Design, synthesis, anti-HIV activities, and metabolic stabilities of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jm049916x

The alkenyldiarylmethane (ADAM) HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) are effective anti-HIV agents in cell culture. However, the potential clinical utility of the ADAMs is expected to be limited by the presence of methyl ester mo

Full text of DOI:10.1021/jm049916x

J . Med. Chem. 2004, 47, 3149-3162
3149
Design , Syn th esis, An ti-HIV Activities, a n d Meta bolic Sta bilities of
Alk en yld ia r ylm eth a n e (ADAM) Non -n u cleosid e Rever se Tr a n scr ip ta se
In h ibitor s
Maximilian A. Silvestri,† Muthukaman Nagarajan,† Erik De Clercq,‡ Christophe Pannecouque,‡ and
Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences,
Purdue University, West Lafayette, Indiana 47907, and Rega Institute for Medical Research, Katholieke Universiteit Leuven,
B-3000 Leuven, Belgium
Received J anuary 29, 2004
The alkenyldiarylmethane (ADAM) HIV-1 non-nucleoside reverse transcriptase inhibitors
(NNRTIs) are effective anti-HIV agents in cell culture. However, the potential clinical utility
of the ADAMs is expected to be limited by the presence of methyl ester moieties that are likely
to be metabolized by nonspecific esterases in blood plasma to biologically inactive carboxylic
acid derivatives. The present investigation was therefore undertaken to investigate the anti-
HIV activities of the ADAMs versus HIV-1IIIB and HIV-2ROD in MT-4 cells and the stabilities
of the biologically active ADAMs in rat plasma. The ADAMs displayed a wide range of metabolic
stabilities in rat plasma, with half-lives ranging from 0.9 to 76.6 min. A wide assortment of
structural modifications was tolerated, with 18 of the 32 compounds tested displaying EC50
values between 0.3 and 3.7 µM versus HIV-1IIIB in MT-4 cells, 3 compounds in the EC50
)
13.2-35.4 µM range, and the remaining compounds inactive. Consistent with the mechanism
of action of the ADAMs as NNRTIs, they were inactive or displayed comparatively low activity
versus HIV-2ROD. The replacement of the two aromatic methyl ester substituents in one of the
most active ADAMs (EC50 ) 0.6 µM) with two methyl thioester groups resulted in an increase
in plasma half-life from 5.8 to 55.3 min, while maintaining the antiviral potency at the EC50
) 1.8 µM level. At the same time, the bis(thioester) modification was less cytotoxic to uninfected
MT-4 cells, with a CC50 of >224 µM versus 160 µM for the parent compound.
In tr od u ction
The alkenyldiarylmethanes (ADAMs) are a novel
class of NNRTIs that was discovered a decade ago.18-26
The interest in the ADAMs in general has resulted from
their anti-HIV activities against AZT-resistant strains
of HIV-1. In addition, certain ADAMs have displayed
synergistic activity with AZT and have shown enhanced
activity when tested against AZT-resistant strains of
HIV-1.19,20,22,23 Although the ADAMs are effective in-
hibitors of the cytopathic effect of HIV-1 in vitro, their
potential therapeutic usefulness is likely to be compro-
mised by their expected metabolic instability. More
specifically, the ester moieties in the ADAM pharma-
cophore are likely to be hydrolyzed by nonspecific
esterases present in human blood plasma. The present
investigation was therefore undertaken to determine the
metabolic stabilities of biologically active ADAMs in
blood plasma, with the goal of finding ADAMs that
retain antiviral activity and have enhanced metabolic
stabilities. In this paper, we report the anti-HIV activi-
ties of some previously existing and newly synthesized
ADAMs versus HIV-1IIIB in MT-4 cells, as well as the
stabilities of the biologically active compounds in rat
blood plasma.
Reverse transcriptase (RT) provides essential enzy-
matic activity for the human immunodeficiency virus,
the active pathogen for acquired immunodeficiency
syndrome (AIDS).1 Thus, the enzyme has been identified
to be a target for the therapeutic treatment of AIDS.2-4
The non-nucleoside reverse transcriptase inhibitors
(NNRTIs) are a structurally diverse class of compounds
that bind to RT and act as allosteric inhibitors.5,6 The
NNRTIs bind near the substrate binding site of RT and
induce a conformational change that results in reduced
enzymatic activity.7 Three NNRTIs (nevirapine, delavir-
dine, and efavirenz) have been approved for the treat-
ment of AIDS.8-11 Although the therapeutic use of the
NNRTIs is complicated by the rapid development of
viral resistance and drug toxicity, they have neverthe-
less proven to be useful in combination therapy with
nucleoside reverse transcriptase inhibitors and protease
inhibitors.12 Employment of NNRTIs with these agents
has resulted in decreased HIV-1 RNA levels, delayed
disease progression, and increased CD4 lymphocyte
counts. However, adverse side effects, cross-resistance,
and drug incompatibilities limit the use of NNRTIs for
combination therapy.13-17 Therefore, new NNRTIs are
needed that do not suffer from these restrictions.
Ch em istr y
In general, the ADAMs having identically substituted
aromatic rings have been synthesized by McMurry
reactions of aldehydes or ketones with symmetrical
benzophenones in the presence of low-valent titanium
species.22,23 Alternatively, in other cases, Wittig ap-
* Author to whom correspondence should be addressed [e-mail
cushman@pharmacy.purdue.edu; telephone (765) 494-1465; fax (765)
494-6790].
† Purdue University.
‡ Katholieke Universiteit Leuven.
10.1021/jm049916x CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/12/2004

3150 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Silvestri et al.
Sch em e 1a
proaches have been used to generate the alkene moi-
ety.19,20 Alkenyldiarylmethanes 1-3,20,22,23 5-19,23,26
and 3025 were synthesized as previously reported.
a
Reagents and conditions: (a) CuCN, DMF, reflux 22 h.
Sch em e 2a
a
Reagents and conditions: (a) (1) DIBAL-H, CH2Cl2, -78 °C,
2 h; (b) (1) TiCl4‚2THF, Zn0, THF, reflux 3 h, (2) 35, THF, reflux
3.5 h.
Sch em e 3a
a
Reagents and conditions: (a) 2,4-bis(4-methoxyphenyl)-1,3-
dithia-2,4-diphosphetane-2,4-disulfide, toluene, reflux, 96 h.
Alkenyldiarylmethane 4 was prepared from alkenyl-
diarylmethane 1 as outlined in Scheme 1. Treatment
of ADAM 122 with cuprous cyanide in N,N-dimethylfor-
mamide yielded ADAM 4.
Reduction of methyl 5-methoxypentanoate (34) to the
corresponding aldehyde (Scheme 2) with diisobutylalu-
minum hydride in dichloromethane and subsequent
McMurry coupling with benzophenone 35 yielded al-
kenyldiarylmethane 20. Methyl 5-methoxypentanoate
(34)27 and benzophenone 3519 were synthesized as
previously described.
The reaction of alkenyldiarylmethanes 36 (Scheme 3)
and 2 (Scheme 4) with 2,4-bis(4-methoxyphenyl)-1,3-
dithia-2,4-diphosphetane-2,4-disulfide (Lawesson’s re-
agent) in either refluxing toluene or xylenes afforded
the corresponding thiono esters 21 and 22.28
As shown in Scheme 5, hydrolysis of benzophenone
37 in methanol with potassium hydroxide as the base
yielded 3,3′-dichloro-4,4′-dimethoxy-5,5′-dicarboxyben-
zophenone (38). Treatment of 38 with thionyl chloride

ADAM Non-nucleoside Reverse Transcriptase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3151
Sch em e 4a
Sch em e 7a
a
Reagents and conditions: (a) 2,4-bis(4-methoxyphenyl)-1,3-
dithia-2,4-diphosphetane-2,4-disulfide, xylenes, reflux 48 h.
Sch em e 5a
a
Reagents and conditions: (a) DIBAL-H, CH2Cl2, -78 °C, 3 h;
(b) CH3I, NaH, THF, 0 °C-ambient temperature, 24 h.
Sch em e 8a
a
Reagents and conditions: (a) KOH, MeOH, reflux, 24 h; (b)
(1) SOCl2, reflux, 8 h; (2) NaSMe, benzene, ambient temperature,
5 h; (c) (1) TiCl4‚2THF, Zn0, THF, reflux 2 h, (2) methyl 5-oxo-
pentanoate (40) and 39, THF, reflux 1.5 h.
Sch em e 6a
Reagents and conditions: (a) CH3I, DMF, K2CO3, ambient
a
temperature, 24 h; (b) (1) TiCl4‚2THF, Zn0, THF, reflux 2.5 h, (2)
35 and 42, THF, reflux 1.5 h.
Reduction of ADAM analogue 2 using diisobutylalu-
minum hydride in dichloromethane yielded alkenyl-
diarylmethane 25 (Scheme 7). O-Alkylation of alkenyl-
diarylmethane 25 with iodomethane in tetrahydrofuran,
using sodium hydride as a base, afforded ADAM ana-
logues 26 and 27, which were separated by column
chromatography. ADAM 26 is an analogue of the active
triester 2 in which the three ester carbonyls are replaced
by methylene groups.
a
Reagents and conditions: (a) [µ-chloro-µ-methylene[bis(cyclo-
pentadienyl)titanium]dimethylaluminum], THF, 0 °C-ambient
temperature, 24 h.
As shown in Scheme 8, dimethyl 5-oxononanedioate
(42) was synthesized via O-alkylation of 5-oxoazelaic
acid (41) with methyl iodide in N,N-dimethylformamide,
using potassium carbonate as a base. McMurry coupling
of benzophenone 35 with dimethyl 5-oxononanedioate
(42)19 afforded ADAM analogue 28, having two ester
side chains instead of the usual single side chain.
The stereoselective synthesis of ADAM 29, having
nonidentical aromatic substituents, is outlined in Scheme
9. The synthesis relies on the Stille reaction of the
aromatic bromide 47 with the tributyltin derivative
51.24,25 Formylation of 4-bromo-2-chlorophenol (43) with
hexamethylenetetramine (HMT) using conditions for the
and subsequent reaction with sodium thiomethoxide
yielded the thioester 39. McMurry coupling of ben-
zophenone 39 with methyl 5-oxopentanoate (40) af-
forded alkenyldiarylmethane 23. Benzophenone 3729
and methyl 5-oxopentanoate (40)30 were synthesized as
previously reported.
Alkenyldiarylmethane 24 (Scheme 6) was synthesized
by treating alkenyldiarylmethane 3619 with [µ-chloro-
µ-methylene[bis(cyclopentadienyl)titanium]dimethyl-
aluminum] (Tebbe reagent). The conversion of a lactone
to a hydroxylated methyl ketone with Tebbe reagent has
recently been reported.31

