An imidazopiperidine series of CCR5 antagonists for the treatment of HIV: The discovery of N -{(1 S)-1-(3-fluorophenyl)-3-[(3- endo)-3-(5-isobutyryl-2- methyl-4,5,6,7-tetrahydro-1 H -imidazo[4,5- c ]pyridin-1-yl)-8-azabicyclo[3.2.1] oct-8-yl]propyl}acetamide (PF-232798)

Article abstract of DOI:10.1021/jm100978n

Preventing entry of HIV into human host cells has emerged as an attractive approach to controlling viral replication. Maraviroc 1 is an approved antagonist of the human CCR5 receptor which prevents the entry of HIV. Herein, we report the design and discovery of a series of imidazopiperidine CCR5 antagonists which retain the attractive antiviral profile and window over hERG activity of maraviroc 1, combined with improved absorption profiles in rat and dog. Furthermore, this series of compounds has been shown to retain activity against a laboratory generated maraviroc-resistant HIV-1 strain, which indicates an alternative resistance profile to that of maraviroc 1. Compound 41f (PF-232798) was selected as a clinical candidate from the imidazopiperidine series and is currently in phase II clinical trials.

Full text of DOI:10.1021/jm100978n

J. Med. Chem. 2011, 54, 67–77 67
DOI: 10.1021/jm100978n
An Imidazopiperidine Series of CCR5 Antagonists for the Treatment of HIV: The Discovery of
N-{(1S )-1-(3-Fluorophenyl)-3-[(3-endo)-3-(5-isobutyryl-2-methyl-4,5,6,7-tetrahydro-
1H-imidazo[4,5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]propyl}acetamide (PF-232798)
Paul A. Stupple,* David V. Batchelor, Martin Corless, Patrick K. Dorr, David Ellis, David R. Fenwick,
‡
§
ꢀ
Sebastien R. G. Galan, Rhys M. Jones, Helen J. Mason, Donald S. Middleton, Manos Perros, Francesca Perruccio,
Michelle Y. Platts, David C. Pryde, Deborah Rodrigues, Nicholas N. Smith, Peter T. Stephenson, Robert Webster,
Mike Westby, and Anthony Wood
Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, United Kingdom.
‡ Current address: AstraZeneca Laboratories, 35 Gatehouse Park, Waltham, Massachusetts 02451, United States. § Current address: Syngenta
Crop Protection, Muenchwilen AG, WST-820.1.15, Schaffhauserstrasse, CH-4332 Stein, Switzerland.
Received July 30, 2010
Preventing entry of HIV into human host cells has emerged as an attractive approach to controlling viral
replication. Maraviroc 1 is an approved antagonist of the human CCR5 receptor which prevents the entry of
HIV. Herein, we report the design and discovery of a series of imidazopiperidine CCR5 antagonists which
retain the attractive antiviral profile and window over hERG activity of maraviroc 1, combined with
improved absorption profiles in rat and dog. Furthermore, this series of compounds has been shown to retain
activity against a laboratory generated maraviroc-resistant HIV-1 strain, which indicates an alternative
resistance profile to that of maraviroc 1. Compound 41f (PF-232798) was selected as a clinical candidate from
the imidazopiperidine series and is currently in phase II clinical trials.
Introduction
and of these maraviroc 1 became the first CCR5 antagonist to
be approved in the USA and Europe for treatment experi-
enced patients in 2007. Compound 1 has more recently been
approved for treatment naive patients in the USA.2,4 The
absence of alternativeapprovedagents within thisdrugclass is
at least in part explained by the challenge of achieving the
required drug-like properties (antiviral activity, absence of
hERG activity, acceptable clearance, suitable bioavailability,
etc.) in molecules which are typically high molecular weight.
Compound 1 is characterized by excellent antiviral (AV)
activity across a range of HIV isolates, a significant window
over any hERG (human ether-a-go-go related gene) mediated
cardiovascular effects and selectivity over a broad panel of
other G-protein coupled receptors.4,5 Compound 1 is dosed
b.i.d. clinicallyandhasmoderatebioavailabilityinman(23%)
with food effects on oral exposure.6,7 During the discovery of
1, achieving the desired pharmacological profile in an orally
biovailable molecule was a significant challenge. In particular,
few compounds combined acceptable oral absorption with an
appropriate window over hERG channel inhibition. To ad-
dress this, we initiated a follow-up program with the aim of
identifying a new series of CCR5 antagonists with improved
oral absorption. Withinsucha template, wehoped tooptimize
metabolic stability to increase the chances of identifying an
agent suitable for clinical q.d. dosing. Additionally, demon-
strating activity against maraviroc-resistant virus was a key
factor during the design of new CCR5 antagonists.8
Highly active antiretroviral therapy (HAARTa) for the
treatment of human immunodeficiency virus (HIV) involves
a combination of antiretroviral drugs which can deliver
sustained reductions in viral load.1 Initial classes of such drugs
were nucleoside reverse transcriptase inhibitors, non-nucleo-
side reverse transcriptase inhibitors, and protease inhibitors.
More recently, a fusion inhibitor (enfuvirtide) and an inte-
grase inhibitor (raltegravir) have been approved. Although
these both target new mechanisms, as with the initial drug
classes, they rely on interactions with viral proteins. The
strategy of preventing viral entry into the host cell by inter-
acting with a human receptor is an attractive alternative which
could deliver an agent with a broad spectrum of activity
against different viral clades.
Antagonism of the chemokine coreceptor CCR5 has re-
ceived the most attention within this arena.2 One of the
reasons for this is the excellent genetic evidence for validation
of this target provided by CCR5 homozygotes with a 32 base
pair deletion in the coding region for the CCR5 gene, which
confers almost complete resistance to HIV infection.3
A
number of compounds have been progressed to clinical trials
*To whom correspondence should be addressed. Phone: þ44 1304
648441. Fax: þ44 1304 651987. E-mail: paul.stupple@pfizer.com.
a Abbreviations: AB, apical to basal; AV, antiviral; BA, basal to apical;
CYP, cytochrome P450; DLM, dog liver microsomes; ECL2, extracellular
loop 2; HAART, highly active antiretroviral therapy; HEK, human em-
bryonic kidney; hERG, human ether-a-go-go related gene; HLM, human
liver microsomes; HIV, human immunodeficiency virus; MVCRES, ex-
panded B-clade maraviroc-resistant HIV-1 isolate CC185; NMP, N-meth-
yl-2-pyrrolidone; PBMC, peripheral blood mononucleocyte cells; RLM, rat
liver microsomes; SAR, structure-activity relationship.
Compound Design
As a result of the attractive profile of 1, we were keen to
retain the tropane core from this series. In particular, we had
observed excellent antiviral activity with this chemotype and
r
2010 American Chemical Society
Published on Web 12/03/2010
pubs.acs.org/jmc

68 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Stupple et al.
incorporation of the tropane had overcome problems such
as CYP inhibition.5 Optimization of other properties was
planned by replacing the N- and C- substituents within the
tropane scaffold.
