Design, synthesis and structure-activity relationships of zwitterionic spirocyclic compounds as potent CCR1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.05.087

A series of zwitterionic spirocyclic compounds were synthesised. In vitro data revealed that these compounds were potent CCR1 antagonists. In particular, 2, 4, 11 and 20 inhibited CCR1 mediated chemotaxis of THP-1 cells in a functional assay.

Full text of DOI:10.1016/j.bmcl.2013.05.087

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4026–4030
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and structure–activity relationships of zwitterionic
spirocyclic compounds as potent CCR1 antagonists
b,
Nafizal Hossain a, , Svetlana Ivanova , Åsa Sjöholm Timén b, , Jonas Bergare a, Tesfaledet Mussie a,
⇑
Lena Bergström a
a Department of Medicinal Chemistry, Respiratory Inflammation and Autoimmunity Innovative Medicines, AstraZeneca R&D, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
b Department of Medicinal Chemistry, AstraZeneca R&D Lund, Scheelevägen 1, SE-221 87 Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of zwitterionic spirocyclic compounds were synthesised. In vitro data revealed that these com-
pounds were potent CCR1 antagonists. In particular, 2, 4, 11 and 20 inhibited CCR1 mediated chemotaxis
of THP-1 cells in a functional assay.
Received 14 May 2013
Revised 24 May 2013
Accepted 28 May 2013
Available online 6 June 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Leukocytes trafficking is an important aspect of immune re-
sponse against pathogens but, to prevent inappropriate response
against healthy host cells, needs to be tightly controlled. The loss
of such tight control can be the cause of inflammatory diseases.
For instance, diseases such as multiple sclerosis1,2 rheumatoid
arthritis,3,4 are characterised by deregulated leukocytes recruit-
ment. Chemokines play an important role in various inflammatory
diseases by mediating leukocytes recruitment.5–8 Chemokines bind
to their target receptors on the cell surface of leukocytes, transmit-
ting intracellular signals by activating G-proteins, resulting in
movement of the leukocytes towards the source of the chemokines
and the desired site of action. Chemokine receptors are thus attrac-
tive targets in the development of drugs against autoimmune dis-
eases due to their vital role in regulation of leukocytes trafficking.
The C-C (cystine–cystine) chemokine receptor-1 (CCR1) and its ma-
metabolites. Pleasingly, replacement of the N-acetyl moiety with
the benzamide moiety in a substituted phenol also resulted in po-
tent CCR1 compounds (Fig. 1).15b However, these compounds are
prone to inhibit the hERG channel which could render them
unsuitable as potential drugs. Thus, we subsequently focused our
efforts on the identification of analogues devoid of hERG activity,
and it was postulated that this may be facilitated by the inclusion
of an acidic group. Thus a series of spirocyclic zwitterionic com-
pounds were designed, such as 6 and 8 (Scheme 1) which were
synthesised as described below.
The para-methoxybenzyl (PMB) protecting group in 315b was
removed by treatment with trifluoroacetic acid (TFA) to afford 4
in 85% yield. The carboxylic acid derivative 215b was coupled sep-
arately with (S)-methyl pyrrolidine-3-carboxylate and (R)-methyl
pyrrolidine-3-carboxylate in the presence of CDI in DMF to afford
5 and 7 which were hydrolysed by treatment with aqueous NaOH
to give the zwitterionic compounds 6 and 8 respectively, in accept-
able yields (Scheme 1).
Compound 3 was coupled with methyl pyrrolidine-3-carboxyl-
ate in the presence of CDI, Et3N in DMF to afford 9 which was
hydrolysed to zwitterionic compound 10. Finally, the para-
methoxybenzyl group in 10 was removed by treatment with triflu-
oroacetic acid to afford final compound 11 in 28% isolated yield
over three steps (Scheme 2). This compound was a mixture of
diastereomers.
