Solid-phase synthesis of the alkenyldiarylmethane (ADAM) series of non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jo0100291

The Sonogashira and Stille cross-coupling reactions have been employed in the synthesis of several non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the alkenyldiarylmethane (ADAM) series. The synthesis has been carried out both in solution and on a solid support. In contrast to previous syntheses of NNRTIs in the ADAM series, the present strategy allows the incorporation of differently substituted aromatic rings in a stereochemically defined fashion. The most potent of the new ADAMs inhibited the cytopathic effect of HIV-1_RF in CEM-SS cell culture with an EC₅₀ value of 20 nM.

Full text of DOI:10.1021/jo0100291

5958
J . Org. Chem. 2001, 66, 5958-5964
Articles
Solid -P h a se Syn th esis of th e Alk en yld ia r ylm eth a n e (ADAM) Ser ies
of Non -Nu cleosid e HIV-1 Rever se Tr a n scr ip ta se In h ibitor s
Guozhang Xu,† Tracy L. Loftus,‡ Heather Wargo,‡ J im A. Turpin,‡
Robert W. Buckheit, J r.,‡ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal
Sciences, Purdue University, West Lafayette, Indiana 47907, and Infectious Disease Research Department,
Southern Research Institute, 431 Aviation Way, Frederick, Maryland 21701
cushman@pharmacy.purdue.edu
Received J anuary 10, 2001
The Sonogashira and Stille cross-coupling reactions have been employed in the synthesis of several
non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the alkenyldiarylmethane (ADAM)
series. The synthesis has been carried out both in solution and on a solid support. In contrast to
previous syntheses of NNRTIs in the ADAM series, the present strategy allows the incorporation
of differently substituted aromatic rings in a stereochemically defined fashion. The most potent of
the new ADAMs inhibited the cytopathic effect of HIV-1RF in CEM-SS cell culture with an EC50
value of 20 nM.
The alkenyldiarylmethanes (ADAMs) are a class of
a need exists for additional NNRTIs that might display
novel resistance mutation profiles, more favorable phar-
macokinetic properties, and limited toxicities. We have
therefore continued to design and synthesize additional
NNRTIs in the ADAM series.
HIV-1 non-nucleoside reverse transcriptase inhibitors
(NNRTIs).1-5 Certain ADAMs (e.g., ADAM 1) have been
found to inhibit of the cytopathic effect of HIV-1 in cell
culture at low nanomolar concentrations.4,5 A number of
HIV-1 strains containing AZT resistance mutations have
shown increased sensitivity to some of the ADAMs,
indicating a possible therapeutic role for the ADAMs in
combination with AZT.4 In general, the clinical use of
NNRTIs in combination with two additional anti-HIV
agents decreases HIV-1 RNA levels, increases CD4
lymphocyte counts, and delays disease progression. How-
ever, several factors continue to limit the selection of
NNRTIs for combination chemotherapy, including drug
incompatibilities, adverse effects, the emergence of re-
sistant viral strains, and cross-resistance.6-10 Therefore,
The previously existing ADAMs (e.g., 1) have been
synthesized by attaching the alkenyl chain to a sym-
metrical benzophenone (e.g., 2) by Wittig,1-4 McMurry,5
or Horner-Emmons4 reactions. Since these methods
proceed from the readily available, symmetrical ben-
zophenones, they are not ideal for preparing ADAMs
having nonidentical aromatic substituents. In addition,
even if unsymmetrical aromatic benzophenones were
used, these methods would be complicated by generation
of cis and trans double bond isomers that would have to
be separated and defined structurally. We have therefore
sought to establish alternative syntheses of the ADAMs
that would allow for the construction of compounds
having nonidentical aromatic substituents with stereo-
chemically defined relationships to the alkenyl side chain.
Another goal of the present research project was to
adapt the resulting solution-phase synthesis to the
solid phase, which could eventually allow automation and
the preparation of combinatorial libraries for lead
optimization.11-13
* Ph: 765-494-1465. Fax: 765-494-6790.
† Purdue University.
‡ Southern Research Institute.
(1) Cushman, M.; Golebiewski, M.; Buckheit, R. W. J .; Graham, L.;
Rice, W. G. Bioorg. Med. Chem. Lett. 1995, 5, 2713-2716.
(2) Cushman, M.; Casimiro-Garcia, A.; Williamson, K.; Rice, W. G.
Bioorg. Med. Chem. Lett. 1998, 8, 195-198.
(3) Cushman, M.; Golebiewski, W. M.; Graham, L.; Turpin, J . A.;
Rice, W. G.; Fliakas-Boltz, V.; Buckheit, R. W. J . J . Med. Chem. 1996,
39, 3217-3227.
(4) Cushman, M.; Casimiro-Garcia, A.; Hejchman, E.; Ruell, J . A.;
Huang, M.; Schaeffer, C. A.; Williamson, K.; Rice, W. G.; Buckheit, R.
W. J . J . Med. Chem. 1998, 41, 2076-2089.
(5) Casimiro-Garcia, A.; Micklatcher, M.; Turpin, J . A.; Stup, T. L.;
Watson, K.; Buckheit, R. W. J .; Cushman, M. J . Med. Chem. 1999, 42,
4861-4874.
(6) Carpenter, C. C. J .; Fischl, M. A.; Hammer, S. M.; Hirsch, M.
S.; J acobsen, D. M.; Katzenstein, D. A.; Montaner, J . S. G.; Richman,
D. D.; Saag, M. S.; Schooley, R. T.; Thompson, M. A.; Vella, S.; Yeni,
P. G.; Volberding, P. A. J . Am. Med. Assoc. 1998, 280, 78-86.
(7) Barry, M.; Mulcahy, F.; Back, D. J . Br. J . Clin. Pharmacol. 1998,
45, 221-228.
(8) Fischl, M. A. AIDS 1999, 13(Suppl. 1), S49-S59.
(9) Vajpayee, M.; Dar, L. Indian J . Pharmacol. 1999, 31, 96-103.
(10) Beach, J . W. Clin. Ther. 1998, 20, 2-25.