The incomplete absorption of 1 in man was not surprising
as this had also been observed preclinically (approximately
20% absorption in rat and 50% absorption in dog).9
We were confident that permeability was limiting exposure,
which was consistent with in vitro data as shown in Table 1
(caco-2 AB/BA < 1/12). Encouragement that optimization
was possible was provided by close structural analogues
with isomeric triazoles, which had shown improved caco-2
profiles, e.g. 2 (caco-2 AB/BA 11/26). The enhancement in
caco-2 AB flux was hypothesized to result from a reduc-
tion in the extent of hydration of the heterocycle due to a
change in dipole moment as the aromatic heterocycle is
varied.5,10,11 This was one of the factors which encouraged us
to consider replacements for the 1,2,4-triazole in future targets.
It was found that changing the nature of the amide sub-
situent within the tropane scaffold had a significant effect on
hERG channel inhibition. Previous data indicated that the
difluoro moiety of 1 was not tolerated by the hERG channel,
which is consistent with the findings that addition of polar
groups, e.g. amides, sulfonamides, and sulfones, in this part of
the molecule can significantly reduce hERG activity.12,13
Furthermore, docking of examples from this series into a
model of the hERG channel suggested that the amide alkyl
substituent overlaps with a lipophilic region of the hERG
pharmacophore. We reasoned that reducing the size of the
Table 1. gp160 Fusion Activity, hERG Channel Affinity, and Perme-
ability for Lead Compounds
a IC50 determinations were the mean of at least two replicates.
b Inhibition of [3H]-dofetilide binding to hERG stably expressed on
HEK-293 cells. c Papp ꢀ 10-6 cms-1
.
Scheme 1. Synthesis of 3-Substituted 1,4,6,7-Tetrahydro-imidazo[4,5-c]pyridine
Reagents and conditions: (a) (1) aq HCl, 0 °C, (2) PhCH2NH2, CO(CH2CO2H)2, NaOAc, H2O, 50 °C, 47% (2 steps); (b) (Boc)2O, 20% Pd(OH)2 on
carbon, H2, EtOAc, RT, quant; (c) PhCH2NH2, NaBH(OAc)3, AcOH, CH2Cl2, RT, 50%; (d) NH4HCO2, 20% Pd(OH)2 on carbon, EtOH, 50 °C, 94%;
(e) K2CO3, MeCN, reflux, 52%; (f) (1) Fe, AcOH, 130 °C, (2) Ac2O, 140 °C, 65% (2 steps); (g) H2, AcOH, 60 °C, 95% yield; (h) PhCHO, Na(OAc)3BH,
AcOH, CH2Cl2, RT, 47% yield; (i) 6N aq HCl, reflux, 87%; (j) NaBH(OAc)3, AcOH, CH2Cl2, RT, 59-74%; (k) 2N HCl, MeOH, 91%; (l) CH3COCl,
CH2Cl2, RT, 90%; (m) 20% Pd on carbon, NH4HCO2, EtOH, reflux, 93%; (n) for 20a and 20b, (CH3)2CO or paraformaldehyde, AcOH, Na(OAc)3BH,
RT, 22-65%, for 20c MeSO2Cl, Et3N, CH2Cl2, RT, 68%, for 20d CH3COCl, CH2Cl2, RT, 94%, for 20e-h RCO2Cl or RCOCl, Et3N, CH2Cl2, RT,
67-86%.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 69
Table 2. Profiles of 3-Substituted 1,4,6,7-Tetrahydro-imidazo[4,5-c]pyridines
compd
R1
gp160, IC50 (nM)a
hERG binding (%)b
Caco-2 AB/BAc
<1/9
HLM Clint (μL/min/mg)
logDd
18
19
PhCH2
H
Me
29
9
20a
20b
20c
20d
20e
20f
20g
20h
5
0
<7
46
0.4
0.8
0.9
0.6
1.2
1.6
1.5
2.7
i-Pr
3
MeSO2
MeCO
EtCO
i-PrCO
MeCO2
i-PrCO2
2
10
0
24
0.4
0.4
0.4
0.3
<0.1
<1/5
<1/17
<1/20
3/16
<7
<7
42
13
0
20
75
18
44
5/28
a IC50 determinations were the mean of at least two replicates. b Inhibition of [3H]-dofetilide binding to hERG stably expressed on HEK-293 cells with
a compound concentration of 300 nM. c Papp ꢀ 10-6 cms-1
.
d Experimentally determined (octanol/water).
amide to acetyl would minimize these lipophilic interactions
and therefore reduce affinity to the channel.
by cyclization, furnished the imidazopyridine 11, which was
accompanied by an exchange of protecting group from Boc to
acetyl. Hydrogenation of the pyridine followed by protection
of the resulting secondary amine and deacetylation gave
imidazopiperidine 12. Alkylation using homochiral aldehyde
1318,19 gave the versatile Boc-protected intermediate 15. Re-
moval of the Boc, acetylation, and debenzylation formed
tropane 19. From intermediate 19, a range of functionalities
(R1) was introduced to give final compounds 20a-h (see
Table 2).
Scheme 2 shows the synthesis of a set of analogues in which
the primary amino substituent was derivatized in the final
step.17 Debenzylation of 15/16 and subsequent introduction
of either an acetyl (23 and 24) or a methyl carbamate (25),
followed by Boc deprotection, gave the primary amine inter-
mediates 26, 27, and 28.
Benzimidazole 314 was identified as an attractive lead with
potent activity in the gp160 fusion assay, which we have
shown to be a good surrogate for antiviral activity.15 This
was combined with moderate activity in a hERG binding
assay consistent with our hypothesis about small amide
substituents. The permeability of 3 was also considered to
be attractive (Table 1), and molecular weight (MW 416) was
relatively low compared with 1 (MW 513). For these reasons,
3 was selected as a suitable template for further work.
The previously reported hERG modeling studies also sug-
gested that the benzimidazole of 3 overlaps with a lipophilic
binding area of the ion channel.12,13 With this in mind, we were
keen to investigate replacements which featured a polar group
to disrupt lipophilic interactions within the binding site.
Furthermore, by reducing the size of the amide substituent
of 1 and introducing a larger tropane C-substituent, we
reasoned that we were introducing the potential for alternative
interactions with the CCR5 receptor, thus increasing our
chances of seeing a different resistance profile to that of 1.
As a result of these hypotheses, the initial targets designed
featured an imidazopiperidine as a replacement for the benzi-
midazole of 3.
