; CCX354 (4): 2.0 nM; BMS-817399 (5): 3.0 nM.
Method A: Non-adherent cells purchased from Chemicon Corporation (HTS005C), stably
expressing recombinant CCR1 and G-alpha-16 are grown in RPMI 1640 medium
(Mediatech 10-080-CM) supplemented with 10% heat-inactivated FBS, 0.4 mg/mL
Geneticin and penicillin/streptomycin. On the day of the assay, the cells are transferred to
a beaker and dye-loaded in bulk using a Fluo-4 NW Calcium Assay Kit with probenecid
(Invitrogen F36205) at 0.8E6 cells/mL for 1 hour at room temperature. After 1 hour, they
are seeded in a 384-well tissue culture-treated plate at a density of 20,000 cells/well.
Appropriately diluted test compound is added to the well to achieve a top concentration
of 3,000 nM (diluted 3-fold with 10 doses total). The final concentration of DMSO is 1%.
The buffer is HBSS (Invitrogen 14025) with 20 mM HEPES at pH 7.4. The cells are
allowed to incubate 1 hour in the dark at room temperature. The plates are transferred to
the FLIPR TETRA where MIP-1 alpha in 1% BSA is added at the EC80 final
concentration. Wells +/- MIP-1 alpha containing diluted DMSO instead of compound
serve as the controls. Intracellular calcium flux is recorded on the FLIPR TETRA, using
excitation at 470/495 nm and emission at 515/575 nm. Data are analyzed using Activity
Base software.
Method B: Non-adherent cells purchased from Chemicon Corporation (HTS005C), stably
expressing recombinant CCR1 and G-alpha-16 are grown in RPMI 1640 medium
(Mediatech 10-080-CM) supplemented with 10% FBS, 0.4 mg/mL Geneticin and
penicillin/streptomycin. On the day of the assay, the cells are loaded with Calcium 4 dye
16

(Molecular Devices R7448) with Probenecid (Invitrogen P346400) at 8E5 cells/mL for 1
hour at room temperature. After 1 hour, they are seeded in a 384-well tissue culture-
treated plate at a density of 20,000 cells/well. Appropriately diluted test compound is
added to the well to achieve a top concentration of 3,000 nM (diluted 4-fold with 10
doses total). The final concentration of DMSO is 1%. The buffer is HBSS (Invitrogen
14025) with 20 mM HEPES at pH 7.4. The cells incubate 30 minutes at 37°C and then 30
minutes at room temperature. The plates are transferred to the HAMAMATSU
FDSS6000 where MIP-1alpha in 1% BSA is added at the EC80 final concentration. All
plates must be read within 4 hours of the start of dye-loading. Wells +/- MIP-1alpha
containing diluted DMSO instead of compound serve as the controls. Data are analyzed
using Activity Base software.
(27) Compounds are assessed for the ability to block the chemotaxis of THP-1 cells. BX-
471 (1): 1.1 nM; CP-481715 (2): 39 nM; MLN-3897 (3): 1.8 nM; CCX354 (4): 4.0 nM;
BMS-817399 (5): 1.5 nM.
THP-1 cells (500,000 cells/well, each plate will use 60 µL of cells per well at 1.1 x 107
cells/mL. Washed cells 3 times in assay buffer (HBSS + 10 mM HEPES pH 7.4 + 0.1%
BSA (Sigma #A7030)) are added to media (Cell Gro #10-040-CM) add L-Glutamine
(Cell-Gro # 25-005-CI (final 2 mM), Pen/strep (Cell-Gro # 30-002-CI) final 100 Units &
FBS (Cell-Gro #35-015-CV) final 10%) in T175 flask and grown in 37˚C incubator with
5% CO2. Cells grown between 2.0 x 105 and 1.0 x 106 cell/mL. Resuspended cells to 1.1
x 107 cells/mL. Compounds received as dry powder are dissolved in 100% DMSO (Dri-
solve EMD #MX 1457-6) at 10 mM or as liquid in 15 µL tubes (10 mM). Added reagents
by using a multidrop with a disposable head: 90 µL assay buffer to 96 well plate (Falcon
35-1190), 60 µL cell suspension to a 96 well plate (Falcon 35-1190), 280 µL of assay
buffer to column 12 of chemotaxis chamber, 280 µL of 6.75 nM RANTES to columns 1-
11 of chemotaxis chamber. Transferred 10 µL compound titration (100% DMSO) into
90 µL assay buffer mix 6 times. Transferred 6.7 µL diluted compound to 60 µL cells in
Falcon 96 well plate. Cover using lids (Falcon 35-1191), mixed by shaking. Incubated
cells for 30 minutes at 37°C and 5% CO2. Changed tips and transferred 32 µL diluted
compound to lower chamber containing 280 µL 6.75 nM RANTES (R&D systems #278-
RN/CF). Placed membrane on lower chamber. After 30 min incubation, mixed cells on
17