3152 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Silvestri et al.
Sch em e 9a
Sch em e 10a
a
Reagents and conditions: (a) methyl 5-hexynoate, CuI, TEA,
(PPh3)2PdCl2, reflux, 8 h; (b) Bu3SnH, (PPh3)4Pd, THF, ambient
temperature, 2 h; (c) 47, Pd2(dba)3, As(Ph)3, CsF, THF, reflux, 24
h.
Sch em e 11a
a
Reagents and conditions: (a) HMT, TFA, 90 °C, 20 h; (b) CH3I,
DMF, K2CO3, ambient temperature, 22 h; (c) KMnO4, acetone,
water, 0 °C-ambient temperature, 24 h; (d) CH3I, DMF, K2CO3,
ambient temperature, 24 h; (e) bromine, acetic acid, ambient
temperature, 24 h; (f) CH3I, DMF, K2CO3, ambient temperature,
26 h; (g) methyl 5-hexynoate, CuI, TEA, (PPh3)2PdCl2, reflux, 24
h; (h) Bu3SnH, (PPh3)4Pd, THF, ambient temperature, 1.5 h; (i)
47, Pd2(dba)3, As(Ph)3, CsF, THF, reflux, 24 h.
a
Reagents and conditions: (a) veratrole (55) and 2,4-dimethoxy-
benzoyl chloride (56), AlCl3, CH2Cl2, reflux, 2 h; (b) (1) TiCl4‚2THF,
Zn0, THF, reflux 2.5 h, (2) methyl 5-oxopentanoate (40) and 57,
THF, reflux 2 h.
Duff reaction yielded the expected aldehyde 44. O-
Alkylation of intermediate 44 with methyl iodide in
N,N-dimethylformamide, utilizing potassium carbonate
as the base, afforded 5-bromo-3-chloro-2-methoxyben-
zaldehyde (45). The corresponding acid 46 was synthe-
sized by oxidation of 45 using potassium permanganate
in acetone. O-Alkylation of the carboxylic acid 46 with
methyl iodide in N,N-dimethylformamide, utilizing
potassium carbonate as the base, provided methyl
5-bromo-3-chloro-2-methoxybenzoate (47). To make the
organotin intermediate 51, 3-methylsalicylic acid (48)
was brominated to yield 49. Methyl 5-bromo-2-methoxy-
3-methylbenzoate (50) was synthesized by O-alkylation
of intermediate 49 with methyl iodide in N,N-dimeth-
ylformamide, utilizing potassium carbonate as the base.
Sonagashira coupling of methyl 5-hexynoate with meth-
yl 5-bromo-2-methoxy-3-methylbenzoate (50), followed
by hydrostannation with tributyltin hydride, yielded
vinyl stannane 51.24,25 Stille coupling of methyl 5-bromo-
3-chloro-2-methoxybenzoate (47) with vinyl stannane 51
afforded alkenyldiarylmethane 29. The E stereochem-
istry of the alkene 29 results from the regiochemically
defined cis addition to the alkyne intermediate during
the hydrostannation reaction to afford 51 and the
retention of the stereochemical integrity of the coupling
partners during the Stille reaction.24,25,32,33
The Stille approach was also employed for the syn-
thesis of ADAM 31 having nonidentical aromatic rings
(Scheme 10). Sonagashira coupling of methyl 5-hex-
ynoate with 4-bromoveratrole (52) yielded intermediate
53. Vinyl stannane 54 was synthesized by hydrostan-
nation of intermediate 53 with tributyltin hydride in
the presence of tetrakis(triphenylphosphine)palladium-
(0). Stille coupling of methyl 5-bromo-3-chloro-2-meth-
oxy-3-benzoate (47) with vinyl stannane 54 afforded
alkenyldiarylmethane 31.
As outlined in Scheme 11, Friedel-Crafts acylation
of veratrole (55) with 3,4-dimethoxybenzoyl chloride
(57), utilizing aluminum chloride in dichloromethane as
a catalyst, yielded benzophenone 57. McMurry coupling
of benzophenone 57 with methyl 5-oxopentanoate (40)
yielded alkenyldiarylmethane 32.
Benzophenone 59 was synthesized via O-alkylation
of commercially available 2,2′,4,4′-tetrahydroxyben-
zophenone (58) with methyl iodide in N,N-dimethyl-
formamide, using potassium carbonate as the base

ADAM Non-nucleoside Reverse Transcriptase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3153
Sch em e 12a
Ta ble 1. Anti-HIV Activities, Cytotoxicities, and Metabolic
Stabilities of ADAM Analogues
HIV-1IIIB
EC50
HIV-2ROD
EC50
HIV-1IIIB
CC50
rat plasma
a
a
b
c
t1/2
compd (µM ( SD) (µM ( SD)
(µM ( SD)
(min ( SD)
1
2
3
4
5
6
7
8
0.3 ( 0.1
0.6 ( 0.1
1.0 ( 0.2
0.8 ( 0.1
0.7 ( 0.2
1.1 ( 0.5
3.0 ( 0.1
3.7 ( 0.1
2.6 ( 0.4
13.2 ( 2.7
2.2 ( 0.0
3.1 ( 0.4
3.5 ( 0.0
2.7 ( 0.1
3.5 ( 0.1
3.7 ( 1.4
NAd
NAd
25.0 ( 9.5
NAd
91 ( 31
160 ( 25
6.1 ( 0.4
>247
6.2 ( 0.4
5.8 ( 0.9
42.9 ( 4.0
12.4 ( 0.7
18.9 ( 1.6
20.6 ( 2.6
58.5 ( 4.9
39.3 ( 0.7
55.0 ( 5.6
76.6 ( 3.8
3.4 ( 0.2
7.4 ( 1.0
5.2 ( 0.1
5.7 ( 0.3
1.7 ( 1.6
9.0 ( 0.4
NTe
NAd
NAd
57.8 ( 16.1
143 ( 32
62.3 ( 4.0
>253
NAd
NAd
26.4 ( 14.0
29.3 ( 10.5
113 ( 121
NAd
9
>198
>230
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
28
29
30
31
32
33
113.9 ( 11.2
159.2 ( 45.5
93.4 ( 36.8
35.3 ( 6.8
59.5 ( 25.5
73 ( 27
a
NAd
Reagents and conditions: (a) CH3I, DMF, K2CO3, ambient
temperature, 24 h; (b) (1) TiCl4‚2THF, Zn0, THF, reflux 2.5 h, (2)
methyl 5-oxopentanoate (40) and 59, THF, reflux 1 h.
NAd
NAd
NAe
NAd
(Scheme 12). McMurry coupling of benzophenone 59
with methyl 5-oxopentanoate (40) afforded alkenyl-
diarylmethane 33.
NAd
>231
14.1 ( 5.5
NAd
>231
37.7 ( 3.7
NAd
NAd
>209
NTe
2.8 ( 0.3
NAd
NAd
28.6 ( 4.0
180.6 ( 47.5
>218
35.1
NAd
NTe
Biologica l Resu lts a n d Discu ssion
35.4 ( 1.9
1.8 ( 0.7
NAd
NAd
NTe
NAd
>224
55.3 ( 5.1
The goal of the present study was to investigate
structural modifications that could reasonably be ex-
pected to enhance the metabolic stability of the ADAMs
while maintaining their anti-HIV activity. Accordingly,
the ADAMs of the present series were tested for
inhibition of the cytopathic effects of both HIV-1IIIB and
HIV-2ROD in MT-4 cells, and the resulting EC50 values
are listed in Table 1, along with their cytotoxicities (CC50
values) in uninfected MT-4 cells. In addition, the
metabolic stabilities of the biologically active ADAMs
were investigated in rat plasma, and the resulting half-
lives of the compounds are also listed in Table 1.
The data in the table indicate that, in general, a wide
variety of structural modifications can be made without
a large decrease in activity. Of the 32 compounds tested,
18 displayed EC50 values versus HIV-1IIIB between 0.3
and 3.7 µM. Of the remaining compounds, three had
intermediate EC50 values from 13.2 to 35.4 µM, and the
rest were inactive. With the exception of the methyl
thioester 23 (EC50 ) 1.8 µM), all of the compounds in
the more active range (0.3-3.7 µM) possess methyl ester
substituents meta to the bridging carbon atom on each
NAd
47.1 ( 6.4
30.7 ( 0.3
34.0 ( 2.6
78.1 ( 3.5
19.1 ( 6.1
0.25 ( 0.07
8.7 ( 0.9
7.4 ( 0.9
5.9 ( 0.2
NTe
NAd
NAd
NTe
NAd
NAd
NTe
NAd
NAd
NTe
0.8 ( 0.5
NAd
9.0 ( 0.7
19.2 ( 1.6
NTe
0.9 ( 0.01
2.6 ( 0.3
NAd
NAd
NAe
NAd
NAd
NAd
NAd
NAd
a
EC50 is the 50% inhibitory concentration for inhibition of
b
cytopathicity of HIV-1IIIB or HIV-1ROD in MT-4 cells. CC50 is the
c
50% cytotoxic concentration in mock-infected MT-4 cells. t1/2 is
the half-life in rat plasma. Not active. e Not tested.
d
sponding compound with two methyl ester groups is
active.19 Other previously reported inactive replace-
ments for the aromatic methyl esters of various com-
pounds in the ADAM series include carboxylic acids,20
primary, secondary, and tertiary amides,19,20 alcohols,22
and aldehydes.22
Although the new ADAMs in the present series of
compounds were not tested for enzyme inhibitory activ-
ity versus isolated RT, one can assume on the basis of
prior studies that the new compounds are probably
acting as RT inhibitors. The prior evidence includes
inhibition of isolated RT, the presence of resistance
mutations located in RT, time of addition studies, and
inactivity or low activity in a wide range of antiviral
assays against targets representing other mechanisms
of action.19,20,22,23 In addition, as observed in the present
study, the new ADAMs were much more potent versus
HIV-1 than versus HIV-2, which is a hallmark of the
NNRTIs.
The design of the potential NNRTIs described in this
study was aided through molecular modeling of their
complexes formed with HIV-1 RT. The hypothetical
models were constructed using Sybyl (Tripos, Inc.,
version 6.9, 2002). The X-ray crystal structure of HIV-1
RT complexed with nevirapine (1VRT)34 was utilized as
a starting point. The structures of the potential NNRTIs
were overlapped with the ligand, which was then
deleted, and the energies of the new complexes mini-
mized using the MMFF94s force field and MMFF94
aromatic ring. Also, except for compound 16 (EC50
)
3.7 µM), which had ethyl ether substituents, all of the
more active compounds had methyl ether substituents
at the para position of each aromatic ring. Compound
18 (EC50 ) 14.4 µM), having both ethyl ester and ethyl
ether substituents, had intermediate activity, whereas
compounds 17 and 19, with propyl ether or both propyl
ether and propyl ester substituents, were inactive,
documenting an apparent steric limitation on biological
activity. The inactive compound 26, in which the three
ester carbonyls of 2 were replaced by methylene groups,
revealed a critical role played by the ester carbonyls on
the antiviral activity of the ADAMs. Replacement of the
three ester groups of 2 with thiono esters in 22 de-
creased the potency from an EC50 of 0.6 to 35.4 µM. On
the other hand, the replacement of the two aromatic
methyl ester groups of 2 with the methyl thioester
groups of 23 only decreased the potency from an EC50
of 0.6 to 1.8 µM. Compound 24, with two methyl
ketones, was totally inactive, even though the corre-

