Spirocyclic compounds, potent CCR1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.12.095

Conformationally constrained spirocycles (17-23) and (31-36) were synthesised. In vitro data revealed that these compounds are CCR1 antagonists with sub-nanomolar potency. In a functional assay 22, 23 and 36 inhibited CCR1 mediated chemotaxis with an IC

Full text of DOI:10.1016/j.bmcl.2012.12.095

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1883–1886
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Bioorganic & Medicinal Chemistry Letters
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Spirocyclic compounds, potent CCR1 antagonists
Nafizal Hossain a, , Svetlana Ivanova a,b, Jonas Bergare a, Tomas Eriksson a,b
⇑
a Department of Medicinal Chemistry, R&I Innovative Medicines, AstraZeneca R&D, Pepparedsleden 1, Mölndal 431 83, Sweden
b Department of Medicinal Chemistry, AstraZeneca R&D Lund, Scheelevägen 1, Lund 221 87, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Conformationally constrained spirocycles (17–23) and (31–36) were synthesised. In vitro data revealed
that these compounds are CCR1 antagonists with sub-nanomolar potency. In a functional assay 22, 23
and 36 inhibited CCR1 mediated chemotaxis with an IC50 value of 2, 2.6 and 68 nM, respectively.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 30 November 2012
Revised 25 December 2012
Accepted 28 December 2012
Available online 23 January 2013
Keywords:
Chemokines
Spirocycles
Antagonists
Chemotaxis
Chemokines are a large superfamily of cytokines which play an
important role in leukocyte recruitment and activation.1–4 Chemo-
kines transmit intracellular signals to their target cells by binding
to the cell surface chemokine receptor and activating G-proteins
coupled to the receptor. Chemokine receptors are viewed as attrac-
tive therapeutic targets for drug developments due to their central
role in regulating leukocyte trafficking. The C–C chemokine recep-
or 9 was thus proposed to investigate the effect of conformational
restriction on CCR1 potency.
The intermediate spirocycles 8, 9 were prepared from disubsti-
tuted phenylbromide according to Scheme 1. Compound 1 was
treated with iso-propylmagnesium chloride in THF at 0 °C to give
intermediate 3 which was treated with commercially available
tert-butyl 1-oxa-6-azaspiro[2.5] octane-6-carboxylate in the pres-
ence of copper(I) bromide dimethyl sulphide complex at 40 °C to
give intermediate 5. Treatment of 5 with potassium tert-butoxide
in 1,2-dimethoxypropane at 50 °C gave 7 in 67% overall yield.
The Boc protecting group in 7 was removed by HCl treatment in
THF to give 8 in 45% isolated yield. Compound 6 was obtained
using similar reaction conditions as described for 5 followed by
treatment with 48% aqueous HBr in acetic acid to afford 9 in 31%
overall yield (Scheme 1).
As the ortho N-acetyl group of compound A (Fig. 1) was shown
to be important for CCR1 potency, a first attempt was made to se-
lect compounds that would mimic the ortho NHAc meta hydroxy
substituted phenyl ring moiety of compound A (Fig. 1) and replac-
ing 6-chlorobenzyl aminopiperidine with spirocycles 8 and 9.
Therefore, compounds 22 and 23 were designed and synthesised
as outlined in Scheme 2. Thus, 2-nitro-5-methoxyphenol was
tor-1 (CCR1) and its major endogenous ligands MIP-1a (CCL3) and
RANTES (CCL5) are believed to play an important role in chronic
inflamatory diseases such as rheumatoid arthritis5,6 and multiple
sclerosis.7,8 Small molecule chemokines receptor antagonists are
of interest as potential therapeutic agents and the discovery of
selective and highly potent chemokine receptor antagonists has
previously been reported in the literature.9–13 CCR1 antagonists
in clinical development have also been reviewed.14 Here, we report
the design and synthesis of a novel class of spirocyclic CCR1 antag-
onists with sub-nanomolar potency and excellent in vitro meta-
bolic stability.