The synthesis of an isomeric imidazopiperidine is shown in
Scheme 3 using a similar set of transformations to those
shown in Scheme 1.17,20 A related series of imidazopiperidine
CCR5 antagonists has recently been reported21 in addition to
the disclosure of alternative series of tropanes.22-24
Results and Discussion
The first indication of activity from the imidazopiperidine
scaffold was provided bybenzylintermediate18, for which the
IC50 in the gp160 fusion assay was an encouraging 29 nM
(Table 2). Trimming back the benzyl group to methyl gave
20a, which showed a 6-fold potency improvement. Further-
more, hERG binding activity was not detected and the in vitro
intrinsic clearance inhuman livermicrosomes(HLM) was low
(< 7 μL/min/mg). However, caco-2 flux was poor (AB/BA <
1/9), which was perhaps not surprising based on the dibasic
nature and the resulting polarity (logD 0.4). Despite the caco-
2 data, 20a provided encouragement that balancing the
required in vitro properties in this series may well be possible.
Although the potency of the more lipophilic isopropyl ana-
logue 20b was similar (IC50 3 nM), metabolic stability was
reduced, which discouraged us from introducing further alkyl
substituents. Sulfonamide 20c showed a similar overall pro-
file. Introduction of an acetyl substituent (20d) showed ex-
quisite potency in the gp160 assay (IC50 0.4 nM), similar for
the first time to that of 1 (IC50 0.2 nM). This level of potency is
On the basis of the SAR determined during the discovery of
1 (logD 1.9), we felt we were most likely to achieve an
appropriate balance of permeability, potency, and metabolic
stability with a logD in the range 1.5-2.5. Final compounds
were therefore designed to lie in appropriate physicochemical
space to achieve a logD within this range. In addition, the
design of compounds with increased molecular weight was
avoided.16
Synthetic Chemistry
Synthesis of the initial imidazopiperidine template is shown
in Scheme 1.17 The tropane core was constructed by reaction
of dimethoxy tetrahydrofuran 4, benzylamine, and a ketodi-
acid, which gave 5. After a change of N-protecting group, a
primary amine was introduced stereoselectively under reduc-
tive amination conditions, followed by debenzylation. Reac-
tion between tropane 8 and pyridine N-oxide 9 gave amino-
pyridine N-oxide 10. Reduction of the nitro group, followed

70 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Stupple et al.
Scheme 2. Synthesis of 3-Substituted 1,4,6,7-Tetrahydro-imidazo[4,5-c]pyridine
Reagents and conditions: (a) NH4HCO2, Pd/C (10% w/w) or 20% Pd(OH)2 on carbon, EtOH, 60 °C, 86%-quant; (b) for 23 and 24, CH3COCl,
CH2Cl2, RT, 90-99%; (c) for 25, ClCO2CH3, CH2Cl2, RT, 93%; (d) HCl(g), MeOH, CH2Cl2, 0 °C-RT, 66-92%; (e) for 29, CH3COCl, Hunig’s base,
CH2Cl2, RT, 86%; (f) for 30-32, CH3CO2Cl, Hunig’s base, CH2Cl2, RT, 78-87%.
Scheme 3. Synthesis of 1-Substituted 1,4,6,7-tetrahydro-imidazo[4,5-c]pyridine
Reagents and conditions: (a) Hunig’s base, NMP, 120 °C, 33%; (b) (1) Fe, AcOH, 60 °C, (2) Ac2O, 120 °C, 91%; (c) (1) ClCO2CH3, EtOH, -70 °C, (2)
NaBH4, -70 °C to RT, 74%; (d) 10% Pd on carbon, H2, EtOH, 50 °C, 91%; (e) 2 M aq H2SO4, reflux, 80%; (f) NaBH(OAc)3, AcOH, CH2Cl2, RT, 67%;
(g) NaOH, H2O, propan-2-ol, reflux, 73%; (h) for 41a and 41b, (i) 1. 4,6-dichloropyrimidine or 2,3-dichloropyrazine, Et3N, n-BuOH, 100 °C, 74-80%, (2) H2,
Pd/C, EtOH, RT, 57%; for 41d, 41e, 41g, and 41h, R1COCl, Hunig’s base, CH2Cl2, RT 55-97%, for 41f, (CH3)2CHCOCl, Et3N, THF, RT, 90%.
particularly impressive when considering the polarity of 20d
(logD 0.4; 1.5 units lower than 1), which is consistent with
significantly improved lipophilic efficiency compared to that
of 1. It was hoped that the increased polarity of 20d would

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 71
Table 3. Profiles of 3-Substituted 1,4,6,7-Tetrahydro-imidazo[4,5-c]pyridines
compd
R1
R2
X
gp160 IC50 (nM)a
hERG binding (%)b
Caco-2 AB/BAc
HLM Clint (μL/min/mg)
logDd
29
30
31
32
Me
OMe
Me
Me
F
H
F
F
0.1
0.6
0.2
0.2
0
57
28
69
2/23
4/27
19
42
24
92
1.9
1.4
1.7
2.6
OMe
OMe
OMe
6/23
17/17
OMe
a IC50 determinations were the mean of at least two replicates. b Inhibition of [3H]-dofetilide binding to hERG stably expressed on HEK-293 cells with
a compound concentration of 300 nM. c Papp ꢀ 10-6 cms-1
.
d Experimentally determined (octanol/water).
Table 4. Profiles of 1-Substituted 1,4,6,7-Tetrahydro-imidazo[4,5-c]pyridines
compd
R
gp160, IC50 (nM)a
hERG binding IC50 ( μM)b
Caco-2 AB/BAc
4/15
HLM Clint (μL/min/mg)
logDd
41a
41b
39
pyrimidin-4-yl
pyrazin-2-yl
CO2Me
COMe
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.01
35
183
<8
<8
11
2.6
3.0
1.9
1.4
1.7
2.0
2.2
2.8
1
2
3/11
<1/6
2/13
2/8
41d
41e
41f
41g
41h
5
COEt
COiPr
COPr
COtBu
5
12
6
13
25
1/26
1/24
5
35
a IC50 determinations were the mean of at least two replicates. b Fluorescence polarization assay using a fluorescently labeled analogue of dofetilide.26
.
c Papp ꢀ 10-6 cms-1 d Experimentally determined (octanol/water).
result in the attractive potency being combined with favorable
HLM stability, which was indeed found to be the case (Clint
the phenyl ring (data not shown), which had little effect on
activity in the gp160 fusion assay. This trend continued, as
inhibition in the hERG binding assay appeared to be reduced
in compound 29 compared with des-fluoro analogue 20g (0%
and 20% at 300 nM, respectively) with the other in vitro
parameters being similar (Table 3). Swapping the substituent
of the primary amine from acetyl to methyl carbamate was
investigated with 30 and 31, which were found to have higher
hERG inhibition than the original compounds (inhibition at
300 nM: 30/20g 57%/20% and 31/29 28%/0%). These data
indicated that once again hERG inhibition was reduced with
the meta-fluoro in 31. A significantly improved caco-2 profile
was achieved in the bis-carbamate 32 (caco-2 AB/BA 17/17),
although this also resulted in unacceptably high hERG activ-
ity (69% at 300 nM) and reduced metabolic stability (Clint 92
μL/min/mg), all of which were presumably driven by the extra
lipophilicity (logD 2.6).
