The design and discovery of novel amide CCR5 antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.01.012

The synthesis of a range of novel amine-containing structures and their primary potency as inhibitors of HIV-1 fusion via blocking of the CCR5 receptor is described. The development of the medicinal chemistry strategy and SAR's which led to the identification of the piperidine amide compounds 33 and 36 as excellent leads for further evaluation is described, along with key physicochemical data which highlighted their lead potential.

Full text of DOI:10.1016/j.bmcl.2009.01.012

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1084–1088
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The design and discovery of novel amide CCR5 antagonists
*
David C. Pryde , Martin Corless, David R. Fenwick, Helen J. Mason, Blanda C. Stammen, Peter T. Stephenson,
David Ellis, David Bachelor, David Gordon, Christopher G. Barber, Anthony Wood, Donald S. Middleton,
David C. Blakemore, Gemma C. Parsons, Rachel Eastwood, Michelle Y. Platts, Keith Statham,
Kerry A. Paradowski, Catherine Burt, Wolfgang Klute
Department of Chemistry, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a range of novel amine-containing structures and their primary potency as inhibitors of
HIV-1 fusion via blocking of the CCR5 receptor is described. The development of the medicinal chemistry
strategy and SAR’s which led to the identification of the piperidine amide compounds 33 and 36 as excel-
lent leads for further evaluation is described, along with key physicochemical data which highlighted
their lead potential.
Received 17 November 2008
Revised 6 January 2009
Accepted 6 January 2009
Available online 10 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CCR5
Antagonist
Chemokine
HIV
AIDS
Antiviral
Chemokines are a large family of pro-inflammatory peptides
which exert their effects through a range of at least 17 G-protein
coupled receptors (GPCR’s) leading to downstream chemotaxis,
angiogenesis, cell differentiation and other biological activities.1
Recently, antagonism of the CCR5 receptor has been targeted as a
potential treatment for HIV infection, based on the finding that
CCR5 antibodies can block infection of macrophages by M-tropic
erature examples of CCR5 antagonists such as E-913 (2),7 SCH-C
(3),8 and TAK-7799 (4) were all being reported to share potent anti-
viral activity within a range of chemotypes. Despite the diversity of
structures represented in Fig. 1, a common feature of all of these
series is the presence of a basic nitrogen atom which is believed
to ‘anchor’ the ligand to E283 within the transmembrane region
of the CCR5 receptor,10 and a proximal lipophilic group, which
likely lies in close contact with a tyrosine residue, Y108.
HIV.2 Further, CCR5
D32 homozygotes are highly resistant to R5-
tropic HIV infection, while heterozygotes do become infected, but
progress more slowly to AIDS and respond better to treatment. It
has become apparent that CCR5 is the coreceptor for the most com-
monly transmitted HIV-1 strains which predominate during the
early stages of infection and remain the dominant form in >50%
of late stage HIV-1 infected patients.3 Blockade of the CCR5 recep-
tor appears not to be associated with any mechanism-related side
effects, making this a highly compelling target for drug discovery.4
Over the past several years, there has been intensive research
interest in small molecule antagonists of the CCR5 receptor result-
ing in the disclosure of several distinct chemical series.5 This has
led to the first launched CCR5 antagonist, maraviroc (1),6 and a
range of other agents at various stages of development (Fig. 1).
As the work described within this paper was being carried out, lit-
Structural overlays of all 4 structures are at best imperfect, and it
is clear that the structures can occupy distinct, but partially over-
lapping regions of the CCR5 receptor. Literature reports suggest that
the TAK series, exemplified by TAK-779 binds in a cavity formed by
transmembrane helices 1, 2, 3 and 7 near the extracellular surface
of CCR5.11 A similar binding cavity has been reported for SCH-C
based on an alanine scan method.12 This site only partially overlaps
with a putative binding site, proximal to the extracellular side of
helices 2, 3, 6 and 7, of a distinct series of compounds reported by
the Merck group, based on a homology model constructed from
the crystal structure of bovine rhodopsin.13 The E913 series of com-
pounds is reported to additionally make contacts with the second
extracellular loop.14 Finally, site directed mutagenesis studies have
supported the maraviroc series binding in a distinct region of the
receptor to SCH-C.10 These data demonstrate that a range of struc-
turally distinct chemotypes can be accommodated by the CCR5
receptor and retain potent antagonism.