Spirocyclic amine 1a was treated with 3-bromopropan-1-ol in
the presence of potassium carbonate in DMF to give 12 in 79% iso-
lated yield. Compound 12 was treated separately with methyl 4-
fluoro-2-hydroxybenzoate, methyl 5-chloro-2-hydroxybenzoate
and ethyl 4-chloro-5-cyano-2-hydroxybenzoate under Mitsunobu
reaction conditions to afford either derivatives 13, 15 and 17
respectively in high yields. The ester group of 13, 15 and 17 was
hydrolysed by treatment with aqueous NaOH to quantitatively
give the corresponding carboxylic acids 14, 16 and 18 which were
jor endogenous ligands MIP-1a (CCL3) and RANTES (CCL5) play an
important role in chronic inflammatory diseases such as rheuma-
toid arthritis and multiple sclerosis. Inhibition of CCR1 is expected
to be beneficial for patients who suffer from such inflammatory
disorders. Thus, small synthetic CCR1 antagonists could be useful
as therapeutic agents. The search for specific and highly potent
chemokine receptor antagonists has recently been an active area
of research9–14 and here-in, we report the discovery of zwitterionic
spirocyclic compounds as potent CCR1 antagonists.
Recently, we have reported compounds 1d, 1e and 1f (Fig. 1)
from a series of conformationally constrained spirocyclic como-
unds15 many of which are highly potent CCR1 antagonists. These
compounds contain either an N-acetyl or a benzamide moiety as
part of a substituted phenol group. Inclusion of the N-acetyl moiety
was important for CCR1 activity but, being pro-anilinic, has the po-
tential to be problematic due to possible formation of toxic aniline
⇑
Corresponding author. Tel.: +46 317761851; fax: +46 317763818.
E-mail address: nafizal.hossain@astrazeneca.com (N. Hossain).
Former employee.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.087

N. Hossain et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4026–4030
4027
O
N
O
HN
OH
OH
O
O
O
O
O
N
N
N
X
X
Cl
OH
OH
1a: X = Cl
1b: X = F
1c: X = H
1d: X = Cl hCCR1 IC50 = 360 pM
1e: X = F hCCR1 IC50 = 520 pM
1f: hCCR1 IC50 = 1.8 nM
Figure 1. Previously reported CCR1 antagonists.
R1
O
O
O
O
R1
O
N
N
O
O
O
R3
OH
O
OH
OH
O
O
O
(a)
O
O
N
N
N
Cl
Cl
Cl
R2
R1
F
F
2: R1 = F R2 = H R3 = H
7: R1 = Me (100%)
8: R1 = H (19%)
5: R1 = Me (100%)
6: R1 = H (34%)
(c)
(c)
3: R1 = OPMB R2 = Cl R3 = H
4: R1 = OH R2 = Cl R3 = H (85%)
(b)
Scheme 1. Reagents and conditions: (a) (S)-methyl pyrrolidine-3-carboxylate/(R)-methyl pyrrolidine-3-carboxylate, CDI, DMF, rt, 20 h (100%); (b) TFA, CH2Cl2, rt 1 h (85%);
(c) Aq. NaOH, EtOH, rt, 3 h (19–34%).
each coupled with (R)-methyl pyrrolidine-3-carboxylate in the
presence of CDI in DMF to afford 19, 21 and 23 in high yields,
whilst 14 was similarly also coupled with (S)-methyl pyrrolidine-
3-carboxylate to give 25 in high yield. Finally, the ester group
was hydrolysed by treatment with aqueous NaOH to afford zwit-
terionic compounds 20, 22, 24 and 26, respectively in good yield
(Scheme 3).
Spirocyclic amine 1a was treated with 3-bromo-2,2-dimethyl-
propan-1-ol in the presence of NaI and Et3N in DMF to give 28 in
low yield. Compound 28 was coupled with methyl 4-fluoro-2-
hydroxybenzoate in a Mitsunobu type reaction to afford 29 in good
yield. The ester 29 was hydrolysed to give corresponding acid
derivative 30 in 70% yield. Compound 30 was coupled with (R)-
methyl pyrrolidine-3-carboxylate/(S)-methyl pyrrolidine-3-car-
boxylate in the presence of CDI in DMF to give 31 and 33 which
were hydrolysed to give zwitterionic compounds 32 and 34 in
30% isolated yield (Scheme 4).
and 4 inhibited CCR1 mediated chemotaxis in a functional assay.