To investigate the effect of altering the vector of the polar
group attached to the bicycle, the 1-substituted 1,4,6,7-tetra-
hydro-imidazo[4,5-c]pyridine was also considered (Table 4).
Early signs that the nature of the polar substituent could
significantly affect the interactions with the hERG channel
were provided by heterocycle 41a, which showed very potent
inhibition (IC50 0.01μM). We wereveryinterestedtosee thata
100-fold reduction in hERG binding was observed with the
isomeric compound 41b. These data encouraged us that the
nature of the polar group could have significant effects on
<
7 μL/min/mg). There were no signs of hERG inhibition, but
once again we were unable to achieve caco-2 AB flux values
above the limit of quantification, which was also the case for
the more lipophilic amides 20e and 20f (caco-2 AB < 1 for all
three examples).
Alternative changes to acetamide 20d were considered that
would result in increased lipophilicity to target improved
permeability. A methyl carbamate was proposed as a less
polar expression of the corresponding acetamide. Carbamate
20g retained excellent activity in the gp160 fusion assay and
good metabolic stability. Significantly, 20g exhibited the
potential for improved oral absorption (caco-2 AB 3) com-
pared with 1 (caco-2 AB < 1). The increase in AB flux
between equilipophilic 20f and 20g can be rationalized by
the weaker H-bond acceptor capacity of the carbamate, which
would reduce the dehydration penalty on entering a mem-
brane. A positive effect onpermeability/transporter activityof
this change has recently been reported elsewhere.25 Unfortu-
nately, there were signs of hERG inhibition, which only
increased with the more lipophilic carbamate 20h, although
low but measurable caco-2 AB flux was once again observed
for 20h.
A thorough analysis of the hERG binding data within the
tropane series revealed a trend that affinity was generally
reduced by incorporation of a meta-fluoro substituent onto

72 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Table 5. Antiviral Activity of Lead Compounds
Stupple et al.
Table 6. Pharmacokinetic Properties of Lead Compounds in Rat and
Dog iv/po Studiesa
compd
gp160 IC50 (nM)a
AV IC90 (nM)b
1b
29c
39d
41f c
1
0.2
0.1
3.2 (n = 4)
1.6 (n = 6)
0.8 (n = 8)
2.0 (n = 5)
rat PPB (%)e
46
74
71
146
68
91
62
29
39
41f
rat Cl (mL/min/kg)
rat Clu (mL/min/kg)f
rat t1/2 (h)
201
526
1.4
<0.1
<0.1
137 518
2.3 1.7
253
2.3
20
a IC50 determinations were the mean of at least two replicates.
b Antiviral activity against HIV BaL in PBMCs.
rat F (%)
6
8
<5
estimated rat absorption (%) 20
complete complete
dog PPB (%)e
dog Cl (mL/min/kg)
dog Clu (mL/min/kg)f
dog Vd (L/kg)
dog t1/2 (h)
60
21
53
62
36
93
73
36
47
36
hERG activity. Carbamate 39, the isomeric analogue of 29,
possessed a very attractive profile. Excellent potency (IC50 <
150
9.9
3.3
50
63
0.1 nM) was combined with what appeared to be an accep-
table window over hERG activity, low but measurable flux
in caco-2 cells (AB/BA 3/11) and excellent metabolic stability
(Clint < 7 μL/min/mg). Encouraged by the difference between
the hERG data for 41a and 41b, we were keen to investigate
alternative substituents to attempt to further reduce hERG
binding activity. The more polar bisacetamide 41d showed
decreased hERG binding but also reduced flux in the caco-2
assay consistent with previous SAR. Significantly, hERG
binding did not increase as the amide substituent became
increasingly more lipophilic (41e-h). A clear disconnect
between the gp160 and hERG binding SARs was identified
in compound 41f, which demonstrated particularly weak
hERG binding affinity (IC50 12 μM). These data are consis-
tent with the introduction of the steric bulk in 41f introducing
a clash with the hERG channel. Furthermore, the elevated
logD of 41f compared to acetamide 41d (2.0 and 1.4, re-
spectively) contributed to improved flux in caco-2 cells (AB/BA
2/8), with sustained metabolic stability (Clint 13 μL/min/mg).
Compounds 29, 39, and 41f were selected for further
screening as these offered the best balance of in vitro proper-
ties. It is worth noting the similarity in logDs (1.9, 1.9, 1.9, and
2.0 for 1, 29, 39, and 41f, respectively), which highlights the
value of having defined a target range prior to compound
design/synthesis.
4.3 11.8
2.3 3.8
10.5
3.4
31
dog F (%)
estimated dog absorption (%) 70
42
44
complete complete complete
a Assuming rat liver blood flow is 100 mL/min/kg and dog is 50 mL/
min/kg. b Rat iv 1 mg/kg, po 10 mg/kg; dog iv 0.5 mg/kg, po 1 mg/kg.
c Rat iv 1 mg/kg, po 2 mg/kg; dog iv 0.5 mg/kg, po 0.5 mg/kg. d Rat iv
2 mg/kg, po 2 mg/kg; dog iv 0.5 mg/kg, po 0.5 mg/kg. e % of drug bound
to plasma proteins; blood:plasma partitioning is 1:1 for all compounds
in rat and dog. f Clu is unbound clearance.
Table 7. Human, Rat and Dog Intrinsic Clearance Values from in Vitro
Liver Microsome Assays
1
29
39
41f
a,b
a
HLM Clint
RLM Clint
DLM Clint
49
<6
<9
17
<6
<9
7
<6
<9
15
32
a
a Units: μL/min/mg protein. b Assay was performed using alternative
batch of mircosomes to those used previously.
Dog pharmacokinetic studies also showed similar clearance
data for the three lead imidazopiperidines compared to 1.
Because of the varying role of transporters in clearance of 1
across species,27 we were cautious when making predictions of
human clearance for 29, 39, and 41f. On the basis of the
translation between the rat, dog, and human clearance data
for 1,27 in addition to the structural/physicochemical similar-
ities with the imidazopiperidines, we felt confident that the
lead imidazopiperidines would display acceptable clearance in
man. When in vitro HLM stability data was generated in the
same assay for the lead compounds (Table 7), reduced in-
trinsic clearances were apparent for 39 and 41f compared with
1, e.g. 49 and 7 μL/min/mg protein for 1 and 41f, respectively.
These data indicated the possibility of reduced clearance in
man should the in vitro assay be predictive for in vivo
clearance. Reduced human clearance was considered benefi-
cial to both reducing dose size and also extending half-life to
increase chances of suitability for q.d. dosing.
