Inhibitors of HIV-1 attachment. Part 2: An initial survey of indole substitution patterns

Article abstract of DOI:10.1016/j.bmcl.2009.02.040

The effects of introducing simple halogen, alkyl, and alkoxy substituents to the 4, 5, 6 and 7 positions of 1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione, an inhibitor of the interaction between HIV gp120 and host cell CD4 receptors, on activity in an HIV entry assay was examined. Small substituents at C-4 generally resulted in increased potency whilst substitution at C-7 was readily tolerated and uniformly produced more potent HIV entry inhibitors. Substituents deployed at C-6 and, particularly, C-5 generally produced a modest to marked weakening of potency compared to the prototype. Small alkyl substituents at N-1 exerted minimal effect on activity whilst increasing the size of the alkyl moiety led to progressively reduced inhibitory properties. These studies establish a basic understanding of the indole element of the HIV attachment inhibitor pharmacophore.

Full text of DOI:10.1016/j.bmcl.2009.02.040

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1977–1981
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of HIV-1 attachment. Part 2: An initial survey of indole
substitution patterns q
a
a
a
a
a
Nicholas A. Meanwell a, , Owen B. Wallace , Haiquan Fang , Henry Wang , Milind Deshpande , Tao Wang ,
Zhiwei Yin a, Zhongxing Zhang a, Bradley C. Pearce a, Jennifer James a, Kap-Sun Yeung a, Zhilei Qiu a,
J. J. Kim Wright a, Zheng Yang a, Lisa Zadjura b, Donald L. Tweedie b, Suresh Yeola b, Fang Zhao c,
Sunanda Ranadive c, Brett A. Robinson d, Yi-Fei Gong d, Hwei-Gene Heidi Wang d, Wade S. Blair d,
Pei-Yong Shi d, Richard J. Colonno d, Pin-fang Lin d
*
a Department of Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
b Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492 and Princeton, NJ 08543,
United States
c Department of Pharmaceutics, Bristol-Myers Squibb Research and Development, One Squibb Drive, New Brunswick, NJ 08903, United States
d 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:
The effects of introducing simple halogen, alkyl, and alkoxy substituents to the 4, 5, 6 and 7 positions of 1-
(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione, an inhibitor of the interaction between
HIV gp120 and host cell CD4 receptors, on activity in an HIV entry assay was examined. Small substitu-
ents at C-4 generally resulted in increased potency whilst substitution at C-7 was readily tolerated and
uniformly produced more potent HIV entry inhibitors. Substituents deployed at C-6 and, particularly,
C-5 generally produced a modest to marked weakening of potency compared to the prototype. Small
alkyl substituents at N-1 exerted minimal effect on activity whilst increasing the size of the alkyl moiety
led to progressively reduced inhibitory properties. These studies establish a basic understanding of the
indole element of the HIV attachment inhibitor pharmacophore.
Received 24 December 2008
Revised 8 February 2009
Accepted 10 February 2009
Available online 13 February 2009
Keywords:
HIV-1 attachment inhibitor
HIV-1 entry inhibitor
HIV-1 gp120 inhibitor
HIV-1 inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
The interaction of the human immunodeficiency virus-1 (HIV-
1) surface glycoprotein gp120 with CD4, a glycoprotein receptor
expressed on mammalian cells, is the critical first step of a series
of several carefully choreographed events that allows virus access
to host cells.2,3 The binding of gp120 to CD4 induces a conforma-
tional change in the virus glycoprotein that exposes binding sites
recognized by coreceptors expressed on the surface of host cells,
either CCR5 or CXCR4, depending on virus tropism. The engage-
ment of coreceptors by gp120 leads to its dissociation from gp41,
the virus membrane-spanning protein that mediates the fusion of
the virus with the host cell. We have recently described the discov-
ery of indole-3-glyoxamide derivatives as the first small molecule
inhibitors of the gp120-CD4 interaction (HIV-1 attachment inhibi-
tion) that demonstrate potent antiviral activity in cell culture.1,4–7
These compounds, of which the 4-fluoro derivative 1b is prototyp-
ical, appear to act by stabilizing a specific conformation of gp120
that is poorly recognized by CD4.4–7 However, under certain cir-
cumstances, compounds of this class have been shown to form a
ternary complex with gp-120 and CD4 and interfere with the
CD4-induced exposure of the gp41 heptad repeats, providing a po-
tential additional mode of action.8,9 Resistance to these attachment
inhibitors has been shown to map to a highly conserved, recessed
binding pocket of gp120 that recognizes Phe43 of CD4.4–7,10,11
Structural optimization to address deficiencies in the physical
chemical profile of 1b afforded BMS-378806 (2) and BMS-488043
(3), a compound that has demonstrated antiviral activity in HIV-
1-infected patients following oral administration.1,6,12 In this Let-
ter, we elaborate the basic structure–activity relationships associ-
ated with the indole ring that identify the preferred patterns of
substitution for this element of the pharmacophore that ultimately
provided a foundation for the design of BMS-488043 (3).