* Corresponding author. Tel.: +44 0 1304 64.
E-mail address: David.Pryde@pfizer.com (D.C. Pryde).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.012

D. C. Pryde et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1084–1088
1085
N
N
O
F
N
S
a
b
O
O
O
N
N
N
N
OH
O
N
F
H
N
N
N
N
1A
1C
c
1B
O
N+
O
N
O
O
3, SCH-C
1, maraviroc
Br
H
N
O
O
N
N
Cl-
O
O
d
N+
N
N
N
N
O
N
N
O
8
N
H
4, TAK-779
2, E-913
1D
N
H
O
Figure 1. Literature examples of potent CCR5 antagonists taken from the time of
the work described within this paper.
Scheme 1. Reagents and conditions: (a) MsCl, Et3N, DCM, 0 °C, 100%. (b) 1-BOC-4-
methylaminopiperidine, Na2CO3, MeCN, rt, 53%. (c) TFA, DCM, rt, 100%. (d) 2,4-
dimethyl-nicotinic acid, WSCDI, HOBt, Hunigs base, DMAP, DMF, rt, 76%.
Following our extensive investigations around piperidine and
tropane amide series of CCR5 antagonists, we were seeking a com-
plementary series of novel structures with high antiviral potency,
good metabolic stability and high oral absorption, primarily to pro-
vide agents with an orthogonal structure and potential resistance
profile to that of maraviroc. One arm of these investigations in-
volved the syntheses of a number of variant heterocyclic core tem-
plates based around the piperidine amide function as represented
in SCH-C,15 but incorporating maraviroc-like amide functionality
which had been shown to impart favourable properties on the
maraviroc series.6b Inspiration for the heterocyclic cores was taken
from the extensive literature which exists around other peptidic
GPCR antagonists,16 with structural templates built around a cen-
tral basic core. For synthetic expedience, it was decided to target
only right hand side amides which would introduce both synthetic
flexibility and the potential for late stage parallel chemistry into
our syntheses. This basic strategy is shown in Figure 2.
For example, the targeted core structures featured mono- and
bicyclic amine groups, with linking groups of variant length, func-
tionality and number of rotatable bonds to cast our net as wide as
possible. Examples of these groups are shown in the below tables.
The synthesis of these compounds was straightforward and lar-
gely derivative. The central linker group was constructed by a com-
bination of reductive amination or alkylation chemistry, from
which the final compounds were synthesised by simple amide for-
mation. A typical synthetic scheme is shown in Scheme 1 below for
the azetidine 8.
basic group is either part of a ring or acyclic. Initial targets covered
a relatively wide range of calculated pKa values (calculated pKas of
approximately 6–9), although it should be stressed that very few of
these calculated values were confirmed by measurement. The
activity measurements in Table 1 reflect the inhibition of cell–cell
fusion between HIV-1 gp160-expressing CHO cells and CD4/CCR5-
expressing HeLa-P4 cells which has been shown to offer an excel-
lent correlation with anti-HIV activity.6,18 Also detailed in Table 1
are cLogP values for these initial targets. While relatively high,
our strategy relied on simple chemistry to rapidly identify suitable
core templates, which we could then return to and optimise phys-
icochemical properties. Monocyclic linkers such as those contained
in compounds 5–7 showed very little activity, although the incor-
poration of a piperidine ring in the centre of a linking group in 16
gave a 56 nM inhibitor of cellular fusion. The azetidine 8 showed
very encouraging 24 nM fusion IC50, and the isomeric analogue
15 showed a similarly encouraging 102 nM.
Structures in which a piperazine, tropane or azatropane formed
bicyclic core templates such as those in 11–14 largely ablated
activity, although isomeric analogues 9 and 10 had significantly
improved activity, and highlighted the need to hit upon just the
right template and functional substituents to ensure CCR5 potency.