Identifying compounds with the potential to address Mar-
aviroc-resistant virus was another key driver for the develop-
ment of second generation CCR5 antagonists. With this in
mind, activity of the new imidazopiperidines was assessed
against expanded B-clade Maraviroc-resistant HIV-1 isolate
CC185 (MVCRES).8 In addition, representatives of alternative
chemotypes were tested (Aplaviroc 4228 and Vicriviroc 4329)
along with examples of previously reported tropanes (44-47)
as shown in Chart 1.
The data from evaluation in an antiviral assay using mitogen-
stimulated peripheral blood mononucleocyte cells (PBMC)
from pooled donors and HIV Ba-L are shown in Table 5. The
three new imidazopiperidine compounds possessed enhanced
antiviral activity compared to that of 1 with up to a 4-fold
improvement for 39.
We were keen to assess if the improved caco-2 profiles of 29,
39, and 41f translated to improved in vivo oral absorption. As
the oral absorption of 1 in rat was estimated to be 20% (see
Table6), wewere verypleased tofind that a similarassessment
for 29 and 39 was consistent with complete absorption.
Unfortunately, the low bioavailability of 41f prevented an
estimation of absorption in rat; however, exposure in hepatic
portal vein cannulated rats was found to be similar to 39,
which gave us confidence that 41f was also well absorbed. On
the basis of oral bioavailabilities of 44%, 50%, and 31%,
respectively, in dog, oral absorption was estimated to be
complete in each case, compared with 70% for 1. The data
from both rat and dog gave us confidence that we had
identified compounds with the potential for complete oral
absorption in man.
Table 7 shows in vitro data for the stability of the lead
imidazopiperidines in human, rat, and dog liver microsomes.
Compounds 39 and 41f showed identical profiles to 1 in rat
and dog, with only compound 29 having intrinsic clearances
above the limit of detection. Despite the attractive metabolic
stability, in vivo rat pharmacokinetic studies revealed high
total/unbound clearances in all cases as had been found for 1.
The compounds were tested in parallel against MVCRES
HIV-1 isolate CC185 and its passaged controlled parental
isolate, i.e. Maraviroc-sensitive “start” isolate CC185. Each
compound was tested at the predetermined IC90 against the
lab adapted isolate HIV BaL (as in Table 5) in addition to
10- and 100-fold above this concentration. The IC90 value

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 73
Figure 1. Relative activity of various CCR5 antagonists against MVCRES HIV-1 (isolate CC185) versus passaged controlled parental virus in
PBL culture. For each compound the first column indicates a concentration of compound equivalent to the IC90 against HIV BaL, the second
10 ꢀ IC90, and the third 100 ꢀ IC90
.
Figure 2. Docking of 1 (green) and 41f (cyan) into a model of CCR5. E283 is indicated in orange, and Y108 is shown in magenta.
Chart 1. Comparator Compounds Tested against Maraviroc-Resistant Virus
against HIV-BaL was deemed an appropriate benchmark for
primary isolates based upon comparative activities for 1 against
lab-adapted versus primary isolates15 and that the same IC90
values were observed for 39 and 41f against CC185 and HIV-
BaL in full dose-response studies (data not shown). The
inhibition against MVCRES virus at the three concentrations
for each compound is shown in Figure 1 as a percentage of the
inhibition against the passaged controlled parental CC185 virus.
The MVCRES virus was found to be sensitive to the alter-
native chemotypes 42 and 43, which demonstrated the potential
for nonidentical resistance profiles for CCR5 antagonists.
Tropanes 45 and 47 were found to be effectively inactive against
the MVCRES virus, which is consistent with their structural
similarity with 1. Replacing the exocyclic triazole substituted
tropane with an endocyclic benzimidazole substituted tropane
was found to have little effect, as benzimidazoles 44 and 46 also
showed weak inhibition of MVCRES virus. We were pleased to
find that all four imidazopiperidines tested (29, 39, 41e and 41f)
demonstrated very similar inhibition of both MVCRES virus and
the parental virus, as was found to be the case for the alternative

74 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Stupple et al.
chemotypes 42 and 43. These data provided evidence that
resistance profiles distinct from that of 1 can be achieved within
the tropane chemotype.
acid (0.025 N, 160 mL) was cooled to 0 °C for 16 h. Benzylamine
hydrochloride (65 g, 453 mmol), ketomalonic acid (55 g, 377
mmol), and an aqueous solution of sodium acetate (300 mL,
0.69 M) were added and the reaction was stirred at room
temperature for 1 h. The mixture was heated to 50 °C for further
90 min, then cooled in an ice bath and basified to pH 12 with 2N
aqueous sodium hydroxide solution. The layers were separated,
and the aqueous phase extracted with ethyl acetate (3ꢀ300 mL).
The combined organic extracts were washed with water, dried
(MgSO4), filtered, and evaporated under reduced pressure. The
residual brown oil was distilled under reduced pressure (126 °C,
3 mmHg) to afford the title compound as an off-white solid
(37.8 g, 47%). LRMS m/z [M þ H]þ 216.
A CCR5 homology model has previously been reported
based on the bovine rhodopsin crystal structure.30 A binding
site for the tropane series was proposed which was consistent
with site directed mutagenesis studies that showed a loss of
functional binding of multiple examples to E238A and Y108A
mutants.31 The proposed binding mode is allosteric with
respect to the gp120-CD4 complex binding site to CCR5.
An ionic interaction between the tropane nitrogen of 1 and the
E283 carboxylate moiety in addition to a hydrophobic inter-
action between the phenyl group and the Y108 phenol moiety
were proposed (see Figure 2).32 Like 1, imidazopiperidine 41f
demonstrated a loss of functional binding to the E238A and
Y108A mutants, which is consistent with these compounds
occupying similar binding sites.31
The computer-assisted overlay shown in Figure 2 indicates
the differential occupation of the transmembrane region of
CCR5. As all of the tropanes which demonstrate activity
against the MVCRES virus possess a substituted imidazopi-
peridine in place of the triazole within 1, it is possible that
additional interactions made by this substituent affect the
resistance profile. Modeling suggests that this group points
toward the hinge region for extracellular loop 2 (ECL2), hence
it may well control the presentation of the receptor to the virus.
tert-Butyl 3-Oxo-8-azabicyclo[3.2.1]octan-8-carboxylate (6).
A mixture of ketone 5 (15.0 g, 69.7 mmol), di-tert-butyl dicar-
bonate (18.2 g, 83.4 mmol), and 20% w/w palladium hydroxide
on carbon (3.0 g) in ethyl acetate (165 mL) was stirred for 4 h at
room temperature under an atmosphere of hydrogen (269 kPa).