10.1021/jo0100291 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/24/2001

ADAM Series of NNRTIs
Sch em e 1
J . Org. Chem., Vol. 66, No. 18, 2001 5959
Sch em e 2a
a
Reagents and conditions: (a) TMSCHN2/MeOH, benzene, 23
°C (2 h); (b) NaI, NaOH/MeOH, 0-3 °C (3 h) then NaOCl (5.25%);
(c) dimethyl sulfate, K2CO3, acetone, reflux (24 h); (d) Bu3Sn-
SnBu3, Pd(PPh3)4, Pd(OAc)2, triethylamine, 95-100 °C (3 h); (e)
N-methyl 5-hexynoamide, (Ph3P)2Pd(OAc)2, CuI, triethylamine,
EtOAc, 23 °C (14 h); (f) Bu3SnH, Pd(PPh3)4, THF, 23 °C (14 h); (g)
I2, CH2Cl2, 23 °C (20 min).
The application of palladium-catalyzed reactions for
C-C bond formation is particularly attractive for solid-
phase combinatorial library syntheses since the condi-
tions employed are mild and many functional groups are
compatible.14-16 The present article describes the applica-
tion of the Sonogashira and Stille cross-coupling reactions
for the solution and solid-phase synthesis of stereochemi-
cally defined ADAMs having differently substituted
aromatic rings. As shown by the retrosynthetic analysis
described in Scheme 1, Stille coupling of 4 and 5 (X and
Y ) I or SnBu3) would be expected to result in the ADAM
3. The coupling partner 5 (in the case of Y ) SnBu3)
could in turn be made by the Sonogashira reaction of the
alkyne 6 and the aryl iodide 7, followed by hydrostan-
nation. Since the Stille reaction proceeds with retention
of the stereochemical integrity of the coupling partners,
and hydrostannation of alkynes results in regiochemically
defined cis addition to the triple bond, the stereochem-
istry (cis vs trans) of the side chain relative to the two
aromatic rings in the final product would be predictable
(i.e., the side chain would be cis to the aromatic ring
containing R2 in structure 3).
TMSCHN2 in a mixture of MeOH and benzene.17 The
methyl ester was then iodinated with sodium iodide in
the presence of sodium hypochlorite as an oxidant.18
Treatment of the iodinated ester 9 with dimethyl sulfate
and potassium carbonate in refluxing acetone produced
methyl 5-iodo-3-methyl-2-methoxybenzoate 10 in 90%
overall yield. The tributyltin derivative 11 was synthe-
sized by heating a mixture of iodo ester 10, bis(tributyl-
tin), tetrakis(triphenylphosphine)palladium, and palla-
dium acetate in triethylamine at 95-100 °C overnight.18
The Sonogashira reaction of N-methyl 5-hexynoamide
with iodo ester 10 afforded alkyne 12,19,20 which under-
went hydrostannation in the presence of Pd(PPh3)4 to
afford the regio- and stereodefined vinylstannane 13 in
87% yield.21,22 Subsequent treatment of 13 with I2 in
CH2Cl2 produced vinyl iodide 14.23
Our initial attempts to prepare ADAM 15 (Scheme 3),
by employing vinyl tributylstanne 13 and aryl iodide 10
(or the alternative combination, vinyl iodide 14 and aryl
Resu lts a n d Discu ssion
(16) Desphande, M. S. Tetrahedron Lett. 1994, 35, 5613-5614.
(17) Hishimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981,
29, 1475-1478.
As shown in Scheme 2, 3-methylsalicylic acid (8) was
quantitatively converted into its methyl ester using
(18) Chumpradit, S.; Kung, M. P.; Mach, R.; Kung, H. F. J . Med.
Chem. 1993, 36, 221-228.
(11) Ellman, J . A. Acc. Chem. Res. 1996, 29, 132-143.
(12) Gallop, M. A.; Barrett, R. W.; Dower, R. W.; Fodor, S. P. A.;
Gordon, E. M. J . Med. Chem. 1994, 37, 1233-1251.
(13) Terrett, N. K.; Gardner, M.; Gordon, D. W.; Kobylecki, R. J .;
Steele, J . Tetrahedron 1995, 51, 8135-8173.
(14) Fagnola, M. C.; Candiani, I.; Visentin, G.; Cabri, W.; Zarini,
F.; Mongelli, N.; Bedeschi, A. Tetrahedron Lett. 1997, 39, 2307-2310.
(15) Dyatkin, A. B.; Rivero, R. A. Tetrahedron Lett. 1998, 39, 3647-
3650.
(19) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett.
1975, 4467-4470.
(20) Hay, L. A.; Koenig, T. M.; Ginah, F. O.; Copp, J . D.; Mitchell,
D. J . Org. Chem. 1998, 63, 5050-5058.
(21) Zhang, H. X.; Guibe´, F.; Balavoine, G. J . Org. Chem. 1990, 55,
1857-1867.
(22) Huang, X.; Zhong, P. J . Chem. Soc., Perkin Trans. 1 1999,
1543-15445.
(23) Piers, E.; Coish, P. D. Synthesis 1995, 47-55.

5960 J . Org. Chem., Vol. 66, No. 18, 2001
Xu et al.
Sch em e 3
Sch em e 4a
tributylstanne 11) in the presence of Pd(PPh3)4, were
unsuccessful because of the low yield of the coupling
reaction.24 In general, densely substituted stannanes
reacts poorly, and their coupling must be carefully
optimized. Considering the Stille coupling catalytic cycle,
a better combination of catalyst, ligand, additive, and
solvent might be beneficial to the cross-coupling reac-
tion.25 It has been shown that ligands with donicity
toward Pd(II) lower than that of PPh3 (i.e., AsPh3) can
lead to major (up to 1000-fold) rate enhancement in the
transmetalation. Besides the ligand, the addition of
fluoride might lead to more efficient coupling, perhaps
by facilitating transmetalation from tin to palladium,
since tin is fluorophilic.26-28 The cross-coupling reactions
of organosilicon compounds are known to be facilitated
by fluoride.29-32 Under the “optimized” conditions (Pd2-
(dba)3/AsPh3/CsF/DMF), Stille cross-coupling of vinyl
tributylstannane 13 and aryl ioide 10 (or vinyl iodide 14
and aryl tributylstannane 11) afforded 15 in 74% (or 80%)
isolated yield. The reactions proceeded exclusively with
the desired ipso substitution, and none of the undesired
cine substitution products were detected in the reaction
mixtures.
(24) Hirama, M.; Fujiwara, K.; Shigematu, K.; Fukazawa, Y. J . Am.
Chem. Soc. 1989, 111, 4120-4122.
(25) Duncton, M. A. J .; Pattenden, G. J . Chem. Soc., Perkin Trans.