The target compounds were synthesized by acylating
a
substituted indole with oxalyl chloride followed by coupling of
the acid chloride with 1-benzoylpiperazine, a process depicted in
Scheme 1.13 For indole derivatives substituted with electron with-
drawing elements an alternative process was employed that com-
prised reaction of the indole with methyl or ethyl chlorooxoacetate
in the presence of a Lewis acid, typically AlCl3,14 followed by alka-
line hydrolysis of the ester moiety and coupling with 1-benzoyl-
piperazine. Alkylation of the indole N atom was accomplished by
q
See Ref. 1.
* Corresponding author.
E-mail address: nicholas.meanwell@bms.com (N.A. Meanwell).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.040

1978
N. A. Meanwell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1977–1981
O
O
O
O
N
Ph
O
O
N
Ph
N
Ph
OCH3
OCH3
F
OCH3
N
N
N
N
O
O
O
CH3
N
N
H
N
H
N
H
1b
2
3
heating with an alkyl halide in DMF at 80 °C in the presence of a
slight excess of the phosphazene base 2-tert-butylimino-2-diethyl-
providing a consistent series with which to systematically probe
the effects of different electronic and hydrophobic elements. The
results for this cluster of compounds, 1b,c,h for C-4, 1k,l,o for C-
5, 1s,t,u for C-6 and 1w,x,ab for C-7, reveal a pattern of struc-
ture-activity correlates in which these substituents when installed
at C-4 and C-7 lead to enhanced potency compared to the parent
1a. However, these substituents produce markedly less active
HIV-1 attachment inhibitors when deployed at C-5 and generally
weaker inhibitors when installed at C-6. The tolerance for substitu-
tion at C-5 is particularly poor, as exemplified by the micromolar
EC50s recorded for 1k, 1l, and 1o, and confirmed by the data asso-
ciated with the additional 5 examples 1m, 1n, and 1p–r. At C-6, a
fluorine atom improves the potency of the prototype by sevenfold
(1s) but replacement by the larger chlorine (1t) and methoxy sub-
stituents (1u) leads to an erosion of antiviral activity that corre-
lates with the size of the substituent.16 However, the single
additional C-6-substituted analog evaluated, the CF3 derivative
1v, exhibits markedly weaker activity than would be anticipated
based simply on steric dimensions, suggestive of the contribution
of an additional electronic component.
