The synthesis of substituted bipiperidine amide compounds as CCR3 ligands: Antagonists versus agonists

Article abstract of DOI:10.1016/j.bmcl.2005.04.054

Structure-activity relationship study of bipiperidine amide 1 has identified the reverse bipiperidine amide 4a as a CC chemokine-3 (CCR3) receptor antagonist. Optimization of the structure-activity relationship of compound 4a has resulted in the identification of a CCR3 antagonist 4i as well as a CCR3 agonist 13.

Full text of DOI:10.1016/j.bmcl.2005.04.054

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3020–3023
The synthesis of substituted bipiperidine amide compounds as
CCR3 ligands: Antagonists versus agonists
Pauline C. Ting,* Shelby P. Umland, Robert Aslanian, Jianhua Cao,
Charles G. Garlisi, Ying Huang, James Jakway, Zhidan Liu, Himanshu Shah, Fang Tian,
Yuntao Wan and Neng-Yang Shih
Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 31 March 2005; revised 19 April 2005; accepted 19 April 2005
Abstract—Structure–activity relationship study of bipiperidine amide 1 has identified the reverse bipiperidine amide 4a as a CC
chemokine-3 (CCR3) receptor antagonist. Optimization of the structure–activity relationship of compound 4a has resulted in the
identification of a CCR3 antagonist 4i as well as a CCR3 agonist 13.
Ó 2005 Elsevier Ltd. All rights reserved.
Asthma is a chronic inflammatory disease of the lungs
which is characterized by the recruitment of eosinophils,
T lymphocytes, basophils, and mast cells into the lung
tissue and results in reduced airflow and bronchial
hyperresponsiveness. Eosinophil chemotaxis is con-
trolled by the CC chemokine-3 (CCR3) receptor which
is a seven transmembrane G-protein coupled receptor
activated by several chemokines including eotaxins,
RANTES, and macrophage chemoattractant protein-
4.1–3 An antibody to eotaxin has been shown to decrease
eosinophil migration to the lung and airway hyperreac-
tivity after allergen challenge in mice.4 Therefore, a
small molecule CCR3 antagonist may inhibit eosinophil
chemotaxis and be effective in the treatment of asthma.
A number of research organizations are investigating
this approach.5–11
paper will describe the results of our SAR investigation
of compound 4a.
We have previously reported the identification of
compound 1 as a CCR3 antagonist.12 In our structure–
activity relationship (SAR) study of compound 1, we
discovered that the bipiperidine core can be reversed
as in compound 2 and retain reasonable CCR3 affinity.
The CCR3 affinity can be optimized first, by moving the
amide moiety to the 3-position of the reversed bipiperi-
dine core as in compound 3 and second, by reposition-
ing the carbonyl moiety as in compound 4a. This
The construction of compounds 2–4 followed the syn-
thetic route depicted in Scheme 1. Alkylation of 4-pip-
eridone (5) produced ketone 6 which undergoes a
reductive amination reaction with methyl isonipecotate
*
Corresponding author. Tel.: +1 908 740 3534; fax: +1 908 740
7152; e-mail: pauline.ting@spcorp.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.054

