Discovery of a small molecule inhibitor through interference with the gp120-CD4 interaction

Article abstract of DOI:10.1016/j.bmcl.2009.06.080

A series of piperazine derivatives were designed and synthesised as gp120-CD4 inhibitors. SAR studies led to the discovery of potent inhibitors in a cell based anti viral assay represented by compounds 9 and 28. The rat pharmacokinetic and antiviral profiles of selected compounds are also presented. Crown Copyright

Full text of DOI:10.1016/j.bmcl.2009.06.080

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5246–5249
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a small molecule inhibitor through interference with the gp120–
CD4 interaction
a
a
b
c
a
David H. Williams a, , Fiona Adam , David R. Fenwick , Juin Fok-Seang , Iain Gardner , Duncan Hay ,
Rawal Jaiessh c, Donald S. Middleton a, Charles E. Mowbray a, Tanya Parkinson b, Manos Perros b,
Christopher Pickford b, Michelle Platts a, Amy Randall a, Daniel Siddle c, Peter T. Stephenson a,
Thien-Duc Tran a, Hannah Vuong a
*
a Department of Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
b Department of Biology, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
c Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Sandwich Laboratories, 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:
A series of piperazine derivatives were designed and synthesised as gp120–CD4 inhibitors. SAR studies
led to the discovery of potent inhibitors in a cell based anti viral assay represented by compounds 9
and 28. The rat pharmacokinetic and antiviral profiles of selected compounds are also presented.
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Received 15 May 2009
Revised 19 June 2009
Accepted 20 June 2009
Available online 25 June 2009
Keywords:
HIV
gp120–CD4
Entry inhibitors
Human immunodeficiency virus (HIV-1) infection remains a
major medical problem with approximately 33 million people
worldwide living with the disease, together with 2.5 million people
newly infected with HIV in 2007.1 Despite advances in combina-
tion therapies and the approval of 29 marketed drugs that cover
5 main classes (nucleotide/nucleoside, non nucleotide reverse
transcriptase inhibitors, protease inhibitors, entry inhibitors and
integrase inhibitors), emergence of drug resistant viral strains,2
and tolerance/toxicity3 remain major issues in the treatment of
HIV infection. As a consequence, the demand for new classes of
anti-retroviral drugs that target different mechanisms remains
high.
The interaction of HIV-Env, the gp160 envelope protein com-
prising gp120–gp41, with CD4 and a co-receptor triggers a cascade
of events that results in the insertion of gp41 into the host cell
membrane and the cell becoming infected. Compounds interfering
with gp120/co-receptor interactions, for example, maraviroc (Selz-
entry), via blocking of the CCR5 co-receptor, have shown strong
antiviral efficacy in patients infected with CCR5-tropic (R5) virus.4
Additionally, the first clinically approved entry inhibitor, Enfuvir-
tide, (Fuzeon) blocks HIV-1 entry into the host cell by binding to
the gp41 subunit of the HIV-Env glycoprotein and has been shown
to reduce viral load in the clinic.5 Targeting the first stage of the fu-
sion process that is, the gp120–CD4 interaction presents an alter-
native, attractive anti-retroviral approach. Targeting such an
early stage of the viral cycle has the advantage of being able to in-
hibit CCR5-tropic (R5), CXCR4-tropic (X4) and dual-tropic (DM)
viruses.6
Bristol-Myers Squibb has developed a series of small molecule
gp120–CD4 inhibitors based on an alpha keto amide structure
(Fig. 1) which specifically inhibit HIV entry.7
These compounds exhibit potent antiviral activity in cell cul-
ture. Additionally, it has been shown that BMS378806 binds di-
rectly to gp120, and is active against a range of lab adapted
strains of HIV-1, irrespective of chemokine receptor tropism.8–10
BMS488043 has been evaluated in healthy volunteers where it
was shown to be safe, tolerable and adequate pharmacokinetic
profile when administered over a fourteen day period.11 Addition-
ally, BMS488043 has been evaluated in a multi-dose study (800 mg
or 1800 mg bid) in HIV-1 infected patient where the preliminary
results established mean maximal changes in plasma HIV-1 RNA
of up to ꢀ1.2 log10
,
8–10 thus establishing proof of concept for block-
ing the gp120–CD4 interaction as being clinically efficacious.
In this Letter we would like to disclose some of our recent find-
ings towards identifying an orally bioavailable gp120–CD4 inhibi-
tor as a novel class of anti-HIV medicines which would also be
complementary with other classes of anti-retroviral drugs.
