Synthesis of novel N-1 (allyloxymethyl) analogues of 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil (MKC-442, emivirine) with improved activity against HIV-1 and its mutants

Article abstract of DOI:10.1021/jm020949r

This paper reports the synthesis and the antiviral activities of a series of 6-arylmethyl-1-(allyloxymethyl)-5-alkyluracil derivatives, which can be viewed as analogues of the anti-HIV-1 drug emivirine (formerly MKC-442) from which they differ in the repl

Full text of DOI:10.1021/jm020949r

J . Med. Chem. 2002, 45, 5721-5726
5721
Syn th esis of Novel N-1 (Allyloxym eth yl) An a logu es of
6-Ben zyl-1-(eth oxym eth yl)-5-isop r op ylu r a cil (MKC-442, Em ivir in e) w ith
Im p r oved Activity Aga in st HIV-1 a n d Its Mu ta n ts
Nasser R. El-Brollosy,† Per T. J ørgensen,† Berit Dahan,† Anne Marie Boel,† Erik B. Pedersen,*,† and
Claus Nielsen‡
Nucleic Acid Center,§ Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M Denmark
and Retrovirus Laboratory, Department of Virology, State Serum Institute, Artillerivej 5, DK-2300 Copenhagen, Denmark
Received May 30, 2002
This paper reports the synthesis and the antiviral activities of a series of 6-arylmethyl-1-
(allyloxymethyl)-5-alkyluracil derivatives, which can be viewed as analogues of the anti-HIV-1
drug emivirine (formerly MKC-442) from which they differ in the replacement of the
ethoxymethyl group with variously allyloxymethyl moieties. The most active compounds N-1
allyloxymethyl- and N-1 3-methylbut-2-enyl substituted 5-ethyl-6-(3,5-dimethylbenzyl)uracils
(12 and 13) showed activity against HIV-1 wild-type in the picomolar range with selective
index of greater than 5 × 106 and activity in the submicromolar range against the clinically
important Y181C and K103N mutant strains known to be resistant to emivirine. Structure-
activity relationship studies established a correlation between the anti-HIV-1 activity and the
substitution pattern of the N-1 allyloxymethyl group.
In tr od u ction
resistance for many NNRTI including the FDA (U.S.
Food and Drug Administration) approved drugs (nevi-
rapine,10 delavirdine,11 and efavirenz12). No resistance
data for TNK-651 are available.
The reverse transcriptase (RT) of the human immu-
nodeficiency virus type 1 (HIV-1) is a key target for
inhibition of HIV-1 replication.1,2 In recent years, much
effort has been put into the design and synthesis
of HIV-1 non-nucleoside RT inhibitors (NNRTIs, re-
viewed in ref 3). Among the representatives of the
NNRTIs, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-
thymine (HEPT, 1)4 constitutes an important inhibitor
and has been extensively studied for many years.
Although HEPT did not show very high activity against
HIV-1, it was considered an interesting lead compound
for the synthesis of new analogues, among them the
6-benzyl-1-(ethoxymethyl)-5-isopropyluracil5 (emivirine,
formerly MKC-442, 2) and the corresponding 1-benzy-
loxymethyl analogue6 (TNK-651, 3). Both compounds
showed high activity against HIV-1, and MKC-442 was
chosen as a candidate for clinical trials with AIDS
patients.7 However, Triangle Pharmaceuticals halted
development of emivirine in J anuary 2002 when a
comparative study showed emivirine to be less potent
than other antiretrovirals.8 A serious disadvantage of
HEPT analogues, like almost all NNRTIs, is develop-
ment of drugs resistance mediated by mutations of
residues that line the NNRTI binding pocket of the viral
RT and reduce drug binding. However, further interest
in the HEPT class has been augmented by the finding
that GCA-1869 (Figure 1, 4), which has 3,5-dimethyl
substituents on its phenyl ring (only difference to MKC-
442), was better able to tolerate the presence of Y181C
or K103N mutations than did emivirine itself. The
above-mentioned single-point mutations give high-level
According to structure-activity relationships (SAR),
studies of several crystal structures of the RT complex
with inhibitors, such as HEPT,13 MKC-442,6 TNK-651,6
and GCA-186,9 indicate that the optimal C-5 and C-6
substituents of the uracil ring with respect to HIV-1
inhibition are already found. The only site where new
important modifications may be tried is the N-1 position
of the uracil.14 The substituent at N-1 may have larger
volume (e.g., TNK-651) and length, and even bulky N-1
substituents may be accommodated because of the
flexibility of the Pro236 loop region.9,14
The rationale of the present study was that the
susceptibility of MKC-442 analogues to drug-resistant
mutations might be reduced by improving the binding
of the inhibitors by exploiting the possibility of ad-
ditional π-stacking to the NNRTI binding site. Inspec-
tion of the above-mentioned crystal structure reveals
the presence of several aromatic amino acid residues
in the NNRTI pocket, especially Tyr318 appears attrac-
tive, situated in a close distance (3.8 Å) to the N-1
ethoxymethyl group of MKC-442. This paper reports the
synthesis and the antiviral activities of a series of
6-arylmethyl-1-(allyloxymethyl)-5-alkyluracil deriva-
tives (8-21) designed by the above criterion, the new
compounds being viewed as a hybrid between MKC-442
and TNK-651. It is a further object of the present paper
to expand our understanding of the structure-activity
relationship among non-nucleoside HEPT analogues.
Ch em istr y
* Towhom correspondence should be addressed.Phone: +4566502555.
Fax: +4566158780. E-mail: EBP@Chem.sdu.dk.
† University of Southern Denmark.
The synthesis of the newly designed products was
straightforward and is described in Scheme 1. The
acetals 6 were prepared according to the method of
Nazaretyan et al.15 by refluxing the appropriate alcohol
‡ State Serum Institute.
§ A research center funded by The Danish National Research
Foundation for studies on nucleic acid.
10.1021/jm020949r CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/23/2002

5722 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
El-Brollosy et al.
respectively, in the presence of TMS triflate to give 11
and 15-21 in 58-73% yield.
The compounds were identified by comparison of
similar NMR data.5,16 N-1 Substitution was proved by
the NOE enhancement in the benzyl protons when
1-CH2 was irradiated. The N-3 regioisomer was not
observed.
Biologica l Resu lts a n d Discu ssion
F igu r e 1. Chemical structure of HEPT, MKC-442, TNK-651,
and GCA-186.