1 1999, 1235-1246.
a
Reactions and conditions: (a) Dimethyl sulfate, K2CO3, ac-
(26) Fouquet, E.; Pereyre, M.; Rodrigues, A. L. J . Org. Chem. 1997,
62, 5242-5243.
etone, reflux (24 h); (b) SO2Cl2, CH2Cl2, 50 °C (14 h); (c) aq. NaOH/
MeOH, 60 °C (3 h); (d) DCC, HOBt/DMF, 23 °C (24 h); (e) methyl
5-hexynoate, Pd(OAc)2, CuI, Et3N, DMF, 23 °C (12 h); (f) Bu3SnH,
Pd(PPh3)4, THF, 23 °C (4 h); (g) Et3N, MeOH (15:85, v/v), 50 °C
(16 h); (h) 10 or 17, Pd2(dba)3, AsPh3, CsF/DMF, 80 °C (12 h); (i)
NaOMe, MeOH, THF (1:2), reflux (20 h).
(27) Vedejs, E.; Haight, A. R.; Moss, W. O. J . Am. Chem. Soc. 1992,
114, 6556-6558.
(28) Littke, A. F.; Fu, G. C. Angew. Chem., Int. Ed. 1999, 38, 2411-
2413.
(29) Metal-Catalyzed Cross-Coupling Reactions; Hiyama, T., Diedrich,
F., Stang, P. J ., Eds.; Wiley-VCH: Weinheim, Germany, 1998; pp 421-
452.
Having explored the performance of the above reaction
sequence in solution, we went on to apply it on a solid
support. The building block 19 was prepared through a
three-step synthesis (Scheme 4). Methylation of both the
(30) Mowery, M. E.; DeShong, P. J . Org. Chem. 1999, 64, 1684-
1688.
(31) Hatanaka, Y.; Ebina, Y.; Hiyama, T. J . Am. Chem. Soc. 1991,
113, 7075-7076.
(32) Hatanaka, Y.; Hiyama, T. J . Org. Chem. 1988, 53, 918-920.

ADAM Series of NNRTIs
Ta ble 1. An ti-HIV-1 Activities of ADAMs
J . Org. Chem., Vol. 66, No. 18, 2001 5961
enzyme inhibition is less than the EC50 of 2.26 µM for
inhibition of the viral cytopathic effect, whereas for
ADAMs 26 and 27, the IC50 values of 0.516 and 0.587
µM are significantly greater than the EC50 values of 0.020
and 0.080 µM. However, our prior examination of the
ADAMs in cells containing mutant HIV-1 reverse tran-
scriptase leave little doubt that the cellular effects of
these compounds as inhibitors of cell killing by the virus
are in fact due to inhibition of reverse transcriptase.3-5
Moreover, the lack of correlation of the enzyme IC50
values determined with poly(rC)‚oligo(dT) as the tem-
plate primer and the EC50 values is not unusual for the
non-nucleoside reverse transcriptase inhibitors.34-37 The
discrepancy may simply reflect the difference between
the enzyme assay, in which a synthetic template/primer
is used, and the cellular system, in which the native viral
RNA serves as the template/primer.36
RT IC50
rCdGa
(µM)
EC50
MTS assayb
(µM)
CC50
MTS assayc
(µM)
compd
TId
AZT
15
26
27
28
0.005
2.26
0.020
0.080
0.013
>1.0
17.10
1.46
2.17
31.6
>201
7
73
27
2430
0.804
0.516
0.587
0.300
a
Inhibitory activity vs HIV-1 reverse transcriptase with rCdG
b
as the template:primer. The EC50 is the 50% inhibitory concen-
tration for cytopathicity of HIV-1RF in CEM-SS cells. c The CC50
is the 50% cytotoxic concentration for mock-infected CEM cells.
d
The TI is the therapeutical index, which is the CC50 divided by
the EC50
.
phenol and carboxylic acid groups of 16 using dimethyl
sulfate/K2CO3 in acetone gave methyl 5-iodo-2-methoxy-
benzoate 17, which was reacted with SO2Cl2 to result in
complete chlorination of the aromatic ring at the C3
position. The resulting ester 18 was then hydrolyzed in
aqueous NaOH to afford the key intermediate 19 in 95%
yield. Coupling of 19 with hydroxymethylpolystyrene
resin (Wang resin) under standard DCC coupling condi-
tions (DCC, 3 equiv; HOBt, 3 equiv in DMF) afforded the
desired support-bound ester 20.33 It was found that a
double-coupling protocol consistently provided much
higher loading of the acid onto the solid support [based
on elemental analysis (I or Cl) of the modified resin].
Palladium-catalyzed Sonogashira reaction of 20 with
5-hexynoate afforded the resin-bound alkyne 21.14,15 The
IR spectrum (KBr) of the derivatized resin 21 revealed a
new absorption at 2232 cm-1 (internal carbon-carbon
triple bond), indicating that the desired Sonogashira
coupling had occurred. The resin-bound alkyne 21 was
then treated with Bu3SnH in the presence of Pd(PPh3)4
to afford 22. The overall efficiency of Sonogashira and
hydrostannation reactions was determined to be 86% on
the basis of the yield of 23 obtained by basic cleavage
(transesterfication, Et3N/MeOH 15:85, v/v). The Stille
coupling of 22 with methyl 3-methyl-5-iodo-2-methoxy-
benzoate (10) or methyl 5-iodo-2-methoxybenzoate (17)
using Pd2(dba)3, AsPh3, and CsF in DMF afforded 24 and
25.16 Cleavage of 24 and 25 with sodium methoxide in
methanol and THF (40:60, v/v) afforded ADAMs 26 and
27, respectively.14 1H NMR analysis of crude products
cleaved from the resin showed purities ranging from 75%
to 85%. Both 26 and 27 were further purified by silica
gel flash chromatography (EtOAc/hexanes 1:3, v/v).
To evaluate the anti-HIV activities of ADAMs 15, 26,
and 27, all three compounds were tested for inhibition
of the cytopathic effect of HIV-1RF in CEM-SS lympho-
cytes, and the resulting EC50 values are displayed in
Table 1. In addition, these compounds were tested for
inhibition of HIV-1 reverse transcriptase with poly(rC)‚
oligo(dG) as the template primer, and the IC50 values are
also reported in Table 1. The cytotoxicities of the com-
pounds in uninfected CEM-SS cells were also investi-
gated, and the 50% cytotoxic concentrations (CC50 values)
are also shown.