These substances were also very stable in both human microsomes
and hepatocytes as well as rat hepatocytes whilst being inactive
against the hERG channel. Compounds 2, 14 and 16 were found
to have high permeabilities as determined in a Caco-2 assay. From
initial evaluation, the zwitterion approach appeared to yield com-
pounds with an attractive overall profile, albeit with modest CCR1
potency. Thus, in an attempt to improve CCR1 potency zwitterionic
amide derivatives 6, 8 and 11 were designed and gratifyingly bio-
logical evaluation revealed these to be very potent CCR1 antago-
nists. In particular, 11 inhibited CCR1 mediated chemotaxis in a
functional assay (Table 1). As for the earlier zwitterionic com-
pounds, these compounds were also very metabolically stable
and free from hERG activity.16 The permeability of the latter series
of compounds was generally very low and, as these compounds
were intended for oral administration, it was considered that bio-
availability would consequently be limited.
Similarly, compound 14 was coupled with methyl 4,4-dimeth-
ylpyrrolidine-3-carboxylate to afford 35 which was hydrolysed to
give racemic mixture of zwitterionic compound 36 in 28% overall
yield (Scheme 5).
Initially the intermediate carboxylic acid derivatives 2, 4, 14
and 16 (also zwitterionic) were evaluated and in vitro data (Table
1) revealed these to be moderately potent CCR1 antagonists, with
the most potent being compound 2 (IC50 = 20 nM). In addition, 2
In order to improve permeability 20 and 26 were designed with
the hydroxyl group removed from the carbon linker. Pleasingly, the
biological data revealed that the hydroxyl group in 20 and 26 was
not be essential for CCR1 binding affinity and potent CCR1 antago-
nist activity was retained in such compounds. Moreover, 20 was
the most potent inhibitor of CCR1 mediated chemotaxis of the
zwitterion series (Table 1). Although stability in both human
microsomes and hepatocytes was similar to previous compounds
R2
O
O
OH
O
N
O
OH
OH
O
(a), (b), (c)
O
O
O
N
N
Cl
Cl
Cl
OPMB
Cl
R1
09: R1 = OPMB R2 = Me (100%)
10: R1 = OPMB R2 = H (100%)
11: R1 = OH R2 = H (28%)
3
Scheme 2. Reagents and conditions: (a) methyl pyrrolidine-3-carboxylate, CDI, Et3N, DMF, rt 20 h (100%); (b) aqueous NaOH, EtOH, rt, 3 h (100%); (c) TFA, CH2Cl2, rt 1.5 h
(28%).

4028
N. Hossain et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4026–4030
O
O
R3
R2
O
O
O
(b)
(a)
O
OH
N
N
N
Cl
Cl
Cl
12 (79%)
R1
13: R1 = F R2 = H R3 = Me (57%)
14: R1 = F R2 = H R3 = H (100%)
1a
15: R1 = H R2 = Cl R3 = Me (100%)
16: R1 = H R2 = Cl R3 = H (41%)
17: R1 = Cl R2 = CN R3 = Et (80%)
18. R1 = Cl R2 = CN R3 = H (70%)
O
O
O
O
R3
R3
N
O
N
O
O
O
(c)
O
O
N
N
Cl
R2
Cl
R2
R1
R1
19: R1 = F R2 = H R3 = Me (100%)
20: R1 = F R2 = H R3 = H (75%)
21: R1 = H R2 = Cl R3 = Me (100%)
22: R1 = H R2 = Cl R3 = H (60%)
23: R1 = Cl R2 = CN R3 = Et (100%)
24: R1 = Cl R2 = CN R3 = H (50%)
25: R1 = F R2 = H R3 = Me (100%)
26: R1 = F R2 = H R3 = H (82%)
Scheme 3. Reagents and conditions: (a) 3-bromo-propan-1-ol, K2CO3, DMF, rt, 2 days (79%); (b) methyl 4-fluoro-2-hydroxybenzoate/methyl 5-chloro-2-hydroxybenzoate/
ethyl 4-chloro-5-cyano-2-hydroxybenzoate, Ph3P, DEAD, THF, rt 20 h (57–100%); (c) (R)-methyl pyrrolidine-3-carboxylate/(S)-methyl pyrrolidine-3-carboxylate, CDI, DMF, rt,
20 h (100%).