The introduction of a halogen at C-4 produced a homologous
series of potent HIV-1 attachment inhibitors 1b–d that offer a
30–60-fold advantage over 1a, with virus entry inhibitory activity
largely independent of the identity of the substituent. A methoxy
(1h) or ethoxy (1i) substituent at C-4 provides a higher level of po-
tency but the isopropoxy analog 1j is over three orders of magni-
tude weaker. This observation indicates high sensitivity to steric
effects in this region of the pharmacophore and can be attributed
to branching since non-branched alkoxy substituents at C-4 have
been shown to exert a less severe negative impact on potency.17
The same principle may underlie the poor entry inhibition ob-
served for the acetoxy derivative 1f, which is over 160-fold weaker
than 1a and almost 50,000-fold weaker than 1h. However, under
the conditions of the assay, this compound may suffer cleavage
to the simple C-4 hydroxy compound 1g which is equally ineffec-
tive, perhaps pointing to a problem with the presence of polarity at
this site. The final compound prepared in this series, the nitro
derivative 1e, exhibits potency comparable to the prototype 1a.17
The introduction of substituents at C-7 in all cases examined
enhanced the potency of the prototype molecule 1a, with alkoxy
amino-1,3-dimethyl-perhydro-1,3,2-diazaphosphorine
(BEMP).
The starting indoles were procured by methods described in the
literature and the compounds surveyed are compiled in Table 1.
The antiviral activity of target compounds was determined by
evaluation in a single-cycle viral infection system using luciferase
activity as the endpoint.4,15 To generate pseudotype virus, pJRFL-
LucDEnv, which contains full length HIV-1JRFL in which the HIV-1
envelope sequences are replaced by the firefly luciferase gene se-
quence, and a plasmid expressing the HIV-1JRFL envelope sequence
were used to transfect HEK-293 cells and incubated for 48 hours.
The titer of pseudovirus was determined by infecting HeLa67 cells
expressing the primary HIV-1 receptor CD4 and the coreceptor
CCR5 with a serial dilution of virus stock and quantifying luciferase
activity (Luciferase Gene Reporter Assay Kit by Roche) on day 3
after infection. To assess the antiviral activity against the pseudo-
virus, fourfold serial dilutions of compounds were prepared in
DMSO and applied to a luminescence plate containing pseudovirus
and HeLa CD4/CCR5 cells. After three days, the resulting luciferase
activity data were used to estimate the EC50 values of individual
compounds in preventing pseudovirus infection. The cytotoxicity
of compounds toward HeLa67 cells was determined in parallel
and an XTT assay performed 3 days after compound addition.
Where the data reported are the average of only 2 experiments,
the individual results are provided as a measure of assay variabil-
ity. In this assay, 1b half-maximally inhibited infection by the
pseudovirus at a concentration of 2.59 nM.
The prototype compound of the series, indole 1a (Table 1), was
discovered as an HIV-1 attachment inhibitor using a cell-based
screening assay designed to query the Bristol-Myers Squibb pro-
prietary collection.1,4–7,15 This compound potently inhibits the en-
try of pseudotype virus and expresses antiviral activity towards a
representative panel of HIV-1 strains in cell culture.1 The initial ef-
fort to study this new chemotype focused on surveying the effect of
introducing substituents to the indole ring with a view to increas-
ing potency and establishing a basic understanding of this aspect of
the pharmacophore, data that is presented in Table 1. Three sub-
stituents, fluorine, chlorine and a methoxy moiety, were intro-
duced individually at each of the positions of the aromatic ring,
O
O
Ph
Ph
N
N
O
O
O
R'
N
N
R
R
R
R
a
b
c
O
O
O
N
H
N
H
N
N
H
R'
1aab-1aaf
1a-1aaa
Scheme 1. Reagents and conditions: (a) oxalyl chloride/THF or Et2O for R0 = Cl; or EtOꢀCOꢀCOꢀCl or MeOꢀCOꢀCOꢀCl/AlCl3/CH2Cl2 for R0 = OEt or OMe when R is electron
withdrawing; (b) N-benzoylpiperazine/iPr2NEt/THF for R0 = Cl or (i) NaOH or K2CO3/MeOH; (ii) N-benzoylpiperazine/DEPBT/iPr2NEt/DMF for R0 = OEt or OMe; (c) R0-X/BEMP/
dry DMF/80 °C.