Examination of the antiviral activities of compounds
26, 27, and the previously synthesized compound 284
allows an assignment of the effect of variation of the R
group as shown in structures 26 and 27. The relative
anti-HIV potencies are Cl > CH3 > H.
In conclusion, we have devised a new synthesis of
ADAMs that allows the incorporation of nonidentical aryl
groups in a stereochemically defined fashion. The syn-
thesis can be performed both in solution and on a solid
support. The greater flexibility of the present route
should allow the future synthesis of non-nucleoside
reverse transcriptase inhibitors in the ADAM series
having optimized biological activities.
Exp er im en ta l Section
Gen er a l. Melting points were determined in capillary tubes
on a Mel-Temp apparatus and are uncorrected. The proton
nuclear magnetic resonance spectra (1H NMR) of compounds
18, 19, 26, and 27 were recorded on a 500 MHz spectrometer,
and the 1H NMR spectra of the remaining compounds were
determined at 300 MHz. The chemical shift values are
expressed in ppm (parts per million) relative to tetramethyl-
silane as internal standard. The indicated J SnH must be
considered as an approximate mean value of J (117SnH) and
J (119SnH). The 13C NMR spectra were recorded at 75 MHz.
As a rule, mass spectra of alkenyltributylstannanes are
characterized by the presence of an important peak (often the
(34) Pauwels, R.; Andries, K.; Desmyter, J .; Schols, D.; Kukla, M.
J .; Breslin, H. J .; Raeymaeckers, A.; Van Gelder, J .; Woestenborghs,
R.; Heykants, J .; Schellekens, K.; J anssen, M. A. C.; De Clercq, E.;
J anssen, P. A. J . Nature 1990, 343, 470-474.
(35) Baba, M.; De Clercq, E.; Tanaka, H.; Ubasawa, M.; Takashima,
H.; Sekiya, K.; Nitta, I.; Umezu, K.; Nakashima, H.; Mori, S.; Shigeta,
S.; Walker, R. T.; Miyasaka, T. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
2356-2360.
(36) Debyser, Z.; Pauwels, R.; Andries, K.; Desmyter, J .; Kukla, M.;
J anssen, P. A. J .; De Clercq, E. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
1451-1455.
(37) Balzarini, J .; Pe´rez-Pe´rez, M.-J .; San-Fe´lix, A.; Camarasa, M.-
J .; Bathurst, I. C.; Barr, P. J .; De Clercq, E. J . Biol. Chem. 1992, 267,
11831.
It is clear from examination of the numbers in Table 1
that there is not an exact correlation between the IC50
values for inhibition of HIV-1 reverse transcriptase and
inhibition of the cytopathic effect of the virus. For
example, for compound 15, the IC50 of 0.804 µM for
(33) Devraj, R.; Cushman, M. J . Org. Chem. 1996, 61, 9368-9373.

5962 J . Org. Chem., Vol. 66, No. 18, 2001
Xu et al.
base peak) at M+ - 57, which corresponds to the loss of an
n-butyl fragment. The M+ peak was, in the case of 23, not
detected. Microanalyses were performed at the Purdue Mi-
croanalysis Laboratory, and all values were within 0.4% of the
calculated compositions. Silica gel used for column chroma-
tography was 230-400 mesh.
under reduced pressure. The resulting residue was dissolved
in dry THF (10 mL) and cooled to 0 °C. Methylamine (10 mL
of 2 M methylamine in THF) and triethylamine (0.9 g, 8.92
mmol) were added. The mixture was stirred for 2 h and then
washed with water (2 × 5 mL). The organic solvent was
evaporated, and the crude residue was further purified by flash
chromatography on silica gel (40 g, column 1.5 cm × 31 cm;
eluent, ethyl acetate) to give N-methyl-5-hexynoamide40 as a
light yellow oil (0.81 g, 73%): 1H NMR (CDCl3) δ 5.72 (br, 1
H), 2.79 (d, J ) 4.47 Hz, 3 H), 2.30 (t, J ) 7.34 Hz, 2 H), 2.23
(td, J ) 6.88 and 2.24 Hz, 2 H), 1.95 (t, J ) 2.23 Hz, 1 H),
1.84 (m, 2 H); 13C NMR (CDCl3) δ 172.76, 83.42, 68.99, 34.80,
26.15, 24.04, 17.70. The amide (375 mg, 3.0 mmol), methyl
5-iodo-2-methoxy-3-methylbenzoate 4 (875 mg, 2.73 mmol),
Pd(Ph3P)2(OAc)2 (145 mg, 0.194 mmol), copper(I) iodide (104
mg, 0.546 mmol), and triethylamine (791 mg, 7.84 mmol) were
stirred in ethyl acetate (25 mL) at room temperature for 14 h
under argon. The mixture was filtered through a pad of Celite,
and the Celite was washed with a mixture of ethyl acetate
and hexanes (20 mL, 1:1, v/v). The combined filtrates were
evaporated, and the brown residue was purified by flash
chromatography (silica gel 45 g, column 1.5 cm × 31 cm),
eluting with ethyl acetate/hexanes (1:1, v/v), to afford a
colorless oil (800 mg, 80%): 1H NMR (CDCl3) δ 7.65 (d, J )
2.04 Hz, 1 H), 7.35 (d, J ) 2.04 Hz, 1 H), 5.63 (br, 1 H), 3.92
(s, 3 H), 3.80 (s, 3 H), 2.81 (d, J ) 4.92 Hz, 3 H), 2.44 (t, J )
6.79 Hz, 2 H), 2.34 (t, J ) 7.41 Hz, 2 H), 2.26 (s, 3 H), 1.91 (m,
2 H); 13C NMR (CDCl3) δ 172.82, 166.14, 157.78, 137.71,
132.85, 132.23, 131.87, 128.48, 128.33, 124.40, 119.01, 88.96,
61.46, 52.13, 35.05, 26.15, 24.35, 18.72, 15.76; CIMS m/z 304
(MH+); HRMS calcd for C17H22NO4 (MH+) 304.1549, found
304.1534. Anal. Calcd for C17H21NO4: C, 67.33; H, 6.93.
Found: C, 67.12; H, 6.87.