The novel MKC-442 derivatives synthesized in this
study were evaluated for cytotoxicity and inhibitory
activity against two different HIV-1 strains induced
cytopathogenicity in MT-4 cells, and the results are
listed in Table 1 along with the results from MKC-442
and the three currently available NNRTIs for the
treatment of AIDS, nevirapine, delavirdine, and efavir-
enz, which are included in the table for comparison.
The majority of the synthesized compounds and the
reference compounds have similar antiviral activities
against wild-type HIV-1 IIIB (a T-cell-tropic strain) and
HIV-1 HxB2 (a molecular clone) in MT-4 cells. In
contrast, compounds 12 and 13 were significantly strain
dependent, showing a 200- and 15-fold, respectively,
increment against the molecular clone than against the
HIV-1 IIIB strain (Table 1). The factors responsible for
the improved inhibitory profile of these compounds
remain to be elucidated. One explanation could be that
HxB2 is a more pure strain having a lower content of
mutated virus; however, one would expect to see the
same trend of all the tested compounds, but this is not
the case in our study.
Sch em e 1
All the present compounds can be divided into three
groups according to the substitution pattern at C-5 and
C-6 position of the uracil ring. (i) series a: the first-
generation derivatives (MKC-442 analogues); com-
pounds 8-11 (C-5 ) isopropyl and C-6 ) benzyl), (ii)
series b: compounds 12-18 (C-5 ) ethyl, and C-6 ) 3,5-
dimethylbenzyl or 1-naphthylmethyl), and (iii) series
c: the second-generation inhibitors (GCA-186 ana-
logues); compounds 19-21 (C-5 ) isopropyl and C-6 )
3,5-dimethylbenzyl).
As seen from examination of the results listed in
Table 1, structural variation in the three series have
resulted in a wide range of biological activity ranging
in ED50 values from 1 to 0.00002 µM against wild-type
HIV-1. When comparing trends among the series of
compounds, we found that greater or equal activity is
obtained from series a through series c. The majority
of the novel compounds were noncytotoxic for MT-4 cells
at doses as high as 100 µM (highest concentration
tested), and the rest of the compounds showed CD50
values comparative to delavirdine.
Compound 8 (the most active compound in the series
a) substituted at R1 by a rigidify vinyl group was in our
study 5 times more potent than MKC-442 (4 nM versus
20 nM). The factors responsible for the significant
activity of compound 8 remain to be elucidated but
suggest either π-stacking to Tyr318 or a repositioning
of Pro236. Increasing the bulkiness around the double
bound of 8 with methyl moieties to 9 and 10 leads to
significant drop in activity compared to 8 (8- and 70-
fold, respectively). However, compound 9 was still
equipotent to the lead compound MKC-442 and efavirenz,
and compound 10 was equipotent to nevirapine. The
5, dibromomethane, and tetrabutylammonium bromide
in benzene to afford the acetals 6 after distillation. It is
important to distill the product to remove tetrabutyl-
ammonium bromide from the product to avoid difficul-
ties in chromatographic work up of the products in the
subsequent condensation reaction. The required 5,6-
disubstituted uracils 7 were easily synthesized accord-
ing to the method of ours16 from the corresponding 3-oxo
estes prepared by reaction of the appropriate arylac-
etonitrile with ethyl 2-bromobutyrate or ethyl 2-bromo-
3-methylbutyrate. The so-formed ethyl 2-alkyl-4-aryl-
3-oxo ester was condensed with thiourea to furnish the
corresponding 2-thiouracil, which in boiling aqueous
chloroacetic acid afforded 5-alkyl-6-(arylmethyl)uracils
7a -d by exchanging sulfur with oxygen. The substi-
tuted uracils 7a -d was silylated at reflux in 1,1,1,3,3,3-
hexamethyldisilazane (HMDS)17 and alkylated by treat-
ment with bis(allyloxy)methane 6a -c in the presence
of trimethylsilyl trifluoromethanesulfonate (TMS tri-
flate) as a Lewis acid catalyst18 to give the MKC-442
analogues 8-10 and 12-14 in 10-77% yield. For
compounds 11 and 15-21, the method was modified by
silylation in situ of 7a -d using N,O-bis(trimethylsilyl)-
acetamide (BSA) and alkylated at the N-1 position with
bis(propargyloxy)methane (6d ) (for compounds 7a , b)
or bis(allyloxy)methane 6a -c (for compounds 7c, d ),

Synthesis of Novel N-1 (Allyloxymethyl) Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5723
Ta ble 1. Anti-HIV-1 Activity of Novel Emivirine Analogues (Compounds 8-21).a MKC-442 (6-Benzyl-1-ethoxymethyl-5-
isopropyluracil), Nevirapine (Viramune), Delavirdine (Rescriptor), and Efavirenz (Sustiva) Are Used for Comparison
ED50 (µM) for HIV-1 IIIB strainb
ED50 (µM ) for HIV-1 HxB2 strainb
wild
type
Y181C + CD50
wild
type
CD50
K103N (µM)c
compd
R1
R2 R3
Y181C
K103N
(µM)c
SId
Y181C
SId
Series a
8
9
10
CH2dCH
(CH3)2CdCH i-Pr H
CH2dC(CH3) i-Pr H
i-Pr H
0.004
0.03
0.28
100
>100
>100
32
40
25 000 0.00 3
1067 0.03
142 0.05
3.3
0.9
3.8
>100
34
> 33 333
1122
> 2000
>100
>100
>100
>100
>100
>100
> 100
> 100
NTe
> 10 0
>100
11
HCtC
i-Pr H
0.007
>100 > 14 285 NTe
NTe
NTe
Series b
12
13
14
15
16
17
18
CH2dCH
(CH3)2CdCH Et CH3 0.0003
CH2dC(CH3) Et CH3 0.0025
HCtC
CH2dCH
Et CH3 0.004
0.32
0.34
0.36
>100
NTe
NTe
NTe
10
>100
28
100
36
25 000 0.00 0 02
120 000 0.00 0 02
0.07
0.45
4.0
0.0 2
0.0 9
2.2
>100 > 5 00 0000
61
3 03 3333
>100 > 40 000 0.00 8
>100
NTe
NTe
NTe
NTe
> 12 500
Et CH3 0.003
3
>100 > 33 333 NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
Et
1
NTe
NTe
NTe
32
>100
35
32 NTe
>333 NTe
35 NTe
(CH3)2CdCH Et
CH2dC(CH3) Et
0.3
1
Series c
19
20
CH2dCH
(CH3)2CdCH i-Pr CH3 0.0002
CH2dC(CH3) i-Pr CH3 0.00015
i-Pr CH3 0.0002
>100
19
15
>100
28
26
30
150 000 NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
>100 > 500 000 NTe
>100 > 667 000 NTe
21
MKC-442 CH3
nevirapine
(Viramune)
delavirdine
(Rescriptor)
efavirenz
i-Pr H
0.02
0.38
44
>100
>100
>100
>100
>100
> 5000 0.01
>263 0.57
3.8
22
2.1
4.3
>100
>100
> 10 000
> 175
0.06
0.01
>100
>100
42
700 0.19
4.5
3.0
>42
> 221
0.3
2.7
>100 > 10 000 0.04
0.04
0.3 5
>100
> 250 0
(Sustiva)
a
All data represent mean values for three separate experiments. b Effective concentration of compound required to achieve 50% inhibition
of HIV-1 multiplication in MT-4-infected cells. c Cytotoxic doses of compound required to reduce the viability of normal uninfected MT-4
d
cells by 50%. The symbol (>) indicates that the CD50 was not reached at the highest concentration tested. Selectivity index: ratio
CD50/ED50 see the Experimental Section for description of assay. e NT: not tested.
