Inhibitors of HIV-1 attachment. Part 4: A study of the effect of piperazine substitution patterns on antiviral potency in the context of indole-based derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.07.076

4-Fluoro- and 4-methoxy-1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (2 and 3, respectively) have been characterized as potent inhibitors of HIV-1 attachment that interfere with the interaction of viral gp120 with the host cell receptor C

Full text of DOI:10.1016/j.bmcl.2009.07.076

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5140–5145
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of HIV-1 attachment. Part 4: A study of the effect of piperazine
substitution patterns on antiviral potency in the context
of indole-based derivatives q
a
a
a
b
b
Tao Wang a, , John F. Kadow , Zhongxing Zhang , Zhiwei Yin , Qi Gao , Dedong Wu ,
Dawn DiGiugno Parker c, Zheng Yang d, Lisa Zadjura d, Brett A. Robinson e, Yi-Fei Gong e, Wade S. Blair e,
Pei-Yong Shi e, Gregory Yamanaka e, Pin-Fang Lin e, Nicholas A. Meanwell a
*
a Department of Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
b Department of Analytical Research and Development, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
c Department of Pharmaceutics, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
d Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
e Department of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Fluoro- and 4-methoxy-1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (2 and 3,
respectively) have been characterized as potent inhibitors of HIV-1 attachment that interfere with the
interaction of viral gp120 with the host cell receptor CD4. As part of an effort to understand fundamental
aspects of this pharmacophore, discovered originally using a high throughput cell-based screen, modifi-
cation and substitution of the piperazine ring was examined in the context of compounds 6a–ah. The
piperazine ring was shown to be a critical element of the HIV-1 attachment inhibiting pharmacophore,
acting as a scaffold to deploy the indole glyoxamide and benzamide in a topographical relationship that
complements the binding site on gp120.
Received 5 June 2009
Revised 13 July 2009
Accepted 15 July 2009
Available online 18 July 2009
Keywords:
HIV
HIV attachment
gp120
Ó 2009 Elsevier Ltd. All rights reserved.
Piperazine
A1,3 strain
The emergence of HIV-1 (human immunodeficiency virus-1) in
1981 foreshadowed an epidemic that has erupted to infect an esti-
mated 40 million individuals worldwide with an annual death rate
from progression to acquired immunodeficiency syndrome (AIDS)
approaching three million.1,2 The development of antiviral agents
specifically targeting HIV has been a successful enterprise that
has spawned 25 marketed drugs with two ‘first-in-class’ drugs,
the integrase inhibitor raltegravir and the CCR5 antagonist marav-
iroc, approved by the FDA in 2008.3 As might be anticipated with
mechanistically novel drugs, raltegravir and maraviroc are not
cross-resistant with existing anti-HIV therapeutics, providing
important new agents for highly antiretroviral drug-experienced
patients, many of whom harbor viruses expressing mutations con-
ferring resistance to one or more of the major drug classes. The
medical need for HIV-1 inhibitors acting by new mechanisms per-
sists and it was against this backdrop that we developed and
implemented a cell-based screening assay designed to identify lead
molecules with novel modes of action.4–8 1-(4-Benzoylpiperazin-
1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (1) emerged from that
initiative as an inhibitor of HIV-1, representative of a series that
was subsequently determined to interfere with the interaction be-
tween the membrane bound HIV glycoprotein 120 (gp120) and the
CD4 receptor expressed on the membrane of human T cells and
macrophages.4–12 The engagement of CD4 by gp120 is the initial
step in the process by which HIV-1 gains entry to the host cell
cytosol where the replication process is initiated.
Indole 1 is a potent and specific inhibitor of HIV-1 in vitro that is
not overtly cytotoxic toward a range of cell lines.8 We have re-
cently described structure–activity relationships associated with
the substitutions patterns for both the indole heterocyclic core
and the benzamide moiety.13,14 Small substituents, including F,
Cl, and MeO, at C-4 of the indole led to significant increases in po-
tency while similar substituents introduced simultaneously at C-7
reinforced this effect, with a 4,7-disubstitution pattern clearly pre-
ferred.13 Representative examples include indoles 2 and 3 which
demonstrate EC50 values of 3 nM and 0.72 nM, respectively, in a
pseudotype assay based on the HIV-1 LAI subtype, which compares
with and EC50 of 86 nM for 1.8,13 In contrast, substitution of the
benzamide moiety generally resulted in compounds with weaker
antiviral activity although replacement by five-membered hetero-
q
See Refs. 8, 13, and 14 for Parts 1–3 of this series.
