Spiropiperidine CCR5 antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.07.122

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.

Full text of DOI:10.1016/j.bmcl.2009.07.122

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5401–5406
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Spiropiperidine CCR5 antagonists
*
David M. Rotstein , Stephen D. Gabriel, Ferenc Makra, Lubov Filonova, Shelley Gleason,
Christine Brotherton-Pleiss, Lina Q. Setti, Alejandra Trejo-Martin, Eun Kyung Lee, Surya Sankuratri,
Changhua Ji, Andre deRosier, Marianna Dioszegi, Gabrielle Heilek, Andreas Jekle, Pamela Berry,
Paul Weller, Cheng-I. Mau
Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening
which were further modified based on insights from competitor molecules. Lead optimization was pur-
sued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with
good pharmacokinetic properties were successfully developed.
Received 25 June 2009
Revised 24 July 2009
Accepted 24 July 2009
Available online 30 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CCR5 antagonist
Chemokine
Anti-HIV-1
Antiviral
Spiropiperidine
The eruption of the AIDS epidemic in the 1980’s catalyzed a
corresponding huge response from the academic, medical and
pharmaceutical communities to identify the responsible pathogen
and develop medications to treat this affliction. Notable progress
has been made in the intervening decades, however significant
challenges remain. The remarkable ability of the virus to rapidly
mutate and generate resistant strains necessitates the continued
development of new pharmaceuticals, particularly ones which em-
ploy novel mechanisms of intervention.1 The observation that a
small percentage of high-risk populations showed either resistance
or delayed development of disease led to the discovery of the key
role played by the chemokine cell surface co-receptors CCR5 and
CXCR4 for successful viral fusion and infection. These discoveries
triggered a search for small molecule antagonists which would
function as viral entry inhibitors.2
low potency hits containing the spiropiperidine template 1-oxa-
3,8-diazo-spiro[4,5]decan-2-one, 1. Researchers at Ono had earlier
disclosed a series of spiropiperidine CCR5 antagonists5 leading to
the clinical candidate aplaviroc 2.6 As illustrated in Figure 1,
combination of our spiropiperidine template 1 with pharmaco-
phore elements from both aplaviroc and from Schering’s CCR5
antagonist program7 (exemplified by 3), led to analog 4, the initial
lead compound in our series. Indeed, as seen in Table 1, analog 4
showed promising activity in both binding and antiviral assays.8
Compound 4 had extremely poor in vitro metabolic stability:
intrinsic clearances in human (HLM) and rat (RLM) liver micro-
somes were 675 and 2000 l
L/min/mg protein, respectively,9 which
corresponded to projected in vivo clearances close to hepatic blood
flow.10 Thus, our objective was to improve the drug-like properties
of the series while improving antiviral potency.
A variety of such antagonists have been disclosed and in 2007
the first CCR5 antagonist, SelzentryÒ, was approved and launched
by Pfizer.3 Development of new, novel CCR5 antagonists continues
both for their antiviral effects and also for potential utility in a vari-
ety of autoimmune indications.4 In this publication we describe
some of our efforts in this field.
The development of our CCR5 antagonist series combined an
information driven approach with leads from high-throughput
assays. Screening of the Roche library uncovered a number of
In describing results from our SAR studies, the benzamide and
cyclohexyl groups are typically referred to as the head and tail
groups of the template respectively. Initial studies with this series
demonstrated that while binding activity was maintained with a
variety of structural modifications, antiviral activity was much more
sensitive,11 requiring conservation of all major pharmacophore ele-
ments. As seen in Table 1, removal of a flanking methyl group on the
benzamide head group 5 led to loss of antiviral activity. Removal or
shrinkage of the cyclohexyl tail substituent to cyclopentyl 6 also led
to diminished activity. Replacement of the cyclohexyl tail substitu-
ent by either heterocyclic or aromaticrings such as pyran 7 or phenyl
8 also led to loss in antiviral potency. The butyl side chain proved
* Corresponding author. Tel.: +1 650 852 1947; fax +1 650 855 6585.