N. A. Meanwell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1977–1981
1979
Table 1
Structure, HIV pseudotype virus inhibitory activity and cytotoxicity associated with indole glyoxamide derivatives
O
N
O
Ph
R1
R4
N
R2
R3
O
N
R5
R1
R2
R3
R4
R5
EC50 (nM)
CC50 (lM)
a
Compd #
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
1s
1t
1u
1v
1w
1x
1y
1z
1aa
1ab
1ac
1ad
1ae
1af
1ag
1ah
1ai
1aj
1ak
1al
1am
1an
1ao
1ap
1aq
1ar
1as
1at
1au
1av
1aw
1ax
1ay
1az
1aaa
1aab
1aac
1aad
1aae
1aaf
H
F
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
Cl
OCH3
CF3
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
152.97 119.0 (n = 21)
2.59 2.46 (n = 17)
4.3 4.2 (n = 6)
338.8 50.7 (n = 11)
>300 (n = 7)
212 67 (n = 4)
142 (n = 1)
>300 (n = 2)
>300 (n = 2)
>300 (n = 2)
>300 (n = 2)
>94.8 (>94.8, 153.4)
150.2 (135.2, 165.2)
>300 (n = 2)
46 (44, 47)
>149 (149.2, >300)
>300 (n = 2)
>300 (n = 2)
44.6 (17.4, 71.9)
>300 (n = 2)
95.2 (70.7, 119.6)
>245 (245.9, >300)
>110 (110.1, >300)
>300 (n = 2)
54.5 (74.0, 35.0)
>152 (152.2, >300)
162.0 (n = 1)
4.5 (7.1, 1.8)
NO2
149.8 (177.9, 121.6)
24,857.5 (30,068.4, 19,646.6)
20,137.4 (20,477.4, 19,7973)
0.52 (0.62, 0.41)
0.45 (0.62, 0.27)
>500 (n = 2)
OCOꢀCH3
OH
OCH3
OCH2CH3
H
H
F
OiPr
H
838.3 (1039.6, 646.9)
395 (572, 218)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
F
OCH3
OCH3
OCH3
OCH3
OCF3
F
Cl
Br
CH3
OCH3
NO2
CN
OCOꢀCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
F
Cl
F
OCH3
F
H
H
H
H
H
H
H
H
H
H
H
H
H
1090.3 (1603.5, 577.1)
1548.6 (1648.2, 1449.0)
21,100 (27,000, 15200)
3,800 (5900, 1700)
120,523.1 (156,613.0, 84,433.1)
952.2 (892.5, 1011.8)
21.1 (34.7, 7.4)
208 (377, 38)
328.8 (418.5, 239.0)
>5000 (n = 2)
7.3 (7.7, 6.9)
4.4 (4.2, 4.5)
17.4 (n = 1)
89.5 48.7 (n = 3)
24.2 23.9 (n = 3)
6.6 2.2 (n = 4)
0.5 (0.5, 0.6)
0.14 (0.09, 0.2)
Cl
Br
CH3
CH3CH2
OCH3
OCH2CH3
OC4H9
CN
H
H
H
H
H
Br
F
OCH3
Cl
Br
CN
Br
Br
CH3
OCH3
OCH2CF3
CN
F
F
CH3
H
F
H
H
H
H
H
64.0 16.3 (n = 3)
>218 (n = 1)
>49 (49.2, >300)
153.6 22.5 (n = 4)
117.4 (163.6, 71.1)
64.5 (48.8, 42.1)
234.3 (184.4, 284.3)
20.9 (28.9, 12.9)
170.4 (112.1, 228.7)
52.3 (49.6, 55.0)
98.5 (145.7, 51.2)
>300 (n = 2)
4.9 (3.7, 6.1)
23.1 (17.3, 28.9)
>290 (292.9, >500)
500 (500, 500)b
F
Cl
Cl
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
>500
46,451.2 (43,771.5, 49,130.8)
73.1 (110.4, 35.8)
0.35 0.24 (n = 8)
0.23 0.33 (n = 7)
0.07 0.05 (n = 4)
0.13 (n = 1)
0.06 (0.06, 0.06)
42.8 (28.1, 57.4)
0.13 0.07 (n = 4)
0.56 0.15 (n = 4)
0.06 (0.008, 0.111)
0.71 (n = 1)
1.9 (3.0, 0.8)
8.2 (10.2, 6.1)
>500 (n = 2)
>500 (n = 2)
0.42 0.04 (n = 3)
1.7 (1.5, 1.9)
265 (310, 220)