* Corresponding author. Tel.: +44 1304 641609; fax: +44 1304 651819.
E-mail address: David.H.Williams@pfizer.com (D.H. Williams).
0960-894X/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.080

D. H. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5246–5249
5247
H
H
N
N
O
O
MeO
N
N
N
N
O
N
O
N
OMe
OMe
O
O
BMS488043
BMS378806
Figure 1.
O
O
Cl
O
HN
N
Cl
O
a
OH
+
N
N
1
2
O
3
O
gp120 Fusion (IC50) 1000 nM
Scheme 1. Reagents and conditions: (a) O-(1H-benzotriazol-1-yl)-N,N,N0,N0-tetramethyluroniumhexafluorophosphate, NEt3, DCM, rt, 24 h, 65%.
Our overall target was to identify an orally bioavailable gp120–
CD4 inhibitor possessing pharmacokinetic properties commensu-
rate with a low dose (<300 mg bid) regimen. At this dose, the Cmin
for this compound should be in excess of the in vitro IC90 for the
majority of primary clinical isolates.1
according to Lipinsky’s rule of five.13 This would maximise our
chances to achieve our desired profile of an orally bioavailable po-
tent gp120–CD4 inhibitor.
All compounds 9–28 were prepared as mixtures of diastereo-
mers by the general method shown in Scheme 2. The versatile
common intermediate 414 was prepared in one step from the cou-
pling of 2-bromopropionylchloride with 2R-4-benzoyl-2-methyl-
piperazine.15
Single diastereomers were synthesised by the general method
exemplified in Scheme 3 for compound 9. Reaction of R-methyl lac-
tate 6 and the appropriate phenol 5 in THF under standard Mitsun-
obu conditions afforded the aryl ether 7 in good general yield.
Alkaline hydrolysis of the methyl ester to furnish the carboxylic
acid 8 followed by a carbodiimide mediated condensation with
2R-4-benzoyl-2-methyl-piperazine 2 afforded the enantiomeri-
cally pure 9 in Scheme 3.
As part of a programme seeking novel inhibitors of the gp120–
CD4 interaction, our work focused initially on the above ketoam-
ides where we looked to replace the potentially reactive and met-
abolically vulnerable ketone. As a consequence of plate based
coupling of acids to 2 to make a range of analogues designed to re-
place the keto acid, it led to the identification of 3 (Scheme 1). This
compound is relatively lipophilic and possessed moderate activity
(IC50 of 3 l
M) in our high throughput cell–cell fusion assay.12 Our
choice of 3 for further optimisation in our hit-to-lead programme
was driven by the fact that the template is amenable to parallel
chemistry. Hence, the goal of the initial hit-to-lead study was to
rapidly optimise this hit by tuning the physiochemical properties
by incorporating small polar groups into lead 3 as a strategy to in-
crease the polarity whilst keeping the molecular weight low
From the initial set of analogues prepared, some intriguing SAR
emerged (Table 1). Most notably, polar substituents on the phenyl
ring were relatively well tolerated with only modest erosion in
O
O
O
OH
Br
(a)
N
N
N
N
R
R
O
O
O
9-28
4
Scheme 2. Reagents and conditions: (a) Cs2CO3, acetone, rt, 24 h, 40–75%.
OH
O
O
(a)
OMe
HO
HN
OMe
HN
HN
OMe
O
OMe
O
5
6
7
8
O
(b)
O
O
O
(c)
N
OH
HN
N
OMe
OMe
O
O
O
9
Scheme 3. Reagents and conditions: (a) Triphenylphosphine, diisopropylazodicarboxylate, THF, 5 °C, rt, 2 h; (b) LiOH, MeOH/THF, rt, 2 h, 75% over two steps; (c) 1-N-
hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideꢁHCl, triethylamine, rt, 17 h, 72%.