E-mail address: david.rotstein@roche.com (D.M. Rotstein).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.122

5402
D. M. Rotstein et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5401–5406
O
N
HO
N
O
N
O
N
N
N
O
O
2
O
3
OH
Br
N
N
N
O
O
O
4
R'
O
N
N
R
O
1
Figure 1.
similarly intolerant to modification as seen by the loss of antiviral
activity with propyl side chain analog 9. Introduction of an angular
methyl group (similar to one utilized in Schering’s CCR5 template)
did improve antiviral potency, as seen with analog 10. Metabolic
stability remained a major issue with this template as exemplified
by 10 with human and rat intrinsic microsomal clearance of 526
and 523 lL/min/mg protein, respectively.
Metabolite profiling of our series showed multiple oxidative
metabolites, particularly on the lipophilic cyclohexyl tail and
phenyl head substituents. To combat this problem, in analogy to
Schering’s approach7, a strategy of heteroatom introduction into
these groups was pursued to block metabolism and lower overall
lipophilicty. As shown in Table 2, replacement of the phenyl head
group with pyrimidine afforded analog 11 which demonstrated
much improved metabolic stability in human liver microsomes.
Further replacement of cyclohexane tail with pyran resulted in
metabolically stable analog 12. Unfortunately, these modifications
also resulted in loss of antiviral activity and the emergence of
active efflux by P-glycoprotein (P-gp) as an issue.
This example is representative of a common trend observed in
our series where reduction of lipophilicity increased metabolic
stability but also led to decreased potency and increased efflux
by P-gp. Moving forward, our objective was to balance these
parameters to achieve the desired profile for a clinical candidate.
A key finding was that expansion of the spiro carbamate from a 5
to a 6 member ring afforded a template which allowed greater
opportunity to balance potency and drug-like properties. As shown
in Table 3, analog 13, containing the 1-oxa-3,9-diaza-spiro
[5.5]undecane-2-one spiropiperidine template demonstrated
impressive antiviral potency, although metabolic stability was still
Table 1
SAR of lead compound 4
O
R2
N
O
O
N
N
R4
R1
R3
#
R1
R2
H
R3
R4
Bindinga IC50
51
Antiviralb IC50
125
4
nBu
nBu
nBu
nBu
nBu
nPr
an issue (HLM intrinsic clearance of 307 lL/min/mg). However,
unlike the 5 member spiro system, introduction of heteroatoms into
the head and tail region was tolerated in the 6 member spiro system
without significant loss in potency. This can be seen with analog 14,
where phenyl has been replaced by pyrimidine. Indeed, analog 14
demonstrated improved metabolic stability in both human and rat
5
6
H
100
18
>625
>625
>625
>625
>625
<1.2
H
H
intrinsic microsomal clearance: 119 and 80 lg/min/mg, respec-
tively. This improved in vitro metabolic stability translated into very
promising oral bioavailability in rats: 43% F, at 10 mg/kg. Of concern
for this compound was the low projected human exposure due to
high metabolic clearance (82% hepatic blood flow). Further efforts
targeted improving this parameter.
Introduction of additional heteroatoms into our template to
improve metabolic stability and lower lipophilicity was explored
next. All modifications of the butyl side chain led to decreased
activity, including longer, shorter or cyclic side chains and hetero-
atom substitution at a variety of positions (data not shown). Intro-
duction of heteroatoms into the tail cyclohexyl group was
tolerated as seen in pyran analog 15. However, as with the 5 mem-
ber spiro system, combination of a pyran tail with a pyrimidine
head group led to loss of antiviral activity, as seen with analog
7
H
24
O
8
H
144
300
10
9
H
10
Me
nBu
a
Competitive binding evaluated vs. RANTES with IC50 values in nM, as mean of
two experiments.
b
Antiviral IC50 values in nM as mean of two experiments.