* Corresponding author. Tel.: +1 203 677 6584; fax: +1 203 677 7702.
E-mail address: Tao.Wang@bms.com (T. Wang).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.076

T. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5140–5145
5141
cycles was better tolerated.14 With a basic understanding of two
critical elements of the HIV-1 attachment inhibitor pharmaco-
phore in hand, attention was directed towards probing the effect
of structural variation of the piperazine moiety of 1. In this Letter,
we describe the results of that study which examined potential
replacements for the piperazine and probed the effect of introduc-
ing both simple alkyl and more polar substituents on HIV-1 inhib-
itory activity. In addition, the effect of some of these changes on
properties in assays conducted in vitro designed to predict aspects
of pharmacokinetic performance in vivo was assessed.
derivatization of substituted piperazines in a selective fashion.
The mono-acylation of symmetrical diamines is typically compli-
cated by the tendency for bis-acylation to occur,15 a problem that
was addressed by two complementary approaches that relied upon
the selective activation16 or deactivation17 of one of the nitrogen
atoms of piperazines 10, as summarized in Scheme 2. The selective
activation of one nitrogen atom in symmetrical piperazines 10 was
accomplished by treatment with 2 equiv of n-BuLi in THF at room
temperature followed by the addition of 1 equiv of benzoyl chlo-
ride, a protocol that furnished the mono-benzoyl derivatives 11
(Scheme 2).16 In those cases where nitrogen deactivation was
exploited, a symmetrical piperazine 10 was complexed with 9-
BBN before the addition of benzoyl chloride, which reacted selec-
tively with the remaining nitrogen atom to afford the target
mono-substituted piperazines 11 (Scheme 2).17
O
O
N
Ph
Ph
5
N
3
6
N
N
2
O
O
When unsymmetrical piperazines were used as substrates, the
lithium dianion typically reacted with benzoyl chloride to generate
a mixture of the two isomers 13 and 14, with low regioselectivity
(Scheme 3, Route A).15 Exceptions were tert-butyl- and 2,6-di-
methyl-piperazine, for which benzoylation under these conditions
occurred exclusively on the less hindered nitrogen. Unsymmetrical
piperazines with alkyl, phenyl, and carboxylic acid-derived substit-
uents could be selectively acyclated on the least hindered nitrogen
atom with benzoyl chloride in CH2Cl2 without added base to afford
R
O
O
N
N
H
H
2: R = F
3: R = OCH3
1
13 initially as the hydrochloride salt (Scheme 3, Route B).18,19
A
Several of the molecules targeted in this survey were prepared
using the protocols outlined in Scheme 1. 4-Fluoro- or 4-meth-
oxy-2-(1H-indol-3-yl)-2-oxoacetyl chloride (4), obtained from the
corresponding acid by treatment with an excess of oxalyl chloride
in Et2O, was coupled with either a mono-benzoyl piperazine deriv-
ative 5 to afford target molecules 6 directly (Scheme 1, Route A) or a
mono-Boc protected piperazine (7) (Scheme 1, Route B) using iPr2-
NEt or Et3N as the base in THF or CH2Cl2.15 In the latter approach
(Route B), the Boc group was removed under acidic conditions, typ-
ically by exposing to excess CF3CO2H acid in CH2Cl2, to provide the
free amine 9 which was then acylated with benzoyl chloride to
afford the final products 6.
However, the use of Route A was dependent on the availability
of mono-benzoylated piperazine derivatives 5 while Route B was
restricted by the limited commercial availability of appropriately
substituted mono-Boc piperazines 7. In order to surmount these
limitations, synthetic methodology was developed that allowed
preparatively convenient strategy that avoided the complication
of a mixture of products was developed for the benzoylation of
some unsymmetrical piperazines 12 in which the more hindered
nitrogen was derivatized selectively.20 Exposure of the lithium
dianion of an unsymmetrical piperazine 12 to triethylsilyl chloride
protected the least hindered N atom in situ, allowing the more
hindered N atom to be selectively acylated by benzoyl chloride
to provide 14 (Scheme 3, Route C).20
Similar synthetic approaches were used to obtain a series of
acyclic diamine derivatives that examined the effect of replacing
the piperazine heterocycle with moieties that either conferred
conformational relaxation or alternative constrained topologies,
compounds (1–9) that are compiled in Table 1. The substituted
piperazines 6a–ah that constitute this survey are compiled in
Table 2.