3154 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Silvestri et al.
F igu r e 1. Hypothetical model of the ADAM 23 docket in the NNRTI binding site of HIV-1 reverse transcriptase. The ligand is
displayed in red. The figure is programmed for walleyed viewing.
charges. During energy minimization, the structure of
the ligand and a 6 Å sphere surrounding the ligand were
allowed to move, whereas the protein was maintained
either semirigid (12 Å sphere) or frozen. Figure 1
displays a model derived from ADAM 23, which is very
similar to the previously published molecular models
derived from several of the other ADAMs.20,22,23 In the
present case, one can conclude that the NNRTI binding
pocket can readily accommodate the increased size of
the sulfurs in 23 relative to the corresponding ester
oxygens of 1 or 2.20,22,23 Previous molecular modeling
studies with ADAM 1 have indicated the possibility for
either bifurcated hydrogen bonding between the side
chain ester carbonyl of the ligand, the terminal amino
group of Lys103, and the backbone amide N-H of
Lys10122 or hydrogen bonding to the backbone amide
N-H of Lys101 only.23 In the present case, the model
displays a hydrogen bond from the side-chain ester
carbonyl and Lys103 only, and this may result from
displacement of the side chain more in the direction of
Lys103 resulting from the larger steric bulk of a
thioester group in 23 versus an ester group in 1.
However, the general features of the model derived from
23 are similar to those seen with 1, and the two have
the general “butterfly shape” seen in the X-ray crystal
structures of nevirapine and a number of other NNR-
TIs.35,36
With regard to stability in blood plasma, the ester
groups of the ADAMs are a potential source of instabil-
ity. However, prior antiviral testing of ADAM 2 was
performed in the presence of fetal calf serum (FCS), and
various tests with the compound in different levels of
FCS or time in FCS did not reveal any decrease in
activity that would result from metabolism.20 These
results seemed to suggest that hydrolysis of 2 by
nonspecific esterases present in FCS is unlikely under
the assay conditions.20 In contrast, the present inves-
tigation has revealed that there is a wide range of
stabilities in rat plasma. First-order disappearance of
the ADAMs was observed, with half-lives spanning from
0.9 min (compound 32) to 76.6 min (compound 10). The
most stable of the more active compounds is the bis-
(thioester) 23, which has an EC50 of 1.8 µM and a
plasma half-life of 55.3 min. In addition, compound 3
(EC50 ) 1.0 µM), having methyl substituents in the meta
position of each aromatic ring, was considerably more
stable metabolically (half-life ) 42.9 min) than the
corresponding dibromo compound 1 (half-life ) 6.2 min),
the dichloro compound 2 (half-life ) 5.8 min), or the
dicyano compound 4 (half-life ) 12.4 min). However, the
replacement of the two bromine atoms of 1 or the two
chlorine atoms of 2 with the two methyl groups of 3
results in greater cytotoxicity in uninfected MT-4 cells
(CC50 ) 6.1 µM). This trend is also present in the
methyl-chloro compound 29 (CC50 ) 19.1 µM) versus the
dichloro compound 2 (CC50 ) 160 µM). The increased
cytotoxicity of these methylated compounds does not
seem to result from the methyl groups per se, because
the effect was not seen in the 11-13 series or with 14
versus 15.
The replacement of the terminal methyl ester of the
side chain with the more hindered propyl esters in 11
and 12, or with isopropyl esters in 13-15, did not result
in an increase in metabolic stability. On the other hand,
replacement with the alkyne, alkene, and phenyl sub-
stituents in the 5-10 series did increase the half-life
in rat blood plasma significantly (see Table 1). Further-
more, with the exception of compound 10, these latter
substitutions did not cause a large decrease in antiviral
activity when tested versus HIV-1IIIB in MT-4 cells.
Some of the results seemed counterintuitive from the
purely chemical point of view. For example, on the basis
of the number of metabolically unstable ester groups
per molecule, one might have expected the triester 3
(half-life ) 49.2 min) to be less stable than the corre-
sponding monoester 32 (half-life ) 0.9 min), but the
observed order was the reverse of the expected order.
Moreover, relatively small structural changes at the end
of the alkenyl side chain were discovered to have a
significant effect on stability. For example, the alkyne
5 had a half-life of 18.9 min, whereas the corresponding
alkene 7 had a half-life of 58.8 min. A similar trend of
lesser magnitude was observed with the corresponding
chloro compounds, the alkyne 6 (half-life ) 20.6 min)
and the alkene 8 (half-life ) 39.3 min). These results
would be difficult to rationalize solely on the basis of
the relative stabilities of the esters to simple chemical
(nonenzymatic) hydrolysis, and they indicate that re-
mote structural changes can affect the propensities of
the esters to undergo enzymatic hydrolysis. Obviously,

ADAM Non-nucleoside Reverse Transcriptase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3155
was then stirred at -78 °C for an additional 2 h. The reaction
solution was quenched with ice-cold 1 M HCl (50 mL). This
solution was poured into a separatory funnel, and dichlo-
romethane (100 mL) was added. The aqueous and organic
layers were separated, and the aqueous layer was extracted
with dichloromethane (3 × 100 mL). The organic extracts were
dried over anhydrous sodium sulfate. The combined organic
layer was filtered and solvent removed in vacuo to yield a crude
colorless oil. The crude oil was then dissolved in dry THF (20
mL) and added to a flame-dried 50 mL pear-shape flask. 3′,3′′-
Dibromo-4′,4′′-dimethoxy-5′,5′′-dimethoxycarbonylbenzophe-
none (35) (0.27 g, 0.5 mmol) was added to the flask under an
argon atmosphere. Titanium tetrachloride bis(tetrahydro-
furanate) (1.24 g, 3.7 mmol) was added to a flame-dried two-
neck 100 mL flask, under an argon atmosphere, equipped with
a condenser, magnetic stir bar, and a rubber septum. Zinc dust
(0.45 g, 6.9 mmol) was then added to the flask. The solids were
suspended in dry THF (20 mL). The suspension was heated
at reflux for 3 h. The solution in the pear-shape flask was
transferred via cannula under an argon atmosphere to the
titanium suspension. The resulting suspension was heated at
reflux for 3.5 h. The suspension was then cooled to ambient
temperature, and 1 M HCl (50 mL) was added. The reaction
mixture was stirred at ambient temperature for 15 min. The
reaction mixture was then poured into a separatory funnel,
and both water (100 mL) and ethyl acetate (100 mL) were
added. The aqueous and organic layers were separated, and
the aqueous layer was extracted with ethyl acetate (3 × 100
mL). The combined organic extracts were washed with brine
(100 mL) and dried over anhydrous sodium sulfate. Solvents
were removed in vacuo yielding a brown oil. The crude oil was
separated by flash column chromatography using silica gel (50
g, 2 × 30 cm) and a gradient eluant from 0 to 20% ethyl acetate
in hexanes. Like fractions were combined, and solvent was
removed in vacuo to yield a crude red-brown oil. The crude oil
was purified again using silica gel (12 g, 2 × 10 cm) and a
gradient eluant of 0-10% ethyl acetate in hexanes. Like
fractions were combined, and solvent was removed in vacuo
to yield a crude red oil. This crude oil was further purified
using silica gel (5 g, 1 × 12 cm) and a gradient eluant of 0-14%
ethyl acetate in hexanes. Like fractions were combined, and
solvent was removed in vacuo to yield a colorless oil (0.076 g,
24%): IR (film) 2933, 2858, 1732, 1473, 1434, 1281, 1260, 1206,
although the esterases present in plasma are relatively
nonspecific, the rates of ester hydrolysis depend largely
on the overall structure of the substrate.
In summary, the structural modifications introduced
in the present series of ADAMs result in some favorable
properties, including a decrease in cytotoxicity and an
increase in metabolic stability. In particular, the thioester
23 displays enhanced stability (t1/2 ) 55.3 min) and
diminished cytotoxicity (CC50 > 224 µΜ) while main-
taining reasonable potency (EC50 ) 1.8 µM) relative to
the corresponding dichloro ADAM 2.
Exp er im en ta l Section
1H NMR spectra were recorded on an ARX300 300 MHz or
a DRX500 500 MHz Bruker NMR spectrometer. IR spectra
were obtained using a Perkin-Elmer 1600 series FTIR or a
Perkin-Elmer Spectrum One spectrometer. IR spectra of neat
solid compounds were obtained with a single reflection hori-
zontal attenuated total reflectance (HATR) device (Pike Tech-
nologies, Inc., Madison, WI). Flash chromatography was
performed with 230-400 mesh silica gel. Thin-layer chroma-
tography was performed using Baker-flex silica gel IB2-F
plates of a 2.5 mm thickness. Melting points were taken in
capillary tubes on a Mel-Temp or Thomas-Hoover apparatus
and are uncorrected. Unless otherwise stated, chemicals and
solvents were of reagent grade and used as obtained from
commercial sources without further purification. Tetrahydro-
furan (THF) was freshly distilled from sodium/benzophenone
ketyl radical prior to use. Dichloromethane was freshly
distilled from calcium hydride prior to use. Microanalyses were
performed at the Purdue University Microanalysis Laboratory.
Meth yl 3′,3′′-Dicya n o-4′,4′′-d im eth oxy-5′,5′′-d im eth oxy-
ca r bon yl-6,6-d ip h en yl-5-h exen oa te (4). Methyl 3′,3′′-di-
bromo-4′,4′′-dimethoxy-5′,5′′-dimethoxycarbonyl-6,6-diphenyl-
5-hexenoate (1) (0.31 g, 0.50 mmol) was added to an oven-dried
10 mL round-bottom flask, under an argon atmosphere,
equipped with a condenser and a magnetic stir bar. The solid
was then dissolved in N,N-dimethylformamide (4 mL). Cu-
prous cyanide (0.24 g, 2.7 mmol) was added to the flask. The
reaction mixture was heated at reflux for 22 h. The reaction
solution was then cooled to ambient temperature, and both
water (100 mL) and ethyl acetate (100 mL) were added. The
solution was then filtered on a Bu¨chner funnel, and the
inorganic salts were washed with ethyl acetate. The filtrate
was poured into a separatory funnel, and the aqueous and
organic layers were separated. The aqueous layer was ex-
tracted with ethyl acetate (3 × 100 mL). The combined organic
extracts were then washed with brine (2 × 100 mL) and dried
over anhydrous magnesium sulfate. The organic extracts were
filtered, and the solvent was removed in vacuo to yield a crude
green oil. The crude oil was purified by flash column chroma-
tography using silica gel (25 g, 2 × 18 cm) and eluted with a
gradient eluant from 0 to 45% ethyl acetate in hexanes. Like
fractions were combined, and solvent was removed in vacuo
to yield a colorless oil (0.04 g, 19%): IR (film) 2952, 2232, 1733,
1479, 1436, 1258, 993 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.80
(d, J ) 2.35 Hz, 1 H), 7.76 (d, J ) 2.10 Hz, 1 H), 7.51 (d, J )
2.23 Hz, 1 H), 7.43 (d, J ) 2.37 Hz, 1 H), 6.14 (t, J ) 7.51 Hz,
1 H), 4.16 (s, 3 H), 4.09 (s, 3 H), 3.96 (s, 3 H), 3.95 (s, 3 H),
3.66 (s, 3 H), 2.33 (t, J ) 7.23 Hz, 2 H), 2.15 (q, J ) 7.41 Hz,
2 H), 1.81 (qn, J ) 7.34 Hz, 2 H); CIMS, m/z (relative intensity)
507 (100, MH+). Anal. (C27H26N2O8) C, H, N.
1
1088, 998, 729 cm-1; H NMR (300 MHz, CDCl3) δ 7.53 (d, J
) 2.28 Hz, 1 H), 7.52 (d, J ) 1.6 Hz, 1 H), 7.49 (d, J ) 2.18
Hz, 1 H), 7.47 (d, J ) 2.35 Hz, 1 H), 6.05 (t, J ) 7.47 Hz, 1 H),
3.99 (s, 3 H), 3.92 (s, 3 H), 3.92 (s, 3 H), 3.91 (s, 3 H), 3.33 (t,
J ) 2.59 Hz, 2 H), 3.31 (s, 3 H), 2.12 (q, J ) 7.35 Hz, 2 H),
1.63-1.41 (m, 4 H); CIMS, m/z (relative intensity) 599/601/
603 (75/100/50, MH+), 567/569/571 (46/74/34, MH+ - MeOH).
Anal. (C25H28Br2O7) C, H.
3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-d im eth oxyth ioca r -
b on yl-1,1-d ip h en yl-1-h ep t en e (21). 3′,3′′-Dichloro-4′,4′′-
dimethoxy-5′,5′′-dimethoxcarbonyl-1,1-diphenyl-1-heptene (36)
(0.40 g, 0.8 mmol) was added to an oven-dried 10 mL round-
bottom flask, under an argon atmosphere, equipped with a
condenser and a magnetic stir bar. Lawesson’s reagent [2,4-
bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disul-
fide; 2.63 g, 6.5 mmol] was then added to the flask. The solids
were suspended in toluene (5 mL). The suspension was heated
at reflux for 96 h. The suspension was then cooled to ambient
temperature, and the contents of the round-bottom flask were
filtered on a Bu¨chner funnel. The filter cake was washed with
hexane (100 mL) and benzene (100 mL). The filtrate was then
collected, and solvent was removed in vacuo to yield a crude
oil. The crude oil was purified by flash column chromatography
using silica gel (25 g, 2 × 19 cm) and a gradient eluant from
0 to 9% ethyl acetate in hexanes. Like fractions were combined,
and solvent was removed in vacuo to yield a yellow oil (0.040
g, 10%): IR (film) 2932, 2361, 2341, 1473, 1244, 998 cm-1; 1H
NMR (500 MHz, CDCl3) δ 7.41 (d, J ) 2.11 Hz, 1 H), 7.39 (d,
J ) 1.85 Hz, 1 H), 7.24 (d, J ) 1.88 Hz, 1 H), 7.20 (d, J ) 2.05
Hz, 1 H), 6.06 (t, J ) 7.53 Hz, 1 H), 4.31 (s, 3 H), 4.29 (s, 3 H),
3.95 (s, 3 H), 3.89 (s, 3 H), 2.11 (q, J ) 7.44 Hz, 2 H), 1.46 (m,
1,1-Bis[3-b r om o-4-d im et h oxy-5-d im et h oxyca r b on yl-
p h en yl]-6-m et h oxyh exen e (20). Methyl 5-methoxypen-
tanoate (34)27 (1.86 g, 12.8 mmol) was added to a flame-dried
50 mL round-bottom flask equipped with a stirring bar and
rubber septum. An inert argon atmosphere was introduced into
the flask. Dry dichloromethane (25 mL) was then added to the
flask via syringe. The flask was cooled to -78 °C in a dry ice
acetone bath. A 1.0 M solution of diisobutylaluminum hydride
(DIBAL-H) in dichloromethane (12.9 mL, 12.9 mmol) was
added to the flask dropwise via syringe. The reaction solution