Met h yl 2-H yd r oxy-5-iod o-3-m et h ylb en zoa t e (9).38
TMSCHN2 (9 mL of 2 M solution in hexanes) was added to a
stirred mixture of 3-methyl salicylic acid (8) (2.28 g, 15 mmol)
in methanol (20 mL) and benzene (20 mL) at room tempera-
ture. The mixture was stirred for 2 h and concentrated to
afford the corresponding methyl ester39 quantitatively. The
methyl ester (2.49 g, 15 mmol) was dissolved in methanol (40
mL). Sodium iodide (2.25 g, 15 mmol) and sodium hydroxide
(0.60 g, 15 mmol) were added, and the solution was cooled to
0 °C. Aqueous sodium hypochlorite (22.0 g, 5.25% NaOCl) was
added dropwise. The brown mixture was stirred for 3 h at 0-3
°C and then treated with 10% aqueous sodium thiosulfate. The
pH of the mixture was adjusted to 6-7 using 1 N HCl. Ether
(100 mL) was added, and the layers were separated. The ether
layer was washed with brine and dried over anhydrous sodium
sulfate. After the ether was evaporated, the crude solid was
crystallized from a mixture of acetone and hexanes (1:1, v/v)
1
to afford 9 as a yellow solid (4.0 g, 91.3%): mp 82-84 °C; H
NMR (CDCl3) δ 10.94 (s, 1 H), 7.95 (d, J ) 2.1 Hz, 1 H), 7.56
(d, J ) 1.53 Hz, 1 H), 3.92 (s, 3 H), 2.19 (s, 3 H); 13C NMR
(CDCl3) δ 169.67, 159.69, 144.33, 135.61, 129.44, 113.56, 79.59,
52.43, 15.24; CIMS m/z 293 (MH+).
Meth yl 5-Iod o-2-m eth oxy-3-m eth yl-ben zoa te (10). Com-
pound 9 (3.0 g, 10.27 mmol) was dissolved in acetone (50 mL).
Anhydrous K2CO3 (4.2 g, 30.47 mmol) and dimethyl sulfate
(3.88 mL, 41.08 mmol) were added. The reaction mixture was
vigorously stirred under reflux for 24 h and then filtered, and
the inorganic salts were washed with acetone (45 mL). The
solvent was evaporated, and the yellow residue was washed
with water (3 × 100 mL) to remove excess dimethyl sulfate
and crystallized from acetone to afford 10 as white crystals
Meth yl 5-[(1E)-5-(N-Meth ylca r ba m oyl)-1-(tr ibu tylsta n -
n yl)p en t-1-en yl]-2-m eth oxy-3-m eth ylben zoa te (13). Bu3-
SnH (1.3 mL, 4.85 mmol) was added dropwise to a stirred
solution of alkyne 12 (1.0 g, 3.3 mmol) and (Ph3P)4Pd (109.4
mg, 0.095 mmol) in dry THF (25 mL) at room temperature.
The reaction mixture was stirred for 40 min under argon. The
solvent was evaporated, and the brown residue was purified
by flash chromatography on silica gel (100 g, EtOAc/hexanes
2:3, v/v) to afford a light brown oil (1.70 g, 86.7%): 1H NMR
(CDCl3) δ 7.21 (d, J ) 2.28 Hz, 1 H), 6.92 (d, J ) 2.07 Hz, 1
1
(3.1 g, 99%): mp 55-57 °C; H NMR (CDCl3) δ 7.93 (d, J )
2.28 Hz, 1 H), 7.65 (d, J ) 2.20 Hz, 1 H), 3.90 (s, 3 H), 3.80 (s,
3 H), 2.27 (s, 3 H); 13C NMR (CDCl3) δ 165.21, 158.26, 143.43,
137.55, 135.34, 126.32, 86.54, 61.49, 52.27, 15.60; EIMS m/z
306 (M+); HRMS calcd for C10H12IO3 (MH+) 306.9831, found
306.9828. Anal. Calcd for
Found: C, 39.01; H, 3.50.
C10H11IO3: C, 39.34; H, 3.61.
3
H), 5.75 (t, J ) 6.96 Hz, 1 H, J SnH ) 33 Hz), 5.40 (br, 1 H),
3.95 (s, 3 H), 3.84 (s, 3 H), 2.74 (d, J ) 4.89 Hz, 3 H), 2.32 (s,
3 H), 2.11 (t, J ) 7.39 Hz, 2 H), 2.08 (m, 2 H), 1.73 (m, 2 H),
1.23-1.51 (m, 18 H), 0.90 (t, J ) 7.0 Hz, 9 H); 13C NMR (CDCl3)
δ 173.30, 166.99, 155.58, 144.86, 141.48, 140.13, 133.60,
132.16, 127.25, 123.84, 61.37, 51.97, 35.82, 29.26, 28.84, 27.14,
26.06, 25.55, 16.00, 13.53, 9.85; ESI m/z 594/596 (MH+). Anal.
Calcd for C29H49NSnO4.CH3COOC2H5: C, 58.15; H, 8.37.
Found: C, 57.86; H, 8.16.