The mixture was filtered through Arbocel and the solvent
removed under reduced pressure. The residue was purified by
column chromatography on silica gel (100:0 to 50:50 hexane:
ether) to afford the title compound as a colorless oil which
1
crystallized on standing (16.2 g, quant). H NMR (400 MHz,
CDCl3) δ 4.58-4.35 (2H, m), 2.82-2.48 (2H, m), 2.34-2.26
(2H, m), 2.11-2.00 (2H, m), 1.68-1.60 (2H, m), 1.48 (9H, s).
tert-Butyl 3-(Benzylamino)-endo-8-azabicyclo[3.2.1]octane-
8-carboxylate (7). A solution of ketone 6 (10.0 g, 44.4 mmol),
benzylamine (4.85 mL, 49.7 mmol), and sodium triacetoxyborohy-
dride (14.11 g, 66.6 mmol) was stirred for 16 h at room temperature
in a mixture of glacial acetic acid and dichloromethane (1:9 v/v,
290 mL). The solvents were evaporated under reduced pressure and
the residue dissolved in ethyl acetate (200 mL) and washed with
saturated aqueous sodium carbonate solution (50 mL) and water
(50 mL). The organic solution was dried (MgSO4), filtered, and
evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel (98:2:0.25 dichloromethane:
methanol:concentrated aqueous ammonia) to afford the title com-
pound as a white solid (7.00 g, 50%). 1H NMR (400 MHz, CDCl3)
δ 7.32-7.26 (4H, m), 7.26-7.20 (1H, m), 4.23-4.03 (2H, m), 3.74
(2H, s), 3.03-2.95 (1H, m), 2.19-1.85 (5H, m), 1.61-1.52 (2H, m),
1.48-1.42 (11H, m).
tert-Butyl 3-endo-Amino-8-azabicyclo[3.2.1]octane-8-carbox-
ylate (8). A mixture of benzylamine 7 (7.00 g, 22.1 mmol),
ammonium formate (7.00 g, 111 mmol), and 20% w/w palladium
hydroxide on carbon (0.70 g) in ethanol (200 mL) was heated to
50 °C until gas evolution ceased. The cooled mixture was filtered
through Arbocel and the filtrate evaporated under reduced pres-
sure. The residue was purified by column chromatography on
silica gel (98:2:0.25 to 95:5:0.5 dichloromethane:methanol:con-
centrated aqueous ammonia) to afford the title compound as a
colorless oil (4.70 g, 94%). LRMS m/z [M þ H]þ 227.
Conclusion
A series of imidazopiperidines has been identified which in
the case of 41f combine complete oral absorption in rat and
dog and improved in vitro metabolic stability compared to 1.
Antiviral activity of 41f against HIV BaL in PBMCs is also
enhanced compared to that of 1. Furthermore, antiviral
activity of 41f is retained against an HIV isolate which has
been shown to be resistant to 1, consistent with a distinct
resistance profile. Because of a favorable profile in preclinical
safety studies, 41f was progressed to clinical studies.33 Phase II
clinical trials of 41f (PF-232798)20 are currently ongoing, and
further data will be reported in due course.
Experimental Procedures
Unless otherwise stated, all reactions were carried out under a
nitrogen atmosphere, using commercially available anhydrous
solvents. Flash column chromatography was carried out using
40-63 μm silica gel. NMR spectra were carried out on a Varian
Mercury 400 or Varian Inova 500 in the solvents specified. HPLC
purity determinations were carried out using a Phenomenex
phenyl hexyl 150 mm ꢀ 4.6 mm column with 5 μm particle size;
gradient: 98-2% A over 18 min, 2 min hold, 1 mL/min flow rate
(A, 10 mM ammonium acetate in H2O; B, 10 mM ammonium
acetate in MeOH), temperature 50 °C. All test compounds were
confirmed to be g95% pure by either combustion analysis or
HPLC. Combustion analyses were conducted by Exeter Analyt-
ical UK, Ltd., Uxbridge, Middlesex, UK. Compounds 42,28 43,29
44,14 45,34 46,14 and 4734 were prepared using methods previously
reported. The FluorAce β-galactosidase kit for the gp-160 fusion
assay was purchased from Bio-Rad (Hemel Hempstead, UK).
Black, clear bottom, 384-well tissue culture-treated plates were
obtained from Greiner (Stonehouse, UK). Cell culture media,
with the exception of serum, was obtained from Gibco (Paisley,
UK). Fetal calf serum was obtained from PAA (Yeovil, UK). All
other reagents for the gp160 fusion assay were purchased from
Sigma (Poole, UK).
tert-Butyl 3-endo-[(3-Nitro-4-pyridinyl)amino]-8-azabicyclo[3.2.1]-
octane-8-carboxylate (34). tert-Butyl 3-amino-endo-8-azabicyclo-
[3.2.1]octane-8-carboxylate 8 (3.0 g, 13.2 mmol), 4-ethoxy-3-
nitropyridine hydrochloride 33 (2.7 g, 13.2 mmol), and N-ethyl-N,
N-diisopropylamine (1.89 g, 14.6 mmol) were dissolved in 1-meth-
yl-2-pyrrolidinone (5 mL) and heated at 120 °C for 18 h. The
cooled reaction mixture was diluted with ethyl acetate (150 mL)
and washed with water (3 ꢀ 50 mL), saturated aqueous sodium
hydrogen carbonate solution (50 mL), and brine (30 mL). The
organic layer was dried (MgSO4), filtered, and the solvent re-
moved under reduced pressure. The residue was triturated with
diethyl ether and filtered to afford the title compound as a yellow
solid (1.5 g, 33%). 1H NMR (300 MHz, CDCl3) δ 9.25 (1H, s),
9.00-8.75 (1H, m), 8.35 (1H, m), 6.60 (1H, m), 4.50-4.15 (2H, m),
3.98 (1H, m), 2.50-1.40 (17H, m). LRMS m/z [M þ H]þ 349
1-endo-(8-Acetyl-8-azabicyclo[3.2.1]oct-3-yl)-2-methyl-1H-
imidazo[4,5-c]pyridine (35). Aminopyridine 34 (4.40 g, 12.6 mmol)
and iron powder (2.11 g, 37.8 mmol) were dissolved in glacial
acetic acid (50 mL), and the mixture was heated to 60 °C for 2 h.
8-Benzyl-8-azabicyclo[3.2.1]octan-3-one (5). A solution of 2,5-
dimethoxytetrahydrofuran 4 (50 g, 378 mmol) in hydrochloric

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 75
Acetic anhydride (8 mL) was then added and the mixtureheated to
140 °C for 18 h. The cooled reaction mixture was filtered through a
pad of Arbocel, and solvent was removed under reduced pressure.