3156 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
2 H), 1.31-1.26 (m, 4 H), 0.89 (t, J ) 6.69 Hz, 3 H); ESIMS,
Silvestri et al.
ice bath, and [µ-chloro-µ-methylene[bis(cyclopentadienyl)-
titanium]dimethylaluminum] (Tebbe reagent) (0.5 M) (3.3 mL,
1.7 mmol) was added to the flask dropwise via syringe. The
reaction mixture was stirred and allowed to warm to ambient
temperature for 24 h. The reaction mixture was then cooled
to 0 °C using an ice bath, and ether (30 mL) was added to the
round-bottom flask. Ten drops of NaOH (0.1 M) were slowly
added over a 10 min period to quench the reaction. Anhydrous
sodium sulfate was added, and the contents of the flask were
stirred for an additional 10 min. The reaction mixture was
then filtered over a pad of Celite on a Bu¨chner funnel. The
filter cake was washed with ether (100 mL). The filtrate was
then added to a separatory funnel, and water (100 mL) was
added. The aqueous layer was extracted with ether (3 × 100
mL). The combined extracts were then dried over anhydrous
sodium sulfate. The organic extracts were then filtered, and
solvent was removed in vacuo to yield a crude oil. The crude
oil was purified by flash column chromatography using silica
gel (25 g, 3 × 14 cm) and a gradient eluant from 0 to 14%
ethyl acetate in hexanes. Like fractions were combined, and
solvent was removed in vacuo to yield a colorless oil (0.169 g,
m/z (relative intensity) 527/529 (100/75, MH+). Anal. (C25H28
-
Cl2O4S2) C, H, S.
Meth yl 3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-d im eth oxy-
th iocar bon yl-6,6-diph en yl-5-h exen e-1-m eth oxyth ion e (22).
Methyl 3′,3′′-Dichloro-4′,4′′-dimethoxy-5′,5′′-dimethoxcarbonyl-
6,6-diphenyl-5-hexenoate (2) (0.42 g, 0.8 mmol) was added to
an oven-dried 25 mL round-bottom flask, under an argon
atmosphere, equipped with a condenser and a magnetic stir
bar. Lawesson’s reagent [2,4-bis(4-methoxyphenyl)-1,3-dithia-
2,4-diphosphetane-2,4-disulfide; 2.10 g, 5.2 mmol] was then
added to the flask. The solids were suspended in xylene (10
mL). The suspension was heated at reflux for 48 h. The
suspension was then cooled to ambient temperature, and both
hexanes (20 mL) and water (20 mL) were added. The contents
of the round-bottom flask were added to a beaker. The round-
bottom flask was rinsed with toluene (20 mL), and this was
added to the beaker. The contents of the beaker were filtered
on a Bu¨chner funnel. The filter cake was washed with hexane
(100 mL) and toluene (100 mL). The filtrate was collected and
solvent removed in vacuo, yielding a crude oily solid. The crude
solid was purified by flash column chromatography using silica
gel (25 g, 2 × 24 cm) and eluted with a gradient eluant from
0 to 10% ethyl acetate in hexanes. Like fractions were
combined, and solvent was removed in vacuo to yield a yellow
oil (0.200 g, 43%): IR (film) 2939, 1592, 1550, 1474, 1439, 1244,
997, 772 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.38 (d, J ) 2.27
Hz, 1 H), 7.35 (d, J ) 2.11 Hz, 1 H), 7.20 (d, J ) 2.14 Hz, 1 H),
7.17 (d, J ) 2.25 Hz, 1 H), 6.02 (t, J ) 7.49 Hz, 1 H), 4.28 (s,
3 H), 4.26 (s, 3 H), 4.02 (s, 3 H), 3.92 (s, 3 H), 3.85 (s, 3 H),
2.73 (t, J ) 7.42 Hz, 2 H), 2.15 (q, J ) 7.31 Hz, 2 H), 1.89 (qn,
J ) 7.42 Hz, 2 H); CIMS, m/z (relative intensity) 573/575/577
(100/79/13, MH+). Anal. (C25H26Cl2O5S3) C, H.
Met h yl 5′,5′′-Dich lor o-4′,4′′-d im et h oxy-3′,3′′-d i(m et h -
ylth ioca r bon yl)-6,6-d ip h en yl-5-h exen oa te (23). Titanium
tetrachloride bis(tetrahydrofuranate) (1.56 g, 4.7 mmol) was
added to a dry 100 mL two-neck flask, under an argon
atmosphere, equipped with a condenser, magnetic stir bar, and
a rubber septum. Zinc dust (0.650 g, 10.0 mmol) was then
added to the flask. The solids were suspended in dry THF (15
mL). The suspension was heated at reflux for 2 h. A solution
of 3,3′-dichloro-4,4′-dimethoxy-5,5′-di(methylthiocarbonyl)ben-
zophenone (39) (0.78 g, 1.6 mmol) and methyl 5-oxopentanoate
(40) (0.436 g, 3.3 mmol) in dry THF (15 mL) was transferred
via cannula under an argon atmosphere to the titanium
suspension. The resulting suspension was heated at reflux for
1.5 h. The suspension was cooled to ambient temperature, and
1 M HCl (50 mL) was added. The reaction mixture was poured
into a separatory funnel, and ethyl acetate (100 mL) and water
(100 mL) were added. The aqueous and organic layers were
separated, and the aqueous layer was extracted with ethyl
acetate (3 × 100 mL). The combined organic extracts were
washed with brine (100 mL) and dried over anhydrous sodium
sulfate. Solvents were removed in vacuo, yielding a yellow oil.
The crude oil was purified by flash column chromatography
using silica gel (50 g, 2 × 34 cm) and eluted with a gradient
eluant from 0 to 15% ethyl acetate in hexanes. Like fractions
were combined, and solvent was removed in vacuo to yield a
colorless oil (0.196 g, 21%): IR (film) 2929, 1736, 1674, 1643,
1473, 1423, 1248, 1136, 992, 882, 780, 747 cm-1; 1H NMR (300
MHz, CDCl3) δ 7.27 (d, J ) 2.37 Hz, 1 H), 7.25 (d, J ) 2.12
Hz, 1 H), 7.13 (d, J ) 2.18 Hz, 1 H), 7.12 (d, J ) 2.12 Hz, 1 H),
5.89 (t, J ) 7.65 Hz, 1 H), 3.84 (s, 3 H), 3.78 (s, 3 H), 3.48 (s,
3 H), 2.29 (s, 3 H), 2.28 (s, 3 H), 2.15 (t, J ) 7.34 Hz, 2 H),
1.99 (q, J ) 7.25 Hz, 2 H), 1.63 (qn, J ) 7.37 Hz, 2 H); CIMS,
m/z (relative intensity) 509/511/513 (100/75/9, MH+ - SMe).
Anal. (C25H26Cl2O6S2) C, H, S.
44%): IR (film) 2920, 2855, 1685, 1473, 1246, 996, 731 cm-1
;
1H NMR (500 MHz, CDCl3) δ 7.35 (d, J ) 2.26 Hz, 1 H), 7.33
(d, J ) 1.97 Hz, 1 H), 7.31 (d, J ) 2.06 Hz, 1 H), 7.29 (d, J )
2.28 Hz, 1 H), 6.08 (t, J ) 7.52 Hz, 1 H), 3.98 (s, 3 H), 3.91 (s,
3 H), 2.67 (s, 3 H), 2.64 (s, 3 H), 2.08 (q, J ) 7.42 Hz, 2 H),
1.45 (m, 2 H), 1.29-1.28 (m, 4 H), 0.89 (t, J ) 6.58 Hz, 3 H);
ESIMS, m/z (relative intensity) 463/465 (100/83, MH+). Anal.
(C25H28Cl2O4) C, H.
3′,3′′-Dich lor o-5′,5′′-d ih yd r oxym eth yl-4′,4′′-d im eth oxy-
1,1-d ip h en yl-1-h exen -6-ol (25). Methyl 3′,3′′-dichloro-4′,4′′-
dimethoxy-5′,5′′-dimethoxcarbonyl-6,6-diphenyl-5-hexenoate (2)
(0.74 g, 1.4 mmol) was added to a flame-dried 50 mL round-
bottom flask, equipped with a stir bar and a rubber septum.
The contents of the flask were then placed under an argon
atmosphere. Dry dichloromethane (25 mL) was added to the
flask via syringe. The flask and its contents were cooled to
-78 °C using a dry ice acetone bath. A 1.0 M DIBAL-H solution
in dichloromethane (8.5 mL, 8.5 mmol) was added dropwise
to the cooled flask via a syringe, while the bath temperature
was maintained at -78 °C. The reaction solution was allowed
to stir at -78 °C for an additional 3 h. The reaction solution
was then poured over ice-cold 1 M HCl (25 mL). The quenched
solution was placed into a separatory funnel, and both water
(100 mL) and dichloromethane (100 mL) were added. The
aqueous layer was extracted with dichloromethane (3 × 150
mL). The combined organic extracts were washed with brine
(2 × 100 mL) and dried over anhydrous magnesium sulfate.
The organic extracts were filtered, and solvent was removed
in vacuo to yield a colorless oil (0.467 g, 74%): IR (film) 3343,
1
2935, 1476, 1430, 1405, 1230, 1002, 883 cm-1; H NMR (500
MHz, CDCl3) δ 7.14 (s, 1 H), 7.10 (s, 3 H), 6.03 (t, J ) 7.54 Hz,
1 H), 4.75 (s, 2 H), 4.68 (d, J ) 2.54 Hz, 2 H), 3.96 (s, 3 H),
3.91 (s, 3 H), 3.57 (t, J ) 5.47 Hz, 2 H), 2.10 (q, J ) 7.43 Hz,
2 H), 1.56 (qn, J ) 5.73 Hz, 4 H); CIMS, m/z (relative intensity)
423/425/427 (100/62/11, MH+ - H2O). Anal. (C22H26Cl2O5) C,
H.
3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-d im eth oxym eth yl-
6-m eth oxy-1,1-d ip h en yl-1-h exen e (26). 3′,3′′-Dichloro-5′,5′′-
dihydroxymethyl-4′,4′′-dimethoxy-1,1-diphenyl-1-hexen-6-ol (25)
(0.16 g, 0.4 mmol) was added to a flame-dried 10 mL round-
bottom flask equipped with a stir bar and a rubber septum.
An inert argon atmosphere was introduced into the flask. Dry
THF (5 mL) was then added to the flask via syringe. Sodium
hydride (0.047 g, 2.0 mmol) was added to the flask. The flask
was cooled to 0 °C in an ice bath. Iodomethane (0.070 mL, 1.1
mmol) was added to the reaction solution dropwise via syringe.
The reaction solution was allowed to warm to ambient tem-
perature for 24 h. The reaction solution was poured over ice
(10 mL). Water (50 mL) was added, and the reaction solution
was added to a separatory funnel, followed by the addition of
ethyl acetate (50 mL). The aqueous and organic layers were
separated, and the aqueous layer was extracted with ethyl
acetate (3 × 50 mL). The organic extracts were dried over
3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-d im eth ylca r bon yl-
1,1-d ip h en yl-1-h ep ten e (24). 3′,3′′-Dichloro-4′,4′′-dimethoxy-
5′,5′′-dimethoxcarbonyl-1,1-diphenyl-1-heptene (36) (0.41 g, 0.8
mmol) was added to an oven-dried 50 mL round-bottom flask,
under an argon atmosphere, equipped with a condenser and
a magnetic stir bar. The solid was dissolved in dry THF (5
mL). The reaction mixture was then cooled to 0 °C using an