1450 (1900, 1000)
2650 (2400, 2900)
6760 (7720, 5800)
13750 (22300, 5200)
22.5 (22.7, 22.3)
>300 (n = 8)
279.0 (277.3, 280.6)
>300 (n = 1)
118 (n = 1)
>275 (275.4, >300)
75.7 (92.6, 58.8)
39.8 29.9 (n = 4)
87.5 19.3 (n = 3)
>300 (n = 2)
196.1 26.9 (n = 3)
>300 (n = 2)
86.0 (98.1, 73.9)
>300 (n = 2)
F
F
F
F
Br
CN
CH3
F
F
H
H
H
F
H
H
H
H
H
201.6 (109.7, 293.5)
>188 (188.3, >300, >244.0)
55.7 (n = 1)
F
H
H
H
H
H
CH3
CH3CH2
CH3(CH2)3
CH2=CH-CH2
PhCH2
226 (300, 152)
H
H
H
H
>300 (>300, >300)
>300 (>300, >300)
201.5 (195, 208)
182 (137, 227)
a
Data are the means of two or more experiments with individual data provided for those experiments conducted twice.
LAI envelope used rather than JRFL.
b

1980
N. A. Meanwell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1977–1981
(1ab–ad) superior to alkyl (1z and 1aa) and both demonstrating a
trend in which potency increases with the size of the substituent.
With a basic understanding of the SAR associated with substitu-
tion at each of the 4 sites established, it became of considerable
interest to examine the effect of combinations, results that are cap-
tured by compounds 1af–1aaa in Table 1. Dual substitution at C-5
and C-6 (1ag–1aj) produced compounds with unimpressive biolog-
ical activity, reflecting an additive combination of the effects of the
individual substituents. However, the poor potency associated
with this pattern could be mitigated quite considerably by the
introduction of a fluorine atom at C-4, as exemplified by 1af, fur-
ther underscoring the marked positive effect observed with this
substituent in the context of the parent molecule. This property ex-
tends to the C-4, C-6 difluoro analog 1az and the tetra-fluoro deriv-
ative 1aaa, both of which are more potent than might be
anticipated based on the data for the individually substituted com-
pounds. The single C-5, C-7 combination studied, the fluoro, bromo
derivative 1ak, performed essentially as expected based on a com-
posite of the individual components. It was anticipated that com-
binations of C-4 and C-7 substituents would be particularly
productive, an aspect of SAR probed with compounds 1am–1aaa
and most effectively realized with analogs 1am–1ap and 1ar–
1au. The most impressive representatives of this substitution pat-
tern are 1an, 1ap and 1at, all of which demonstrate EC50s in the
pseudotype virus assay of 60–70 picomolar. The results with 1ax
and 1ay provide some insight into what may be expected of either
a C-4 CN or CH3 substituent, compounds not explicitly investigated
but anticipated to show poor antiviral activity.