The residue was partitioned between dichloromethane (200 mL)
and water (200 mL) and the mixture adjusted to pH 9 with 2N
aqueous sodium hydroxide solution. The mixture was filtered
through a pad of Arbocel and the organic phase separated. The
aqueous layer was extracted with dichloromethane (100 mL) and
the combined organic extractsdried (MgSO4) and filtered. Solvent
was evaporated under reduced pressure and the residue triturated
with ethyl acetate, filtered, and dried to give the title compound as
a white solid (3.27 g, 91%). 1H NMR (400 MHz, CDCl3) δ 8.95
(1H, s), 8.35 (1H, m), 7.22 (1H, m), 4.90 (1H, m), 4.35 (1H, m),
4.25-4.10 (1H, m), 2.70-2.45 (5H, m), 2.35-1.80 (9H, m).
LRMS m/z [M þ H]þ 285.
methanol:concentrated aqueous ammonia) to give the desired
product as a solid (7.4 g, 80%). H NMR (400 MHz, CDCl3) δ
1
4.50-4.35 (3H, m), 3.82-3.65 (7H, br s), 2.80-2.10 (5H, m), 2.40
(3H, s), 1.95-1.55 (6H, m). LRMS (APCI) m/z [M þ H]þ 305.
Methyl 1-endo-{8-[(3S)-3-(Acetamido)-3-(3-fluorophenyl)propyl]-
8-azabicyclo[3.2.1]oct-3-yl}-2-methyl-4,5,6,7-tetrahydro-1H-imidazo-
[4,5-c]pyridine-5-carboxylate (39). Sodium triacetoxyborohydride
(11.0 g, 51.9 mmol) was added portionwise to a solution of N-[(1S)-
1-(3-fluorophenyl)-3-oxopropyl]acetamide 3817 (11.32 g, 54.1 mmol)
and secondary amine 37 (13.73 g, 45.1 mmol) in dichloromethane
(150 mL) and the mixture stirred for 1 h at room temperature. The
solution was then washed with water (100 mL), brine (50 mL), dried
(MgSO4), and filtered. Solvent was evaporated under reduced
pressure and the resulting white solid was dissolved in ethyl acetate
(100 mL). Slight cooling induced crystallization, which was allowed
to complete at room temperature overnight. The resulting white solid
was isolated by filtration and recrystalized from acetone (4 mL/g) to
give the title compound as a white crystalline solid (15 g, 67%). 1H
NMR (400 MHz, CDCl3) δ 7.30-7.25 (1H, m), 7.10-6.90 (3H, m),
6.80-6.50 (1H, m), 5.15 (1H, m), 4.65-4.38 (3H, m), 3.80-3.60
(5H, m), 3.50-3.30 (2H, m), 2.80-1.90 (16H, m), 1.70-1.40 (4H,
m). LRMS (ES) m/z [M þ Hþ] 498. Found C, 62.80; H, 7.48;
Methyl 1-(8-Acetyl-8-azabicyclo[3.2.1]oct-3-yl)-2-methyl-1,
4-dihydro-5H-imidazo[4,5-c]pyridine-5-carboxylate (36). A solution
of imidazopyridine 35 (10.0 g, 35.2 mmol) in ethanol (95 mL) and
water (5 mL) was degassed twice by application of a vacuum and
then flushing with nitrogen gas prior to cooling to -70 °C. To the
cooled, stirred solution was added methyl chloroformate (3.3 mL,
42.2 mmol) dropwise over 10 min. After a further 45 min, sodium
borohydride (4.0 g, 105.7 mmol) was added portionwise. The
reaction mixture was allowed to warm to room temperature over
1 h. Crushed ice was carefully added, and stirring was continued for
10 min. The supernatant was decanted from the white precipitate,
and the ethanol was removed under reduced pressure. The resulting
aqueous mixture was recombined with the white precipitate and
was diluted with 2 M aqueous HCl (100 mL) to give a solution
which was then extracted with ethyl acetate (100 mL). The organic
layer was extracted with 2 M aq HCl (50 mL). The two acidic layers
were then combined and basified to pH 9 by careful addition of
solid potassium hydroxide. The aqueous mixture was extracted
with dichloromethane (100 mL and then 2 ꢀ 50 mL). The combined
organic phases were dried (MgSO4), filtered, and concentrated. The
residue was triturated with ethyl acetate, filtered, and dried to give a
solid (7.0 g). The mother liquors were concentrated and purified by
silica column chromatography (99:1 to 95:5 dichloromethane:
methanol) to give a second batch of the title compound as a solid
(2.0 g, combined yield 74%). 1H NMR (400 MHz, CDCl3) δ 6.82
and 6.65 (1H, 2 ꢀ m), 5.48 (1H, m), 4.90 (2H, s), 4.84 (1H, m),
4.35-4.25 (1H, m), 4.00-3.80 (4H, m), 3.80 (3H, br s), 2.40 (3H, br
s), 2.70-2.35 (2H, m), 2.15 (3H, s), 2.30-2.10 (2H, m), 2.00-1.70
(4H, m). LRMS (APCI) m/z [M þ H]þ 345
N, 13.55. C27H37FN5O3 (H2O) requires C, 62.89; H, 7.43; N, 13.58%
3
N-{(1S)-3-[3-endo-(2-Methyl-4,5,6,7-tetrahydro-1H-imidazo-
[4,5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-(3-fluorophenyl)-
propyl}acetamide (40). To a stirred solution of carbamate 39 (13.27 g,
26.7 mmol) in propan-2-ol (80 mL) was added 2 M aqueous sodium
hydroxide solution (120 mL), and the mixture was heated at reflux
for 48 h. After cooling to room temperature, the mixture was
extracted with ethyl acetate (2 ꢀ 200 mL). The combined organic
components were washed with brine (150 mL), dried (MgSO4),
filtered, and concentrated under reduced pressure. The crude
product mixture was purified by silica column chromatography
(90:10:1 then 80:20:1 dichloromethane:methanol:concentrated
aqueous ammonia) to give the title compound as a white foam
(8.54 g, 73%). 1H NMR (400 MHz, CD3OD) δ7.38-7.29 (1H, m),
7.18-7.12 (1H, m), 7.10-7.04 (1H, m), 7.00-6.91 (1H, m),
5.20-5.12 (1H, m), 4.63-4.52 (1H, m), 3.70 (2H, br s),
3.41-3.33 (2H, m) 3.08-3.02 (2H, m), 2.78-2.72 (2H, m),
2.52-2.41 (2H, m), 2.39 (3H, s), 2.30-2.25 (2H, m), 2.19-2.08
(2H, m), 1.98 (3H, s), 1.95-1.85 (2H, m), 1.70-1.60 (4H, m).
LRMS (APCI) m/z [M þ H]þ 440. Found C, 66.19; H, 7.99; N,
15.44. C25H34FN5O 0.8(H2O) requires C, 66.14; H, 7.90; N,
3
15.43%.