Previously, CCR1 antagonists of such as compound A (Fig. 1)
were investigated.14,15 Compounds of this class were obtained
from a parallel synthesis approach by reaction of 6-chlorobenzyl
aminopiperidine with a variety of epoxides of type B (Fig. 1). In vi-
tro data indicated that the NHAc substituent ortho to the phenol
was important for CCR1 potency. The introduction of conforma-
tional constraint16a into the drug molecule to minimize the confor-
mational entropy loss16b upon binding to the receptor may lead to
compounds with enhanced potency. The replacement of 6-chloro-
benzyl aminopiperidine of compound A (Fig. 1) with spirocycle 8
OH
X
O
O
N
N
H
O
Cl
HO
NHAc
Y
A
B
⇑
Corresponding author. Tel.: +46 317761851; fax: +46 317763818.
E-mail address: Nafizal.Hossain@astrazeneca.com (N. Hossain).
Figure 1.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.095

1884
N. Hossain et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1883–1886
O
R
MgBr
R
R
N
O
(ii)
(i)
X
X
Br
X
OH
1. X = Cl R = F
5. X = Cl R = F
3. X = Cl R = F
2. X = F R = OMe
6. X = F R = OMe
4. X = F R = OMe
O
(iii)/(iv)
X
N
R
7. X = Cl R = Boc (67%)
8. X = Cl R = H (45%)
9. X = F R = H (31%)
9a. X = H R = H
Scheme 1. Reagents and conditions: (i) iso-propylmagnesium chloride in THF, 0 °C, 0.5 h for 3, 30 °C, 16 h for 4 (quant.); (ii) tert-butyl 1-oxa-6-azaspiro [2.5] octane-6-
carboxylate, copper (I) bromide dimethyl sulphide complex, 40 °C, 18 h for 5 30 °C , 3 h for 6 (iii) potassium tert-butoxide, 1,2-dimethoxypropane, 40–50 °C, 40 h (67%); (iv)
48% aqueous HBr in acetic acid, reflux, 24 h (31%).
R2
NHAc
NHAc
R1
O
O
HO
O
(i)
(ii)
N
O
OH
NHAc
X
R1
17. X = Cl R2 = OMe (97%)
18. X = Cl R2 = F (35%)
19. X = Cl R2 = H (36%)
20. X = F R2 = F (30%)
21. X = F R2 = H (70%)
22. X = Cl R2 = OH (42%)
23. X = F R2 = OH (30%)
10. R1 = OMe (80%)
11. R1 = F (87%)
12. R1 = H
13. R1 = OMe (63%)
14. R1 = F (60%)
15. R1 = H (64%)
16. R1 = OAc
(iii)
Scheme 2. Reagents and conditions: (i) (2S)-oxiran-2-yl-methyl-3-nitrobenzene sulfonate, CS2CO3, DMF, rt, 24 h (60–64%); (ii) Spirocycles 8, 9, EtOH, 80 °C, 24 h, (30–97%);
(iii) BBr3, CH2Cl2, 0–22 °C, 2.5 h (42%).
hydrogenated in the presence of Pd/C in THF and acetic anhydride
to give 10 in high yield. Compound 10 was treated with (2S)-oxi-
ran-2-yl-methyl-3-nitrobenzene sulphonate and Cs2CO3 to give
corresponding epoxide 13 in 63% isolated yield. Epoxides 13 was
opened by spirocycle 8 in ethanol to give 17 (97%). The methyl
group in 17 was removed by treatment with BBr3 in CH2Cl2 to af-
ford 22 in 42% isolated yield. Compound 23 was prepared in one
pot two steps reaction. Thus, 16 was opened by spirocycle 9 in
ethanol to give 23 (30%) and the acetyl protecting group was re-
moved during the course of the reaction without addition of any
external base. In vitro biological data (Table 1) revealed 22 and
23 to be very potent CCR1 antagonists, nearly 10-fold more potent
than non-conformationally restricted compound A. Compounds 22
and 23 were stable both in human liver microsomes and human
hepatocytes and exhibited good permeability. However 22 and
23 were unstable in rat hepatocytes (Table 1).