Having established several lead compounds within new tem-
plates which possessed encouraging activity, we set about reduc-
ing the relatively high LogP of these leads primarily through the
incorporation of an amide group in the left hand side portion of
the molecules, initially via small alkyl or cycloalkyl amides which
we knew had precedented antiviral potency in the maraviroc ser-
ies. These structures are shown in Table 2 and incorporate both
pyridyl and phenyl right hand side amides. The monocyclic struc-
ture of 16 was reproduced in the amide 17, but this resulted in sig-
All other targets represented in the data tables below were ac-
cessed using analogous chemistry, starting from amine/carbonyl
combinations. In the cases where the amine component was not
commercially or easily available, the synthesis of that component
is detailed in the Supplementary Information section.
Table 1 shows the initial examples made in this investigation,
all based on a left hand side benzhydryl group and a 2,6-dimethyl
pyridyl amide on the right hand side.17 The linking groups exam-
ined included monocycles and bicycles, contained bridging hetero-
cycles and contained both monobasic or dibasic amines where the
nificant loss of potency. The azetidine structure of
8 was
incorporated into the amides 21 and 22, albeit with the addition
of an extra methylene group to avoid the introduction of an unsta-
ble aminal functional group, and again this resulted in a loss of fu-
sion potency. Building upon the finding that some bicyclic
structures in Table 1 were potent, we set about incorporating sev-
eral bicyclic core templates into the basic target structures using
combinations of azetidine, pyrrolidine and piperidine rings. This
was largely an unsuccessful exercise viz 19–20, 23–25 and 27–
29, until the bis-piperidine 30 was made, and showed to have very
encouraging potency and moderate lipophilicity (IC50 145 nM,
cLogP 3.5). These results showed the importance of suitably ori-
enting the left hand side functionality to achieve good primary po-
tency, and that presumably very few of our test structures
achieved a correct orientation. The only other compound in Table
heterocyclic amine
containing templates
R1
R1
various
O
substituents
N
N
R2
N
R2
target structures
amide right hand side;
heterocycle or amide
phenyl containing
left hand side
central amine pKa 6-9
MWt. <500
2 which had an IC50 below 3
lM in the fusion assay was the azeti-
dine analogue 26 albeit some 20 fold weaker.
Figure 2. Design strategy for a novel series of CCR5 antagonists.

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D. C. Pryde et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1084–1088
Table 1
Table 2
Initial examples of linking groups in a benzhydryl–pyridyl amide series
Left-hand side amide analogues of compounds from Table 1
Compound Structurea
gp160 fusion IC50
,
cLogP
2.7
nMb
O
linker
O
N
N
H
N
N
17c
20% @10
lM
N
H
Compound #
Linker
gp160 fusion IC50, nMa
cpkab (cLogP)
6.5 (5.0)
O
N
N
5
0%@10 lM
O
N
N
H
N
H
N
6
15%@100 nM
6.1 (4.4)
18c
12% @10
28% @10
l
M
M
2.7
2.6
N
H
O
Me
N
O
7
13%@100 nM
8.7 (4.3)
8.0 (4.4)
7.8 (4.4)
8.9 (4.4)
9.4 (4.5)
7.7 (4.5)
7.3 (4.4)
7.3 (4.4)
8.2 (4.9)
N
N
N
19c
l
8
24
N
N
N
N
N
N
N
N
O
N
H
9
313
41
N
N
O
N
10
11d
12
13
14
15
N
N
N
N
N
N
N
20c
0% @10
l
M
2.6
O
25%@10 lM
N
N
H
12%@100 nM
N
O
N
H
Me
N
N
21%@10 lM
N
N
21
20% @10
l
M
4.2
3.5
N
6%@100 nM
102 nM
N
N
O
O
Me
N
N
H
Me
N
N
N
22
50% @10
lM
N
O
16c
N
56
8.5 (5.9)
H
N
a
The concentration of test compound required to inhibit the cell–cell fusion of
HeLa-P4 cells expressing recombinant human CCR5 and CD4 at the cell surface and
encoding an HIV-1 long terminal repeat regulated B-Gal reporter gene with CHO
cells expressing cell surface Tat and HIV-1 gp160 by 50%. IC50 determinations were
the mean of at least two replicates.18
O
N
N
H
N
23d
20% @10
l
M
4.2
2.1
O
b
Calculated using the ACD Labs pKa prediction software.