N-{(1S )-3-[3-endo-(5-Isobutyryl-2-methyl-4,5,6,7-tetrahydro-
1H-imidazo[4,5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-(3-
fluorophenyl)propyl}acetamide (41f). To a stirred solution of
secondary amine 40 (19.9 g, 45.3 mmol) in tetrahydrofuran
(500 mL) was added triethylamine (7.0 mL, 50.0 mmol), fol-
lowed by dropwise addition of isobutyryl chloride (5.3 mL,
50 mmol). After 1 h, a second portion of isobutyryl chloride was
added dropwise (0.5 mL, 5.0 mmol). After 0.5 h, the reaction
mixture was concentrated to approximately 300 mL and ethy-
lacetate (200 mL) was added. The reaction mixture was washed
with 10% aqueous potassium carbonate solution (200 mL; w/v).
The aqueous phase was separated and extracted with ethylace-
tate (100 mL). The organic components were combined, washed
with brine (100 mL), dried (MgSO4), filtered, and concentrated
until a mobile oil was obtained which had just started to form a
foam. The residue was dissolved in ethyl acetate (100 mL) and
heated to 90 °C. Water (0.5 mL) was added to the hot solution,
and the mixture was allowed to slowly cool to room tempera-
ture. The precipitate was collected by filtration, washed with
ethyl acetate (50 mL), and dried under reduced pressure to give
the title compound as a white solid (20.9 g, 90%). 1H NMR (400
MHz, CD3OD) δ 7.35-7.29 (1H, m), 7.14-7.12 (1H, m),
7.07-7.05 (1H, m), 6.97-6.92 (1H, m), 5.17-5.13 (1H, m),
4.63-4.52 (1H, m), 4.44-4.43 (2H, m), 3.89-3.78 (2H, m),
3.40-3.31 (2H, m), 3.06-2.90 (1H, m), 2.84-2.77 and
2.75-2.69 (2H, 2 ꢀ m), 2.51-2.39 (2H, m), 2.36 and 2.35 (3H,
Methyl 1-[(3-endo)-8-Azabicyclo[3.2.1]oct-3-yl]-2-methyl-1,4,6,7-
tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylate (37) . Part 1:
Methyl 1-(8-Acetyl-8-azabicyclo[3.2.1]oct-3-yl)-2-methyl-1,4,6,7-
tetrahydro-5H-imidazo[4,5-c]pyridine-5-carboxylate (50). To a so-
lution of olefin 36 (6.75 g, 19.6 mmol) in ethanol (60 mL) at room
temperature was added 10% palladium on carbon (500 mg), and
the mixture was stirred at 50 °C under an atmosphere of hydrogen
gas (60 psi) for 5 h. After cooling to room temperature, the catalyst
was removed by filtration and the filtrate was evaporated. The
residue was dissolved in dichloromethane, dried (MgSO4), filtered,
and concentrated. The residue was dissolved in ethyl acetate
(10 mL), and the flask was scratched to induce crystallization.
The suspension was diluted with diethyl ether, the precipitate was
collected by filtration, washed with diethyl ether, and dried to give
the desired product as a white solid (6.2 g, 91% yield). 1H NMR
(400 MHz, CDCl3) δ 4.85 (1H, m), 4.50 (2H, br, s), 4.30 (1H, m),
4.00-3.87 (1H, m), 3.85-3.70 (m, 3H), 3.70 (3H, s) 2.80-2.40 (7H,
m), 2.30-2.05 (2H, m), 2.15 (3H, s), 1.90-1.70 (4H, m). LRMS
(APCI) m/z [M þ H]þ 347. Part 2: A mixture of the acetamide 50
(10.58 g, 30.0 mmol) in 2 M aqueous sulfuric acid (150 mL) was
heated at 100 °C for 18 h. After cooling to room temperature, the
mixture was basified to pH 11-12 using solid sodium hydroxide
and extracted three times with dichloromethane. The combined
organic layers were dried (MgSO4), filtered, and concentrated to
give a yellow/brown gum. The crude product was purified by silica
column chromatography (99:1:0.1 to 90:10:1 dichloromethane:

76 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Stupple et al.
2ꢀs), 2.30-2.20 (2H, m), 2.13-2.03 (2H, m), 1.95 (3H, s), 1.90-
1.84 (2H, m), 1.65-1.55 (4H, m), 1.11-1.08 and 1.06-1.04 (6H,
2 ꢀ m). Rotamers apparent in spectrum. LRMS m/z [MHþ]
requires C, 67.16; H, 7.97; N, 13.50%. [R]D25 -23.4° (1.64 mg/mL in
MeOH).
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3
Gp160 Fusion Assay.35 CHO-Tat10 and HeLa-P4 cells were
grown in 225 cm2 flasks or roller bottles to confluence. Cells were
washed twice with PBS and harvested using Trypsin EDTA. Cells
were pooled into two separate 1 L sterile spinner flasks, one for each
cell line. A cell count was performed, and the cell suspension was
diluted in medium (DMEM with 2 mM L-glutamine and 2% FCS
for HeLa-P4 or RPMI1640 with 2 mM L-glutamine and 2% FCS
for CHO-Tat10) to give a suspension at 6.6 ꢀ 105 cells per mL. The
spinner flasks were connected to a 5% CO2 gas supply to maintain
pH and stirred at 30 rpm at room temperature during dispensing.
Then 15 μL of CHO-Tat10 and 15 μL of HeLa-P4 were added
sequentially to a sterile tissue culture-treated 384-well plate con-
taining 10 μL compound/control solution (360 wells of test com-
pound and 8 wells each of a maximum [no inhibitor], minimum
[saturating concentration of fusion inhibitor], and standard [an
IC50 concentration of a fusion inhibitor] controls). Plates were
lidded and incubated at 37 °C and 5% CO2 in a humidified
(Cytomat) incubator. After 20 h, 20 μL of reaction buffer (13.4%
glycerol, 1.3% Triton-X100, 0.34 M Tris [pH7.8], 200 μM 4-methy-
lumbelliferyl-galactoside (MUG), 0.028% β-mercapto-ethanol,
33% Bio-Rad 1ꢀ reaction buffer) was added to each well. Plates
were incubated at 25 °C for a further 2 h, during which time cell lysis
occurred, and then 10 μL of Bio-Rad 10 ꢀ stop buffer was added to
each well. The fluorescent signal was detected, after a further
45-min incubation, using a Tecan Spectrafluor Plus (Tecan)
(λex = 360 nm, λem = 460 nm; bottom reading; 3 flashes; gain =
50-60).
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Acknowledgment. We thank Paul Griffin, Becky Irvine,
Alex Martin, Julie Mori, Hannah Perkins, Nikki Robas,
Graham Rickett Caroline Smith-Burchnell, Malcolm Macartney,
and Roy Mansfield for their contributions to the biology
studies.
Supporting Information Available: Additional experimental
procedures and analytical data for intermediates and final
compounds. Further pharmacokinetic data from rat and dog
po studies are also provided. This material is available free of
charge via the Internet at http://pubs.acs.org.
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