Table 1
In vitro data of CCR1 antagonists.19
Entry
hCCR1 IC50
M)
hheps (
cells)
l
l/min/106
hmics (
mg)
l
l/min/
Caco2 (cm/
rheps (
cells)
l
l/min/106
rmics (
mg)
l
l/min/
Chemotaxis MIP-1
M)
a IC50
(
l
s  106)
(l
17
18
19
20
21
22
23
31
32
33
34
35
36
0.0009
0.00064
0.00096
0.00036
0.00096
0.00036
0.00052
0.006
0.009
0.032
0.00079
0.0014
0.018
n.a
6.1
2.5
9.3
11
55
24.4
17
26
n.a
17.5
26
n.a
n.a
n.a
29
n.a
n.a
n.a
n.a
n.a
n.a
<10
<10
14
13.5
<17
17.8
25.5
<10
<10
12.6
17.4
18.8
<10
<10
<10
18
12
16.5
13.5
6.6
n.a
n.a
n.a
2.7
2.7
1.6
<3
25.7
17.6
n.a
n.a
n.a
4.2
<3
6.6
0.002
0.0026
<3.6
n.a
3.5
n.a
<3
<3
<3
0.068
Data represent the average values of duplicates or triplicates or multiplets standard deviation.
N.a: not available.
hheps: human hepatocytes.
hmics: human microsomes.
rheps: rat hepatocytes.
rmics: rat microsomes.

N. Hossain et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1883–1886
1885
OMe
R1
NO2
O
O
O
O
(i)
N
N
O
(ii)
X
OH
X
OMe
25. X = Cl R1 = NO2 (88%)
26. X = F R1 = NO2 (99%)
27. X = H R1 = NO2 (73%)
8. X = Cl
24
9. X = F
9a. X = H
28. X = Cl R1 = NHAc (86%)
29. X = F R1 = NHAc (90%)
30. X = H R1 NHAc ((85%)
R2
NH2
NHAc
O
O
(iii)
(iv)
O
N
O
N
NH2
NHAc
X
X
OMe
31. X = Cl R2 = OMe (13%)
32. X = F R2 = OMe (13%)
33. X = H R2 = OMe (19%)
34. X = Cl R2 = OH (32%)
35. X = F R2 = OH (39%)
36. X = H R2 = OH (40%)
31a: X = Cl (14%)
32a: X = F (9%)
33a: X = H (12%)
Scheme 3. Reagents and conditions: (i) EtOH, 88 °C, 4–20 h (73–99%); (ii) Pt/C, EtOAc, Ac2O, rt, 4 h (85–90%); (iii) N(Boc)2, Ph3P, DEAD, 0–22 °C, 24 h (12–19%); (iv) TFA,
CH2Cl2, rt 1 h (100%).
Subsequently, we wanted to investigate if the meta hydroxy
phenyl substitution of 22 and 23 is essential for CCR1 potency.
Thus, we designed 18–21, synthesised according to Scheme 2.
The intermediate 11 was prepared by hydrogenation of 2-nitro-
5-fluorophenol in the presence of Pd/C in THF and acetic anhydride
in high yield. Phenols 11 and 12 were treated with (2S)-oxiran-2-
yl-methyl-3-nitrobenzene sulphonate and Cs2CO3 to give the cor-
responding epoxides 14 (60%) and 15 (64%) which were opened
by the spirocycles 8 and 9 to give 18–21 in moderate yields. Biolog-
ical data of 18–21 revealed that the meta hydroxy phenyl substitu-
ent in 22 and 23 could be replaced either by fluorine or a hydrogen
without loss of CCR1 potency (Table 1). This indicates that meta hy-
droxy substitution of the phenyl is not required for CCR1 potency
and it can provide a tool to modulate pharmacokinetic properties
if needed. Spirocycles 17–21 are metabolically unstable. Nonethe-
less all compounds 17–21 have good permeability (Table 1).
Next, we wanted to investigate whether the replacement of the
aliphatic hydroxy group in the linker by other polar group such as
primary amine would be tolerated with regard to CCR1 potency
and what effect such a change would have on pharmacokinetic
properties. Thus, we designed 34–36 which were synthesised
according to Scheme 3. The epoxide 24 was prepared in good yield
in a similar manner as described for 13–16. The ring opening reac-
tion of 24 with spirocyle 8 or 9 or 9a17 in ethanol gave 25–27 in
high yields. The hydrogenation of 25–27 in the presence of Pt/C
and subsequent acetylation in the presence of acetic anhydride in
ethyl acetate afforded 28–30 in excellent yields. The reaction of
28, 29 or 30 with N(Boc)2 in the presence of Ph3P and DEAD gave
31–33 along with 31a–33a18 in low yield after removal of the
Boc group under acidic condition. Finally, BBr3 mediated demethyl-
ation of 31–33 gave 34–36 in low yield. In vitro data revealed that
34 and 35 were very potent CCR1 antagonists with good in vitro
pharmacokinetic properties. Compounds 22, 23 and 36 were tested
in a functional assay in which they inhibited CCR1 mediated che-
motaxis with an IC50 value of 2, 2.6 and 68 nM, respectively
(Table 1).