Meth yl 5-(Tr i-n -bu tylsta n n yl)-2-m eth oxy-3-m eth ylben -
zoa te (11). Compound 10 (612 mg, 2.0 mmol) was dissolved
in triethylamine (20 mL). Palladium(II) acetate (34 mg, 0.148
mmol) and tetrakis(triphenylphosphine)palladium(0) (86 mg,
0.074 mmol) were added, followed by bis(tri-n-butyltin) (1.75
mL, 3.45 mmol). The mixture was heated at 95-100 °C for 3
h. The black reaction mixture was filtered, and the black solid
was washed with triethylamine (3 × 10 mL). The filtrate was
evaporated under reduced pressure. The oily residue was
purified by flash chromatography on silica gel (120 g, column
5 cm × 31 cm), eluting with a mixture of ethyl acetate and
hexanes (1:5, v/v), to afford a colorless oil (600 mg, 64%): 1H
NMR (CDCl3) δ 7.67 (s, 1 H), 7.39 (s, 1 H), 3.92 (s, 3 H), 3.82
(s, 3 H), 2.31 (s, 3 H), 1.62-1.27 (m, 16 H), 1.05 (t, J ) 8.11
Hz, 2 H), 0.92 (t, J ) 7.30 Hz, 3 H), 0.89 (t, J ) 7.24 Hz, 6 H);
13C NMR (CDCl3) δ 167.44, 158.17, 143.01, 136.56, 131.72,
124.01, 118.33, 61.29, 51.99, 29.02, 28.90, 27.19, 26.51, 16.29,
15.88, 13.52, 13.45, 9.52; ESI m/z 469 (MH+); HRMS calcd for
Meth yl 5-[(1E)-1-Iod o-5-(N-m eth ylca r ba m oyl)p en t-1-
en yl]-2-m eth oxy-3-m eth ylben zoa te (14). The vinyl tribu-
tylstannane 13 (594 mg, 1.0 mmol) was dissolved in dry CH2Cl2
(20 mL). Finely divided I2 (305 mg, 1.2 mmol) was added, and
the mixture was stirred vigorously at room temperature for
20 min. Saturated aqueous Na2S2O3 (15 mL) was added, the
phases were separated, and the aqueous phase was extracted
with CH2Cl2 (3 ×10 mL). The combined organic extracts were
dried (Na2SO4) and concentrated. The residue was purified by
flash chromatography on silica gel (30 g, EtOAc as eluent) to
afford a brown oil (310 mg, 71.9%): 1H NMR (CDCl3) δ 7.51
(d, J ) 2.31 Hz, 1 H), 7.23 (d, J ) 2.01 Hz, 1 H), 6.43 (t, J )
7.60 Hz, 1 H), 5.50 (br, 1 H), 3.91 (s, 3 H), 3.82 (s, 3 H), 2.71
(d, J ) 4.87 Hz, 3 H), 2.29 (s, 3 H), 2.08 (t, J ) 7.48 Hz, 2 H),
2.10 (dt, J ) 7.34 and 7.33 Hz, 2 H), 1.71 (m, 2 H); 13C NMR
(CDCl3) δ 172.75, 166.32, 157.78, 142.98, 136.81, 135.08,
132.85, 129.12, 124.08, 93.59, 61.43, 52.22, 35.37, 31.36, 26.12,
C
C
22H39SnO3 (MH+) 469.1915, found 469.1934. Anal. Calcd for
22H38SnO3: C, 56.41; H, 8.12. Found: C, 56.22; H, 7.90.
N-Meth yl-6-[4-m eth oxy-5-(m eth oxyca r bon yl)-3-m eth -
ylp h en yl]h ex-5-yn oa m id e (12). 5-Hexynoic acid (1.0 g, 8.92
mmol) was dissolved in dry THF (15 mL). Oxalyl chloride (1.17
mL, 13.38 mmol) was added, followed by a catalytic amount
of DMF (1 drop). The mixture was stirred for 45 min at room
temperature. Excess solvent and oxalyl chloride were removed
24.77, 16.03; CIMS m/z 432 (MH+); HRMS calcd for C17H22
-
NIO4 432.0672, found 432.0671. Anal. Calcd for C17H22NIO4:
C, 47.33; H, 5.10; N, 3.25; I, 29.47. Found: C, 47.52; H, 5.22;
N, 3.21; I, 29.36.
(38) Takalo, H.; Kankare, J .; Hanninen, E. Acta Chem. Scand., Ser.
B 1988, B42, 448-454.
(39) Filler, R.; Lin, S.; Zhang, Z. J . Fluorine Chem. 2000, 74, 69-
75.
(40) Maier, M. E.; Schoeffling, B. Chem. Ber. 1989, 122, 1081-1087.

ADAM Series of NNRTIs
J . Org. Chem., Vol. 66, No. 18, 2001 5963
Meth yl 3,3′-Dim eth yl-4,4′-d im eth oxy-5,5′-bis(m eth oxy-
ca r bon yl)-6,6-d ip h en ylh exen oa m id e (15). Meth od A. Un-
der an atmosphere of argon, a solution of iodo ester 10 (60
mg, 0.2 mmol) in dry THF (5 mL) and a solution of AsPh3 (24.5
mg, 0.08 mmol) in dry THF (5 mL) were added in turn to a
two-necked flask charged with Pd2(dba)3 (18.3 mg, 0.02 mmol)
and CsF (67 mg, 0.44 mmol). The organostannane 13 (120 mg,
0.2 mmol in 5 mL of dry THF) was then added by syringe,
and the flask was placed in an oil bath (70-80 °C) and stirred
for 14 h under argon. The reaction mixture was cooled to room
temperature, diluted with Et2O, and filtered through a pad of
Celite. The Celite was washed throughly with Et2O, and the
combined filtrates were concentrated by rotary evaporation.
The product was purified by flash chromatography on silica
gel (30 g), eluting with ethyl acetate, to afford the desired
ADAM-amide 15 (71 mg, 74%): 1H NMR (CDCl3) δ 7.49 (d, J
) 2.12 Hz, 1 H), 7.44 (d, J ) 1.75 Hz, 1 H), 7.13 (m, 2 H), 6.01
(t, J ) 7.38 Hz, 1 H), 3.96 (s, 3 H), 3.94 (s, 3 H), 3.91 (s, 3 H),
3.79 (s, 3 H), 2.78 (d, J ) 5.02 Hz, 3 H), 2.35 (s, 3 H), 2.28 (s,
3 H), 2.21 (t, J ) 7.55 Hz, 2 H), 2.16 (dt, J ) 7.60 and 6.11
Hz, 2 H), 1.76-1.85 (m, 2 H); CIMS m/z 484 (MH)+; HRMS
calcd for C27H33NO7 483.2257, found 483.2263. Anal. Calcd for
sion was filtered, and the resin was washed with pyridine (25
mL) and DMF, CH2Cl2, and MeOH (3 × 25 mL each) and then
dried under reduced pressure over P2O5. Elemental analysis
of the resin 20 (first cycle): I, 3.21; Cl, 0.93. This procedure
was repeated again on this resin to afford 20. IR (KBr) 1726.3
cm-1, 1492.2 cm-1. Elemental analysis of the resin 20 (second
cycle): I, 12.70; Cl, 3.80.
P d(0)-Catalyzed Son ogash ir a Cou plin g to Affor d Resin
21. The resin 20 (950 mg) was suspended in a mixture of dry
DMF and THF (30 mL, 5:1, v/v). Methyl 5-hexynoate (434 mg,
3.44 mmol), Pd(OAc)2 (21 mg, 0.092 mmol), PPh3 (60.3 mg,
0.23 mmol), CuI (44 mg, 0.23 mmol), and triethylamine (337
mg, 3.34 mmol) were sequentially added to a round-bottomed
reaction flask containing the resin 20. The reaction flask was
then stoppered, and the reaction mixture was stirred overnight
under argon. The solvent and excess reactants were then
removed by filtration, and the remaining light brown solid
support was washed with DMF, THF, MeOH, and CH2Cl2 (3
× 25 mL of each) and dried in vacuo. IR (KBr) 2232 cm-1
;
1731.4 cm-1
.