The antiviral properties of target compounds were assessed in a
pseudotype virus infection system using an engineered virus.4–10,13
A proviral clone of LAI virus in which the env gene is replaced by
the firefly luciferase gene was co-transfected into HEK-293 cells
with a plasmid expressing either a JRFL (CCR5-specific) or LAI
(CXCR-4-specific) virus envelope. After 48 h, the supernatant con-
taining the recombinant pseudovirus was harvested and the titer
determined by performing serial dilutions in HeLa67 cells, which
expresses the primary receptor CD4 and the HIV-1 coreceptors
CXCR4 and CCR5. Virus growth was quantified by measuring lucif-
erase activity (Luciferase Gene Reporter Assay Kit by Roche) 3 days
post-infection. For the analysis of the antiviral activity of test
O
Ph
N
O
R
R
HN
N
Ph
N
Cl
O
O
X
X
O
O
5
iPr2NEt or Et3N,THF
Route A
N
N
H
H
6: X = F, OCH3
PhCOCl
4: X = F, OCH3
R
HN
N
Boc
7
Route B
iPr2NEt or Et3N,THF
9-BBN
N
H
N
H
N
R
iPr2NEt or Et3N,THF
R
R
PhCOCl
9-BBN
Boc
N
THF/r.t.
N
R
H
N
N
H
R
N
H
R
R
R
CF3CO2H/CH2Cl2
O
Ph
N
10
11
N
O
O
X
X
Li
N
O
BuLi (2eq.)
THF/r.t.
O
PhCOCl
R
N
N
H
H
N
R
8: X = F, OCH3
9: X = F, OCH3
Li
Scheme 1.
Scheme 2.

5142
T. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5140–5145
Resolution of the enantiomers by chiral chromatography revealed
Route B
PhCOCl, CH2Cl2
some preference for the (R)-isomer in both series, more pro-
nounced for the 4-fluoro-substituted pair of compounds 6c and
6e. However, progressive homologation of the methyl moiety to
ethyl (6g) and n-propyl (6h) substituents was associated with an
almost linear reduction in potency that fell substantially with the
n-pentyl analogue 6i. The effects of branching were even more det-
rimental, with the iso-propyl derivative 6j determined to be 70-
fold weaker than the ethyl derivative 6g while the addition of a
second methyl group to 6j gave a compound with an EC50 above
R = alkyl, Ph, COOR
Route A
PhCOCl
Li
N
O
Ph
H
N
H
N
BuLi (2eq.)
N
+
R
N
THF, r.t.
N
H
R
N
R
N
H
13
R
Li
R = alkyl
Ph
O
12
14
SiEt3
N
Route C
PhCOCl
5 lM, as examined with the tert-butyl derivative 6l. Introducing
Et3SiCl
branching remote from the piperazine core appeared to be better
tolerated with 6k only 10-fold less potent than 6g. A comparable
survey conducted at C-3 of the piperazine, compounds 6m–6s, pro-
duced qualitatively similar results.
N
R
Li
Scheme 3.
The effect of dual substitution with methyl groups was exam-
ined in the context of three compounds, 6t–6w. The cis-C-2,C-6-di-
methyl derivative 6t exhibits comparable activity toward the LAI
(CXCR4) envelope as the prototype 2 and the C-3 methyl derivative
6m but is several fold weaker than the C-2 methyl derivative 6a.