9, to give 22 and 23, respectively, the CCR1 binding affinity was in-
creased nearly 10-fold. When the meta hydroxy group in a substi-
tuted phenol (22 or 23) was replaced by fluorine (18 or 20) or with
a hydrogen (19, 21) CCR1 potency was retained although in vitro
metabolic stability was decreased. The meta hydroxy phenyl sub-
stitution on compound 22 could be methylated (17) without any
loss of CCR1 potency but when meta hydroxy group in 34 was
methylated (31) it lost some potency. The substitution pattern in
the spirocycle 8 or 9 seems important for CCR1 potency. When
chlorine or fluorine atom in 34 or 35 was replaced by a hydrogen
(36) the CCR1 potency decreased more than tenfold probably due
to reduced lipophilicity. The replacement of the hydroxy group
(22 and 23) in the linker with a polar group such as a primary
amine (34 and 35) was possible without losing CCR1 potency. In
addition, rat hapatocyte stability was improved compared to 22
and 23. Compounds 22, 23, 34 and 35 are very potent CCR1 antag-
onists which exhibit good in vitro metabolic stability (22 and 23
are less stable in rat hepatocytes) and have moderate to good per-
meability. In addition these compounds inhibited CCL3 induced
chemotaxis of THP-1 cells in a functional assay.20 In vivo results
will be reported elsewhere. This investigation demonstrates the
possible effectiveness of conformational restriction as an approach
to improve drug binding affinity and functional potency.
Acknowledgments
We thank to our colleagues at the Departments of Biosciences
and DMPK for generating data, Drs. Soren Andersen, Magnus Nils-
son for critical reading of this manuscript and Matin Cooper for lin-
guistic revision of the manuscript. We also thank Dr. Peter Sjö,
Director of Department of Medicinal Chemistry, for his suggestions
in writing this manuscript.
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human CCR1 binding assay: 100 of HEK-CCR1 membranes
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diluted in assay 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
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filter plate (0.45 lM opaque Millipore), 12 ll of compound in assay buffer
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1 Â 10À5.5 to 1 Â 10À9.5 M. 12
ll old human recombinant MIP-1a (R&D
systems), 10 nM final concentration in assay buffer supplemented with 10%
DMSO, was included in certain wells (without compound) as non specific
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binding control (NSB). 12
wells (without compound) to detect maximal binding (B0). 12
diluted in assay buffer to a final concentration 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, Millipore) and washed once
l
l assay buffer with 10% DMSO was added to certain
l
l [125I] MIP-1
a
with 200
l
l assay buffer. After the wash, all wells received an addition of 50
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was
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a
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measured using a wallac Trilux 1450 MicroBeta counter.Calculation of percent
displacement and IC50. The following equation was used to calculate percent
displacement.
Percent
displacement = 1 À {(cpm
test À cpm
NSB)/
(cpmB0 À cpmNSB)} where cpm test = average cpm in wells with
membranes and compound and
wells with membranes and MIP-1
[
125I] MIP-1
and [125I] MIP-1
a,
NSB = average cpm in the
(non specific binding).
a
a
B0 = average cpm in well with membranes and assay buffer and [125I] MIP-1
a
(maximum binding). The molar concentration of compound producing 50%
displacement (IC50) was derived using the Excel XL fit (version 2.0.9) to fit data
to a 4-parameter logistics function.
20. Culture of THP-1 cells: 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
Cells were removed from the flask and washed by centrifugation in
RPMI + 10%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 centrifugation at 400Âg. Labelled cells were
then resuspended at a cell concentration of 1 Â 107 cells/ml and incubated
with an equal volume of MIP-1
a M M final
antagonist (10À10 to 10À6
concentration) for 30 min at 37 °C in a humidified CO2 incubator. Chemotaxis
was performed using Neuroprobe 96-well chemotaxis plates employing 8
filters (cat no.101–8). Thirty l of chemoattractant supplemented with various
lm
l
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 corresponding concentration of antagonist
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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
19. 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