D. M. Rotstein et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5401–5406
5403
Table 2
SAR of the 1-oxa-3,8-diazo-spiro[4,5]decan-2-one template
O
O
O
N
N
N
R4
R1
n-Bu
#
R1
R4
Binding aIC50
10
Antivirala IC50
<1.2
clog P
HLMb
526
Cacoc AB/ER
14/0.4
10
6.4
11
12
7
46
4.4
2
162
27
NDd
N
N
N
N
58
375
0.6/31
O
aDefined as in Table 1b human liver microsomal intrinsic clearance (
l
l/min/mg protein) c Permeability in Caco-2 cells AB, apical to basolateral and BA, basolateral to apical
d
movement of test compound in 21 day cultured Caco-2 cells (cm/sec  10EÀ6). ER, efflux ratio of BA to AB not determined.
16. Fortunately, it was found that the introduction of an angular
methyl group into the 6 member spiro template restored antiviral
potency, even with the incorporation of heteroatoms necessary to
enhance metabolic stability. This can be seen with analog 17,
which possesses a promising combination of antiviral potency
and microsomal stability.
Compound 17 is a racemate and as shown in Table 4, demon-
strated that antiviral activity resided in the (S) enantiomer 19.
As SAR studies on this template progressed, a wide tolerance for
substitution was demonstrated. These studies focused on the
active (S) enantiomers.
viral activity. In contrast, all activity is lost when a methyl tail is
combined with a pyrimidine head group (data not shown). Unfor-
tunately, metabolic stability decreased with fully elaborated
cyanopyridine analogs, as seen with analog 22. The effect of mod-
ification on the tail group was examined next. Lipophilic groups
such as difluorocyclohexyl 26 maintain excellent antiviral activity,
but with concurrent loss of metabolic stability. Movement of the
pyran oxygen around the ring, as in analog 27, results in a loss of
potency, as does removal of the methylene tail linker as shown
in 28 which had an antiviral IC50 of 84 nM. Extension of the linker
to ethyl, 29, is tolerated, but at a cost of microsomal stability (AV
Analogs 20–25 show modifications to the head group which
maintain good potency, including the pyridine, pyridine, N-oxide,
cyanopyridine, pyrone and 2-trifluoromethyl pyrimidine groups.
Introduction of the cyanopyridine head group (as in analog 22),
consistently provided excellent antiviral activity across many
examples. This can be clearly seen in the case of compound 23,
where despite trimming of the tail group down to only methyl,
incorporation of a cyanopyridine head group maintains good anti-
IC50 8 nM, HLM clearance 37
matic or heteroaromatic groups led to lower potency.
ll/min/mg). Incorporation of aro-
O
N
O
N
O
N
O
O
O
O
N
N
N
N
O
N
N
N
28
29
Table 3
SAR of the 1-oxa-3,9-diaza-spiro[5.5]undecane-2-one template
O
R2
N
O
N
O
R4
N
R1
nBu
R1
R2
H
R4
Binding IC50
30
Antiviral IC50
29
clog P
HLMa
307
a
a
#
13
6.5
4.5
4.1
2.1
2.6
14
15
16
17
H
68
89
76
25
31
24
119
164
ND
17
N
N
H
O
O
O
H
405
14
N
N
N
N
Me
a
Defined as in Tables 1 and 2.

5404
D. M. Rotstein et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5401–5406
Table 4
SAR of the chiral 1-oxa-3,9-diaza-spiro[5.5]undecane-2-one template
O
N
O
O
R4
N
N
R1
nBu
R1
nBu
R4
Binding IC50
Antiviral IC50
>625
HLMa
17
a
a
#
18
R
100 (n = 1)
O
N
N
N
19
20
21
S
S
S
25
61
80
6
12
33
ND
O
O
O
N
4
N
O
N+
52
N
N
22
23
24
25
26
27
Racemate
24
22
12
97
27
10
4
42
28
36
46
106
53
O
N
N
S
S
S
S
S
Me
12
3
O
O
O
O
N
3
N
F3C
F
F
2
N
N
N
55
N
O
a
Defined as in Tables 1 and 2.