shaker. On the Cybio added 50 µL cells to top of membrane. Incubated 3 hours at 37°C
and 5% CO2. In a 96 well (Falcon 35-1190) added 260 µL assay buffer to column 1 row
A-D and 100 µL to columns 2-8 in Rows A-D. Added 50 µL cell suspension to column 1,
rows A-D. Titrate 1:2 to column 7. Added lid (Falcon 35-1191) place in incubator for 3
hours. On Cybio mixed % chemotaxis plate and added 40 µL to white half area plate
containing 40 µL Promega Cell Titer-Glo reagent (#G-7572). On cybio mixed lower
chamber and added 40 µL to white half area plate containing 40 µL Promega Cell Titer-
Glo reagent (#G-7572). Mixed on shaker. Incubated at room temperature for 15 min.
Read luminescence on LJL Analyst. % Chemotaxis generated by excel template.
(28) Cook, B. N.; Harcken, C.; Lee, T. W-H.; Liu, P.; Mao, C., Lord, J.; Mao, W.;
Raudenbush, B. C.; Razavi, H.; Sarko, C. R.; Swinamer, A. D. Pyrazole Compounds as
CCR1 Antagonists. PCT Int. Appl., WO2009137338, 2009.
(29) Carter, P. H.; Hynes, J. N-aryl pyrazoles, indazoles and azaindazoles as antagonists
of CC chemokine receptor 1: patent cooperation treaty applications WO2010/036632,
WO2009/134666 and WWO2009/137338. Expert Opin. Ther. Pat. 2010, 20, 1609-1618.
(30) Disalvo, D.; Kuzmich, D.; Mao, C.; Razavi, H.; Sarko, C.; Swinamer, A. D.;
Thomson, D.; Zhang, Q. Indazoles compounds as CCR1 receptor antagonist. PCT Int.
Appl., WO2009134666, 2009.
(31) Chelucci, G.; Salvatore, B.; Chessa, S.; Pinna, G. A.; Soccolini, F. An easy route to
optically active 1-substituted-1-pyridyl-methylamines by diastereoselective reduction of
enantiopure N-tert-butanesulfinyl ketimines. Tetrahedron: Asymmetry 2006, 17, 3163-
3169.
(32) Reeves, J. T.; Tan, Z.; Reeves, D. C.; Song, J. J.; Han, Z. S.; Xu, Y.; Tang, W.;
Yang, B.-S.; Razavi, H.; Harcken, C.; Kuzmich, D.; Mahaney, P. E.; Lee, H.; Busacca, C.
A.; Senanayake, C. H. Development of an Enantioselective Hydrogenation Route to (S)-
1-(2-(Methylsulfonyl)pyridin-4-yl)propan-1-amine. Org. Process Res. Dev. 2014, 18,
904-911.
18

Highlights:




A HTS screen for CCR1 antagonists afforded a novel hit containing a pyrazole core
Multi parameter optimization led to advanced candidate compounds
Candidates show 250-fold improved CCR1 potency compared to initial hit
Candidates show excellent off-target selectivity, attractive drug-like properties
19

Graphical Abstract:
N
Benzylic Amines
SO2Me
CF3
NH
NH
O
O
Pyrazole Substitution
and/or Replacement
N
N
N
N
Cl
Cl
Cl
Aryl Substitution
Pyrazole CCR1 Antagonist
Development Candidate
Pyrazole CCR1 Antagonist
Screening Hit
20