ADAM Non-nucleoside Reverse Transcriptase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3157
anhydrous sodium sulfate. The organic layer was filtered, and
solvent was removed in vacuo to yield a crude oil. The crude
oil was purified by flash column chromatography using silica
gel (25 g, 3 × 8 cm) and a gradient eluant from 0 to 30% ethyl
acetate in hexanes. Like fractions were combined, and solvent
removed in vacuo to yield a colorless oil (0.07 g, 41%): IR (film)
2933, 1478, 1376, 1232, 1119, 1100, 1001, 880 cm-1; 1H NMR
(300 MHz, CDCl3) δ 7.14 (d, J ) 2.28 Hz, 1 H), 7.11 (d, J )
2.09 Hz, 1 H), 7.09 (d, J ) 2.28 Hz, 1 H), 7.07 (d, J ) 2.07 Hz,
1 H), 6.00 (t, J ) 7.44 Hz, 1 H), 4.51 (s, 2 H), 4.46 (s, 2 H),
3.92 (s, 3 H), 3.86 (s, 3 H), 3.41 (s, 3 H), 3.40 (s, 3 H), 3.33 (t,
J ) 6.11 Hz, 2 H), 3.31 (s, 3 H), 2.11 (q, J ) 7.38 Hz, 2 H),
1.62-1.44 (m, 4H); CIMS, m/z (relative intensity) 451/453 (100/
80, MH+ - MeOH). Anal. (C25H32Cl2O5‚0.5H2O) C, H.
3′,3′′-Dich lor o-4′,4′′-d im eth oxy-5′,5′′-d im eth oxym eth yl-
1,1-d ip h en yl-1-h exen -6-ol (27). This compound (0.039 g,
22%) was obtained as a side product from the reaction to
synthesize (26): IR (film) 3440, 2926, 1478, 1377, 1229, 1095,
1000, 875 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.15 (d, J ) 2.25
Hz, 1 H), 7.11 (d, J ) 2.15 Hz, 1 H), 7.10-7.09 (m, 2 H), 6.01
(t, J ) 7.50 Hz, 1 H), 4.51 (s, 2 H), 4.45 (s, 2 H), 3.92 (s, 3 H),
3.86 (s, 3 H), 3.56 (t, J ) 5.85 Hz, 2 H), 3.42 (s, 3 H), 3.41 (s,
3 H), 2.11 (q, J ) 6.96 Hz, 2 H), 1.60-1.49 (m, 4H); CIMS,
m/z (relative intensity) 437/439 (100/75, MH+ - MeOH). Anal.
(C24H30Cl2O5) C, H.
dipalladium(0) (0.02 g, 0.02 mmol) were added to a flame-dried
50 mL two-neck flask equipped with a condenser, stirbar, and
rubber septum under an argon atmosphere. Dry THF (10 mL)
was added via syringe to the round-bottom flask. Methyl 6-(4-
methoxy-5-methoxycarbonyl-3-methylphenyl)-6-(tributylstan-
nyl)hex-5-enoate (51) (0.1 g, 0.2 mmol) was dissolved in dry
THF (10 mL) and added to the round-bottom flask via syringe.
The reaction mixture was heated at reflux for 24 h. Solvent
was removed in vacuo to yield a crude oily solid. The crude
oily solid was purified via flash column chromatography using
silica gel (15 g, 2 × 10 cm) and a gradient eluant of 0-20%
ethyl acetate in hexanes. Like fractions were combined, and
solvents were removed in vacuo to yield a crude brown oil. The
crude oil was purified again via flash column chromatography
using silica gel (5 g, 1 × 10 cm) and a gradient eluant of 0-25%
ethyl acetate in hexanes. Like fractions were combined, and
solvent was removed in vacuo to yield a light brown oil (0.031
g, 28%): IR (film) 2951, 1737, 1514, 1475, 1255, 1027, 764
cm-1; 1H NMR (300 MHz, CDCl3) δ 7.50 (d, J ) 2.34 Hz, 1 H),
7.38 (d, J ) 2.19 Hz, 1 H), 7.27 (d, J ) 2.35 Hz, 1 H), 7.07 (d,
J ) 2.22 Hz, 1 H), 5.98 (t, J ) 7.43 Hz, 1 H), 3.90 (s, 3 H),
3.89 (s, 6 H), 3.86 (s, 3 H), 3.61 (s, 3 H), 2.30 (s, 3 H), 2.28 (t,
J ) 7.51 Hz, 2 H), 2.11 (q, J ) 7.33 Hz, 2 H), 1.75 (qn, J )
7.25 Hz, 2 H); CIMS, m/z (relative intensity) 505/507 (83/33,
MH+), 473/475 (100/39, MH+ - MeOH). Anal. (C26H29ClO8) C,
H.
Dim eth yl-5-[bis(3-br om o-4-m eth oxy-5-m eth oxyca r bo-
n ylp h en yl)m eth ylen e]n on a n e-1,9-d ioa te (28). Titanium
tetrachloride bis(tetrahydrofuranate) (1.67 g, 5.00 mmol) was
added to a dry 50 mL two-neck flask, under an argon
atmosphere, equipped with a condenser, magnetic stir bar, and
a rubber septum. Zinc dust (0.495 g, 7.57 mmol) was then
added to the flask. The solids were suspended in dry THF (10
mL). The suspension was heated at reflux for 2.5 h. A solution
of 5,5′-dibromo-4,4′-dimethoxy-3,3′-dimethoxycarbonylben-
zophenone (35) (0.468 g, 0.91 mmol) and dimethyl 5-ox-
ononanedioate (42) (0.217 g, 0.91 mmol) in dry THF (10 mL)
was transferred via cannula under an argon atmosphere to
the titanium suspension. The resulting suspension was heated
at reflux for 1.5 h. The suspension was then cooled to ambient
temperature, and 1 M HCl (20 mL) was added. The reaction
solution was then poured into a separatory funnel, and ethyl
acetate (100 mL) was added. The aqueous and organic layers
were separated, and the aqueous layer was extracted with
ethyl acetate (3 × 50 mL). The combined organic extracts were
washed with brine (100 mL) and dried over anhydrous sodium
sulfate. Solvents were removed in vacuo, yielding a yellow oil.
The crude oil was purified by flash column chromatography
using silica gel (40 g, 2 × 24 cm) and eluted with a gradient
eluant from 0 to 30% ethyl acetate in hexanes. Like fractions
were combined, and solvent removed in vacuo to yield a crude
colorless oil. The crude oil was purified a second time using
silica gel (50 g, 2 × 38 cm) and eluted with a gradient eluant
from 0 to 25% ethyl acetate in hexanes. Like fractions were
combined, and solvent was removed in vacuo to yield another
crude colorless oil. This crude oil was purified a third time
using silica gel (25 g, 2 × 18 cm) and eluted with a gradient
eluant from 0 to 10% acetone in dichloromethane. Like
fractions were combined, and solvent was removed in vacuo
to yield a crude colorless oil. The crude oil was purified one
more time using silica gel (5 g, 1 × 14 cm) and eluted with a
gradient eluant from 0 to 1% acetone in dichloromethane. Like
fractions were combined, and solvent was removed in vacuo
to yield a pure colorless oil (0.060 g, 9%): IR (film) 2951, 1732,
6-(3,4-Dim eth oxyph en yl)-6-(3-ch lor o-4-m eth oxy-5-m eth -
oxyca r bon ylp h en yl)h ex-5-en oa te (31). Methyl 5-bromo-3-
chloro-2-methoxybenzoate (47) (0.058 g, 0.2 mmol), triphenyl-
arsine (0.03 g, 0.1 mmol), cesium fluoride (0.078 g, 0.5 mmol),
and tris(dibenzylideneacetone)dipalladium(0) (0.02 g, 0.02
mmol) were added to a flame-dried 50 mL two-neck flask
equipped with a condenser, stirbar, and rubber septum under
an argon atmosphere. Dry THF (10 mL) was added via syringe
to the round-bottom flask. Methyl 6-(3,4-dimethoxyphenyl)-
7,7-dibutyl-7-stannaundec-5-enoate (54) (0.2 g, 0.2 mmol) was
dissolved in dry THF (10 mL) and added to the round-bottom
flask via syringe. The reaction mixture was heated at reflux
for 24 h. Solvent was removed in vacuo to yield a crude oily
solid. The crude oily solid was purified via flash column
chromatography using silica gel (25 g, 2 × 20 cm) and a
gradient eluant of 0-30% ethyl acetate in hexanes. Like
fractions were combined, and solvent was removed in vacuo
to yield a light brown oil (0.020 g, 21%): IR (film) 2951, 1733,
1477, 1436, 1258, 1003, 741 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.52 (d, J ) 2.30 Hz, 1 H), 7.30 (d, J ) 2.32 Hz, 1 H), 6.86
(d, J ) 8.17 Hz, 1 H), 6.66 (dd, J ) 8.12, 1.73 Hz, 1 H), 6.57
(d, J ) 1.82 Hz, 1 H), 5.97 (t, J ) 7.50 Hz, 1 H), 3.89 (s, 6 H),
3.87 (s, 3 H), 3.81 (s, 3 H), 3.60 (s, 3 H), 2.27 (t, J ) 7.43 Hz,
2 H), 2.13 (q, J ) 7.36 Hz, 2 H), 1.74 (qn, J ) 7.46 Hz, 2 H);
GC-MS m/z (relative intensity) 463/465 (100/35, MH+). Anal.
(C24H27ClO7) C, H.
Met h yl 3′,3′′,4′,4′′-Tet r a m et h oxy-6,6-d ip h en yl-5-h ex-
en oa te (32). Titanium tetrachloride bis(tetrahydrofuranate)
(3.33 g, 10.0 mmol) was added to a flame-dried two-neck 100
mL flask, under an argon atmosphere, equipped with a
condenser, magnetic stir bar, and a rubber septum. Zinc dust
(1.04 g, 15.9 mmol) was then added to the flask. The solids
were suspended in dry THF (15 mL). The suspension was
heated at reflux for 2.5 h. A solution of 3,3′,4,4′-tetramethoxy-
benzophenone (57) (0.41 g, 1.36 mmol) and methyl 5-oxopen-
tanoate (40) (0.41 g, 3.10 mmol) in dry THF (10 mL) was
transferred via cannula under an argon atmosphere to the
titanium suspension. The resulting suspension was stirred at
reflux for 2 h. The suspension was then cooled to ambient
temperature, and 1 M HCl (50 mL) was added. The reaction
solution was stirred at ambient temperature for 15 min. The
reaction solution was then poured into a separatory funnel,
and both water (100 mL) and ethyl acetate (100 mL) were
added. The aqueous and organic layers were separated, and
the aqueous layer was extracted with ethyl acetate (3 × 50
mL). The combined organic extracts were washed with brine
(100 mL) and dried over anhydrous sodium sulfate. The
organic extracts were filtered, and solvents were removed in
1
1471, 1435, 1259, 1202, 1089, 997 cm-1; H NMR (300 MHz,
CDCl3) δ 7.44 (d, J ) 2.15 Hz, 2 H), 7.40 (d, J ) 2.19 Hz, 2 H),
3.91 (s, 12 H), 3.60 (s, 6 H), 2.21 (t, J ) 7.49 Hz, 4 H), 2.13 (t,
J ) 6.65 Hz, 4 H), 1.72 (qn, J ) 7.65 Hz, 4 H); GC-MS, m/z
(relative intensity) 712/714/716 (43/100/49, M+). Anal. (C30H34
Br2O10) C, H.
-
6-(3-Ch lor o-4-m et h oxy-5-m et h oxyca r bon ylp h en yl)-6-
(4-m et h oxy-5-m et h oxyca r b on yl-3-m et h ylp h en yl)h ex-5-
en oa te (29). Methyl 5-bromo-3-chloro-2-methoxybenzoate (47)
(0.06 g, 0.2 mmol), triphenylarsine (0.02 g, 0.1 mmol), cesium
fluoride (0.081 g, 0.5 mmol), and tris(dibenzylideneacetone)-