The synthesis of analogs 4–12 from the 1-oxa-3,8-diazo-
An analysis of our data pointed to analog 19 as having the most
spiro[4,5]decan-2-one series is outlined in Scheme 1.13
promising profile and subsequent efforts focused on further profil-
ing of this compound. None of the major hepatic cytochrome P450
enzyme isoforms (3A4, 1A2, 2C9, 2C19 and 3D6) were inhibited by
analog 19. Plasma protein binding was moderate across species:
the free fraction in rat, dog, monkey and human plasma was 32%,
13%, 4% and 17%, respectively. Table 5 shows the favorable phar-
macokinetic data generated for compound 19 in rat,12 dog and
monkey.
Generation of the dianion of hexanoic acid with LDA followed
by reaction with 4-oxo-piperidine-1-carboxylic acid benzyl ester
30 gave hydroxy acid 31. Treatment of 31 with diphenylphospho-
Table 5
Pharmokinetic profile of analog 9a
Pharmacokineticsb
Rat
Dog
Monkeyc
Analog 19 proved very selective for the human CCR5 receptor.
Screening of 19 for binding versus a panel of other chemokine
receptors (CCR1, 2b, 3, 4, 6 and CXCR4) showed no cross reactivity
Cmax (ng/mL)
AUC (ng h/mL)
Cl (mL/min/kg)
T1/2 (h)
Vdss(L/kg)
F%
13
54
52
1.4
3.8
10
225
700
20
2.9
3.2
85
42
396
19
3.5
3.0
42
*
with IC50’s >50
lM. An assessment of in vitro hERG inhibition for
19 at 37 °C gave IC50 and IC20 values of 11.9 and 2.9
l
M, respec-
tively. The biological activity of analog 19 was further assessed
by screening in a peripheral blood mononuclear cell (PBMC) viral
a
Doses were 1 mg/kg IV and PO for all species.
Cmax, AUC and %F were determined after the oral dose and Cl, Vdss, t1/2 were
b
replication assay with 10% FBS using an R5-tropic HIV-Ba-L
strain.
determined from the iv dose.
The excellent potency of this compound was confirmed, affording
an IC50 of 0.18 nM.
c
Cynomolgus monkey.

D. M. Rotstein et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5401–5406
5405
b
b
a
a
OH
OH
CO2H
Bz
N
O
Bz
N
CBZ
CBZ
N
O
CBZ
N
CN
38
39
30
31
O
OH
d, e
c
O
O
NH2
O
Bz
N
c
d, e
Bz
N
N
O
N
N
O
N
CBZ
N
N
R4
40
41
32
33
O
N
O
N
O
f
R2
R2
N
O
O
O
R2
d, f
HN
R4
O
O
N
CBZ
O
N
R4
CBZ
N
N
N
N
R1
R4
R4
42
43 R2 = H
44 R2 = CN
45 R2 = Me
37
34 R2 = H
35 R2 = CN
36 R2 = Me
O
O
R2
Scheme 1. Reagents and conditions: (a) CH3(CH2)4CO2H, LDA, THF, À78 °C to rt,
99%; (b) (PhO)2P(O)N3, TEA, toluene, reflux, 75%; (c) R4Br, NaH, DMF, 70 °C, yield
range: 20–85%;(d) 10% Pd/C, H2ÁEtOH, rt, 95%; (e) (i) 30, Ti(i-OPr)4, ClCH2CH2Cl, rt;
(ii) NaHB(OAc)3, rt, 56% or (i) 30, Ti(i-OPr)4, Et2AlCN, THF; (ii) MeMgBr, THF, rt, 61%
for two steps; (f) R1CO2H, EDCI, HOBT, iPr2NEt, DMF, rt, yield range: 25–90%.