ginally reduced clearance and lower oral bioavailability, data that
were predicted by rat liver microsomal studies where the rate of
metabolic degradation predicted clearance in rats of 52 mL/min/
kg, close to the observed figure reported in Table 2. However, com-
pound 1b is considerably more stable in human liver microsomes
with clearance predicted to be 8 mL/min/kg, a figure that places
this compound in the intermediate range of clearance. The 4,7-
difluoro derivative 1al performed similarly to 1b in the rat, with
high clearance also predicted well by rat liver microsomal stability
data. For all three of these compounds, the terminal half life fol-
lowing oral dosing was longer than that measured after intrave-
nous (IV) administration, indicative of an absorption rate that is
slower than the rate of elimination. This appears to be a function
of the dissolution properties of the drug in the gut since all three
compounds readily permeate a confluent Caco-2 cell layer with
rates of 228, 100 and 219 nm/s for 1a, 1b and 1al, respectively, data
consistent with high membrane permeability based on comparison
to standard agents. This was supported by subsequent studies with
1b in higher species, where bioavailability was complete after oral
dosing of a solution of the drug in poly(ethylene glycol) 400 (PEG
400) and ethanol (90:10 v/v) to cynomolgus monkeys and dogs.1
The 4,7-dimethoxy derivative 1am offered superior pharmacoki-
netic properties in the rat, with low clearance (5.4 mL/min/kg),
an observation predicted by good in vitro rat liver microsomal sta-
bility, and excellent absorption, anticipated based on the high
Caco-2 cell permeability of 204 nm/s. Taken together, these prop-
erties predict the complete bioavailability of this compound that
is observed in the rat. Whilst the potency of 1am represents a
10-fold improvement compared to 1b, further profiling of this
compound was not pursued after in vitro studies with human liver
microsomal preparations revealed that demethylation of each of
the methoxy moieties were prevalent metabolic pathways, arous-
ing concern for the potential of para-quinone formation in vivo
should further oxidative metabolism of this electron-rich ring sys-
tem occur. Quinones are chemically reactive electrophiles that
have been shown to form covalent adducts with proteins and
nucleotides and are associated with a number of toxicological ef-
fects.18–20 Indoloquinones demonstrate a range of biological prop-
erties,21 including cytotoxic acitvity in vitro, and metabolic
The effect of alkylating the indole N atom in the context of the
parent molecule 1a, surveyed with 1aab–1aaf, indicates that only
a CH3 substituent (1aab) preserves significant HIV inhibitory activ-
ity with potency progressively eroded as the size of the alkyl group
increases within this short series.17
The pharmacokinetic properties of compounds 1a, 1b, 1al and
1am were evaluated in the rat, data that is summarized in Table
2. The parent compound 1a, selected to provide a basic under-
standing of the chemotype, demonstrated high clearance in the
rat after intravenous administration and modest oral bioavailabil-
ity of 29% after oral dosing. The 4-fluoro analog 1b exhibited mar-
Table 2
Pharmacokinetic parameters of compounds 1a, 1b, 1al and 1am in the rat
1a
1b
1al
1am
Rat IV: dosea
CL (mL/min/kg)
Vss (L/kg)
Terminal T1/2 (min)
MRT (min)
5 mpk (n = 3)
57 11
1.5 0.3
30 12
5 mpk (n = 3)
1 mpk (n = 2)
46.9 (44.5/49.2)
0.85 (0.9/0.79)
17 (20/14)
1 mpk (n = 2)
5.2 (5.4/5.1)
0.52 (.39/0.65)
138 (120/156)
102 (72/126)
48
6
0.99 0.08
28
21
7
3
26
4
19 (23/16)
Rat PO: dosea
Cmax (ng/mL)
Tmax (min)
Terminal T1/2 (min)
F (%)
25 mpk, n = 3
1037 871
33 12
163 131
29 15
25 mpk, n = 3
649 324
53 59
5 mpk, n = 2
55 (80/30)
20 (10/30)
551 (684/416)
11 (8.3/13)
5 mpk, n = 2
3200 (3100/3400)
180 (240/120)
111 (108/114)
107 (113/102)
72 20
17
9
a
Dosed as solutions in PEG400/EtOH (90:10).
Table 3
Pharmacokinetic parameters for different formulations of compound 1b in the rat and dog
Entry #
Species
Dose
Formulation
Cmax (ng/mL)
Tmax (min)
F (%)
1
2
3
4
5
6
Rat
Rat
Rat
Dog
Dog
Dog
5 mpk (n = 3)
25 mpk (n = 3)
5 mpk (n = 3)
1 mpk (n = 3)
1 mpk (n = 3
1 mpk (n = 3)
PEG 400/EtOH (90:10)
PEG 400/EtOH (90:10)
Nanosuspension (184 nm)
PEG 400/EtOH (90:10)
Micronized suspension (46
Nanosuspension (184 nm)
57 13
649 324
37 46
53 59
170 165
72 17
9
17
4
89 14
12
36
2
9
0.2
18
7
560 84
60 26
160 32
lm)
240
0
6
6
108 17

N. A. Meanwell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1977–1981
1981
activation of 1am in vivo may lead to toxicity, possibly idiosyn-
cratic in nature.22–24
bility that would demonstrate enhanced dissolution properties, the
results of which will be communicated in due course.