Interestingly, 6t exhibits reduced potency toward the CCR5-recog-
nizing JRFL virus, indicative of some variation between the enve-
lopes. The complementary topology, in which cis-disposed
dimethyl substitution is deployed at C-3 and C-5 adjacent to the
benzamide nitrogen atom of the piperazine, results in a compound
6u that is 20-fold weaker than the mono-methyl compound 6m to-
ward both viral envelopes. The alternate C-2,C-5-dimethyl substi-
tution afforded a very different pattern of antiviral activity, with
the potency of the trans-disposed isomer 6v markedly better than
that of the cis isomer 6w. In order to gain a deeper understanding
of the effects of methyl substitution, particularly on piperazine ring
conformation, single crystal X-ray structures were determined for
compound 6u and its debenzoylated precursor 9u.21 The solid state
data indicate that the piperazine heterocycle in both molecules
adopts a chair conformation but there are notable differences be-
tween the two compounds with respect to the disposition of the
methyl groups, as depicted in Figure 1. For the unsubstituted NH
derivative 9u, the methyl substituents are equatorial while in the
benzamide 6u they adopt an axial configuration, establishing a
strong 1,3-diaxial repulsive interaction estimated to be ꢀ5.5 kcal/
mol given the similarity between the dimethyl piperazine sub-
structure of compound 6u and 1,3-cis-dimethyl cyclohexane.22
An alteration in configuration from an equatorial to axial disposi-
tion is a common observation for six-membered nitrogen-contain-
ing ring systems when the nitrogen adjacent to the carbon carrying
the substituent is acylated.23,24 This effect originates from the sp2
hybridization of the amide nitrogen25 which constrains the car-
compounds, fourfold serial dilutions of compounds were added to
pseudovirus-infected (containing either JRFL or LAI envelopes)
HeLa67 cells at the time of infection. After 3 days, the extent of
luciferase activity was compared to controls where no compound
was added and the data used to calculate the EC50 values for indi-
vidual compounds. The cytotoxicity of test compounds toward
HeLa67 cells was determined in parallel using an XTT assay per-
formed 3 days after compound addition. The results are presented
in Tables 1 and 2 where individual results are provided as a mea-
sure of assay variability when the data reported are the average
of two experiments.
The survey of potential piperazine replacements examined a
series of simple alkylene linkers, either acyclic or cyclic, that ex-
plored a range of topologies. However, as summarized in Table 1,
of these analogues only the cis-cyclohexane-1,2-diamine derivative
(Table 1, entry 4) provided detectable antiviral activity and this
compound is 10,000-fold weaker than the prototype 2. These re-
sults are indicative of the importance of both the structural rigidity
and precise topology conferred by the piperazine heterocycle, with
the cis-cyclohexane-1,2-diamine presenting the closest approxi-
mation. The complementary survey summarized in Table 2 probes
the effect of substitution of the piperazine ring on antiviral activity
and correlates the structural changes with effects on metabolic sta-
bility in human and rat liver microsomes and permeability across a
monolayer of Caco-2 cells grown to confluence. The introduction of
a methyl group at C-2 of the piperazine ring, designated as the car-
bon atom proximal to the glyoxamide moiety, essentially pre-
served antiviral activity with a trend towards improved potency
in the 4-F indole (6a) compared to the 4-methoxy congener (6b).
Table 1
Piperazine replacements
bonyl group of the amide to be coplanar with both carbons
the nitrogen atom. This leads to significant allylic 1,3 (A1,3
strain26,27 between the amide carbonyl moiety and the substituent
installed at the carbon atom
-to the nitrogen atom.28 In the case
a-to
)
O
O
F
Ph
X
a
of 6u, A1,3 strain is relieved by both methyl groups adopting an ax-
ial configuration, which preserves the topographical relationship
between the benzamide and glyoxamide elements inherent to 2.
However, since potency is eroded by 16-fold for the LAI virus
and over 50-fold for the JRFL virus, these data suggest that the
two axial methyl groups in this topographical relationship to the
amide moieties are poorly tolerated by HIV-1 gp120. This phenom-
enon also provides a potential explanation for the large difference
in antiviral potency observed between compounds 6v and 6w. For
compound 6v, the trans-relationship allows the two methyl groups
to adopt an axial disposition without altering the preferred chair
conformation of the piperazine ring,29,30 leading to the retention
of biological activity, EC50 = 4.72 nM against the LAI subtype
although the JRFL envelope is again more sensitive to structural
changes. However, for the cis-dimethyl derivative 6w, the pipera-
O
N
H
Entry no.