3158 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Silvestri et al.
vacuo yielding a crude oil. The crude oil was purified by flash
column chromatography using silica gel (25 g, 2 × 19 cm) and
a gradient eluant from 0 to 25% ethyl acetate in hexanes. Like
fractions were combined, and solvent was removed in vacuo
to yield a colorless oil (0.383 g, 70%): IR (film) 2950, 1737,
added to the suspension, and this was poured into a separatory
funnel. Ethyl acetate (50 mL) and water (50 mL) were added.
The aqueous and organic layers were separated, and the
aqueous layer was extracted with ethyl acetate (3 × 100 mL).
The combined organic extracts were washed with brine (100
mL) and dried over anhydrous sodium sulfate. Solvents were
removed in vacuo, yielding a pale brown glassy oil (0.914 g,
73% over two steps): IR (film) 2929, 1667, 1588, 1473, 1300,
1273, 1137, 990, 782 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.98
(d, J ) 2.15 Hz, 2 H), 7.97 (d, J ) 2.11 Hz, 2 H), 4.03 (s, 6 H),
2.47 (s, 6 H); GC-MS m/z (relative intensity) 459/461 (100/84,
MH+). Anal. (C19H16Cl2O5S2) C, H, S.
1600, 1579, 1513, 1463, 1247, 1169, 1136, 1027, 810, 762 cm-1
;
1H NMR (300 MHz, CDCl3) δ 6.94-6.71 (m, 6 H), 6.09 (t, J )
6.47 Hz, 1 H), 4.33 (s, 3 H), 4.29 (s, 3 H), 4.26 (s, 3 H), 4.25 (s,
3 H), 4.09 (s, 3 H), 2.94 (t, J ) 6.47 Hz, 2 H), 2.81 (q, J ) 6.46
Hz, 2 H), 2.49 (qn, J ) 6.30 Hz, 2 H); CIMS, m/z (relative
intensity) 401 (100, MH+). Anal. (C23H28O6) C, H.
2,2′,4,4′-Tetr a m eth oxy-6,6-d ip h en yl-5-h exen oa te (33).
Titanium tetrachloride bis(tetrahydrofuranate) (3.2 g, 9.6
mmol) was added to a dry 100 mL two-neck flask, under an
argon atmosphere, equipped with a condenser, magnetic stir
bar, and a rubber septum. Zinc dust (0.91 g, 14.0 mmol) was
then added to the flask. The solids were suspended in dry THF
(20 mL). The suspension was heated at reflux for 2.5 h. A
solution of 2,2′,4,4′-tetramethoxybenzophenone (59) (0.59 g,
1.94 mmol) and methyl 5-oxopentanoate (40) (0.53 g, 4.1 mmol)
in dry THF (10 mL) was transferred via cannula under an
argon atmosphere to the titanium suspension. The resulting
suspension was heated at reflux for 1 h. The suspension was
then cooled to ambient temperature, and 1 M HCl (30 mL)
was added. The reaction mixture was then poured into a
separatory funnel, and ethyl acetate (100 mL) was added. The
aqueous and organic layers were separated, and the aqueous
layer was extracted with ethyl acetate (3 × 50 mL). The
combined organic extracts were washed with brine (100 mL)
and dried over anhydrous sodium sulfate. Solvents were
removed in vacuo, yielding a yellow oil. The crude oil was
purified by flash column chromatography using silica gel (50
g, 2 × 38 cm) and eluted with a gradient eluant from 0 to 20%
ethyl acetate in hexanes. Like fractions were combined, and
solvent was removed in vacuo to yield a colorless oil (0.400 g,
52%): IR (film) 2949, 2835, 1734, 1605, 1576, 1503, 1454, 1437,
Dim eth yl 5-oxon on a n ed ioa te (42). 5-Oxoazelaic acid (41)
(0.51 g, 2.54 mmol) was added to an oven-dried 100 mL round-
bottom flask equipped with a stir bar and a rubber septum.
N,N-Dimethylformamide (25 mL) was added to the flask. An
inert argon atmosphere was introduced in the reaction flask.
Potassium carbonate (3.59 g, 26.0 mmol) was added to the
flask. Iodomethane (0.45 mL, 7.2 mmol) was then added to
the flask via syringe. The reaction solution was stirred at
ambient temperature for 24 h. Water (75 mL) was added to
the reaction flask. The reaction solution was added to a
separatory funnel, and both water (100 mL) and ethyl acetate
(100 mL) were added. The aqueous layer was separated and
extracted with ethyl acetate (3 × 150 mL). The combined
organic extracts were washed with both water (3 × 200 mL)
and brine (2 × 100 mL) and dried over anhydrous sodium
sulfate. The organic extracts were filtered, and the solvent was
removed in vacuo to yield a white solid (0.217 g, 37%): mp
1
33-34 °C (lit.38 mp 29-30 °C); H NMR (300 MHz, CDCl3) δ
3.64 (s, 6 H), 2.45 (t, J ) 7.21 Hz, 4 H), 2.32 (t, J ) 7.23 Hz,
4 H), 1.87 (qn, J ) 7.18 Hz, 4 H).
5-Br om o-3-ch lor osa licyla ld eh yd e (44). 4-Bromo-2-chlo-
rophenol (43) (5.40 g, 26.0 mmol) was added to a 100 mL
Teflon-coated bomb apparatus. Trifluoroacetic acid (20 mL)
was added to the reaction vessel. Hexamethylenetetramine
(7.41 g, 52.9 mmol) was then added to the bomb apparatus.
The bomb apparatus was placed in a preheated oven at 90 °C
for 20 h. The reaction mixture was cooled to ambient temper-
ature, and water (30 mL) was added. An aqueous 50% sulfuric
acid solution (14 mL) was added, and the reaction solution was
stirred for an additional 2 h at ambient temperature. Water
(50 mL) was added, and yellow precipitate formed. The
precipitate was collected by vacuum filtration on a Bu¨chner
funnel. The crude yellow solid was then recrystallized from a
20% aqueous ethanol solution to yield yellow crystals (2.22 g,
1414, 1362, 1301, 1259, 1207, 1158, 1127, 1034, 937, 831 cm-1
;
1H NMR (300 MHz, CDCl3) δ 6.99 (d, J ) 9.04 Hz, 1 H), 6.92
(d, J ) 8.19 Hz, 1 H), 6.45 (d, J ) 2.33 Hz, 1 H), 6.42 (d, J )
2.38 Hz, 1 H), 6.40-6.35 (m, 2 H), 5.90 (t, J ) 7.29 Hz, 1 H),
3.79 (s, 3 H), 3.76 (s, 3 H), 3.71 (s, 3 H), 3.62 (s, 3 H), 3.60 (s,
3 H), 2.27 (t, J ) 7.49 Hz, 2 H), 2.01 (q, 7.27 Hz, 2 H), 1.71
(qn, J ) 7.55 Hz, 2 H); ESIMS, m/z (relative intensity) 401
(100, MH+). Anal. (C23H28O6) C, H.
5,5′-Dica r boxy-3,3′-d ich lor o-4,4′-d im eth oxyben zop h e-
n on e (38). 3,3′-Dichloro-4,4′-dimethoxy-5,5′-dimethoxycarbo-
nylbenzophenone (37) (0.58 g, 1.3 mmol) was added to a 250
mL round-bottom flask equipped with a condenser. The solid
was dissolved in methanol (100 mL). Potassium hydroxide
(0.75 g, 13.3 mmol) was added to the flask. The reaction
mixture was heated at reflux for 24 h. The reaction mixture
was added to a separatory funnel, and water (200 mL) and
dichloromethane (200 mL) were added. The aqueous layer was
separated and extracted with ethyl acetate (3 × 125 mL). The
combined organic extracts were dried over anhydrous magne-
sium sulfate and filtered. Solvents were removed in vacuo,
yielding a white solid (0.363 g, 67%): mp 194-195 °C (lit.37
1
36%): mp 100-102 °C (lit.39 mp 86 °C); H NMR (300 MHz,
CDCl3) δ 11.36 (s, 1 H), 9.83 (s, 1 H), 7.72 (d, J ) 2.25 Hz, 1
H), 7.61 (d, J ) 2.19 Hz, 1 H).
5-Br om o-3-ch lor o-2-m eth oxyben za ld eh yd e (45). 5-Bro-
mo-3-chlorosalicylaldehyde (44) (2.28 g, 9.68 mmol) was added
to a flame-dried 100 mL round-bottom flask equipped with a
stir bar and a rubber septum. N,N-Dimethylformamide (50
mL) was added to the flask. An inert argon atmosphere was
then introduced in the reaction flask. Potassium carbonate
(14.5 g, 105 mmol) was added to the flask. Iodomethane (0.900
mL, 14.5 mmol) was then added to the flask via syringe. The
reaction solution was stirred at ambient temperature for 22
h. Water (75 mL) was added to the reaction flask. A white
precipitate formed and was collected by vacuum filtration on
a Bu¨chner funnel. The solid was washed with water (500 mL).
The solid was then dissolved in dichloromethane (100 mL) and
added to a separatory funnel. Water (100 mL) was added, and
the aqueous and organic layers were separated. The organic
layer was washed with brine (100 mL) and dried over
anhydrous sodium sulfate. The organic extracts were then
filtered, and solvent was removed in vacuo to yield a white
solid (2.22 g, 92%): mp 96-98 °C; IR (film) 2881, 1690, 1466,
1421, 1216, 977, 872 cm-1; 1H NMR (500 MHz, CDCl3) δ 10.30
(s, 1 H), 7.87 (d, J ) 2.35 Hz, 1 H), 7.78 (d, J ) 2.36 Hz, 1 H),
4.01 (s, 3 H); ESIMS, m/z (relative intensity) 249/251/253 (85/
100/20, MH+). Anal. (C8H6BrClO2) C, H, Br.
1
mp 224-226 °C); H NMR (300 MHz, DMSO-d6) δ 7.94 (d, J
) 2.17, 2 H), δ 7.89 (d, J ) 2.17 Hz, 2 H), δ 3.93 (s, 6 H).
3,3′-Dich lor o-4,4′-d im eth oxy-5,5′-d i(m eth ylth ioca r bo-
n yl)b en zop h en on e (39). 5,5′-Dicarboxy-3,3′-dichloro-4,4′-
dimethoxybenzophenone (38) (1.02 g, 2.5 mmol) was added to
an oven-dried 10 mL round-bottom flask equipped with a
stirbar and a rubber septum. An inert argon atmosphere was
introduced into the flask. Thionyl chloride (10 mL) was then
added to the flask via syringe. The reaction mixture was
heated at reflux for 8 h. The reaction mixture was cooled to
ambient temperature, and benzene (10 mL) was added. Solvent
was removed in vacuo to yield a crude colorless oil. Additional
benzene (4 × 25 mL) was added to the crude oil and evaporated
to azeotrope any excess thionyl chloride. The crude oil was then
dissolved in benzene (10 mL), and sodium thiomethoxide (0.41
g, 5.8 mmol) was added to the flask. The suspension was
stirred at ambient temperature for 5 h. Water (10 mL) was
5-Br om o-3-ch lor o-2-m eth oxyben zoic Acid (46). 5-Bromo-
3-chloro-2-methoxybenzaldehyde (45) (3.78 g, 15.2 mol) was