Hyd r osta n n a tion of Resin -Bou n d Alk yn e 21 to Yield
Resin 22. Pd(PPh3)4 (35 mg, 0.03 mmol) was added to a
degassed suspension of polymer-bound alkyne 21 (0.83 g, ∼1
mmol) in a mixture of dry THF and DMF (30 mL, 2:1 v/v).
The mixture was stirred for 5 min, and Bu3SnH (0.41 mL, 1.5
mmol) was then slowly added via syringe over 10 min. The
reaction mixture was allowed to stir at room temperature for
4 h under argon, transferred to a glass filter, and thoroughly
washed with CH2Cl2, MeOH, DMF, and then CH2Cl2 (3 × 25
mL each) to afford the desired solid support 22.
Clea va ge of Com p ou n d 23 fr om Solid Su p p or t 22. The
cleavage cocktail was prepared by degassing a solution of
MeOH (17 mL), THF (5 mL), and Et3N (3.0 mL) with argon.
This solution was added to the dried resin 22 (100 mg), and
the mixture was heated at 50 °C under argon for 16 h at room
temperature. The resin was filtered and washed with MeOH
(2 mL). The resin was subjected to this procedure a total of
six times. The combined filtrates were evaporated to afford
compound 23 (64 mg): 1H NMR (CDCl3) δ 7.16 (d, J ) 2.17
Hz, 1 H), 7.02 (d, J ) 2.18 Hz, 1 H), 5.68 (t, J ) 6.95 Hz, 1 H,
3J SnH ) 31.1 Hz), 3.86 (s, 3 H), 3.85 (s, 3 H), 3.56 (s, 3 H), 2.19
(t, J ) 7.55 Hz, 2 H), 1.99 (dt, J ) 7.04 and 7.33 Hz, 2 H),
1.61 (m, 2 H), 1.14-1.45 (m, 18 H), 0.90 (t, J ) 7.0 Hz, 9 H).
Anal. Calcd for C28H45ClSnO5: C, 54.61; H, 7.37; Cl, 5.76.
Found: C, 54.43; H, 7.31; Cl, 5.68.
P d (0)-Ca ta lyzed Stille Cou p lin g to Affor d Resin s 24
a n d 25. A solution of AsPh3 (24.5 mg, 0.08 mmol) in THF (5
mL) was added to a degassed suspension of polymer-bound
vinylstannane 22 (400 mg) in anhydrous DMF (15 mL)
containing Pd2(dba)3 (19.6 mg, 0.0214 mmol) and CsF (67 mg,
0.44 mmol). After 5 min, aryl iodide 10 (391 mg, 1.28 mmol)
in anhydrous DMF (10 mL) was added, and the reaction
mixture was heated at 80 °C overnight under argon. The
reaction mixture was then transferred to a fritted syringe and
thoroughly washed with DMF, CH2Cl2, EtOH, and CH2Cl2 (3
× 25 mL each) to afford solid support 24, which was dried
under reduced pressure over P2O5. A similar procedure was
employed to prepare solid support 25 via Stille coupling of aryl
iodide 17 with resin-bound vinylstannane 22.
C
27H33NO7: C, 67.08; H, 6.83. Found: C, 66.97; H, 6.79.
Meth od B. Under an atmosphere of argon, a solution of
vinyl iodide 14 (54.2 mg, 0.13 mmol) in dry THF (3 mL) and
a solution of AsPh3 (15.9 mg, 0.05 mmol) in dry THF (3
mL) were added in turn to a two-necked flask charged with
Pd2(dba)3 (11.9 mg, 0.01 mmol) and CsF (43.6 mg, 0.29 mmol).
The organostannane 11 (61.9 mg, 0.13 mmol) in dry THF (4
mL) was then added by syringe, and the flask was placed in
an oil bath (70-80 °C) and stirred overnight under argon. The
reaction mixture was cooled to room temperature, diluted with
Et2O, and filtered through a pad of Celite. The Celite was
washed thoroughly with Et2O, and the combined filtrates were
concentrated by rotary evaporation. The product was purified
by flash chromatography on silica gel (25 g), eluting with ethyl
acetate, to afford the desired ADAM-amide 15 (47 mg, 84%).
Meth yl 3-Ch lor o-5-iod o-2-m eth oxyben zoa te (18). To a
solution of methyl 5-iodo-2-methoxybenzoate 1741 (2.92 g, 10
mmol) in CH2Cl2 (10 mL) was gradually added SO2Cl2 (5.4 g,
40 mmol). The reaction mixture was heated at 50 °C for 14 h
and then cooled to room temperature. It was poured into a
large amount of water (200 mL) and extracted with CH2Cl2 (3
× 40 mL). The CH2Cl2 extracts were combined, dried over
Na2SO4, and evaporated in vacuo to afford a white solid (3.20
g, 98%). A small amount of the sample was further purified
by flash chromatography on silica gel (10 g, hexanes as
1
eluent): mp 41-43 °C; H NMR (CDCl3) δ 7.97 (d, J ) 2.12
Hz, 1 H), 7.84 (d, J ) 2.08 Hz, 1 H), 3.90 (s, 6 H); 13C NMR
(CDCl3) δ 164.25, 156.14, 142.05, 138.42, 130.79, 128.33, 86.15,
62.00, 52.63. Anal. Calcd for C9H8IClO3‚0.6 H2O: C, 32.05; H,
2.75. Found: C, 31.90; H, 2.35.
3-Ch lor o-5-iod o-2-m eth oxyben zoic Acid (19). A 1 N
NaOH solution (10 mL) was added to a MeOH solution (8 mL)
containing benzoate 18 (0.9 g, 2.76 mmol). The mixture was
heated at 60 °C for 3 h and then acidified with concentrated
HCl solution (the pH was adjusted to 1-2). The white
precipitate was filtered and recrystallized from acetone to
1
afford compound 19 (0.82 g, 95%): mp 189-191 °C (dec); H
Clea va ge of ADAMs 26 a n d 27 fr om Resin s 24 a n d 25.
The appropriate resin 24 or 25 (200 mg) was added to a
degassed MeOH/THF solution (60 mL, 1:2 v/v) containing
NaOMe (167 mg, 3 mmol). The reaction mixture was heated
under reflux for 20 h. The resin was filtered and washed with
MeOH (10 mL), and the filtrate was evaporated under reduced
pressure to afford crude ADAM 26 (73 mg) or 27 (92 mg) as a
light yellow liquid. Both compounds were further purified by
flash chromatography on silica gel (EtOAc/hexanes 1:3, v/v).