e, g
O
N
N
N R4
R1
46
ryl azide gave the intermediate acyl azide, which upon heating
underwent a Curtius rearrangement and subsequent cyclization
to afford the spiro system 32. Alkylation of the carbamate in the
presence of sodium hydride provided intermediate 33. The second
piperidine ring was incorporated by a Strecker reaction of the
deprotected amine via titanium isoproxide mediated condensation
with ketone 30, followed by sodium triacetoxyborohydride reduc-
tion of the intermediate imine to give 34. Alternatively, treatment
of the imine with diethylaluminum cyanide afforded nitrile 35
which can be converted to methyl analog 36 under Bruylant condi-
tions with methyl magnesium bromide. Final amide formation was
accomplished by deprotection of 36 followed by treatment with
requisite carboxylic acids, HOBT, EDCI and base to give product 37.
The synthesis of analogs 13–29 from the 1-oxa-3,9-diaza-
spiro[5,5]undecan-2-one series is outlined in Scheme 2.13 The an-
ion from hexanenitrile was generated by treatment with n-butyl
lithium, then reacted with 1-benzyl-4-oxo-piperidine 38 to afford
cyanoalcohol 39. Nitrile reduction with LAH afforded amino
alcohol 40. Intramolecular cyclization of the amino alcohol with
CDI afforded carbamate 41. In a process analogous to Scheme 1,
N-alkylation, deprotection of the piperidinyl nitrogen, amination
with N-protected 4-oxo-piperidine, deprotection and amide cou-
pling lead to final product 46.
The synthetic sequence used to prepare chiral analogs from the
1-oxa-3,9-diaza-spiro[5,5]undecan-2-one series is illustrated in
Scheme 3 for the preparation of inhibitor 19. Wittig olefination
of N-Boc piperidone 47 afforded the 4-pentylidene intermediate
48. Asymmetric dihydroxylation using AD-mix-D afforded chiral
diol 49, which was selectively mesylated on the secondary alcohol,
then converted to chiral epoxide 50 upon basic treatment. Epoxide
opening with diethylaluminum cyanide afforded hydroxynitrile
51, which was reduced to amino alcohol 52 under Suzuki reduction
conditions with cobalt(II) chloride hexahydrate and sodium boro-
hydride. Cyclic carbamate 53 was then formed by treatment of
the amino alcohol with CDI. After BOC removal with TFA, a Strecker
reaction, via titanium isopropoxide mediated condensation of the
amine with ketone 47 and reaction of the intermediate imine with
diethylaluminum cyanide afforded nitrile 54. Subsequent
treatment of 54 under Bruylant conditions with methyl magne-
sium bromide afforded the methyl analog 55. Alkylation of 55 with
2-bromomethyl tetrahydropyran and sodium hydride, followed by
N-BOC removal with TFA afforded the penultimate intermediate
56. Final amide formation with 4,6-dimethyl-pyrimidine-5-car-
boxylic acid using HOBt, EDCI and base gave final product 19.
Scheme 2. Reagents and conditions: (a) CH3(CH2)4CN, nBuLi, THF À78 °C, 53%; (b)
LAH, THF, 0 °C; (c) CDI, THF, rt, 62% for two steps; (d) R4Br, NaH, DMF, yield range:
25–75%; (e) 10% Pd/C, H2ÁEtOH, rt, 79%; (f) (i) 30, Ti(i-OPr)4, ClCH2CH2Cl, rt; (ii)
NaHB(OAc)3, rt, 77% for two steps or (i) 30, Ti(i-OPr)4, Et2AlCN, THF; (ii) MeMgBr,
THF, rt, 60% for two steps; (g) R1CO2H, EDCI, HOBT, iPr2NEt, DMF, rt, yield range:
30–85%.
In summary, we have discovered a novel series of potent CCR5
small molecule antagonists, exemplified by analog 19, possessing
good selectivity and pharmacokinetic properties.