ADAM Non-nucleoside Reverse Transcriptase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3159
added to a 100 mL round-bottom flask. Acetone (50 mL) and
water (10 mL) were added to the flask. The reaction mixture
was cooled to 0 °C in an ice bath. Potassium permanganate
(0.80 g, 17.0 mol) was then slowly added to the flask. The
reaction mixture was allowed to warm to ambient temperature
for 24 h. The inorganic salts were removed by filtration on a
Bu¨chner funnel and washed with acetone (400 mL). The
filtrate was concentrated in vacuo to yield a white solid. The
solid was dissolved in water (100 mL) and added to a
separatory funnel. Ethyl acetate (100 mL) was added to the
funnel. The aqueous layer was separated and acidified to pH
1.0 using concentrated hydrochloric acid. The white precipitate
(0.924 g, 23%) that formed was collected by vacuum filtration
using a Bu¨chner funnel: mp 177-179 °C; IR (film) 3073, 2955,
2660, 1710, 1679, 1468, 1416, 1305, 1238, 988 cm-1; 1H NMR
(300 MHz, acetone-d6) δ 7.87 (d, J ) 2.50 Hz, 1 H), 7.85 (d, J
) 2.50 Hz, 1 H), 3.92 (s, 3 H); ESIMS, m/z (relative intensity)
263/265/267 (65/100/20, M-). Anal. (C8H6BrClO3) C, H.
1156, 1007, 875, 849, 795 cm-1; 1H NMR (300 MHz, CDCl3) δ
7.76 (d, J ) 2.58 Hz, 1 H), 7.47 (d, J ) 2.58 Hz, 1 H), 3.91 (s,
3 H), 3.81 (s, 3 H), 2.30 (s, 3 H); CIMS, m/z (relative intensity)
259/261 (100/97, MH+). Anal. (C10H11BrO3) C, H.
Meth yl 6-[4-Meth oxy-5-m eth oxycar bon yl-3-m eth ylph en -
yl]-6-(tr ibu tylsta n n yl)h ex-5-en oa te (51). Cuprous iodide
(0.50 g, 2.6 mmol) and dichlorobis(triphenylphosphine)-
palladium(II) (1.7 g, 2.4 mmol) were added to a flame-dried
100 mL round-bottom flask under an argon atmosphere.
Triethylamine (75 mL) was added to the flask. Methyl 5-bromo-
2-methoxy-3-methylbenzoate (50) (3.05 g, 11.8 mmol) and
methyl 5-hexynoate (3.08 g, 24.4 mmol) were then added to
the flask. The round-bottom flask was equipped with
a
condenser, and the reaction mixture was heated at reflux for
24 h. The reaction mixture was cooled, and solvent was
removed in vacuo to yield a crude black oily solid. The crude
solid was purified by flash column chromatography using silica
gel (125 g, 5 × 15 cm) and a gradient eluant from 0 to 10%
ethyl acetate in hexanes. Like fractions were combined, and
solvent was removed in vacuo to yield a crude red oil. The
crude oil was purified a second time using silica gel (125 g, 5
× 15 cm) and a gradient eluant from 0 to 8% ethyl acetate in
hexanes. Like fractions were combined, and solvent was
removed in vacuo to yield a crude yellow oil. The crude oil was
dissolved in dry THF (200 mL) and added to a flame-dried 500
mL round-bottom flask under an argon atmosphere. Tetrakis-
(triphenylphoshpine)palladium(0) (0.20 g, 0.17 mmol) was
added to the flask. Tributyltin hydride (2.37 mL, 8.14 mmol)
was added dropwise via syringe to the round-bottom flask over
a 5 min period. The reaction solution was stirred at ambient
temperature for 1.5 h. Solvent was removed in vacuo to yield
a crude black oil. The oil was purified by flash column
chromatography using silica gel (75 g, 3 × 38 cm) and a
gradient eluant from 0 to 10% ethyl acetate in hexanes. Like
fractions were combined, and solvent was removed in vacuo
to yield the product 5125 as a colorless oil (2.39 g, 49%, over
two steps): 1H NMR (500 MHz, CDCl3) δ 7.15 (d, J ) 2.06
Hz, 1 H), 6.85 (d, J ) 2.06 Hz, 1 H), 5.69 (t, J ) 6.91 Hz, 1 H),
3.87 (s, 3 H), 3.79 (s, 3 H), 3.60 (s, 3 H), 2.26 (s, 3 H), 2.23 (t,
J ) 7.68 Hz, 2 H), 2.04 (q, J ) 7.17 Hz, 2 H), 1.66 (qn, J )
7.46 Hz, 2 H), 1.45-1.20 (m, 11 H), 0.86-0.69 (m, 16 H).
Met h yl 5-Br om o-3-ch lor o-2-m et h oxyb en zoa t e (47).
5-Bromo-3-chloro-2-methoxybenzoic acid (46) (1.75 g, 6.59
mmol) was added to an oven-dried 250 mL round-bottom flask
equipped with a stirbar and rubber septum. N,N-Dimethyl-
formamide (100 mL) was added to the flask. An inert argon
atmosphere was introduced in the reaction flask. Potassium
carbonate (9.62 g, 69.6 mmol) was added to the flask. Iodo-
methane (0.41 mL, 6.6 mmol) was then added to the flask via
syringe. The reaction mixture was stirred at ambient temper-
ature for 24 h. Water (200 mL) was added, and a white
precipitate formed. The precipitate was collected by vacuum
filtration on a Bu¨chner funnel. The solid was washed with
water (400 mL). The solid was dissolved in ethyl acetate (300
mL) and added to a separatory funnel. Water (200 mL) was
added to the separatory funnel, and the aqueous layer was
separated from the organic layer. The organic layer was
washed with water (3 × 150 mL) and brine (2 × 100 mL) and
dried over anhydrous sodium sulfate. The organic layer was
filtered and solvent removed in vacuo to yield a white solid
(0.910 g, 49%): mp 37-39 °C; IR (film) 2950, 1738, 1466, 1416,
1282, 1240, 997 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.80 (d, J
) 2.49 Hz, 1 H), 7.66 (d, J ) 2.48 Hz, 1 H), 3.91 (s, 3 H), 3.90
(s, 3 H); GC-MS, m/z (relative intensity) 279/281/283 (80/100/
24, MH+). Anal. (C9H8BrClO3) C, H.
Meth yl 6-(3,4-Dim eth oxyp h en yl)h ex-5-yn oa te (53). Cu-
prous iodide (0.56 g, 3.0 mmol) and dichlorobis(triphenylphos-
phine)palladium(II) (1.0 g, 1.5 mmol) were added to a flame-
dried 100 mL round-bottom flask under an argon atmosphere.
Triethylamine (50 mL) was added to the flask. 4-Bromovera-
trole (52) (1.2 mL, 8.3 mmol) and methyl 5-hexynoate (2.10 g,
16.6 mmol) were then added to the flask. The round-bottom
flask was equipped with a condenser, and the reaction mixture
was heated at reflux for 8 h. The reaction mixture was cooled
and solvent was removed in vacuo to yield a crude black oily
solid. The crude solid was purified by flash column chroma-
tography using silica gel (125 g, 5 × 14 cm) and a gradient
eluant from 0 to 20% ethyl acetate in hexanes. Like fractions
were combined, and solvent was removed in vacuo to yield a
brown oily solid. The solid was purified a second time using
silica gel (50 g, 3 × 22 cm) and a gradient eluant from 0 to
10% ethyl acetate in hexanes. Like fractions were combined,
and solvent was removed in vacuo to yield a brown solid (0.620
g, 28%): mp 53-55 °C; IR (film) 2952, 1736, 1513, 1440, 1208,
5-Br om o-3-m eth ylsa licylic Acid (49). 3-Methylsalicylic
acid (48) (2.06 g, 13.6 mmol) was added to an oven-dried 100
mL round-bottom flask equipped with a stir bar and rubber
septum. Acetic acid (30 mL) was added to the flask. Bromine
(0.700 mL, 13.7 mmol) was added to the flask via syringe. The
contents of the flask were stirred at ambient temperature for
24 h. Water (30 mL) was added to the flask, and a white
precipitate formed. The white precipitate was collected by
vacuum filtration on a Bu¨chner funnel (2.51 g, 80%): mp 236-
1
237 °C (lit.40 mp 231-232 °C); H NMR (300 MHz, acetone-
d6) δ 7.84 (d, J ) 2.55 Hz, 1 H), 7.55 (d, J ) 2.45 Hz, 1 H),
2.22 (s, 3 H).
Met h yl 5-Br om o-2-m et h oxy-3-m et h ylb en zoa t e (50).
5-Bromo-3-methylsalicylic acid (49) (2.12 g, 9.19 mmol) was
added to an oven-dried 100 mL round-bottom flask equipped
with a stirbar and a rubber septum. N,N-Dimethylformamide
(50 mL) was added to the flask. An inert argon atmosphere
was then introduced in the reaction flask. Potassium carbonate
(12.9 g, 93.4 mmol) was added to the flask. Iodomethane (1.5
mL, 24 mmol) was then added to the flask via syringe. The
reaction solution was stirred at ambient temperature for 26
h. Water (50 mL) was added to the reaction flask. A white
precipitate formed and was collected by vacuum filtration on
a Bu¨chner funnel. The solid was washed with water (400 mL).
The solid was then dissolved in dichloromethane (200 mL) and
added to a separatory funnel. Water (200 mL) was added to
the separatory funnel, and the aqueous layer was separated
from the organic layer. The organic layer was washed with
brine (100 mL) and dried over anhydrous sodium sulfate. The
organic layer was then filtered and solvent removed in vacuo
to yield a white solid (2.06 g, 86%): mp 59-60 °C; IR (film)
2951, 1731, 1578, 1470, 1434, 1417, 1293, 1251, 1225, 1194,
1
1169, 1025, 762 cm-1; H NMR (300 MHz, CDCl3) δ 6.97 (dd,
J ) 1.75 and 8.25 Hz, 1 H), 6.88 (d, J ) 1.68 Hz, 1 H), 6.75 (d,
J ) 8.28 Hz, 1 H), 3.85 (s, 6 H), 3.66 (s, 3 H), 2.49 (t, J ) 7.40
Hz, 2 H), 2.45 (t, J ) 6.90 Hz, 2 H), 1.91 (qn, J ) 7.18 Hz, 2
H); GC-MS, m/z (relative intensity) 263 (100, MH+). Anal.
(C15H18O4) C, H.
Met h yl 6-(3,4-Dim et h oxyp h en yl)-7,7-d ib u t yl-7-st a n -
n a u n d ec-5-en oa te (54). Methyl 6-(3′,4′-dimethoxyphenyl)-
hex-5-ynoate (53) (0.1 g, 0.4 mmol) was added to a flame-dried
100 mL round-bottom flask under an argon atmosphere. The
solid was dissolved in dry THF (40 mL). Tetrakis(tri-
phenylphoshpine)palladium(0) (0.02 g, 0.02 mmol) was added
to the flask. Tributyltin hydride (0.17 mL, 0.63 mmol) was
added dropwise via syringe to the round-bottom flask over a