P. C. Ting et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3020–3023
3021
Table 1. In vitro CCR3 membrane binding activity of amide bipiperi-
dine analogues 4a–q
Compd
R
Ki (nM)
4a
60 15
4b
4c
Me
533 83
107 25
Scheme 1. Reagents and conditions: (a) 3,4-dichlorobenzyl chloride,
Et3N, ClCH2CH2Cl, D, 65%; (b) 3-(tBOC-aminomethyl)piperidine,
CH2Cl2, AcOH, NaB(OAc)3H, 64% for X = H; (c) TFA, CH2Cl2,
100%; (d) 3-(tBOC-aminomethyl)-piperidine, Ti(OiPr)4, CH2Cl2,
Et2AlCN, 96% for X = CN; (e) MeMgBr, THF, 0 °C, 88% for
X = Me; (f) RCOOH, Et3N, EDCI, HOBT, DMF.
4d
4e
92 13
27%a
for the formation of compound 2, ethyl nipecotate for
the formation of compound 3, or 3-(tBOC-amino-
methyl)-piperidine for the formation of compound 4.
Since the amide moiety was introduced at the last step,
a variety of amides were surveyed and selected results
are summarized in Table 1. All analogues were studied
as a mixture of stereoisomers. Each compound has been
assayed for in vitro CCR3 membrane binding activity13
as well as agonist activity in a [35S]GTPcS assay.14–17 All
of these analogues were found to be antagonists. The
spatial shape of the phenyl ring appears to be important
for activity since the acetamide 4b is less potent while the
cyclohexyl amide 4c retains activity. In addition, the
methyl group on the phenyl ring can be replaced by a
methoxy group as in 4d, but the electron withdrawing
chloro substituent in 4e reduces CCR3 activity. Inser-
tion of a methylene linker in the toluyl amide 4f is also
tolerated. In particular, the phenyl ring can be replaced
with a number of bicyclic rings including naphthalene,
quinoline, and indole and retain biological activity.
The 6-quinolinyl amide 4i is the most potent CCR3
antagonist in this subseries.
4f
4g
4h
4i
86 23
48
9
58 14
23
1
4j
30
34
2
6
4k
4l
80 12
99 11
4m
4n
4o
4p
Next, we decided to examine the SAR of the 3,4-dichlo-
robenzyl moiety. These analogues were synthesized
according to the route outlined in Scheme 2. 1-BOC-3-
aminomethylpiperidine (8) was converted into the 6-
quinolinyl amide 9. Reductive amination introduced
the second piperidine ring of intermediate 10 and varia-
tion of the 3,4-dichlorobenzyl subunit is accomplished at
the last synthetic step.
29
36
47
1
6
9
Selected dichlorobenzyl analogues are tabulated in
Table 2. The 3,4-dichloro substitution pattern is critical
and exhibits the optimal binding interaction in this lipo-
philic region of the CCR3 receptor. This key moiety has
also been established in the published SAR studies of
CCR3 antagonists from Gong et al.7 and Naya et al.8
Interestingly, the 3,5-dichloro analogue 11a and the
2,5-dichloro analogue 11b are less active as well as exhi-
bit a degree of agonist activity in the [35S]GTPcS as-
say.15 In terms of characterization of agonist activity,
since the Emax% of the numerous natural chemokine li-
gands of human CCR3 ranged from approximately 40
a % inhibition at 1 lM (n = 2).
to 115% in our [35S]GTPcS assay, compounds with stim-
ulatory activity in this assay of >40% inhibition are con-
sidered to have significant agonist activity. The
monosubstituted 2-chloro analogue 11c is inactive while
the 4-chloro analogue 11d shows reasonable CCR3
affinity. The saturated cyclohexylmethyl analogue 11e
is completely inactive. Methyl substitution at the

