Novel CXCR3 antagonists with a piperazinyl-piperidine core

Article abstract of DOI:10.1016/j.bmcl.2009.07.020

High-throughput screening of an encoded combinatorial aryl piperazine library led to the identification of a novel series of potent piperazinyl-piperidine based CXCR3 antagonists. Analogs of the initial hit were synthesized via solid and solution phase me

Full text of DOI:10.1016/j.bmcl.2009.07.020

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5205–5208
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel CXCR3 antagonists with a piperazinyl-piperidine core
a
a
a
a
Brian F. McGuinness a, , Carolyn DiIanni Carroll , Lisa Guise Zawacki , Guizhen Dong , Cangming Yang ,
Doug W. Hobbs a, Biji Jacob-Samuel a, James W. Hall III a, Chung-Her Jenh b, Joseph A. Kozlowski b,
Gopinadhan N. Anilkumar b, Stuart B. Rosenblum b
*
a Ligand Pharmaceuticals , 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
b Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
High-throughput screening of an encoded combinatorial aryl piperazine library led to the identification of
a novel series of potent piperazinyl-piperidine based CXCR3 antagonists. Analogs of the initial hit were
synthesized via solid and solution phase methods to probe the influence of structure on the CXCR3 bind-
ing of these molecules. Various functional groups were found to contribute to the overall potency and
essential molecular features were identified.
Received 4 May 2009
Revised 20 June 2009
Accepted 2 July 2009
Available online 9 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CXCR3 receptor antagonists
Piperazinyl-piperidine
Combinatorial chemistry
Solid-phase synthesis
Chemokine
The chemokines IP-10 (CXCL10), MIG (CXCL9), and I-TAC
(CXCL11) interact with the CXCR3 receptor, facilitating the recruit-
ment of Th1 cells during a variety of inflammatory responses.1
Hence, a CXCR3 antagonist offers promise as an anti-inflammatory
agent for conditions, such as multiple sclerosis,2 psoriasis,3 and
transplant rejection,4 where upregulation of CXCR3+ T lympho-
cytes is implicated. A variety of chemotypes have been disclosed
recently as small molecule CXCR3 antagonists5 including 2,3-
disubstituted quinazolinones and 8-azaquinazolinones,6 2-imino-
benzimidazoles,7 aryl-[1,4]diazepane ureas,8 aminopiperidine and
aminotropane homotropene amides,9 and benzetimide deriva-
tives.10 High-throughput screening of a portion of an encoded com-
binatorial library collection11 revealed the piperazinyl-piperidine
compound 1a as a moderately potent binder (110 nM) of the
CXCR3 receptor (Table 1). Characterization in a chemotaxis assay12
confirmed 1a as an antagonist and led to further follow-up efforts
based on this hit.
bound amine 2 via HATU resulted in Boc-protected 4 and deprotec-
tion generated intermediate 5. Reductive amination with N-Boc-
piperidinone and subsequent removal of the newly added Boc
protecting group led to secondary amine resin 6. This key interme-
diate was treated with carboxylic acids (amide formation) or alde-
Table 1
Human CXCR3 receptor antagonist hit 1a and right side analogs
O
O
Cl
N
N
H
N
N
N
R2
Compds
R2
hCXCR3 binding Ki (nM)14
Since 1a was identified from an encoded combinatorial library,
a solid-phase pathway to construct analogs probing the right and
left hand sides of the lead was in place (Scheme 1). Nucleophilic
loading of a variety of amines to Tentagel resin harboring a photo-
cleavable bromomethyl-ortho-nitrophenyl linker15 afforded
secondary amine resins 2. Coupling of carboxylic acid 3 to resin-
1a
1b
1c
1d
1e
1f
1g
1h
1i
4-Cyanobenzyl
Benzyl
110 10
1900 50
4100 650
8900 3100
320 50
70 20
3-Cyanobenzyl
2-Cyanobenzyl
4-Cyanobenzoyl
4-Chlorobenzyl
4-Bromobenzyl
4-Fluorobenzyl
4-Trifluoromethylbenzyl
4-Methoxybenzyl
4-Dimethylamino
85 10
160 40
510 100
120 10
2900 50
* Corresponding author. Tel.: +1 609 452 3779; fax: +1 609 452 3671.
E-mail address: bmcguinness@ligand.com (B.F. McGuinness).
In 2008, Pharmacopeia, Inc. was acquired by Ligand Pharmaceuticals.