X
EC50
(
l
M) (JRFL)
CC50 (lM)
1
2
3
4
5
6
7
8
–NH–CH2–CH2–NH–
>50 (n = 1)
>50 (n = 1)
>50 (n = 1)
30.5 (25.5, 35.6)
>50 (n = 1)
>50 (n = 1)
>50 (n = 1)
>50 (n = 1)
>300 (n = 1)
>300 (n = 1)
>300 (n = 1)
65 (51, 79)
>300 (n = 1)
>300 (n = 1)
>300 (n = 1)
>300 (n = 1)
–N(CH3)H–CH2–CH2–NH(CH3)–
trans-Cyclohexane-1,2-diamine
cis-Cyclohexane-1,2-diamine
–NH–CH2–CH2–CH2–NH–
–N(CH3)H–CH2–CH2–CH2NH(CH3)–
Cyclohexane-1,3-diamine
1,4-Diazepane (homopiperazine)
9
>50 (n = 1)
>300 (n = 1)
N
N

Table 2
Substituted piperazines
R3
O
R4
O
O
N
Ph
R
N
R2
R1
N
H
Compd no.
R
F
R1
H
R2
H
R3
H
R4
H
EC50 (nM)
CC50
(lM)
HLM% rem at 10 min
98
RLM% rem at 10 min
51
Caco-2 (nm/s)
47
c log P
2
2.9 (LAI)
>300
2.43
2.4 (JRFL)
3
6a
OCH3
F
H
CH3
H
H
H
H
H
H
0.72 (0.87, 0.57) (LAI)
0.56 (0.53, 0.57) (LAI)
0.37 n = 5 (JRFL)
>300 (n = 2)
>300 (n = 2)
100
82
88
12
43
72
2.23
2.95
6b
6c
OCH3
F
CH3
(R)-CH3
H
H
H
H
H
H
1.57 (1.78, 1.37) (LAI)
<0.16 (LAI)
>300 (n = 2)
>300 (n = 2)
84
74
72
2
N/A
54
2.75
2.95
0.42 (0.43, 0.41) (JRFL)
0.93 (0.91, 0.96) (LAI)
0.58 (0.86, 0.30) (JRFL)
1.38 (1.54, 1.23) (LAI)
1.58 (2.13, 1.03) (JRFL)
3.33 (2.67, 3.99) (LAI)
0.58 (0.75, 0.42) (JRFL)
7.10 (5.73, 8.47) (LAI)
15.4 (14.0, 16.9) (LAI)
855 (1316, 394) (LAI)
471 (510, 432) (LAI)
79.2 (104.3, 54.1) (LAI)
>5000 (n = 2) (LAI)
2.60 (3.38, 1.83) (LAI)
2.14 (2.12, 2.16) (JRFL)
5.38 (6.21, 4.54) (LAI)
3.55 (3.55, 3.56) (LAI)
>5000 (n = 2) (LAI)
23.3 (16.7, 30.0) (LAI)
303 (280, 326) (LAI)
>5000 (n = 2) (LAI)
6d
6e
6f
OCH3
F
(R)-CH3
(S)-CH3
(S)-CH3
H
H
H
H
H
H
H
H
H
>300 (n = 3)
>300 (n = 2)
>300 (n = 3)
84
85
98
56
32
75
46
2.75
2.95
2.75
88
OCH3
N/A
6g
6h
6i
6j
6k
6l
F
F
F
F
F
F
F
CH3CH2
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
157 (154, 159)
94 (81, 107)
33 (33, 34)
90 (85, 95)
28 (27, 30)
55
52
29
59
38
68
100
8
11
0
31
7
49
32
150
172
154
165
171
170
36
3.48
4.01
5.06
3.88
4.40
4.27
2.95
CH3CH2CH2
CH3(CH2)4
(CH3)2CH
(CH3)2CHCH2
(CH3)3C
105 (98, 112)
>300 (n = 2)
6m
H
6n
6o
6p
6q
6r
6s
6t
F
F
F
F
F
F
F
H
H
H
H
H
H
CH3
CH3CH2
H
H
H
H
H
H
H
H
H
H
H
H
H
188 (183, 193)
100 (97, 103)
45 (43, 47)
130 (127, 135)
69 (69, 69)
63
50
45
63
35
58
55
25
25
3
39
13
44
2
171
154
69
161
190
190
<15
3.48
4.01
5.06
3.88
4.40
4.27
3.47
CH3CH2CH2
CH3(CH2)4
(CH3)2CH
(CH3)2CHCH2
(CH3)3C
127 (127, 127)
206 (131, 281)
H
cis-CH3
3.3 (0.97, 6.33) (LAI)
23.7 (JRFL)
6u
6v
F
F
H
CH3
H
cis-CH3
H
H
48.4 (18.7, 102.1) (LAI)
125 (80, 169) (JRFL)
4.7 (3.90, 5.53) (LAI)
23.1 (15.4, 27.8) (JRFL)
676 (650, 702) (LAI)
>5000 (n = 2) (LAI)
3400 (2933, 3875) (LAI)
>5000 (n = 2) (LAI)
>500 (n = 2) (LAI)
>5000 (n = 2) (LAI)
>500 (n = 2) (LAI)
>5000 (n = 2) (LAI)
3.1 (2.10, 4.03) (LAI)
>5000 (n = 2) (LAI)
200 53 (n = 4)
102 56 (n = 4)
69
43
76
16
0
73
50
96
3.47
3.47
3.47
CH3
trans-CH3
6w
6x
6y
F
F
F
F
F
F
F
F
F
F
F
CH3
Ph
H
H
H
Ph
H
(S)-PhCH2
H
CONH2