3022
P. C. Ting et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3020–3023
Figure 1. CCR3 35S-GTPcS binding for eotaxin and compound 13.
Scheme 2. Reagents and conditions: (a) 6-quinolinecarboxylic acid,
EDCI, HOBT, CH2Cl2, 96%; (b) TFA, CH2Cl2, 100%; (c) N-BOC-4-
piperidone, AcOH, NaB(OAc)3H, CH2Cl2, 70%; (d) RCHO, AcOH,
NaB(OAc)3H, CH2Cl2 or RCOCl, Et3N, CH2Cl2.
Modification of the reverse bipiperidine core by addition
of a methyl group afforded an interesting biological re-
sult. A methyl group is tolerated at the 4-position as
in compound 12, but at the 3-position produced a very
potent agonist, compound 13. The synthesis of com-
pound 12 is included in Scheme 1 (X = Me) while the
synthesis of compound 13 follows a similar pathway
beginning with the commercially available N-benzyl-3-
methyl-4-piperidone. The dose-response study of ago-
nist 13 in the [35S]GTPcS assay is depicted in Figure 1.
This experience of unexpectedly discovering an agonist
profile from a structural modification of an antagonist
has precedence in the CCR3 research of De Lucca et
al.6 and Anderskewitz et al.10 De Lucca and co-workers
found that quaternary piperidinium salts were potent
functional agonists while their corresponding parent
piperidine derivatives were antagonists. Anderskewitz
and co-workers reported that their lead cyclic amidine
structure was a CCR3 antagonist, but addition of an
imine moiety to the cyclic amidine in order to modulate
the basicity of the compound produced a potent agonist.
A second example of a structural modification inducing
an agonist profile in our series is illustrated below. The
8-quinolinyl amide 4j is a potent CCR3 antagonist,
and replacement of the 3,4-dichlorobenzyl moiety with
the 2,3-dichlorobenzoyl moiety generates the potent
agonist 14.
Table 2. In vitro CCR3 membrane binding and agonist (GTPcS)
activity of benzyl bipiperidine analogues 4i and 11a–j
Compd
R
Ki (nM)
23 1
Emax% GTPcSa
À7
4i
3,4-DiCl-PhCH2
3,5-DiCl-PhCH2
2,5-DiCl-PhCH2
2-Cl-PhCH2
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
398 59 45
391 44 45
36%a
95
17%b
74
NT
4-Cl-PhCH2
CyclohexylCH2
7
À8
NT
À12
2
3,4-DiCl-PhCHMe
3,4-DiCl-PhCH2CH2
3,4-DiCl-PhCO
3
180 32
20%b
NT
3,4-DiCl-PhCH2CO
3,4-DiCl-PhNHCONH
767 16 NT
6%b
NT
NT = not tested.
a Emax% at 10 lM (n = 2).
b % inhibition at 1 lM (n = 2).
benzylic position as in 11f or extension to the 3,4-dichloro-
phenethyl as in 11g also decrease affinity. Replacement
of the 3,4-dichlorobenzyl moiety with the corresponding
amide moieties as in compounds 11h and 11i or the urea
moiety as in compound 11j also produces inactive
analogues.
In conclusion, compound 4i was identified as a potent
CCR3 antagonist from this structural series and exhib-
ited minimal activity at the CCR1 (17% at 1 lM),
CCR4 (37% at 1 lM), and CCR8 (À8% at 1 lM) recep-
tors. Compound 4i was also active in the calcium flux