5248
D. H. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5246–5249
Table 1
SAR Summary: in vitro inhibition gp120 (JRFL) mediated cell–cell fusion,a clog P calculations,b log D7.4 and in vitro metabolic stabilityc
R1
O
R2
R3
O
N
N
O
3, 9-26
Compd
R1
R2
R3
gp120a IC50
(
lM)
clog Pb
Log D
HLMc Clint (
ll/min/mg)
3
H
Cl
H
H
H
H
1
4.2
2.3
2.4
2.4
2.1
2.1
2.1
4.3
2.5
2
ND
1.5
1.3
1.1
1.1
1
0.9
ND
1.2
1
ND
23
<8
<8
31
12
<8
158
<8
<8
10
11
12
13
14
15
16
17
18
CONHMe
H
H
CONMe2
H
H
Me
Me
>25
1.4
2.6
>25
>25
>25
0.1
12
CONHMe
H
H
CONMe2
H
Me
CONHMe
H
H
CONMe2
H
H
H
CONHMe
CONHMe
OMe
>10
O
N
19
20
Me
H
H
H
0.075
0.035
3.1
3.7
2.1
2.3
141
61
N
N
N
21
22
23
9d
H
Me
H
OMe
OMe
OEt
F
Me
H
OMe
H
H
H
CONHMe
CONHMe
CONHMe
CONHMe
CONHMe
CONHMe
CONHMe
1
2.53
2.9
2.3
2.1
2.1
2.6
2.5
1.1
1.8
ND
1
ND
ND
1.4
<8
<8
2.9
2.7
0.017
>2
>2
2
ND
<8
ND
ND
<8
24e
25
26
H
ND denotes not determined.
a
The concentration required to inhibit gp120 (JRFL) mediated cell–cell fusion.12
Biobyte Corporation, 201 W. 4th St., #204, Claremont CA 91711-4707, cLOG P version 4.3.
Human Liver Microsomes, minimum measurable in vitro intrinsic clearance was 8
Single S,R diastereomer.
Single R,R diastereomer.
b
c
ll/min/mg protein.
d
e
potency as exemplified by the carboxamides 11 and 12, but signif-
icant drop in lipophilicity. Tertiary amide substitution 13, 14 and
15 and the ortho secondary amide in 10 were not well tolerated.
Introduction of modestly lipophilic substituents on the phenyl ring
16 proved to be advantageous affording improvements in potency
over the starting 3-chlorophenyl group in 2. Unfortunately, these
changes resulted in increased metabolic vulnerability of 16 and
precluded further advancement of the compound.
The strategy of introducing polar substituents at the meta posi-
tion while incorporating the favourable substitution of 16 met with
limited overall success (17–20). Analogues 17 and 18, whilst
achieving improved levels of in vitro metabolic stability, displayed
disappointing potency. The oxadiazole 19 and pyrazole 20 gave
highly encouraging levels of potency however, the increased LogD
of these compounds resulted in unacceptably high metabolic rates
in human liver microsomal incubations. For this series large in-
creases in metabolic instability were observed with compounds
having clogP >2.5 and LogD >1.5, so efforts were made to keep lipo-
philicty below these values in further analogues. We next turned
our attention to the synthesis of para secondary amide analogues
(9, 21–26). This strategy proved to be highly rewarding with the
identification of amide 9. A homochiral synthesis of the two diaste-
reoisomers (9 and 24) unambiguously established that the activity
resided with the S, R diastereomer 9 (Table 1).
amide. Previous SAR suggested that methyl substituents in the
ortho and meta position were advantageous for potency.
Replacement of the ortho-methyl in 23 for a methoxy (9) re-
sulted in a dramatic 100 fold improvement in potency as well as
approximately a unit reduction in overall lipophilicity relative to
22.
Encouraged by the latter data a number of substituents at the
ortho position were investigated as exemplified by 25–26. Disap-
pointingly, these latter derivatives (25, 26) resulted in a precipi-
tous loss in potency.
With the identification of amide 9 as our most promising lead,
we sought to further improve the physicochemical properties of
the series through the introduction of a ring nitrogen (Table 2). A
series of pyridyl analogues based around 9 were synthesised (27,
28). The pyridyl derivative 27 showed a significant drop off in po-
tency in our assay, however, compound 28 retained a high level of
potency and excellent metabolic stability.
Based on their overall in vitro profiles 9 and 28 were progressed
for full pharmacokinetic (Table 3) and antiviral assay15 profiling.
Encouragingly, 9 demonstrated moderate oral bioavailability and
excellent anti viral activity with an IC50 of 3 nM. Excitingly, the full
pharmacokinetic profile of 28 showed the compound to posses im-
proved clearance and oral bioavailability and excellent antiviral
activity of IC50 2 nM. Projected human pharmacokinetics suggested
that 28 would exhibit an oral bioavailability of greater than 90%
and a predicted half life of around 5 h.