detrimental effect of the bulkiness of compounds 9 and
especially with 10 could be due to a steric clash with
adjacent residues and thus to weakened interaction with
the NNRTI pocket. To confirm the above-mentioned
findings, we found it interesting to synthesize the
corresponding ethynyl compound 11 to verify our hy-
potheses. Indeed, the present of an ethynyl group
restored the activity to a level comparable to that of
compound 8. Furthermore, 8 and 11 had larger selectiv-
ity indices (SIs, ratios of CD50 to ED50) than MKC-442
(25 000 and >14 000, respectively, versus >5000).
Encouraged by the promising results for compounds
8 and 11, we next investigated the influence of the
substituent at C-5 and C-6 for the activity against HIV-
1. It is known from the literature that the corresponding
MKC-442 analogue with the same substitution at C-5
and C-6 as in the series b are equipotent to MKC-442
itself.5 In our study compounds, 12 and 15 followed the
trend of MKC-442 with similar activities as their benzyl
counterparts (8 and 11). Surprisingly, compound 12 and
13 was significantly (100-fold) more potent against
HIV-1 replication compared to the corresponding benzyl
analogues 9 and 10. Compound 13 showed the highest
activity and selectivity (0.3 nM, SI 120 000) in the series
b, 67 times more active as MKC-442; however, com-
pound 12 was equipotent to compound 13 when tested
against wild-type HIV-1 HxB2 strain, with ED50 ) 20
pM and SI > 5 000 000. In contrast to the observation
made in series a, bulky substituent at R1 seems to be
essential for high activity in the series b.
According to molecular modeling study on MKC-442,
it was postulated by Hopkins et al.6 that replacement
of the 6-benzyl group with a naphthylmethyl might be
favorable. To confirm the above-mentioned findings from
molecular modeling, we synthesized the closely related
1-naphthylmethyl derivatives 16-18. The effect was
rather dramatic as can be seen by comparing 12-14
with a decrease in activity against HIV of about 3
powers, thus demonstrating that the naphthylmethyl
group is unfavorably positioned to make interactions
with the π-clouds of the aromatic chain of Tyr181 and
Tyr188. However, compound 17 showed similar activity
and SI as nevirapine.
Like previous data reported in the HEPT series,5,9
introduction of an isopropy group at C-5 and a 3,5-
dimethylbenzyl substituent at C-6 (e.g., AGC-186) does
potentiate the antiviral activity. Thus, compounds 19,
20, and 21 were about 20-, 150-, and 1900-fold more
active against HIV-1 than their unsubstituted benzyl
counterparts 8-10 (series a versus c). Furthermore, 19-
21 had very large selectivity indices which were 150 000,
>500 000, and >667 000, respectively. The results in
series a and b suggested that the substitution pattern
of the vinyl group at R1 is an important criterion for
activity, which surprisingly is not the case in series c.
An tivir a l Activity of th e MKC-442 An a logu es
a ga in st HIV-1 Mu ta tion s. The novel MKC-442 de-
rivatives were tested in cell culture for their ability to
inhibit the multiplication of HIV-1 strains carrying the
Y181C, K103N, and Y181C + K103N mutations. The

5724 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
El-Brollosy et al.
extracted with ether (2 × 100 mL). The ether phase was dried
(MgSO4) and evaporated under reduced pressure. The acetals
6a -d were isolated by distillation.
Y181C mutation is associated with high-level resistance
to MKC-442, nevirapine, and delavirdine but remain
susceptible to efavirenz. The K103N mutant confers
resistance to MKC-442, nevirapine, delavirdine, and
efavirenz.
Bis(a llyloxy)m eth a n e (6a ). Yield 25.9 g containing 5%
benzene (55%); bp 115-125 °C/760 mmHg; lit.15 138-139 °C/
680 mmHg; 1H NMR (CDCl3): δ 5.92 (m, 2H, 2 × CH)), 5.23
(m, 4H, 2 × dCH2), 4.72 (s, 2H, OCH2O), 4.09 (dd, 4H, J )
5.6 and 1.5 Hz, 2 × OCH2CH)); 13C NMR (CDCl3): δ 134.41
(CH)), 117.02 (CH2)), 93.74 (OCH2O), 68.26 (OCH2CH)).
Bis(3-m eth yl-2-bu ten yloxy)m eth a n e (6b). Yield 27.84 g
(44%); bp 112-114 °C/23 mmHg; 1H NMR (CDCl3): δ 5.38 (t,
2H, J ) 7.0 Hz, 2 × CH)), 4.69 (s, 2H, OCH2O), 4.07 (d, 4H,
J ) 7.0 Hz, 2 × OCH2CH)), 1.76 (s, 6H, 2 × CH3), 1.69 (s,
6H, 2 × CH3); 13C NMR (CDCl3): δ 137.37 ((CH3)2C), 120.54
(CH)), 93.59 (OCH2O), 63.69 (OCH2CH)), 25.69 (2 × CH3),
17.85 (2 × CH3); MS m/z 184 (M+). Anal. (C11H20O2) H; C: calcd
71.70; found, C, 71.11.