O
O
R1
O
O
O
(b)
(a)
OH
O
N
N
N
Br
OH
Cl
Cl
Cl
29: R1 = Me (69%)
30: R1 = H (70%)
1a
28: (20%)
27:
F
R1
O
R1
O
O
O
N
O
N
O
(c)
O
O
O
O
N
N
Cl
Cl
F
F
31: R1 = Me (100%)
32: R1 = H (30%)
33: R1 = Me (100%)
34: R1 = H (30%)
Scheme 4. Reagents and conditions: (a) NaI, Et3N, DMF, 78 0C 60 h (20%); (b) methyl 4-fluoro-2-hydroxybenzoate, Ph3P, DEAD, THF, rt 20 h (69%); (c) (R)-methyl pyrrolidine-
3-carboxylate/(S)-methyl pyrrolidine-3-carboxylate, CDI, DMF, rt, 20 h (100%).
R1
O
O
OH
N
O
O
O
O
(a)
O
O
N
N
Cl
Cl
14
35: R1 = Me (100%)
36: R1 = H (30%)
F
F
(b)
Scheme 5. Reagents and conditions: (a) methyl 4,4-dimethylpyrrolidine-3-carboxylate, CDI, DMF, rt, 20 h (100%); (b) aqueous NaOH, EtOH, rt, 3 h (30%).
in this series, regrettably moderate activity against the hERG chan-
nel was observed whilst improvement in permeabilities for these
compounds was somewhat modest. The removal of hydroxyl group
from the carbon linker and incorporation of a para chlorine instead
of a meta fluorine in a substituted phenol as in 22 resulted in a
nearly 26-fold less potent CCR1 antagonist compared to 20. In
addition, 22 was a potent inhibitor of hERG channel and the per-
meability was moderate.
Next, we wanted to investigate whether the substitution of para
chloro with a nitrile group in a substituted phenol would be toler-
ated with regard to CCR1 potency. Thus, 24 was designed and syn-
thesised. The biological data revealed that CCR1 binding affinity
was lost completely in such compound. In an attempt to investi-
gate the tolerability with regard to CCR1 potency and the effect
on pharmacokinetic properties 32 and 34 were designed with
gem dimethyl substitution incorporated into the carbon linker.

N. Hossain et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4026–4030
4029
Table 1
potent CCR1 antagonists17
Entry
hCCR1 MIP1
a
IC50
(
lM)
hheps
hmics
Caco2
rheps
hERG IC50
(lM)
Chemotaxis MIP1
a
IC50
(l
M)
PSA (Å2)
clogP
2
4
6
8
0.02
<3
<3
<3
<3
<3
<3
N.a
N.a
<3
<3
N.a
<3
N.a
N.a
N.a
N.a
<3
9
<3
<3
N.a
<3
<3
>25.6
61.7
81.9
>81.9
>100
1.3
N.a
7.9
14.1
1.9
N.a
10.7
N.a
N.a
N.a
N.a
0.25
0.093
N.a
N.a
0.024
N.a
N.a
N.a
0.0067
N.a
N.a
83
106
102
102
125
60
60
79
80
80
98
80
60
80
80
80
1.67
1.53
0.92
0.92
0.92
2.83
3.4
2.93
2.08
2.65
2.11
2.08
3.63
2.88
2.88
3.12
0.022
0.0045
0.0032
0.0056
0.089
0.24
<10
<10
<10
<10
<10
<10
<10
<10
10
<10
<10
<10
<10
<10
<10
1.1
0.2
0.35
1.8
22
11
14
16
18
20
22
24
26
30
32
34
36
<3
27
N.a
N.a
4.3
N.a
N.a
N.a
N.a
N.a
N.a
N.a
>3.2
2.5
1.4
2.4
1.6
1.2
N.a
N.a
N.a
1.2
0.0014
0.037
1.7
0.0092
>3.2
1.6
2.5
0.3
N.a
N.a
N.a
N.a
N.a
Data represents average value of at least two experiments.