1j
1k
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.020

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B. F. McGuinness et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5205–5208
NH
R1
O
O
O
Cl
N
2
Cl
Cl
Cl
N
a,b
N
MeO
HO
R1
N
N
N
c
NBoc
Cl
NBoc
4
3
O
O
O
f,h or g,h
d
e, d
Cl
N
Cl
N
R1
N
N
N
R1
R1
H
N
N
N
N
NH
N
N
5
6
1
NH
N
R2
Scheme 1. Synthesis of analogs 1a–p. Reagents and conditions: (a) Boc-piperazine, K2CO3, DMF, 90 °C; (b) LiOH, MeOH, H2O, rt; (c) HATU, DIEA, DMF, rt; (d) TFA, DCM, rt; (e)
Boc-piperidinone, Na(OAc)3BH, DCE, rt; (f) substituted benzaldehyde, Na(OAc)3BH, DCE, rt; (g) substituted benzoic acid, HATU, DIEA, DMF, rt; (h) h
m (365 nm), 3%TFA/MeOH
(v/v), 50 °C.
hydes (reductive alkylation) to generate upon resin cleavage, ana-
logs 1a–q.16
Table 2
Human CXCR3 receptor antagonist binding of left side analogs
Testing of the initial set of analogs synthesized by this method-
ology (Table 1) indicated a clear preference for a para-substituted
benzyl functionality on the piperidine as indicated by the dramatic
loss of potency exhibited by unsubstituted benzyl analog 1b. Both
the 3-cyano (1c), and 2-cyano (1d) benzyl derivatives were also
less active than 1a. Replacement of the benzyl linkage with an
amide (1e) was tolerated, albeit with a threefold drop in potency.
Finally, the 4-chlorobenzyl moiety (1f) was identified as preferable
to benzyl groups bearing either more electron withdrawing (1h, 1i)
or electron donating (1j, 1k) para-substituents. The SAR of this re-
gion of the molecule appears to be similar to the trend exhibited by
the benzyl-substituted piperidine moiety in the recently published
benzetimide series.10 It is intriguing to speculate that both inhibi-
tor classes may share a similar binding mode with the CXCR3
receptor.17
Attention was next drawn to the left hand side nicotinyl amide.
Attempts to lower the molecular weight of 1a by complete removal
of the amide moiety led to a loss of activity (data not shown). Var-
ious amide substituents, however, were tolerated (1f, 1l–1q, Table
2) with a preference for electron-deficient benzyl moieties. More-
over, molecular weight reduction by truncation of the benzylamide
to a methylamide (1l) resulted in a 15-fold decrease in potency.
Therefore, the most potent analog 1q was chosen as the lead struc-
ture moving forward.
The combinatorial library’s original solid-phase attachment
point generated a residual secondary amide in the analogs dis-
cussed so far. Hybrid approaches of solution-phase synthesis
(Scheme 2) and alternative solid-phase syntheses (Scheme 3) were
employed to prepare analogs which explored the importance of
this secondary amide (Table 2). Inversion of the amide to an acyl-
ated 3-aminopyridine resulted in a loss of activity (12a). The urea
analog of this 3-amino pyridine (12b) did not rescue receptor affin-
ity. Binding affinity was also lost by reduction of the secondary
amide 1q to the secondary amine 12c or tertiary amine 12d. Relo-
cation of the amide in these reduced analogs could not recover the
lost activity (12e). Hence, the nicotinyl amide linkage was identi-
fied as an essential part of the pharmacophore. The combinatorial
library from which 1a was decoded contained multiple un-de-
coded (inactive) aryl piperazine cores, indicating the 5-substituted
nicotinamide was essential for CXCR3 receptor binding. Indeed,
replacement of the pyridine ring with a benzene caused a 10-fold
drop in affinity (21).18 Remarkably, as indicated by the comparison
of analogs 22 and 23 (Table 3), removal of the 5-chloro moiety
caused a 100-fold drop in potency.19
R1
Cl
N
X
N
Cl
N
Compds
R1
X
hCXCR3 binding Ki (nM)
O
O
1f
N
70 20
N
H
O
1l
N
N
560 40
N
H
O
O
1m
370 160
N
H
1n
N
140 10
N
H
O
1o
1p
N
N
470 120
410 40
N
H
O
O
O
N
H
Cl
1q
N
35 10
N
H
Cl
H
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
12a
12b
N
N
1900 400
>50,000
O
O
H
N
H
N
N
H
12c
12d
N
N
870 10
N
3700 700
O
Cl
12e
21
N
3800 600
330 50
N
H
Cl
Cl
O
The role of the piperazinyl-piperidine was next explored. The
required analogs were synthesized via slight variations on the ori-
ginal solid-phase route as outlined in Scheme 4. Removal of a basic
CH
N
H
Cl

B. F. McGuinness et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5205–5208
5207
O
BocN
BocN
HN
a
b,c
NFmoc
d
N
N
Cl
N
Cl
N
N
N
O
Fmoc
7
8
9
Z
O2N
Cl
N
H
f
g or h
e
H2N
Cl
Z
Cl
Cl
X
N
Cl
Z
N
N
Cl
N
N
N
12a X = CH2
12b X = NH
11
10
Scheme 2. Solution synthesis of aminopyridine analogs 12a,b. Reagents and conditions: (a) Boc-piperazine, Na(OAc)3BH, AcOH, DCE, rt; (b) 20% piperidine in THF, rt;
(c) 4-chlorobenzaldehyde, Na(OAc)3BH, AcOH, DCE, rt; (d) HCl, Dioxane, rt; (e) 2,3-dichloro-5-nitropyridine, K2CO3, DMF, 90 °C; (f) Na2S2O4, EtOH, H2O; rt;
(g) 3,4-dichlorophenylacetyl chloride, DCM; rt; (h) 3,4-dichlorophenyl isocyanate, DCM, rt.