As seen from examination of the results listed in
Table 1, only compounds 12-14 showed activity against
the drug-resistant strain Y181C with activity in the
submicromolar range. The Y181C mutation provides a
2200-fold reduced sensitivity to MKC-442, but com-
pound 12 and 14 are only 80- and 114-fold less potent
toward this mutant, respectively, and compound 13
shows a greater loss of potency (1100-fold). Surprisingly,
compound 15 was inactive against Y181C despite the
structural similarity with compound 12. Additionally,
compound 12 was also significantly more potent (20 nM)
against the K103N mutant than MKC-442 (105-fold)
and efavirenz (18-fold).
Unfortunately, none of the analogues in series c
showed any inhibition to the above-mentioned mutants
despite the structural similarity to GCA-186 which has
showed greater resilience to Y181C and K103N mutants
than MKC-442 itself.9
Surprisingly, the concentration of compound 12 re-
quired to inhibit any of the viruses with single muta-
tions in type HIV-1 HxB2 strain was comparable to or
less than the concentration of MKC-442, nevirapine,
delavirdine, and efavirenz required to inhibit the wild-
type HIV-1 HxB2 strain.
Bis(2-m eth yl-2-p r op en yloxy)m eth a n e (6c). Yield 20.29
1
g (38%); bp 68-70 °C/18 mmHg; H NMR (CDCl3): δ 5.00 (s,
2H, 2 × CHH)), 4.91 (s, 2H, 2 × CHH)), 4.74 (s, 2H, OCH2O),
4.01 (s, 4H, 2 × OCH2C), 1.77 (s, 6H, 2 × CH3); 13C NMR
(CDCl3): δ 141.75 (CH2)C), 112.10 (CH2dC), 93.53 (OCH2O),
71.18 (OCH2C), 19.52 (2 × CH3); MS m/z 156 (M+).
Bis(p r op a r gyloxy)m eth a n e (6d ). Yield 17.1 g (40%); bp
110-115 °C/35 mmHg; lit.15 85-87 °C/30 mmHg; 1H NMR
(CDCl3): δ 4.86 (s, 2H, OCH2O), 4.09 (s, 4H, 2 × OCH2C), 2.45
(s, 2H, 2 × CH); 13C NMR (CDCl3): δ 91.94 (OCH2O), 78.97
(Ct), 74.49 (OCH2Ct) 54.54 (HCt).
Acyclic Ur a cil Der iva tives 8-10, 12-14. Gen er a l P r o-
ced u r e. A mixture of compound 7 (3 mmol), 1,1,1,3,3,3-
hexamethyldisilazane (HMDS) (30 mL) and (NH4)2SO4 (10 mg)
was heated under reflux overnight. The mixture was concen-
trated at room temperature under reduced pressure to obtain
the silylated base as translucent oil or as a pale yellow solid.
The resulting residue was dissolved in anhydrous CH3CN (10
mL) and cooled to -40 °C. Trimethylsilyl trifluoromethane-
sulfonate (TMS triflate) (3 mmol) was added in one portion
followed by the dropwise addition of the appropriate acetal
6a -c (3 mmol). When the reaction was finished (TLC analy-
sis), the mixture was quenched with ice-cold saturated solution
of NaHCO3 (20 mL) and evaporated to near dryness by
coevaporation with ethanol (2 × 50 mL). The resulting solid
was suspended in Et2O (200 mL), and the mixture was stirred
for 1 h. After filtration, the residue was extracted with Et2O
(100 mL) and the combined organic fractions were evaporated
under reduced pressure to give 8-10 and 12-14 after silica
column chromatography (10 f 25% EtOAc in petroleum ether).
1-(Allyloxym eth yl)-6-ben zyl-5-isop r op ylu r a cil (8). Yield
518 mg (55%); mp 98-100 °C; 1H NMR (DMSO-d6): δ 1.13 (d,
6H, J ) 6.8 Hz, CH(CH3)2) 2.78 (sep, 1H, J ) 6.9 Hz,
CH(CH3)2), 4.02 (dd, 2H, J ) 5.5, 1.4 Hz, OCH2CH)), 4.14 (s,
2H, CH2Ph), 5.08 (s, 2H, NCH2O), 5.19 (m, 2H, dCH2), 5.82
Con clu sion
In summary, we have described the synthesis and
structure-activity relationship of a new series N-1
allyloxymethyl analogues of MKC-442. The most active
compounds 12 and 13 showed activity against wild-type
HIV-1 HxB2 strain in the picomolar range with selective
index of greater than 5 000 000. These compounds are
among the most active NNRTIs ever described in the
literature. Both compounds showed greater inhibitory
effect than MKC-442 to the clinically important Y181
and K103N mutant strains.
On the basis of the promising anti-HIV-1-data, the
next step will consist in optimizing the N-1 allyoxym-
ethyl linker of the new MKC-442 analogues. These
experiments are currently in progress.
(m, 1H, CH)), 7.17-7.38 (m, 5H, aryl), 11.36 (s, 1H, NH); 13
C
Exp er im en ta l Section
NMR (DMSO-d6): δ 20.04 (CH(CH3)2), 27.41 (CH(CH3)2), 33.00
(CH2Ph), 69.19 (OCH2CH)), 72.20 (OCH2N), 116.96 (C5),
118.18 (CH2)), 126.76, 127.39, 128.93, 134.34, 136.38 (aryl,
CH)), 148.15 (C2), 151.61 (C6), 162.42 (C4); MS m/z 314 (M+).
Anal. (C18H22N2O3) C, H, N.
NMR spectra were recorded on a Varian Gemini 2000 NMR
spectrometer at 300 MHz for 1H and 75 MHz for 13C with
tetramethylsilane as an internal standard. Chemical shifts are
reported in parts per million (δ), and signals are expressed as
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
or br (broad). EI mass spectra were recorded on a Finnigan
Mat SSQ 710, FAB mass spectra on a Kratos MS50RF
instrument. Melting points were determined on a Bu¨chi
melting point apparatus. Elemental analyses were performed
at Atlantic Microlab, Inc., Atlanta, Georgia; found values
agreed favorably with the calculated ones. The progress of
reactions was monitored by TLC (analytical silica gel plates
60 F254). Merck silica gel (0.040-0.063 mm) was used for
column chromatography.