Racemic mixture.
Mixture of diastereoisomers.
c
d
O
N
Our efforts now focussed entirely around the lead structure 30,
summarised in Table 3. These designs kept the rightmost piperi-
dine ring invariant (either as a simple piperidine or a tropane
equivalent), and sought to alter the remainder of the structure.
The most successful design feature identified was a benzyl amino
group appended to the amide N atom, which provided all of the
most active compounds, and importantly simplified any synthesis
and stereochemistry issues significantly. Compounds 32, 33, 34, 36
and 38 all possessed very similar structures and shared modest to
excellent activity. A 3-linked piperidine 35 was significantly poorer
than its 4-linked equivalent, as was a left hand ring tropane in 31
and it was notable that rigidifying the central region of the bis-
piperidine system in 37 gave a further potency enhancement.
O
N
24c
18% @1 lM
N
H
O
N
O
N
25c
0% @ 10
l
M
2.1
N
H

D. C. Pryde et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1084–1088
1087
Table 2 (continued)
Table 3
Analogues designed around compound 30
Compound Structurea
gp160
cLogP
fusion IC50
,
Compound Structure
gp160
cLogP
nMb
fusionIC50, nMa
O
O
O
N
N
N
HN
N
26
2490
1.5
31
32
10%@1
l
M
3.3
3.9
O
N
O
O
O
N
N
O
O
1
N
N
N
N
HN
27
20%
@10
1.1
O
l
M
N
N
O
33
34
35b
36
37
1
3.4
3.4
4.2
3.3
3.5
4.6
O
N
N
O
N
N
N
H
28
18% @1
l
M
1.9
N
N
1230
N
O
O
N
HN
N
N
29
20%
@10
2.7
O
l
M
O
19%@10 lM
N
N
O
N
O
O
N
N
N
H
30
145
3.5
O
O
N
N
N
N
3
O
O
O
N
N
N
a
Compounds 17–23, 26 and 29 were all tested as racemates.
The concentration of test compound required to inhibit the cell–cell fusion of
HeLa-P4 cells expressing recombinant human CCR5 and CD4 at the cell surface and
encoding an HIV-1 long terminal repeat regulated B-Gal reporter gene with CHO
cells expressing cell surface Tat and HIV-1 gp160 by 50%. IC50 determinations were
the mean of at least two replicates.18
b
c
Single, unassigned diastereoisomer.
0.5
d
Mixture of diastereoisomers.
It is interesting to note the difference in potency between the
very similar structures represented by 3-linked piperidine 35, 4-
linked piperidine 36, pyrrolidine 33 and the related architecture
of 25 which may have been difficult to fully elucidate had we not
set out with a flexible design approach to synthesising a wide
range of targets.
O
N
N
38
5800
The result of these investigations was the identification of sev-
eral highly potent and promising novel agents. Physicochemical
and in vitro ADME data was obtained on two compounds, 33 and
36, along with confirmation of competitive antagonism by 33 of
the CCR5 receptor using a MIP-1b displacement binding assay (Ta-
ble 4).
a
The concentration of test compound required to inhibit the cell–cell fusion of
HeLa-P4 cells expressing recombinant human CCR5 and CD4 at the cell surface and
encoding an HIV-1 long terminal repeat regulated B-Gal reporter gene with CHO
cells expressing cell surface Tat and HIV-1 gp160 by 50%. IC50 determinations were
the mean of at least two replicates.18
b
Compound 35 was tested as a racemate.
Both compounds were moderately lipophilic, which partly ex-
plained their very poor metabolic stability in hepatic microsomes.