H
cis-CH3
H
H
H
H
F
H
H
H
H
H
H
>300 (n = 2)
28 (27, 30)
109 (103, 116)
47 (46, 47)
>100 (n = 2)
>300 (n = 2)
>300 (n = 2)
207 (191, 223)
>300 (n = 2)
128 (127, 128)
>300 (n = 4)
59
H
H
H
F
H
H
H
H
H
H
6z
(S)-PhCH2
H
CONH2
H
(S)-CO2CH3
H
CO2CH2CH3
CO2H
6aa
6ab
6ac
6ad
6ae
6af
6ag
(S)-CO2CH3
H
H
>5000 (n = 2) (LAI)
836 (398, 1275) (JRFL)
640 (467, 814) (JRFL)
6ah
F
CH2OH
H
H
H
>300 (n = 2)

5144
T. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5140–5145
90.75O
Me
N
Me
Me
NH
Me
87.44O
O
101.45O
O
N
O
F
O
N
O
F
N
H
N
H
6u
9u
Figure 1.
zine ring is unable to adopt a chair conformation that avoids A1,3
strain between one of the methyl groups and either the proximal
benzamide or glyoxylamide carbonyl moiety. This compels the het-
erocyclic ring to distort from the idealized chair conformation in an
attempt to position the two methyl groups in pseudo-axial orien-
tations if it is to effectively preserve amide resonance.31,32 The
resulting deformation of the topographical projections of the
amide moieties is presumably responsible for the large reduction
in potency observed with 6w towards the LAI strain envelope,
EC50 = 676 nM. Reinforcing the importance of the relative disposi-
tion of the two amide moieties, both the homopiperazine and the
2,5-diazabicyclo[2.2.1]heptane derivatives, with the latter con-
strained to a boat conformation, are inactive (Table 1, entries 8
and 9, respectively). Collectively, these data suggest that the chair
conformation of the piperazine ring is essential for optimal HIV-1
attachment inhibition and that there is limited tolerance for sub-
stitution of the piperazine ring, with a single methyl group de-
ployed at C-2 that adopts an axial disposition preserving or
enhancing antiviral activity.33
phoric elements, in a topographical relationship that complements
the binding site of gp120.34,35 An examination of substitution pat-
terns has illuminated fundamental aspects of the SAR and conforma-
tional preferences. The incorporation of a simple methyl substituent
to the piperazine ring proximal to the glyoxamide offers some
advantage and this element was incorporated into BMS-378806
(15), a compound that was evaluated clinically. However, BMS-
378806 (15) failed to achieve targeted plasma exposure levels36
and antiviral efficacy in HIV-1-infected subjects was subsequently
demonstrated with an optimized analogue, BMS-488043 (16).36
Nevertheless, BMS-378806 (15) protected macaque monkeys
against a vaginally administered challenge with simian-human
immunodeficiency virus (SHIV) when administered topically.37
O
N
O
N
Ph
Ph
N
N
O
O
OCH3
OCH3
CH3
O
O
The final aspect of this survey examined a range of larger sub-
stituents that encompassed the potential to establish hydrogen
N
N
H
N
N
bonding and
p–p interactions, probed with compounds 6x–6ah.