Aceta ls 6a -d . Gen er a l P r oced u r e. A mixture of KOH
(38.8 g, 692 mmol), alcohol 5 (691 mmol), dibromomethane
(60.1 g, 24.2 mL, 346 mmol), and tetrabutylammonium bro-
mide (12.0 g, 37 mmol) in anhydrous benzene (200 mL) was
heated under reflux for 4 h. The reaction mixture was left to
cool to room temperature, and H2O (200 mL) was added and
6-Ben zyl-5-isop r op yl-1-(3-m eth ylbu t-2-en yloxym eth yl)
1
u r a cil (9). Yield 100 mg (10%); H NMR (CDCl3): δ 1.29 (d,
6H, J ) 7.1 Hz, CH(CH3)2), 1.65, 1.73 (2 × s, 6H, CdC(CH3)2),
2.88 (sep, 1H, J ) 6.8 Hz, CH(CH3)2), 4.09 (d, 2H, J ) 6.9 Hz,
OCH2CH), 4.19 (s, 2H, CH2Ph), 5.13 (s, 2H, NCH2O), 5.30 (t,
1H, J ) 7.0 Hz, OCH2CH)), 7.17-7.38 (m, 5H, aryl), 11.36
(s, 1H, NH); 13C NMR (CDCl3): δ 18.02, 25.79 (CdC(CH3)2),
20.39 (CH(CH3)2), 28.29 (CH(CH3)2), 33.35 (CH2Ph), 65.99
(OCH2CH)), 72.66 (OCH2N), 119.69 (C5), 119.99 (OCH2CH)),
127.24, 129.14, 135.37 (aryl), 138.33, ((CH3)2C)), 148.55 (C6),
151.81 (C2), 162.38 (C4).
6-B e n zy l-5-is o p r o p y l-1-(2-m e t h y la lly lo x y m e t h y l)-
u r a cil (10). Yield 760 mg (77%); mp 108-110 °C; 1H NMR
(CDCl3): δ 1.29 (d, 6H, J ) 7.1 Hz, CH(CH3)2), 1.72 (s, 3H,
CH2dCCH3), 2.88 (sep, 1H, J ) 6.9 Hz, CH(CH3)2), 4.04 (s,
2H, OCH2C), 4.21 (s, 2H, CH2Ph), 4.89, 4.96 (2 × s, 2H,

Synthesis of Novel N-1 (Allyloxymethyl) Analogues
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5725
CH2)C), 5.16 (s, 2H, NCH2O), 7.12 (d, 2H, J ) 7.3 Hz, aryl),
7.26-7.39 (m, 3H, aryl), 11.36 (s, 1H, NH); 13C NMR (CDCl3):
δ 19.45 (CH2dCCH3), 20.39 (CH(CH3)2), 28.30 (CH(CH3)2),
33.46 (CH2Ph), 72.85 (OCH2N), 73.54 (CCH2O), 112.51 (CH2)),
119.80 (C5), 127.23, 129.20, 135.32 (aryl), 141.26, (CH2C)),
148.45 (C6), 151.97 (C2), 162.50 (C4); MS m/z 328 (M+). Anal.
(C19H24N2O3‚0.25H2O) C, H, N.
1-(Allyloxym e t h yl)-6-(3,5-d im e t h ylb e n zyl)-5-e t h yl-
u r a cil (12). Yield 514 mg (55%); mp 115-116 °C; 1H NMR
(DMSO-d6): δ 0.91 (t, 3H, J ) 7.3 Hz, CH2CH3), 2.24 (s, 6H,
2 × CH3), 2.30 (q, 2H, J ) 7.2 Hz, CH2CH3), 4.01 (s, 2H, OCH2-
CH)), 4.03 (s, 2H, CH2Ph), 5.04 (s, 2H, NCH2O), 5.17 (m, 2H,
dCH2), 5.84 (m, 1H, CH)), 6.78-6.88 (m, 3H, aryl), 11.47 (s,
1H, NH); 13C NMR (DMSO-d6): δ 13.46 (CH2CH3), 18.48 (CH2-
CH3), 20.75 (2 × CH3), 32.78 (CH2Ph), 69.26 (OCH2CH)),
72.00 (OCH2N), 115.58 (C5), 116.90 (CH2)), 138.10, 135.98,
134.40, 128.39, 125.04 (aryl, CH)), 148.47 (C2), 151.67 (C6),
163.16 (C4); MS m/z 328 (M+). Anal. (C19H24N2O3‚0.25H2O) C,
H, N.
6-(3,5-Dim eth ylben zyl)-5-eth yl-1-(3-m eth ylbu t-2-en yl-
oxym eth yl)u r a cil (13). Yield 310 mg (29%); mp 136-138 °C;
1H NMR (CDCl3): δ 1.08 (t, 3H, J ) 7.4 Hz, CH2CH3), 1.66,
1.74 (2 × s, 6H, CdC(CH3)2), 2.28 (s, 6H, 2 × CH3), 2.48 (q,
2H, J ) 7.4 Hz, CH2CH3), 4.09 (m, 4H, CH2Ph, OCH2CH)),
5.13 (s, 2H, NCH2O), 5.31 (t, 1H, J ) 7.0 Hz, OCH2CH)), 6.70
(s, 2H, aryl), 6.90 (s, 1H, aryl), 9.68 (s, 1H, NH); 13C NMR
(CDCl3): δ 13.76 (CH2CH3), 19.10 (CH2CH3), 18.02, 25.77 (2
× dCCH3), 21.23 (2 × CH3), 33.19 (CH2Ph), 65.99 (OCH2CH)),
72.54 (OCH2N), 116.74 (C5), 120.03 (OCH2CH)), 124.99,
128.88, (aryl), 134.94, ((CH3)2C)), 138.23, 138.79 (aryl), 149.22
(C2), 151.93 (C6), 163.50 (C4); MS m/z 356 (M+). Anal.
(C21H28N2O3‚0.1H2O) C, H, N.
6-(3,5-Dim eth ylben zyl)-5-eth yl-1-(2-m eth yla llyloxym e-
th yl)u r a cil (14). Yield 120 mg (13%); mp 142-144 °C; 1H
NMR (CDCl3): δ 1.08 (t, 3H, J ) 7.4 Hz, CH2CH3), 1.73 (s,
3H, CH2dCCH3), 2.29 (s, 6H, 2 × CH3), 2.51 (q, 2H, J ) 7.4
Hz, CH2CH3), 4.03, 4.10 (2 × s, 4H, CH2Ph, OCH2C), 4.90, 4.98
(2 × s, 2H, CH2)C), 5.16 (s, 2H, NCH2O), 6.71 (s, 2H, aryl),
6.91 (s, 1H, aryl), 9.58 (s, 1H, NH); 13C NMR (CDCl3): δ 13.74
(CH2CH3), 19.12 (CH2CH3), 19.45 (CH2dCCH3), 21.25 (2 ×
CH3), 33.22 (CH2Ph), 72.74 (OCH2N), 73.56 (OCH2CH)),
112.47 (CH2)C), 116.81 (C5), 124.99, 128.94, (aryl), 134.89,
138.87 (aryl), 141.29 (CH2C)), 149.34, (C2), 151.96 (C6),
164.91 (C4); MS m/z 342 (M+). Anal. (C20H26N2O3‚0.1H2O) C,
H, N.