Examination of the route by which compound 33 was metabo-
lised20 revealed that a facile oxidation of the benzylic methylene
of the N-benzyl group was responsible for the very short micro-
somal half-life. This was mirrored in the predominant metabolic
route of compound 36.

1088
D. C. Pryde et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1084–1088
4. (a) Dorr, P.; Perros, M. Ex. Op. Dr. Discov. 2008, 3, 1345; (b) Perros, M. Adv.
Table 4
Antiviral Drug Design 2007, 5, 185.
Selected properties of compounds 33 and 36
5. Barber, C. G. Curr. Opin. Invest. Drugs 2004, 5, 851.
Compound
MWt.
33
36
6. (a) Dorr, P.; Westby, M.; Dobbs, S.; Griffin, P.; Irvine, B.; Macartney, M.; Mori, J.;
Rickett, G.; Smith-Burchnell, C.; Napier, C.; Webster, R.; Armour, D.; Price, D.;
Stammen, B.; Wood, A.; Perros, M. Antimicrob. Agents Chemother. 2005, 49,
4721; (b) Wood, A.; Armour, D. Progress Med. Chem. 2005, 43, 239.
7. Maeda, K.; Yoshimura, K.; Shibayama, S.; Habashita, H.; Tada, H.; Sagawa, K.;
Miyakawa, T.; Aoki, M.; Fukushima, D.; Mitsuya, H. J. Biol. Chem. 2001, 276,
35194.
8. Strizki, J. M.; Xu, S.; Wagner, N. E.; Wojcik, L.; Liu, J.; Hou, Y.; Endres, M.; Palani,
A.; Shapiro, S.; Clader, J. W.; Greenlee, W. J.; Tagat, J. R.; McCombie, S.; Cox, K.;
Fawzi, A. B.; Chou, C.-Chu.; Pugliese-Sivo, C.; Davies, L.; Moreno, M. E.; Ho, D.
D.; Trkola, A.; Stoddart, C. A.; Moore, J. P.; Reyes, G. R.; Baroudy, B. M. PNAS
2001, 98, 12718.
9. Baba, M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.; Sawada, H.; Iizawa, Y.;
Shiraishi, M.; Aramaki, Y.; Okonogi, K.; Ogawa, Y.; Meguro, K.; Fujino, M. PNAS
1999, 96, 5698.
10. Dorr, P. K.; Todd, K.; Irvine, R.; Robas, N.; Thomas, A;, Fidock, M.; Sultan, H.;
Mills, J.; Perrucio, F.; Burt, K.; Rickett, G.; Perkins, H.; Griffin, P.; Macartney, M.;
Hamilton, D.; Westby, M.; Perros, M. 45th Interscience Conference on
Antimicrobial Agents and Chemotherapy, 2005, Washington DC, USA. b.
Kondru, R.; Zhang, J.; Ji, C.; Mirzadegan, T.; Rotstein, D.; Sankuratri, S.;
Dioszegi, M. Mol. Pharm., 2008, 73(3), 789.
460
2.6
195
2
487
3.5
205
40
ND
3410
ND
44
aLogD
bHLM Clint
lL/min/mg
cBaL IC90, nM
dMIP-1b IC50, nM
ehERG IC50, nM
Caco-2 AB/BA
TPSA19
8
9800
23/26
44
a
Partition coefficient measured in 1-octano/aqueous buffer at pH 7.4
b
Determined using pooled liver microsomes in phosphate buffer at pH7.4 and
M substrate concentration, with sampling made at the 30min timepoint.
The concentration at which replication of the R5 HIV-1 viral strain Ba-L is 90%
1
l
c
inhibited by test compounds in the PM-1 cell line as measured by p24 antigen
output.
d
The concentration of test compound required to inhibit binding of [
125I]-
labelled MIP-1b to CCR5 stably expressed on MIP34.10 cells by 50%.
e
The concentration of test compound required to inhibit binding of [3H]dofeti-
lide to HERG stably expressed on HEK293 cells by 50%.
11. Dragic, T.; Trkola, A.; Thompson, D. A.; Cormier, E. G.; Kajumo, F. A.; Maxwell,
E.; Lin, S. W.; Ying, W.; Smith, S. O.; Sakmar, T. P.; Moore, J. P. PNAS 2000, 97,
5639.