Consequently, from these four compounds attention was fo-
cused on the 4-fluoro analog 1b, a compound that demonstrated a
sufficiently promising antiviral profile towards a panel of HIV-1
viruses replicating in cell culture to be a useful vehicle for a more de-
tailed evaluation. In probing 1b in greater depth, the objective was
to develop a greater appreciation for the potential of the chemotype
and to identify any issues that might interfere with a drug develop-
ment campaign. Whilst the pharmacokinetic profile of 1b in higher
species was quite encouraging following oral administration as a
solution in PEG 400/ethanol and increases in exposure were ob-
served with increased dosage in rats, problems were encountered
when the compound was dosed to rats as a suspension in water con-
taining 0.75% w/w of Methocel 4AM and 0.5% w/w of Pluronic F68.1
The results of experiments conducted with different formulations in
rats and dogs are summarized in Table 3. After oral administration
of a solution of 1b to rats at 5 mpk, a Cmax of 57 ng/mL was observed
at 37 min after dosing and bioavailability was 9% (Table 3, entry 1).
Increasing the dose to 25 mpk as a solution resulted in an 11-fold in-
crease in Cmax with a Tmax delayed to 53 min and an increase in bio-
Acknowledgement
We thank Beata Nowicka-Sans for generating the antiviral data
for compound 1ah.
References and notes
1. For Part 1 of this series see: Wang, T.; Zhang, Z.; Wallace, O. B.; Deshpande, M.;
Fang, H.; Yang, Z.; Zadjura, L. M.; Tweedie, D. L.; Huang, S.; Zhao, F.; Ranadive,
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H.-W. H.; Rose, R.; Yamanaka, G.; Robinson, B.; Li, C.-B.; Fridell, R.; Deminie, C.;
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L.; Biorn, A. C.; Cocklin, S.; Chaiken, I.; Freire, E.; Smith, A. B., III; Sodroski, J. G.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5036.
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availability to 17% (Table 3, entry 2). However, dosing
a
nanosuspension in water gave a Cmax that was only 31% of that of
the solution formulation and the significant increase in Tmax is indic-
ative of slowed absorption (Table 3, entry 4). An analogous series of
experiments in the dog revealed a similar pattern of exposure with
the Cmax after a micronized suspension ꢁ10% of the solution formu-
lation and the Tmax markedly extended (Table 3, entries 5 and 6). A
nanosuspension formulation of 1b increased the Cmax almost three-
fold relative to the micronized form whilst reducing Tmax. The rela-
tive oral bioavailabilities of the micronized suspension and the
nanosuspension compared to the solution formulation were 13%
and 40%, respectively. These findings were reflected in the physical
properties of 1b which exhibited a melting point of 236 °C and sol-
ubility ranging from 5 ng/mL in water to 4.6 mg/mL in PEG 400. Ta-
ken together, these data indicated that the poor performance in vivo
following oral administration of suspension formulations could be
attributed to poor dissolution in the gut, a problem only partially
addressed by reducing particle size.
In summary, structure–activity relationships associated with
this series of indole-based HIV-1 attachment inhibitors provide
fundamental insights into the optimal indole substitution patterns
and compounds with increased potency were readily obtained by
the introduction of relatively simple substituents at C-4 and C-7.
Whilst representative compounds demonstrated good pharmaco-
kinetic properties in the rat and dog following oral administration
as solutions in PEG-400/ethanol, the physical properties associated
with the chemotype resulted in poor performance in vivo following
dosing of aqueous suspensions. As a consequence, attention was
directed towards identifying analogs with improved intrinsic solu-