H
OCH3
These substituents produced uniformly weak inhibitors of HIV-1
attachment with the exception of the ester 6ae which performed
comparably to the prototype 2.
16
15
References and notes
The effect of these structural modifications on metabolic stabil-
ity and membrane permeability revealed interesting trends with
all compounds examined exhibiting reduced stability in rat liver
microsomes compared to human liver microsomes, a profile inher-
ent to 4-fluoro prototype 2.8 In general, liver microsomal stability
was inversely related to lipophilicity, which increased in concert
with the size of the substituents explored. Not surprisingly, mem-
brane permeability increased in parallel with enhanced lipophilic-
ity, as estimated by c log P values (Table 2), with several
compounds achieving permeability coefficients of >100 nm/s, pre-
dictive of good intestinal permeability based on comparison to
standards. However, none of these compounds provided a combi-
nation of potency, metabolic stability, and membrane permeability
that would qualify for a more detailed examination of the pharma-
cokinetic properties in vivo.
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Crystal data for 9u: colorless prism crystals grown from CH2Cl2/EtOAc are a
mono-hydrate,
Monoclinic,
space
group
P21/c,
a = 8.7886(1) Å,
b = 12.2014(2) Å, c = 15.5672(3) Å, b = 98.449(1), V = 1651.20(5) Å3, Z = 4,
dx = 1.293 g cmÀ3, 14,606 reflections measured, 2739 independent reflections
(Rint = 0.0254), 225 parameters refined with 2246 reflections (I ꢁ 2
r,
R(F2) = 0.0377, wR(F2) = 0.0984, S = 1.017, w = 1/[r2 (F2o ) + (0.050 P)2 + 0.425 P]
where P = (F2o + 2 F2c )/3, À0.155 ꢂ
D
q
ꢂ 0.160 e/Å3. Full crystallographic data
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CCD diffractometer equipped with graphite-monochromated Cu K
(k = 1.54056 Å) or Kappa-CCD diffractometer equipped with graphite-
monochromated Mo K radiation (k = 0.71073 Å). An empirical absorption
correction utilized the SADABS routine (Bruker AXS. 1998, SMART and SAINTPLUS
a radiation
a
a
.
Area Detector Control and Integration Software, Bruker AXS, Madison,
Wisconsin, USA). The unit cell parameters were determined using the entire
data set. The structures were solved by direct methods and refined by the
full-matrix, least-squares techniques, using the SHELXTL software package
(Sheldrick, GM. 1997, SHELXTL. Structure Determination Programs. Version
5.10, Bruker AXS, Madison, Wisconsin, USA.). Non-hydrogen atoms were
refined with anisotropic thermal displacement parameters. Hydrogen atoms
involved in hydrogen bonding were located in the final difference Fourier
maps, while the positions of the other hydrogen atoms were calculated from an
idealized geometry with standard bond lengths and angles. They were assigned
isotropic temperature factors and included in structure factor calculations with
fixed parameters. Crystal data for 6u: colorless plate crystals grown from ethyl
acetate are
a hemi-EtOAc solvate, Orthorhombic, space group P212121,
a = 6.3659(1) Å,
b = 16.7942(4) Å,
c = 21.7178(6) Å,
a = b = c = 90°,
V = 2321.85(9) Å3, Z = 4, dx = 1.292 g cmÀ3, 12936 reflections measured, 3988
independent reflections (Rint = 0.0297), 273 parameters refined with 3197
reflections (I ꢁ 2
r
,
R(F2) = 0.0496, wR(F2) = 0.1398, S = 1.071, w = 1/[r2
(F2o ) + (0.094 P)2 + 0.088 P] where P = (Fo2 + 2 F2c )/3, À0.161 ꢂ
D
q
ꢂ 0.148 e/Å3.