As shown in Table 1 the para carboxamide series is character-
ised by some very subtle SAR, introduction of the 3-methyl and
the 2-methyl substituent 21 and 22, respectively, resulted in no
significant improvement in potency relative to the parent carbox-
The potency of 28 against a panel of viral strains is listed in
Table 4. The compound shows encouraging antiviral potency

D. H. Williams et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5246–5249
5249
Table 2
Additionally, compound 28 showed potent antiviral activity
against a panel of 25 clade B isolates17 where 70% of these viruses
were inhibited at an IC90 below Cmin of 500 nM.
SAR Summary: in vitro inhibition gp120 (JRFL) mediated cell–cell fusion,a clog P
calculations,b log D7.4 and in vitro metabolic stabilityc
O
Modelling of the predicted human pharmacokinetics of 28 (t1/2
5 h, F 90% and Vd 1 L/kg) suggests that the Cmin of 500 nM could be
achieved with a twice daily dose of 200 mg, hence, enabling good
antiviral coverage against many relevant isolates. Based on these
data and further profiling, 28 was selected for clinical development
as a broad spectrum gp120–CD4 inhibitor for the treatment of HIV.
In summary, a series of 2R-methyl-piperazinylbenzamide deriv-
atives were successfully designed and efficiently synthesised,
incorporating polar functionality as a strategy to improve overall
metabolic stability. These neutral low molecular weight inhibitors
that interfere with the interaction of gp120 to CD4 displayed very
good cell based efficacy against a broad spectrum of viral strains.
Overall analogue 28 displayed the most attractive profile and
was found to possess good rat pharmacokinetics commensurate
with its physicochemical properties.
O
R
N
N
O
Compd
ROH
gp120a IC50
M)
clog Pb
Log D
HLMc Clint
l/min/mg)
(
l
(l
OH
27
0.257
0.011
2.3
ND
ND
<8
O
N
OMe
NHMe
OH
N
28
2.2
0.7
O
OMe
References and notes
NHMe
1. UNAIDS/WHO 2008 AIDS epidemic updates. http://www.unaids.org/en/.
2. Vaclavikova, J.; Weber, J.; Machala, L.; Reinis, M.; Linka, M.; Bruckova, M.;
Vandasova, J.; Stankova, K.; Konvalinka, J. Acta Virol. 2005, 49, 29.
3. Haugaard, S. B. Exp. Opin. Drug Metab. Toxicol. 2006, 2, 429.
4. (a) Markovic, I. Curr. Pharm. Des. 2006, 12, 1105; (b) Fatkenheuer, G.; Pozniak,
A. L.; Johnson, M. A.; Plettenberg, A.; Staszewski, S.; Hoepelman, A. I.; Saag, M.
S.; Goebel, F. D.; Rockstroh, J. K.; Dezube, B. J.; Jenkins, T. M.; Medhurst, C.;
Sullivan, J. f.; Ridgway, C.; Abel, S.; James, I. T.; Youle, M.; van der Ryst, E. Nat.
Med. 2005, 11, 1170.
ND denotes not determined.
a
The concentration required to inhibit gp160 (JRFL) mediated cell–cell fusion.12
b
Biobyte Corporation, 201 W. 4th St., #204, Claremont CA 91711-4707, CLOGP
version 4.3.
c
Human Liver Microsomes, minimum measurable in vitro intrinsic clearance
was 8 l/min/mg protein.
l
5. (a) Hughes, A.; Barber, T.; Nelson, M. J. Infect. 2008, 57, 1; (b) Lalezari, J. P.;
Luber, A. D. Drugs of Today 2004, 3, 259.
Table 3
6. (a) Doms, R. W. Top. HIV Med. 2004, 100; (b) Rusconi, |S.; Scozzafava, A.;
Mastrolorenzo, A.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 1273.
7. Wang, T.; Zhang, Z.; Wallace, O. B.; Deshpande, M.; Fang, H.; Yang, Z.; Zadujura,
L. M.; Tweedie, D. L.; Hunag, S.; Zhao, F.; Ranadive, S.; Robinson, B. S.; Gong, Y.-
F.; Ricarrdi, K.; Spicer, T. P.; Deminie, C.; Rose, R.; Wang, H.-D. H.; Blair, W. S.;
Shi, P.-Y.; Lin, P.-F.; Colono, R. J.; Meanwell, N. A. J. Med. Chem. 2003, 46, 4236.