Nonetheless, the series represented by compounds 33 and 36
offered the potential for high permeability, relatively weak binding
to the hERG ion channel,21 and highly promising levels of antiviral
activity. They therefore represented excellent starting points for a
lead development programme with the aims of further driving
down antiviral potency and increasing metabolic stability, the re-
sults of which will be disclosed elsewhere.
12. Tsamis, F.; Gavrilov, S.; Kajumo, F.; Seibert, C.; Kuhmann; Shawn; Ketas, T.;
Trkola, A.; Palani, A.; Clader, J. W.; Tagat, J. R.; McCombie, S.; Baroudy, B.;
Moore, J. P.; Sakmar, T. P.; Dragic, T. J. Virol. 2003, 77, 5201.
13. Castonguay, L. A.; Weng, Y.; Adolfsen, W.; Di Salvo, J.; Kilburn, R.; Caldwell, C.
G.; Daugherty, B. L.; Finke, P. E.; Hale, J. J.; Lynch, C. L.; Mills, S. G.; MacCoss, M.;
Springer, M. S.; DeMartino, J. A. Biochemistry 2003, 42, 1544.
14. Maeda, K.; Das, D.; Ogata-Aoki, H.; Nakata, H.; Miyakawa, T.; Tojo, Y.; Norman,
R.; Takaoka, Y.; Ding, J.; Arnold, G. F.; Arnold, E.; Mitsuya, H. J. Biol. Chem. 2006,
281, 12688.
15. For a description of SAR within this series, see: Palani, A.; Shapiro, S.; Josien, H.;
Bara, T.; Clader, J. W.; Greenlee, W. J.; Cox, K.; Strizki, J. M.; Baroudy, B. M. J.
Med. Chem. 2002, 45, 3143.
Supplementary data
16. For a recent review, see: Blakeney, J. S.; Reid, R. C.; Le, G. T.; Fairlie, D. P. Chem.
Rev. 2007, 107, 2960.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.012.
17. For a discussion of rotameric properties of this structural class, see: Palani, A.;
Shapiro, S.; Clader, J. W.; Greenlee, W. J.; Blythin, D.; Cox, K.; Wagner, N. E.;
Strizki, J.; Baroudy, B. M.; Dan, N. Bioorg. Med. Chem. Lett. 2003, 13, 705.
18. Bradley, J.; Gill, J.; Bertelli, F.; Letafat, S.; Corbau, R.; Hayter, P.; Harrison, P.; Tee,
A.; Keighley, W.; Perros, M.; Ciaramella, G.; Sewing, A.; Williams, C. J. Biomol.
Screen. 2004, 9, 516.
19. For comparison, maraviroc has a TPSA of 63.
20. Carried out by incubation with human liver microsomes, and analysis of the
supernatant by mass spectrometry.
References and notes
1. Viola, A.; Luster, A. D. Ann. Rev. Pharm. Toxicol. 2008, 48, 171.
2. Cocchi, F.; DeVico, A. L.; Garzino-Demo, A.; Arya, S. K.; Gallo, R. C.; Lusso, P.
Science 1995, 270, 1811.
3. Samson, M.; Libert, F.; Doranz, B. J.; Rucker, J.; Liesnard, C.; Farber, C. M.;
Saragosti, S.; Lapoumeroulie, C.; Cognaux, J.; Forceille, C.; Muyldermans, G.;
Verhofstede, C.; Burtonboy, G.; Georges, M.; Imai, T.; Rana, S.; Yi, Y.; Smyth, R.
J.; Collman, R. G.; Doms, R. W.; Vassart, G.; Parmentier, M. Nature 1996, 382,
722.
21. For a discussion of working around hERG issues in the maraviroc series, see:
Price, D. A.; Armour, D.; De Groot, M.; Leishman, D.; Napier, C.; Perros, M.;
Stammen, B. L.; Wood, A. Bioorg. Med. Chem. Lett. 2006, 16, 4633.