O
a
c
b
Cl
Z
Cl
Z
Cl
Cl
Cl
Z
Cl
Cl
Cl
Z
N
N
N
N
H
N
N
N
N
Cl
Cl
d
13
14
12c
12d
Cl
Cl
Cl
Cl
Cl
e,f,g
Cl
h
Cl
N
HO
HO
H2N
N
H
N
N
N
N
N
N
NBoc
NBoc
NBoc
15
16
17
18
Cl
N
Cl
N
N
N
j, k, l
NBoc
m.n
i
12e
O
N
O N
N
Cl
Cl
Cl
Cl
NH
19
20
Scheme 3. Synthesis of aminomethylpyridine analogs. Resin bound 13 was synthesized via the method outlined in Scheme 1. Reagents and conditions: (a) BH3–THF, 65 °C;
(b) h (365 nm), 0.2 M methylamine in MeOH, 50 °C; (c) formaldehyde (37 wt % in H2O), Na(OAc)3BH, AcOH, DCE, rt; (d) Boc-piperazine, Pd2(dba)3, dppf, K2CO3, DMF, 95 °C;
(e) methanesulfonyl chloride, Et3N, DCM, rt; (f) NaN3, DMSO, 65 °C; (g) Ph3P, THF; rt; (h) bromomethyl-ortho-nitrophenyl linked Tentagel resin, DMF; (i) 3,4-dichlorobenzoyl
m
chloride, 2,6-lutidine, DCM, rt; (j) TFA, DCM, rt; (k) Boc-piperidinone, Na(OAc)3BH, DCE, rt; (l) TFA, DCM, rt; (m) p-chlorobenzaldehyde, Na(OAc)3BH, AcOH, DCE, rt; (n) h
m
(365 nm), 3%TFA/MeOH (v/v), 50 °C.
O
O
Cl
N
Cl
Cl
Cl
N
N
N
H
a, b
R1
N
N
Cl
Cl
24
NH
c,d
N
N
28
Ki = 2.7 µM
O
O
O
Cl
N
Cl
N
Cl
Cl
Cl
a, b
NH
N
N
N
H
R1
R1
N
N
N
N
O
N
N
29
30
25
Ki = 4.5 µM
O
O
O
Cl
N
Cl
N
Cl
Cl
Cl
N
N
N
H
e,d
f, d
a, b
NH
R1
R1
N
N
N
N
NH
N
N
N
Cl
Cl
31
26
32
N
Ki = 23 µM
O
O
O
Cl
N
Cl
N
O
Cl
a, g, b
NH2
N
N
N
R1
R1
H
N
N
N
N
NH
N
33
34
27
N
Ki = 2 µM
Scheme 4. Synthesis of piperazinyl-piperidine analogs. Resin bound starting compounds were synthesized via the method outlined in Scheme 1. Reagents and conditions: (a)
p-chlorobenzaldehyde, Na(OAc)3BH, AcOH, DCE, rt; (b) h (365 nm), 3%TFA/MeOH (v/v), 50 °C; (c) Boc-piperazine, Na(OAc)3BH, DCE, rt; (d) TFA, DCM, rt; (e) Boc-piperidinone,
Na(OAc)3BH, AcOH, DCE, rt; (f) 3-(Boc-amino)-1-propanal, Na(OAc)3BH, DCE, rt; (g) formaldehyde, Na(OAc)3BH, DCE, rt.
m

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B. F. McGuinness et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5205–5208
Table 3
References and notes
Effect of Y substituent on CXCR3 binding affinity
1. (a) Liu, L.; Callahan, M. K.; Huang, D.; Ransohoff, R. M. Curr. Top. Dev. Biol. 2005,
O
68, 149; (b) Turner, J. E.; Steinmetz, O. M.; Stahl, R. A.; Panzer, U. Mini-Rev. Med.