7.4 Hz, CH2CH3), 4.07 (s, 2H, CH2Ph), 4.28 (d, 2H, J ) 2.4
Hz, OCH2Ct), 5.20 (s, 2H, OCH2N), 6.71 (s, 2H, aryl), 6.90 (s,
1H, aryl), 9.49 (s, 1H, NH); 13C NMR (CDCl3): δ 13.78
(CH2CH3), 19.14 (CH2CH3), 21.25 (2 × CH3), 33.25 (CH2Ph),
57.22 (OCH2Ct), 72.56 (OCH2N), 74.64 (HCtC), 79.11 (HCt
C), 116.97 (C5), 125.01, 129.00, 134.74, 138.89 (aryl), 149.16
(C6), 152.01 (C2), 163.32 (C4). HRMS-MALDI (M + Na+), calcd
for C19H22N2O3Na, 349.1523; found, 349.1521.
1-Allyloxym et h yl-5-et h yl-6-(n a p h t h a len -1-ylm et h yl)-
u r a cil (16). Yield 253 mg (72%); mp 119-123 °C; 1H NMR
(DMSO-d6): δ 0.90 (t, 3H, J ) 7.4 Hz, CH2CH3), 2.27 (q, 2H,
J ) 7.4 Hz, CH2CH3), 3.97 (dd, 2H, J ) 4.3, 1.4 Hz, OCH2-
CH)), 4.52 (s, 2H, CH2Naph), 4.96 (s, 2H, NCH2O), 5.12 (m,
2H, dCH2), 5.76 (m, 1H, CH)), 7.11-8.21 (m, 7H, Naph),
11.54 (s, 1H, NH); 13C NMR (DMSO-d6): δ 13.49 (CH2CH3),
18.50 (CH2CH3), 30.19 (CH2Naph), 69.24 (OCH2CH)), 72.21
(OCH2N), 116.13 (C5), 116.96 (CH2)), 123.12, 123.68, 125.84,
126,26, 126.71, 127.49, 128.79, 130.96, 131.95, 133.44, 134.27
(Naph, CH)), 148.50 (C2), 151.72 (C6), 163.11 (C4); MS m/z
350 (M+). Anal. (C21H22N2O3‚0.5 H2O) C, H, N.
5-Eth yl-1-(3-m eth ylbu t-2-en yloxym eth yl)-6-(n a p h th a -
len -1-ylm eth yl)u r a cil (17). Yield 276 mg (73%); mp 179-
181 °C; 1H NMR (CDCl3): δ 1.03 (t, 3H, J ) 7.3 Hz, CH2CH3),
1.62, 1.65 (2 × s, 6H, CdC(CH3)2), 2.39 (q, 2H, J ) 7.3 Hz,
CH2CH3), 4.06 (d, 2H, J ) 6.9 Hz, OCH2CH), 4.55 (s, 2H, CH2-
Naph), 5.04 (s, 2H, NCH2O), 5.23 (t, 1H, J ) 7.0 Hz,
OCH2CH)), 7.02-8.08 (m, 7H, Naph), 9.84 (s, 1H, NH); 13C
NMR (CDCl3): δ 13.73 (CH2CH3), 18.00 (CH3), 19.10 (CH2CH3),
25.64 (CH3), 30.41 (CH2Naph), 66.07 (OCH2CH)), 72.70
(OCH2N), 117.36 (C5), 119.96 (OCH2CH)), 122.57, 123.42,
125.53, 126.19, 126.70, 127.98, 128.96, 131.03, 131.21, 133.80
(Naph), 138.13, ((CH3)2C)), 149.29 (C6), 151.92 (C2), 163.33
(C4); MS m/z 378 (M+). Anal. (C23H26N2O3‚0.75 H2O) C, H, N.
5-E t h yl-1-(2-m et h yla llyloxym et h yl)-6-(n a p h t h a len -1-
ylm eth yl)u r a cil (18). Yield 258 mg (71%); mp121-123 °C;
1H NMR (CDCl3): δ 1.04 (t, 3H, J ) 7.3 Hz, CH2CH3), 1.70 (s,
3H, CH2dCCH3), 2.40 (q, 2H, J ) 7.3 Hz, CH2CH3), 4.01 (s,
2H, OCH2C), 4.57 (s, 2H, CH2Naph), 4.86, 4.94 (2 × s, 2H,
CH2)C), 5.07 (s, 2H, NCH2O), 7.03-8.08 (m, 7H, Naph), 9.84
(s, 1H, NH); 13C NMR (CDCl3): δ 13.73 (CH2CH3), 19.13 (CH2-
CH3), 19.41 (CH2dCCH3), 30.31 (CH2Naph), 72.94 (OCH2N),
73.54 (OCH2CH)), 112.43 (CH2dC), 117.46 (C5), 122.49,
123.49, 125.56, 126.23, 126.74, 128.06, 129.01, 130.92, 131.22,
133.84 (Naph), 141.18 (CH2C)), 149.25, (C6), 152.00 (C2),
163.37 (C4); MS m/z 364 (M+). Anal. (C22H24N2O3‚0.25 H2O)
C, H, N.
1-(Allyloxym eth yl)-6-(3,5-d im eth ylben zyl)-5-isop r op yl-
u r a cil (19). Yield 245 mg (72%); mp 104-105 °C; 1H NMR
(CDCl3): δ 1.29 (d, 6H, J ) 6.7 Hz, CH(CH3)2), 2.29 (s, 6H, 2
× CH3), 2.82 (sep, 1H, J ) 6.7 Hz, CH(CH3)2), 4.11-4.14 (m,
4H, OCH2CH, CH2Ph), 5.16 (s, 2H, NCH2O), 5.21-5.33 (m,
2H, dCH2) 5.81-5.92 (m, 1H, CH)) 6.71 (s, 2H, aryl), 6.90 (s,
1H, aryl), 9.47 (s, 1H, NH); 13C NMR (CDCl3): δ 20.41 (CH-
(CH3)2), 21.26 (2 × CH3), 28.34 (CH(CH3)2), 33.35 (CH2Ph),
70.52 (OCH2CH)), 72.72 (OCH2N), 117.66 (CH2)), 119.68
(C5), 125.01, 128.84, 133.69, 138.83 (aryl), 135.01 (CH)),
148.72 (C6), 152.04 (C2), 162.53 (C4); MS m/z 342 (M+). Anal.