O
O
O
O
b
a
N
O
N
48
47
O
O
OH OH
c
O
d
O
N
N
O
49
50
OH
O
CN
e
g
O
OH
f
NH2
N
N
O
O
52
51
O
NH
O
h, i
O
O
O
R
O
O
N
N
N
NH
O
54 R = CN
55 R = Me
53
O
O
j
O
O
O
N
N
N
HN
N
N
N
O
N
O
56
19
Scheme 3. Reagents and conditions: (a) CH3(CH2)4PPh3Br, nBuLi, THF, À78 °C, 36%;
(b) AD-mix-b, CH3SO2NH2, t-BuOH, H2O, 91%; (c) (i) CH3SO2Cl, TEA, EtOAc,
CH2Cl2,0 °C; (ii) NaOMe, MeOH, 95% for two steps; (d) Et2AlCN, THF, 0 °C, 58%; (e)
CoCl2Á6H2O, NaBH4, 86%; (f) CDI, THF, 69%; (g) (i) CF3CO2H, CH2Cl2; (ii) 47, Ti(i-
OPr)4, Et2AlCN, THF; (iii) MeMgBr, THF, 71% for three steps; (h) 2-bromomethyl
tetrahydropyran, NaH, THF, 86%; (i) CF3CO2H, CH2Cl2; (j) 4,6-dimethylpyrimidine-
5-carboxylic acid, EDCI, HOBT, iPr2NEt, DMF, 92%.

5406
D. M. Rotstein et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5401–5406
5. Habashita, H.; Kikubo, M.; Hmano, S.; Hamanaka, N.; Masaaki, T.; Shibayama,
Acknowledgments
S.; Tada, H.; Sagawa, K.; Fukushima, D.; Maeda, K.; Mitsuya, H. J. Med. Chem.
2006, 49, 4140.
The authors thank Rebecca Stephenson, Gary Cooper, Ann
Petersen, Fernando Padilla, Chris Melville, Remy Lemoine, Lijing
Chen, Jason Manka and David Loughhead for synthetic support;
Rama Kondru, Nidhi Arora and Taraneh Mirzadegan for computa-
tional support; Yanzhou Liu for expert NMR analysis; Marquis L.
Cummings Jr. for chiral HPLC purifications; Naina Patel for for-
mulation development; Joe Muchowski, Counde Oyang, David
Goldstein, David Swinney, Denis Kertesz and Eric Sjogren for
helpful discussions.
6. McIntyre, J. A.; Castñer, J. Drugs Future 2004, 29, 677.
7. Palani, A.; Shapiro, S.; Clader, J. W.; Greenlee, W. J.; Cox, K.; Strizki, J.; Endres,
M.; Baroudy, B. M. J. Med. Chem. 2001, 44, 3339.
8. For assay protocols see: Melville, C.R.; Rotstein, D. M. WO2008119663.
9. The thresholds above which intrinsic mircrosomal clearance values were
considered to be high were P90 ll/min/mL for RLM and P35 ll/min/mL for
HLM. These scaled to in vivo clearance values P70% of liver blood flow using
the well-stirred model. Intrinsic microsomal clearance values corresponding to
630% of liver blood flow (615
HLM) were considered to be low.
10. Obach, R. S. Curr. Opin. Drug Discovery Dev. 2001, 4, 36.
ll/min/mL for RLM and 66.5 ll/min/mL for
11. Ernst, J.; Dahl, R.; Lum, C.; Sebo, L.; Urban, J.; Miller, S. G.; Lundström J. Bioorg.
Med. Chem. Lett 2008, 18, 1498.
References and notes
12. The relatively low exposure observed in rat was due to active transport, in
addition to metabolic clearance. Pre-treatment with the P-gp inhibitor
elacridar gave higher exposures due to reduced biliary excretion and
intestinal secretion. Saturation of transporter effects upon higher dosing also
gave improved exposures.
1. Wainberg, M. A.; Jeang, K.-T. BMC Med. 2008, 6, 1.
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3. Kuritzkes, D.; Kar, S.; Kirkpatrick, P. Nat. Rev. Drug Disc. 2008, 7, 15.
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13. For more detailed experimental data, see: Gabriel, S. D.; Rotstein, D. M. US
Patent 2005, 176703, Ref. 8.