Meth yl (5Z)-6-[3-Ch lor o-4-m eth oxy-5-(m eth oxyca r bo-
n yl)p h en yl]-6-[4-m eth oxy-5-(m eth oxyca r bon yl)-3-m eth -
ylp h en yl]h ex-5-en oa te (26). Yield 54.7 mg; 1H NMR (CDCl3)
δ 7.62 (d, J ) 2.40 Hz, 1 H), 7.56 (d, J ) 2.06 Hz, 1 H), 7.37
(d, J ) 2.30 Hz, 1 H), 7.34 (d, J ) 2.20 Hz, 1 H), 5.93 (t, J )
7.46 Hz, 1 H), 3.92 (s, 3 H), 3.87 (s, 3 H), 3.83 (s, 3 H), 3.73 (s,
3 H), 3.57 (s, 3 H), 2.24 (t, J ) 7.32 Hz, 2 H), 2.19 (s, 3 H),
NMR (CD3COCD3) δ 8.04 (d, J ) 2.24 Hz, 1 H), 7.98 (d, J )
2.13 Hz, 1 H), 3.90 (s, 3 H); 13C NMR (CD3COCD3) δ 169.14,
160.87, 146.72, 143.67, 134.32, 91.00, 66.60; CIMS m/z 313.
Anal. Calcd for C8H6IClO3: C, 30.72; H, 1.92; Cl, 11.36; I,
40.64. Found: C, 30.42; H, 1.68; Cl, 11.08; I, 40.33.
Ester ifica tion of Wa n g Resin w ith 3-Ch lor o-5-iod o-2-
m eth oxyben zoic Acid (19). Solid DCC (630 mg, 3.05 mmol)
and HOBt (621 mg, 3.05 mmol) were added to a suspension of
Wang resin (1.21 mmol -OH, 1 g) in a mixture of dry DMF
and pyridine (30 mL, 9:1 v/v). 3-Chloro-5-iodo-2-methoxyben-
zoic acid (19) (406 mg, 1.30 mmol) was added, and the mixture
was gently stirred at room temperature for 24 h. The suspen-
(41) Robertson, D. W.; Schober, D. A.; Krushinski, J . H.; Mais, D.
E.; Thompson, D. C.; Gehlert, D. R. J . Med. Chem. 1990, 33, 3124-
3126.

5964 J . Org. Chem., Vol. 66, No. 18, 2001
Xu et al.
2.06 (dt, J ) 7.58 and 7.43 Hz, 2 H), 1.68 (m, 2 H). Anal. Calcd
for C26H29ClO8: C, 61.84; H, 5.79; Cl, 7.02. Found: C, 62.13;
H, 5.77; Cl, 6.83.
tetrazolium, inner salt (MTS) and the electron-coupling agent
phenazine ethosulfate in a colorless stable solution, which
upon reduction by viable cells forms a colored solution with
absorbance at 490 nm. Data are presented as the percent
control of MTS values for the uninfected, drug-free control.
EC50 values reflect the drug concentration that provides 50%
protection from the cytopathic effect of HIV-1 in infected
cultures, while the CC50 reflects the concentration of drug that
causes 50% cell death in the uninfected cultures. MTS-based
results were confirmed by measurement of cell-free superna-
tant RT and p24 levels. All MTS cytoprotection data were
derived from triplicate tests on each plate, with two separate
sister plates. Thus, the EC50 value from each plate represents
the average of triplicates, and the two EC50 values from sister
plates were averaged. The variation from the mean averaged
less than 10%.
Meth yl (5Z)-6-[3-Ch lor o-4-m eth oxy-5-(m eth oxyca r bo-
n yl)p h en yl]-6-[4-m eth oxy-3-(m eth oxyca r bon yl)p h en yl]-
h ex-5-en oa te (27). Yield 79.0 mg; 1H NMR (CDCl3) δ 7.63
(d, J ) 2.38 Hz, 1 H), 7.52 (d, J ) 2.46 Hz, 1 H), 7.30 (d, J )
2.12 Hz, 1 H), 7.15 (dd, J ) 8.59 and 2.34 Hz, 1 H), 6.84 (d, J
) 8.79 Hz, 1 H), 5.97 (t, J ) 7.47 Hz, 1 H), 3.96 (s, 3 H), 3.92
(s, 3 H), 3.89 (s, 3 H), 3.86 (s, 3 H), 3.61 (s, 3 H), 2.29 (t, J )
7.07 Hz, 2 H), 2.12 (dt, J ) 7.43 and 7.23 Hz, 2 H), 1.79 (m, 2
H); EIMS m/z 490 (M+). Anal. Calcd for C25H27ClO8: C, 61.10;
H, 5.50; Cl; 7.24. Found: C, 61.28; H, 5.49; Cl, 7.12.
HIV Cyt op r ot ect ion Assa y. Anti-HIV screening of test
compounds was performed as previously described42,43 with
minor changes. Briefly, the assays involve the killing of T4
lymphoid cells (CEM-SS cell line) by HIV-1 and inhibition of
cell killing by active compounds. Experimental antiviral
compounds were diluted as appropriate and added to a 96-
well microtiter plate. Cells were infected in the well at a
multiplicity of infection (MOI) of approximately 0.2, and
viability was assayed at 6 days after infection by reduction of
the CellTiter 96 Reagent (Promega, Madison, WI). This
reagent contains the tetrazolium compound 3-(4,5-dimeth-
ylthiazol-2-yl)-5-(carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
Assa y for In h ibition of HIV-1 RT. The effects of the
compounds on the HIV-1 RT enzyme were performed using
recombinant purified p66/51 RT (a kind gift of S. Hughes, NCI-
FCRDC). Inhibition of reverse transcription was measured by
the level of incorporation of [32P]GTP into the poly(rC)‚oligo-
(dG) (rCdG) homopolymer template primer system.3
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. We thank David
Allen for the creation of the cover art.
(42) Weislow, O. S.; Kiser, R.; Fine, D. L.; Bader, J .; Shoemaker, R.
H.; Boyd, M. R. J . Natl. Cancer Inst. 1989, 81, 577-586.
(43) Rice, W. G.; Bader, J . P. Adv. Pharmacol. (San Diego) 1995,
33, 389-438.
J O0100291