3160 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Silvestri et al.
5 min period. The reaction solution was stirred at ambient
temperature for 2 h. Solvent was removed in vacuo to yield a
crude black oil. The oil was purified by flash column chroma-
tography using silica gel (10 g, 2 × 7 cm) and a gradient eluant
from 0 to 10% ethyl acetate in hexanes. Like fractions were
combined, and solvent was removed in vacuo to yield a
colorless oil (0.101 g, 43%); IR (film) 2954, 2927, 1740, 1508,
1463, 1439, 1252, 1234, 1136, 1030 cm-1; 1H NMR (300 MHz,
CDCl3) δ 6.76 (d, J ) 8.63 Hz, 1 H), 6.44-6.41 (m, 2 H), 5.67
(t, J ) 6.95 Hz, 1 H), 3.84 (s, 3 H), 3.83 (s, 3 H), 3.60 (s, 3 H),
2.23 (t, J ) 7.46 Hz, 2 H), 2.05 (q, J ) 7.32 Hz, 2 H), 1.67 (qn,
J ) 7.39 Hz, 2 H), 1.47-1.19 (m, 11 H), 1.0-0.70 (m, 16 H);
ESIMS, m/z (relative intensity) 551/553/555 (44/78/100, MH+).
Anal. (C27H46O4Sn) C, H.
3,3′,4,4′-Tetr a m eth oxyben zop h en on e (57). Veratrole (55)
(0.64 mL, 5.0 mmol) and 3,4-dimethoxybenzoyl chloride (56)
(1.0 g, 5.1 mmol) were added to a flame-dried two-neck 25 mL
round-bottom flask equipped with a condenser, magnetic stir
bar, and rubber septum. An argon atmosphere was introduced,
and dry dichloromethane (10 mL) was added via syringe.
Aluminum chloride (0.92 g, 6.87 mmol) was slowly added in
three equal portions. The reaction solution was then heated
at reflux in an oil bath for 2 h. The reaction solution was cooled
to ambient temperature and poured over ice (5 mL) and 1 M
HCl (5 mL). This solution was added to a separatory funnel,
and water (100 mL) and dichloromethane (100 mL) were
added. The aqueous layer was separated and extracted with
dichloromethane (3 × 50 mL). The combined extracts were
washed with brine (100 mL) and dried over anhydrous sodium
sulfate. The extracts were then filtered, and solvent was
removed in vacuo to yield a crude white solid. The oily solid
was recrystallized from ethanol to yield a pure white solid
(0.990 g, 65%): mp 143-144 °C (lit.41 mp 142-144 °C); 1H
NMR (300 MHz, CDCl3) δ 7.38 (d, J ) 1.69 Hz, 2 H), 7.34 (dd,
J ) 1.71, 7.19 Hz, 2 H), 6.92 (d, J ) 7.23 Hz, 2 H), 4.37 (s, 6
H), 4.36 (s, 6 H).
effect of test compound on uninfected cells in order to assess
the cytotoxicity of the test compound. Exponentially growing
MT-4 cells46 were centrifuged for 5 min at 1000 rpm, and the
supernatant was discarded. The MT-4 cells were resuspended
at 6 × 105 cells/mL, and 50 µL volumes were transferred to
the microtiter tray wells. Five days after infection, the viability
of mock- and HIV-infected cells was examined spectrophoto-
metrically in the MTT assay.
The MTT assay is based on the reduction of yellow 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Acros Organics, Geel, Belgium) by mitochondrial dehydroge-
nase of metabolically active cells to a blue-purple formazan
that can be measured spectrophotometrically. The absorbances
were read in an eight-channel computer-controlled photometer
(Multiscan Ascent Reader, Labsystems, Helsinki, Finland), at
two wavelengths (540 and 690 nm). All data were calculated
using the median OD (optical density) value of three wells.
The 50% cytotoxic concentration (CC50) was defined as the
concentration of the test compound that reduced the absor-
bance (OD540) of the mock-infected control sample by 50%.
The concentration achieving 50% protection from the cyto-
pathic effect of the virus in infected cells was defined as the
50% effective concentration (EC50).
In Vitr o Ra t P la sm a Sta bility Assa y. The alkenyldiaryl-
methanes were tested for their hydrolytic stability, utilizing
rat plasma in vitro using methods as previously described.47-54
The alkenyldiarylmethanes (4.6-15.8 mg) and 1,1-diphenyl-
ethylene (1.6-4.3 µL), an internal standard, were dissolved
in DMSO (1.0 mL). This solution was filtered through a 0.45
µM filter (Millex-HN). This solution became the “stock solu-
tion” of the alkenyldiarylmethane and 1,1-diphenylethylene.
Lyophilized rat plasma (1.0 mL) (lot 119H7505, Sigma Chemi-
cal Co., St. Louis, MO) was reconstituted with Milli-Q water
(1.0 mL). This plasma solution was incubated at 37 °C for 15
min. The plasma solution was diluted with 0.01 M phosphate-
buffered saline (0.250 mL), pH 7.0, to afford an 80% plasma
solution. The solution was incubated at 37 °C for an additional
5 min. An aliquot (100 µL) of the stock solution was added to
the rat plasma (1.0 mL, 80% plasma). The final concentrations
for the drug in the plasma solution ranged from 0.8 to 2.2 mM.
Aliquots (10 µL) of this solution were then collected at various
time intervals and diluted with acetonitrile (90 µL) to precipi-
tate any protein. The aliquots were thoroughly mixed and then
centrifuged at 10000 rpm for 5 min to pellet the precipitated
protein. After centrifugation, the supernatant (20 µL) of these
aliquots was analyzed via HPLC to determine the residual
amount of alkenyldiarylmethane. The HPLC system consisted
of a Rainin solvent delivery system (model HPXL), a Dynamax
absorbance detector (model UV-1) set at a wavelength of 254
nM, and a Rheodyne injector (model 7125) with a 20 µL
injection loop. Data were collected and processed using the
Rainin Dynamax HPLC controller and data acquisition soft-
ware (version 1.4) on an Apple Computer, Inc. 7600 Power
Macintosh personal computer. The mobile phase consisted of
either 75:25 (v/v %) acetonitrile/water or 80:20 (v/v %) aceto-
nitrile/water. The mobile phase flow rate was 1.0 mL/min, and
the HPLC column (150 mm × 4.6 mm) was packed with 5 µm
Kromasil C18 from Phenomenex (Torrance, CA). A Security
Guard C18 (ODS, octadecyl, 4 mm × 3 mm) column from
Phenomenex preceded the analytical column. The column
temperature was maintained at ambient temperature.
2,2′,4,4′-Tetr a m eth oxyben zop h en on e (59). 2,2′,4,4′-Tet-
rahydroxybenzophenone (58) (2.0 g, 8.1 mmol) was added to
an oven-dried 250 mL round-bottom flask equipped with a
stirbar and rubber septum. N,N-Dimethylformamide (100 mL)
was added to the flask. An inert argon atmosphere was then
introduced in the reaction flask. Potassium carbonate (11.7 g,
85.0 mmol) was added to the flask. Iodomethane (2.5 mL, 40
mmol) was then added to the flask via syringe. The reaction
solution was stirred at ambient temperature for 24 h. Water
(300 mL) was added to the reaction flask. A white precipitate
formed and was collected by vacuum filtration on a Bu¨chner
funnel. The solid was washed with water (400 mL). The solid
was then dissolved in ethyl acetate (200 mL) and added to a
separatory funnel. Water (200 mL) was added to the separa-
tory funnel, and the aqueous layer was separated from the
organic layer. The organic layer was washed with brine (200
mL) and dried over anhydrous sodium sulfate. The organic
layer was then filtered and solvent removed in vacuo to yield
an orange-white solid (1.93 g, 79%): mp 135-139 °C (lit.42 mp
1
135-136 °C); H NMR (300 MHz, CDCl3) δ 7.45 (d, J ) 7.39
Hz, 2 H), 6.58 (dd, J ) 1.95, 7.40 Hz, 2 H), 6.51 (d, J ) 1.91
Hz, 2 H), 4.29 (s, 6 H), 4.12 (s, 6 H).
In Vitr o An tivir a l Assa ys. Evaluation of the antiviral
activity of the compounds against HIV-1 strain IIIB and HIV-2
strain (ROD) in MT-4 cells was performed using the MTT
assay as previously described.43 Stock solutions (10 × final
concentrations) of test compounds were added in 25 µL
volumes to two series of triplicate wells so as to allow
simultaneous evaluation of their effects on mock- and HIV-
infected cells at the beginning of each experiment. Serial 5-fold
dilutions of test compounds were made directly in flat-bottom
96-well microtiter trays using a Biomek 2000 robot (Beckman
Instruments, Fullerton, CA). Untreated control HIV- and
mock-infected cell samples were included for each sample.
Ack n ow led gm en t. This investigation was made
possible by Grant RO1-AI-43637, awarded by the Na-
tional Institutes of Health, DHHS, and support from the
Geconcerteerde Onderzoeksacties (GOA-00/12) and ISEP/
FORTIS. We are grateful to Liesbet De Dier, Kristien
Erven, and Cindy Heens for excellent technical as-
sistance.
HIV-1(IIIB)44 or HIV-2 (ROD)45 stock (50 µL) at 100-300
CCID50 (50% cell culture infectious dose) or culture medium
was added to either the infected or mock-infected wells of the
mictotiter tray. Mock-infected cells were used to evaluate the
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
of compounds 4, 20-29, 31-33, 39, 45-47, 50, 53, and 54.
This material is available free of charge via the Internet at
http://pubs.acs.org.

ADAM Non-nucleoside Reverse Transcriptase Inhibitors
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