Chem. 2007, 7, 1089.
Y
N
H
2. Szczucin´ ski, A.; Losy, J. Acta Neurol. Scand. 2007, 115, 137.
3. Rottman, J. B.; Smith, T. L.; Ganley, K. G.; Kikuchi, T.; Krueger, J. G. Lab. Invest.
2001, 81, 355.
F
N
N
N
CN
N
4. For alternative views on the role of CXCR3 in allograft rejection see: (a)
Hancock, W. W.; Lu, B.; Gao, W.; Csizmadia, V.; Faia, K.; King, J. A.; Smiley, S. T.;
Ling, M.; Gerard, N. P.; Gerard, C. J. Exp. Med. 2000, 192, 1515; (b) Zerwes, H.-G.;
Li, J.; Kovarik, J.; Streiff, M.; Hofmann, M.; Roth, L.; Luyten, M.; Pally, C.; Loewe,
R. P.; Wieczorek, G.; Banteli, R.; Thoma, G.; Luckow, B. Am. J. Transplant. 2008, 8,
1604; (c) Schnickel, G. T.; Bastani, S.; Hsieh, G. R.; Shefizadeh, A.; Bhatia, R.;
Fishbein, M. C.; Belperio, J.; Ardehali, A. J. Immunol. 2008, 180, 4714.
5. For reviews of small molecule CXCR3 antagonists see: (a) Wijtmans, M.; Verzijl,
D.; Leurs, R.; de Esch, I. J. P.; Smit, M. J. ChemMedChem 2008, 3, 861; (b) Collins, T.
L.; Johnson, M. G.; Medina, J. C. Chemokine Biol. Basic Res. Clin. Appl. 2007, 2, 79; (c)
Medina, J. C.; Johnson, M. G.; Collins, T. L. Annu. Rep. Med. Chem. 2005, 40, 215.
6. Johnson, M.; Li, A.; Liu, J.; Fu, Z.; Zhu, L.; Miao, S.; Wang, X.; Xu, Q.; Huang, A.;
Marcus, A.; Xu, F.; Ebsworth, K.; Sablan, E.; Danao, J.; Kumer, J.; Dairaghi, D.;
Lawrence, C.; Sullivan, T.; Tonn, G.; Schall, T.; Collins, T.; Medina, J. Bioorg. Med.
Chem. Lett. 2007, 17, 3339.
Compds
Y
hCXCR3 binding Ki (nM)
22
23
Cl
H
90 30
7200 550
Table 4
Effect of 2-methyl substitution of the piperidine ring
O
R1
H
Cl
N
N
7. Hayes, M. E.; Breinlinger, E. C.; Wallace, G. A.; Grongsaard, P.; Miao, W.;
McPherson, M. J.; Stoffel, R. H.; Green, D. W.; Roth, G. P. Bioorg. Med. Chem. Lett.
2008, 18, 2414.
8. Cole, A. G.; Stroke, I. L.; Brescia, M.; Simhadri, S.; Zhang, J. J.; Hussain, Z.; Snider,
M.; Haskell, C.; Ribeiro, S.; Appell, K. C.; Henderson, I.; Webb, M. L. Bioorg. Med.
Chem. Lett. 2006, 16, 200.
N
N
Cl
1
R
N
Compds
R1
R
hCXCR3 binding Ki (nM)
9. (a) Knight, R. L.; Allen, D. R.; Birch, H. L.; Chapman, G. A.; Galvin, F. C.; Jopling, L.
A.; Lock, C. J.; Meissner, W. G.; Owen, D. A.; Raphy, G.; Watson, R. J.; Williams, S.
C. Bioorg. Med. Chem. Lett. 2008, 18, 629; (b) Watson, R. J.; Allen, D. R.; Birch, H.
L.; Chapman, G. A.; Galvin, F. C.; Jopling, L. A.; Knight, R. L.; Meier, D.; Meissner,
J. W. G.; Oliver, K.; Owen, D. A.; Thomas, E. J.; Tremayne, N.; Williams, S. C.
Bioorg. Med. Chem. Lett. 2008, 18, 147.