(C20H26N2O3) C, H, N.
6-(3,5-Dim et h ylb en zyl)-5-isop r op yl-1-(3-m et h ylb u t -2-
en yloxym eth yl)u r a cil (20). Yield 240 mg (65%); mp 133-
134 °C; 1H NMR (CDCl3): δ 1.29 (d, 6H, J ) 6.9 Hz, CH(CH3)2),
1.66, 1.74 (2 × s, 6H, CdC(CH3)2), 2.28 (s, 6H, 2 × CH3), 2.84
(sep, 1H, J ) 6.9 Hz, CH(CH3)2), 4.09-4.10 (m, 4H, OCH2CH,
CH2Ph), 5.14 (br.s, 2H, NCH2O), 5.31 (t, 1H, J ) 6.4 Hz,
OCH2CH)), 6.70 (s, 2H, aryl), 6.89 (s, 1H, aryl), 9.47 (s, 1H,
NH). 13C NMR (CDCl3); δ 18.03, 25.80 (CdC(CH3)2) 20.41 (CH-
(CH3)2), 21.25 (2 × CH3), 28.30 (CH(CH3)2), 33.27 (CH2Ph),
65.99 (OCH2CH)), 72.67 (OCH2N), 119.57 (C5), 120.06
(OCH2CH)), 125.00, 128.78, 135.08, 138.75 (aryl), 138.23
((CH3)2C)), 148.80 (C6), 151.94 (C2), 162.51 (C4); MS m/z 370
(M+). Anal. (C22H30N2O3‚0.5 H2O) C, H, N.
Acyclic Ur a cil Der iva tives 11, 15-21. Gen er a l P r oce-
d u r e. The appropriate uracil 7 (1 mmol) was stirred in dry
CH3CN (15 mL) under N2, and N,O-bis(trimethylsilyl)aceta-
mide (BSA) (0.87 mL, 3.5 mmol) was added. The mixture
became clear after stirring at room temperature for 10 min.
The reaction mixture was cooled to -50 °C and TMS triflate
(0.18 mL, 1 mmol) was added followed by dropwise addition
of the appropriate acetal (2 mmol). The reaction mixture was
stirred at room temperature for 3 h, quenched with ice-cold
saturated solution of NaHCO3 (5 mL), and evaporated under
reduced pressure. The residue was extracted with Et2O (3 ×
50 mL), and the combined organic fractions were dried
(MgSO4), evaporated under reduced pressure, and chromato-
graphed on silica gel with CHCl3 to afford the product.
6-Ben zyl-5-isop r op yl-1-(p r op -2-yn yloxym eth yl)u r a cil
1
(11). Yield 180 mg (58%); mp 103-104 °C; H NMR (CDCl3):
δ 1.29 (d, 6H, J ) 6.9 Hz, CH(CH3)2), 2.44 (t, 1H, J ) 2.3 Hz,
tCH), 2.85 (sep, 1H, J ) 6.9 Hz, CH(CH3)2), 4.18 (s, 2H, CH2-
Ph), 4.29 (d, 2H, J ) 2.3 Hz, OCH2Ct), 5.21 (s, 2H, NCH2O),
7.11-7.37 (m, 5H, aryl), 9.34 (s, 1H, NH); 13C NMR (CDCl3):
δ 20.40 (CH(CH3)2), 28.32 (CH(CH3)2), 33.48 (CH2Ph), 57.18
(OCH2Ct), 72.64 (OCH2N), 74.72 (HCtC), 79.05 (HCtC),-
119.98 (C5), 127.25, 127.29, 129.21, 135.15 (aryl), 148.25 (C6),
151.97 (C2), 162.31 (C4); HRMS-MALDI (M + H+) calcd for
C
18H21N2O3, 313.1547; found, 313.1552.
6-(3,5-Dim et h ylb en zyl)-5-et h yl-1-(p r op -2-yn yloxym e-
th yl)u r a cil (15). Yield 195 mg (60%); mp 154-156 °C; 1H
NMR (CDCl3): δ 1.06 (t, 3H, J ) 7.4 Hz, CH2CH3), 2.28 (s,
6H, 2 × CH3), 2.44 (t, 1H, J ) 2.5 Hz, dCH), 2.45 (q, 2H, J )
6-(3,5-Dim et h ylb en zyl)-5-isop r op yl-1-(2-m et h yla llyl-
oxym eth yl)u r a cil (21). Yield 356 mg (67%); mp 137-139 °C;
1H NMR (CDCl3): δ 1.29 (d, 6H, J ) 6.9 Hz, CH(CH3)2), 1.73

5726 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
El-Brollosy et al.
(10) Grob, P. M.; Wu, J . C.; Cohen, K. A.; Ingraham, R. H.; Shih, C.
K.; Hargrave, K. D.; McTague, T. L.; Merluzzi, V. J . Nonnucleo-
side inhibitors of HIV-1 reverse transcriptase: Nevirapine as a
prototype drug. AIDS Res. Hum. Retroviruses 1992, 8, 145-152.
(11) Romero, D. L.; Olmsted, R. A.; Poel, T. J .; Morge, R. A.; Biles,
C.; Keiser, B. J .; Kopta, L. A.; Friis, J . M.; Hosley, J . D.;
Stefanski, K. J .; Wishka, D. G.; Evans, D. B.; Morris, J .; Stehle,
R. G.; Sharma, S. K.; Yagi, Y.; Voorman, R. L.; Adams, W. J .;
Tarpley, W. G.; Thomas, R. C. Targeting delavirdine/atevirdine
resistant HIV-1: Identification of (alkylamino)piperidine-con-
taining bis(heteroaryl)piperazines as broad spectrum HIV-1
reverse transcriptase inhibitors. J . Med. Chem. 1996, 39, 3769-
3789.