8. Yang, Z.; Zadjura, L.; D’Arienzo, C.; Marino, A.; Santone, K.; Klunk, L.; Greene, D.;
Lin, P.-F.; Colonno, R.; Wang, T.; Meanwell, N.; Hansel, S. Biopharm. Drug Dispos.
2005, 26, 387.
9. (a) Hanna, G.; Lalezari, L.; Hellinger, J.; Wohl, D.; Masterson, T.; Fiskel, W.;
Kadow, J.; Lin, P.; Giordano, M.; Colonnol, R.; Grasela, D. 11th Conference on
Retroviruses, Opportunistic Infection, Feb 8–11, 2004, San Francisco, CA, 2004;
abstract 141.; (b) Guo, Q.; Ho, H.-T.; Dickeer, I.; Fan, L.; Zhou, N.; Friborg, J.;
Wang, T.; McAuliffe, B. V.; Wang, H.-G. H.; Rose, R. E.; Fang, H.; Sarnati, H. T.;
Langley, D. R.; Meanwell, N. A.; Abraham, R.; Collono, . J.; Lin, P.-F. J. Virol. 2003,
77, 10528.
10. Wang, H.-G.; Williams, R. E.; Lin, P.-F. Curr. Pharm. Des. 2004, 10, 1785.
11. Hanna, G.; Yan, J. -H.; Fiske, W.; Masterson, T.; Zhang, D.; Grasela, D 11th
Conference on Retroviruses, Opportunistic Infection, Feb 8–11, 2004, San
Francisco, CA, 2004; abstract 535.
12. (a) gp120 fusion assay. The HIV fusion assay is based on fusion of CHO cells
expressing HIV-1 gp160 and tat, with HeLa-P4 cells. Fusion is measured by tat
transactivation of the HIV LTR-beta galactosidase reporter gene.; (b) 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.
Screening 2004, 9, 516.
Pharmacokinetic (iv 1 mg/kg, po 1 mg/kg (n = 2)), in vitro metabolic stability,
physiochemical and potency data associated with compounds 9 and 28
9
28
Cla (mL/min/kg)
Vdb (L/kg)
ppbc (%)
75
4.3
19
0.7
48
>120
3
18
1.5
31
1
65
>120
2
t1/2 (h)
Fd (%)
RLMe t1/2 (min)
AVf IC50 (nM)
a
Clearance.
Volume of distribution.
Plasma protein binding.
Bioavailability.
b
c
d
e
Rat Liver Microsomes, maximum measurable half life (t1/2) was 120 min.
f
The concentration required to inhibit viral entry of lab adapted HIV NL4-3 in
HeLa-P4 cells to 50%.16 All compounds had negligible cytotoxicity with CC50
>10 M.
l
Table 4
HIV-1 Inhibitory data against lab adapted (LA) and primary isolate (PI) strains tested
in HeLa-P4 reporter cell line or in human PBMCs for compound 28
13. Lipinski, C. A. Drug Discovery Today 2004, 1, 337.
14. Middleton, D. S.; Mowbray, C. E.; Stephenson, P. T.; Williams, D. H. U.S. Patent.
Appl. US 2005/043300A1, 2005.
15. Fenwick, D. R.; Gillmore, A. T.; Platts, M. Y. PCT Int. Appl., WO2006/085199A1,
2006.
Virus
Tropism
Cells
Geo. Mean IC50 (nM)
n
NL4-3 (LA)
IIIB (LA)
BaL (LA)
BR92004 (PI)
BR92023 (PI)
X4
X4
R5
R5
R5
HeLa-P4
PBMCs
PBMCs
PBMCs
PBMCs
2
3
3
6
3
2
16. Antiviral assays: activity against NL4-3 virus in HeLa-P4 cells was determined
as follows: Diluted compound was added to wells of a sterile 96-well flat-
bottomed black tissue culture plate with clear base and lid (Costar). HeLa-P4
cells were mixed with NL4-3 virus and added to the wells at a concentration of
10
25
6
105 cells/mL in a volume of 90
lL. The plates were incubated for 5 days at 37 °C
0.6
in a 5% CO2 humidified incubator, then virus infection was quantified using the
FluorAce b-galactosidase reporter assay kit (Bio-Rad) according to the
manufacturer’s instructions.
17. Antiviral assay was carried out by Monogram Biosciences using their
Phenosense assay.
against a number of lab adapted strains and primary isolates as-
sayed in human PBMCs (peripheral blood mononuclear cells).