1l
Methyl
H
H
R–Me
S–Me
S–Me
560 40
35 10
86 20
1q
35
36
37
3,4-Dichlorobenzyl
3,4-Dichlorobenzyl
3,4-Dichlorobenzyl
Methyl
16
32
2
6
10. Bongartz, J.-P.; Buntinx, M.; Coesemans, E.; Hermans, B.; Van Lommem, G.; Van
Wauwe, J. Bioorg. Med. Chem. Lett. 2008, 18, 5819.
11. Burbaum, J. J.; Ohlmeyer, M. H. J.; Reader, J. C.; Henderson, I.; Dillard, L. W.; Li,
G.; Randle, T. L.; Sigal, N. H.; Chelsky, D.; Baldwin, J. J. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 6027.
12. In an in vitro chemotaxis assay using a recombinant Ba/F3 cell line expressing
hCXCR3 receptor,13 1a exhibited similar IC50 values for chemotaxis induced by
three ligands: 45 nM (vs 5 nM hIP-10), 20 nM (vs 50 nM hMIG), and 85 nM (vs
5 nM hI-TAC).
13. Jenh, C.-H.; Cox, M. A.; Hipkin, W.; Lu, T.; Pugliese-Sivo, C.; Gonsiorek, W.;
Chou, C.-C.; Narula, S. K.; Zavodny, P. J. Cytokine 2001, 15, 113.
14. A scintillation proximity binding assay was used to measure the affinity of the
nitrogen by replacement of the core piperazine with a piperidine
(24) was not tolerated. In fact, relocation of the basic nitrogen by
the synthesis of the inverted piperidinyl-piperazine analog (25)
depleted binding affinity. Opening either ring to an acyclic analog
(26, 27) also substantially lowered binding activity. Clearly, the
pyridinyl piperazinyl-piperidine core was essential for CXCR3
binding activity.
The goal of lowering molecular weight was only achieved after a
significant increase in potency was obtained via methyl substitu-
tion on the piperazine ring (Table 4).20 While the 20-(R)-methyl
piperazine analog 35 was slightly weaker than 1q, the 20-(S)-
methyl piperazine derivative (36) boosted receptor affinity two-
fold. More importantly, 20-(S)-methyl piperazine analogs main-
tained their affinity (32 nM) after truncation of the benzylamide
group to a simplified methyl amide (37).
compound to CXCR3. Ba/F3-CXCR3 membranes (0.5–2
preincubated for 0.5–20 h with wheat germ agglutinin-coated scintillation
proximity assay beads (300 g per assay). The beads and membranes were
lg per assay) were
l
incubated in the absence and presence of various concentrations of compound
and 25 pM 125I-IP10 in 50 mM Hepes (pH 7.3), 1 mM CaCl2, 5 mM MgCl2,
125 mM NaCl, 0.2% BSA and 1% DMSO. After 1–4 h at room temperature, the
membrane bound 125I-IP10 was measured in a Wallac Microbeta. The values
are reported as the mean of at least two determinations SD.
15. Rich, D. H.; Gurwara, S. K. J. Am. Chem. Soc. 1975, 97, 1575.
16. All final compounds throughout the paper were purified by either silica gel
chromatography or reverse-phase semi-preparative HPLC to a purity of at least
95%.
In conclusion, high-throughput screening of an encoded com-
binatorial library uncovered a novel CXCR3 antagonist hit 1a,
with moderate CXCR3 affinity. Initial studies revealed a tight
SAR around this lead structure. Ultimately, 20-(S)-methyl pipera-
zine substitution led to 37, a lead CXCR3 inhibitor with improved
affinity and reduced molecular weight. Future communications
will further develop the SAR of these CXCR3 antagonists as well
as examine their ADME profile and in vivo disease model
activity.
17. Wijtmans, M.; Verzijl, D.; van Dam, C. M. E.; Bosch, L.; Smit, M. J.; Leurs, R.; de
Esch, I. J. P. Bioorg. Med. Chem. Lett. 2009, 19, 2252.
18. Analog 21 was synthesized via the route outlined in Scheme 1 starting with
3-chloro-4-fluorobenzoic acid.
19. The des-5-chloropyridyl analog 23 was synthesized via the solid-phase route
described in Scheme 1 starting with 6-(4-(tert-butoxycarbonyl)piperazin-1-
yl)nicotinic acid which was synthesized by palladium-catalyzed amination
(Pd(OAc)2, dppf, Cs2CO3, 1,4-dioxane, 100 °C) of methyl 6-chloronicotinate
with Boc-piperazine.
20. Compounds in Table 4 were synthesized via the route outlined in Scheme 1
starting with Boc-protected R and S 2-methylpiperazine.