N.a: not available.
hheps: Human hepatocytes (
rheps: Rat hepatocytes (
l/min/106cells).
l/min/mg).
l
l/min/106cells).
l
hmic: Shuman microsomes (
l
Caco-2: human colonic adenocarcinoma (cm/s Â106).
hERG: human ether-a-go-go-related-gene.
MIP-1a: macrophage inflammatory protein-1-alfa.
PSA: molecular polar surface area.
The in vitro biological evaluation revealed these compounds to
have greatly reduced CCR1 binding affinity without the anticipated
improvement in permeability. Next, we introduced a gem dimethyl
group in the pyrrolidine moiety to investigate the tolerability with
regard to CCR1 potency and impact on pharmacokinetic properties
of such a compound. Thus, 36 was designed and synthesised. The
incorporation of a gem dimethyl group in the pyrrolidine ring
yielded less potent CCR1 antagonist and the permeability did not
improve.
Acknowledgments
We thank to our colleagues at the Departments of Biosciences
and DMPK for generating data, Dr. Soren Andersen for reading
the manuscript. We also thank Martin E. Cooper for linguistic revi-
sion and some suggestions in writting this manuscript.
References and notes
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In conclusion, there was no clear correlation between perme-
ability and clogP for this class of zwitterionic compounds (Table
1). For example, the clogP of 2 was lower than, 18, 20 and 22
but the permeability was good despite the molecular polar surface
area (PSA) being the same for these compounds. The clogP of 14
was comparable to 18 but the permeability of 14 was much higher.
There was no correlation between permeability and PSA either. The
PSA of 2, 18, 20, 22, 26 and 36 was similar but only 2 had good per-
meability. The lipophilicity of 6, 8 and 11 was same (clogP, 0.92)
but the permeability of 6 and 8 was very low despite 11 had higher
PSA. The permeability of 14 (clogP, 2.83) was remarkably higher
than that of 18 (clogP, 2.93) and 22 (clogP, 2.65) despite the clogP
of these compounds being comparable. The high permeability of 14
could be attributed to low PSA (60 Å2) whilst that of 18 and 22 with
PSA 79 Å2 and 80 Å2, respectively had lower permeability. There
was a tendency to exhibit low hERG channel activity with low
clogP. The only exception was observed for compound 18 which
exhibited to some extent lower hERG channel activity compared
to 14 despite clogP was comparable for these compounds.
In this study a series of zwitterionic compounds which were
very potent CCR1 antagonists in vitro, with excellent metabolic
stabilty and devoid of hERG channel activity were designed and
synthesised. In addition, this class of compounds inhibited CCL3
mediated chemotaxis of THP-1 cells in a functional assay. In partic-
ular, 11 and 20 were very potent CCR1 antagonists with excellent
in vitro metabolic stability and these compounds were also the
most potent inhibitor of chemotaxis18 of THP-1 cells in this zwit-
terionic series. The hERG channel activity of 20 was modest whilst
11 was totally inactive in hERG though the permeabilty was bor-
derline. Thus, further investigations will be required to optimise
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4030
N. Hossain et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4026–4030
P.; Glen, A.; Howe, T.; Keller, T.; Davies, J. L.; Li, A.; McCarthy, C.; Mocquet, C.;
displacement (IC50) was derived using the Excel XL fit (version 2.0.9) to fit data
to a 4-parameter logistics function.
18. Culture of THP-1 cells:
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electrophysiology, for detail see Ref. 15b.
17. Human CCR1 Membrane: HEK293 cells, from ECACC, stably expressing
recombinant human CCR1 (HEK-CCR1) were used to prepare cell membranes
containing CCR1. The membranes were stored at À70 °C. The concentration of
membranes of each batch was adjusted to 10% specific binding of 33 pM [125I]
Cells were thawed rapidly at 37 °C from frozen aliquots and resuspended in a
25 cm flask containing 5 ml of RPMI-1640 medium supplemented with
Glutamax and 10% heat inactivated fetal calf serum without antibiotics
(RPMI+10%HIFCS). At day 3 the medium is discarded and replaced with fresh
medium. THP-1 cells are routinely cultured in RPMI-1640 medium
supplemented with 10% heat inactivated fetal calf serum and glutamax but
without antibiotics. Optimal growth of the cells requires that they are passaged
every 3 days and the minimum subculture density is 4 Â 105 cells/ml.