P. C. Ting et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3020–3023
3023
assay18 with IC50 = 215 84 nM and in the recombinant
CCR3 (BaF3 cells) chemotaxis assay19 with IC50
136 38 nM. Unfortunately, in a rat pharmacokinetic
study, compound 4i exhibited a very low AUC of
264 ng h/mL at 3 mpk (iv) which precluded advancing
into in vivo studies.
12. Ting, P. C.; Lee, J. F.; Wu, J.; Umland, S. P.; Aslanian,
R.; Cao, J.; Dong, Y.; Garlisi, C. G.; Gilbert, E. J.;
Huang, Y.; Jakway, J.; Kelly, J.; Liu, Z.; McCombie, S.;
Shah, H.; Tian, F.; Wan, Y.; Shih, N. Y. Bioorg. Med.
Chem. Lett. 2005, 15, 1375.
13. Membrane binding assay protocol: This assay was done
using 125I eotaxin and membranes from CREM3 cells (a
human CCR3 transfected rat Y3 cell line) on PVT-WGA-
SPA beads in binding buffer (0.5% bovine serum albumin,
25 mM HEPES buffer, 75 mM NaCl, 1 mM CaCl2, 5 mM
MgCl2, adjusted to final pH 7.6). Compounds were tested
from 100 lM to 10 pM final concentrations in 96-well
plates. The plates were shaken briefly and incubated at
room temperature for 5 h before counting on a scintilla-
tion counter.
14. Activation of G-protein-coupled receptors with agonists
stimulates the exchange of GTP for GDP on the active site
of the Ga protein. This activation can be measured by the
binding of [35S]GTPcS, a non-hydrolyzable GTP ana-
logue, to receptor-membrane preparations in the presence
of excess GDP. Emax% is the percent increase over basal
binding of [35S]GTPcS achieved for each compound at
10 lM and is expressed as a percent of maximal eotaxin
response at 100 nM.
15. Wan, Y.; Jakway, J. P.; Qiu, H.; Shah, H.; Garlisi, C. G.;
Tian, F.; Ting, P.; Hesk, D.; Egan, R. W.; Billah, M. M.;
Umland, S. P. Eur. J. Pharm. 2002, 456, 1.
16. Gilman, A. G. Annu. Rev. Biochem. 1987, 56, 615.
17. Wieland, T.; Jakobs, K. H. Methods Enzymol. 1994, 237,
3.
18. Calcium flux assay protocol: This assay used CREM3 cells
at 37 °C. Compounds were dissolved in DMSO and
diluted with buffer (pH 7.4 HBSS containing HEPES,
BSA, and probenecid). Intracellular calcium levels were
=
References and notes
1. Daugherty, B. L.; Siciliano, S. J.; DeMartino, J. A.;
Malkowitz, L.; Sirotina, A.; Springer, M. S. J. Exp. Med.
1996, 183, 2349.
2. Ponath, P. D.; Qin, S.; Post, T. W.; Wang, J.; Wu, L.;
Gerard, N. P.; Newman, W.; Gerard, C.; MacKay, C. R.
J. Exp. Med. 1996, 183, 2437.
3. Umland, S. P.; Wan, Y.; Shortall, J.; Shah, H.; Jakway, J.;
Garlisi, C. G.; Tian, F.; Egan, R. W.; Billah, M. M. J.
Leukocyte Biol. 2000, 67, 441.
4. Barnes, P. Eur. Respir. J. Suppl. 2001, 34, 67s.
5. Rupprecht, K. M.; Daugherty, B.; Mudgett, J.; Parsons,
W. H. In Annual Reports in Medicinal Chemistry; Doherty,
A. M., Ed.; Elsevier: Oxford, 2003; Vol. 38, pp 131–140.
6. De Lucca, G. V.; Kim, U. T.; Johnson, C.; Vargo, B. J.;
Welch, P. K.; Covington, M.; Davies, P.; Solomon, K. A.;
Newton, R. C.; Trainor, G. L.; Decicco, C. P.; Ko, S. S. J.
Med. Chem. 2002, 45, 3794.
7. Gong, L.; Hogg, J. H.; Collier, J.; Wilhelm, R. S.;
Soderberg, C. Bioorg. Med. Chem. Lett. 2003, 13, 3597.
8. Naya, A.; Kobayshi, K.; Ishikawa, M.; Ohwaki, K.; Saeki,
T.; Noguchi, K.; Ohtake, N. Chem. Pharm. Bull. 2003, 51,
697.
9. Varnes, J. G.; Gardner, D. S.; Santella, J. B., III; Duncia,
J. V.; Estrella, M.; Watson, P. S.; Clark, C. M.; Ko, S. S.;
Welch, P.; Covington, M.; Stowell, N.; Wadman, E.;
Davies, P.; Solomon, K.; Newton, R. C.; Trainor, G. L.;
Decicco, C. P.; Wacker, D. A. Bioorg. Med. Chem. Lett.
2004, 14, 1645.
10. Anderskewitz, R.; Bauer, R.; Bodenbach, G.; Gester, D.;
Gramlich, B.; Morschhauser, G.; Birke, F. W. Bioorg.
Med. Chem. Lett. 2005, 15, 669.
measured with
a fluorometric imaging plate reader
(FLIPR from Molecular Devices, Sunnyvale, CA) at 1 s
intervals for 60 s then at 2 s intervals for 60 s.
19. Eotaxin-mediated chemotaxis assay protocol: This assay
used BAF3 cells which expressed recombinant human
CCR3. The Neuroprobe ChemoTx microplate system with
a 5 lm pore size filter was used according to the
manufacturerÕs specifications. Cell chemotaxis in response
to 1 nM human eotaxin in the presence and absence of the
compound was measured after 3 h incubation at 37 °C.
Cells which had migrated in response to eotaxin were
quantitated using the LDH assay (Promega).
11. Batt, D. G.; Houghton, G. C.; Roderick, J.; Santella, J. B.,
III; Wacker, D. A.; Welch, P. K.; Orlovsky, Y. I.;
Wadman, E. A.; Trzaskos, J. M.; Davies, P.; Decicco, C.
P.; Carter, P. H. Bioorg. Med. Chem. Lett. 2005, 15, 787.