Chemotaxis assay:
MIP-1
a
Human CCR1 binding assay: 100
l
l of HEK-CCR1 membranes diluted in assay
Cells were removed from the flask and washed by centrifugation in RPMI+10%
buffer pH 7.4 (137 mM NaCl (Merck), 5.7 mM glucose (Sigma) 2.7 mM KCl
(Sigma), 0.36 mM NaH2PO4 Â H2O (Merck), 10 mM HEPES (Sigma), 0.1% (w/v)
Gelatine (Sigma) with the addition of 17500 units/l Bacitracin (Sigma) were
HIFCS + glutamax. The cells were then resuspended at 2 Â 107 cells/ml in fresh
medium (RPMI+10% HIFCS + glutamax) to which was added calcein-AM (5 ll
of stock solution to 1 ml to give a final concentration of 5 Â 10À6 M). After
gentle mixing the cells were incubated at 37 °C in a CO2 incubator for 30 min.
The cells were then diluted to 50 ml with medium and washed twice by
added to each well of the 96-well filter plate (0.45 lM opaque Millipore), 12 ll
of compound in assay buffer containing 10% DMSO was added to give final
compound concentration of 1 Â 10À5.5–1Â10À9.5 M. 12
l
l
Old human
centrifugation at 400Âg. Labelled cells were then resuspended at
a cell
recombinant MIP-1
a
(R & D systems), 10 nM final concentration in assay
concentration of 1 Â 107 cells/ml and incubated with an equal volume of MIP-
buffer supplemented with 10% DMSO, was included in certain wells (without
compound) as non specific binding control (NSB). 12 l Assay buffer with 10%
DMSO was added to certain wells (without compound) to detect maximal
binding (B0). 12 diluted in assay buffer to a final concentration
l [125I] MIP-1
1a
antagonist (10À10–10À6 M final concentration) for 30 min at 37 °C in a
l
humidified CO2 incubator. Chemotaxis was performed using Neuroprobe 96-
well chemotaxis plates employing 8 m filters (cat no.101-8). Thirty l of
l
l
l
a
chemoattractant supplemented with various concentrations of antagonists or
vehicle were added to the lower wells of the plate in triplicate. The filter was
then carefully positioned on top and then 25 ll of cells preincubated with the
in the wells of 33 pM, was added to all wells. The plates with lid were then
incubated for 1.5 h at room temperature. After incubation the wells were
emptied by vaccum filtration (MultiScreen Resist Vacuum Manifold System,
corresponding concentration of antagonist or vehicle were added to the surface
of the filter. The plate was then incubated for 2 h at 37 °C in a humidified CO2
incubator. The cells remaining on the surface were then removed by
adsorption and whole plate was centrifused at 2000 rpm for 10 min. The
filter was then removed and the cells that had migrated to the lower wells
were quantified by the fluorescence of cell associated calcein-AM. Cell
migration was then expressed in fluorescence units after subtraction of the
reagent blank and values were standardized to% migration by comparing the
fluorescence values with that of a known number of labelled cells. The effect of
antagonists was calculated as % inhibition when the number of migrated cells
was compared with vehicle.
Millipore) and washed once with 200
received an addition of 50 l of scintillation fluid (OptiPhase Supermix, Wallac
Oy). Bound [125I] MIP-1
was measured using a wallac Trilux 1450 MicroBeta
ll assay buffer. After the wash, all wells
l
a
counter.Calculation of percent displacement and IC50. The following equation
was used to calculate percent displacement. Percent displacement = 1À{(cpm
testÀcpm NSB)/(cpmB0ÀcpmNSB)} where cpm test = average cpm in wells
with membranes and compound and [125I] MIP-1
a
, NSB = average cpm in the
(non specific binding).
B0 = average cpm in well with membranes and assay buffer and [125I] MIP-1
(maximum binding). The molar concentration of compound producing 50%
wells with membranes and MIP-1
a a
and [125I] MIP-1
a