(12) Young, S. D.; Britcher, S. F.; Tran, L. O.; Payne, L. S.; Lumma,
W. C.; Lyle, T. A.; Huff, J . R.; Anderson, P. S.; Olsen, D. B.;
Carroll, S. S.; Pettibone, D. J .; O’Brien, J . A.; Ball, R. G.; Balani,
S. K.; Lin, J . H.; Chen, I.-W.; Schleif, W. A.; Sardana, V. V.; Long,
W. J .; Byrnes, V. W.; Emini, E. A. L-743,726 (DMP-266): a novel,
highly potent nonnucleoside inhibitor of the human immuno-
deficiency virus type 1 reverse transcriptase. Antimicrob. Agents
Chemother. 1995, 39, 2602-2605.
(13) Ren, J . S.; Esnouf, R.; Garman, E.; J ones, Y.; Somers, D.; Ross,
C.; Kirby, I.; Keeling, J .; Darby, G.; Stuart, D.; Stammers, D.
High-resolution structure of HIV-1 RT: insights from four RT-
inhibitor complexes. Nat. Struct. Biol. 1995, 2, 293-302.
(14) Kireev, D. B.; Chre´tien, J . J .; Grierson, D. S.; Monneret, C. A
3D QSAR study of a series of HEPT analogues: The influence
of conformational mobility on HIV-1 reverse transcriptase
inhibition. J . Med. Chem. 1997, 40, 4257-4264.
(15) Nazaretyan, A. Kh.; Torosyan, G. O.; Babayan, A. T. Quarternary
ammonium salts in alkylation reactions (synthesis of formalde-
hyde acetals). J . Appl. Chem. USSR 1985, 58, 2396-2400.
(16) (a) Danel, K.; Larsen, E.; Pedersen, E. B. Easy synthesis of 5,6-
disubstituted acyclouridine derivatives. Sythesis 1995, 8, 934-
936. (b) Danel, K.; Larsen, E.; Pedersen, E. B.; Vestergaard, B.
F.; Nielsen, C. Synthesis and potent anti-HIV-1 activity of novel
6-benzyluracil analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phe-
nylthio)thymine. J . Med. Chem. 1996, 39, 2427-2431. (c) Danel,
K.; Nielsen, C.; Pedersen, E. B. Anti-HIV active Naphthyl
analogues of HEPT and DABO. Acta Chem. Scand. 1997, 51,
426-430.
(17) Wittenburg, E. A new synthesis of nucleosides. Z. Chem. 1964,
4, 303-304.
(18) Vorbru¨ggen, H.; Krolikiewiecz, K.; Bennua, B. Nucleoside syn-
thesis with trimethylsilyl triflate and perchlorate as catalysts.
Chem. Ber. 1981, 114, 1234-1255.
(19) Harada, S.; Koyanagi, Y.; Yamamoto, N. Infection of HTLV-
III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and applica-
tion in a plaque assay. Science 1985, 229, 563-566.
(20) Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C.
Detection, isolation and continuous production of cytophathic
retroviruses (HTLV-III) from patients with AIDS and pre-AIDS.
Science 1984, 224, 497-500.
(21) Mosmann, T. Rapid colorimetic assay for cellular growth and
survival. Application to proliferation and cytotoxicity assays. J .
Immunol. Methods 1983, 65, 55-63.
(22) Shaw, G. M.; Hahn, B. H.; Arya, S. K.; Groopman, J . E.; Gallo,
R. C.; Wong-Staal, F. Molecular characterization of human T-cell
leukemia (lymphotropic) virus type III in the acquired immune
deficiency syndrome. Science 1984, 226, 1165-1171.
(s, 3H, CH2dCCH3), 2.29 (s, 6H, 2 × CH3), 2.83 (sep, 1H, J )
6.9 Hz, CH(CH3)2), 4.04 (s, 2H, OCH2C), 4.13 (s, 2H, CH2Ph),
4.89, 4.97 (2 × s, 2H, CH2)C), 5.17 (s, 2H, NCH2O), 6.71 (s,
2H, aryl), 6.90 (s, 1H, aryl), 9.64 (s, 1H, NH); 13C NMR
(CDCl3): δ 19.45 (CH2dCCH3) 20.39 (CH(CH3)2), 21.24 (2 ×
CH3), 28.30 (CH(CH3)2), 33.28 (CH2Ph), 72.88 (OCH2N), 73.51
(CCH2O), 112.42 (CH2)), 119.65 (C5), 124.99, 128.81, 135.02,
138.78 (aryl), 141.31 (CH3C)), 148.71 (C6), 152.06 (C2), 162.63
(C4); MS m/z 356 (M+). Anal. (C21H28N2O3) C, H, N.
Vir u s a n d Cells. The inhibitory activity against HIV-1
infection was evaluated using MT-4 cells20 as target cells and
the HIV-1 strain HTLV-IIIB21 as infectious virus. The virus
was propagated in H9 cells20 at 37 °C, 5% CO2 using RPMI
1640 with 10% heat-inactivated fetal calf serum (FCS) and
antibiotics (growth medium). Culture supernatant was filtered
(0.45 nm), aliquotted, and stored at -80 °C until use.
In h ibition of HIV-1 Rep lica tion . Compounds were ex-
amined for possible antiviral activity against both strains of
HIV-1 using MT4 cells as target cells. MT4 cells were
incubated with virus (0.005 MOI) and growth medium con-
taining the test dilutions of compound for 6 days in parallel
with virus-infected and uninfected control cultures without
compound added. Expression of HIV in the cultures was
indirectly quantified using the MTT assay.21 Compounds
mediating less than 30% reduction of HIV expression were
considered without biological activity. Compounds were tested
in parallel for cytotoxic effect in uninfected MT-4 cultures
containing the test dilutions of compound as described above.
A 30% inhibition of cell growth relative to control cultures was
considered significant. The 50% inhibitory concentration (EC50
)
and the 50% cytotoxic concentration (CC50) were determined
by interpolation from the plots of percent inhibition versus
concentration of compound. The test for activity against HIV-1
was performed in MT-4 cell cultures infected with either wild-
type HIV-1 (strain IIIB21 and strain HxB222) or NNRTI
resistant HIV-1 (strain N119,23 strain A17,23b,24 strain ARP1013,
and strain ARP1010 were obtained from J . P. Kleim through
the Centralised Facility for AIDS Reagents,U.K.).
Refer en ces
(1) Mitsuya, H.; Yarchoan, R.; Broder, S. Molecular targets for AIDS
therapy. Science 1990, 249, 1533-1544.
(2) De Clercq, E. Targets and strategies for the antiviral chemo-
therapy of AIDS. Trends Pharmacol. Sci. 1990, 11, 198-205.
(3) Pedersen, O. S.; Pedersen, E. B. Non-nucleoside reverse tran-
scriptase inhibitors: the NNRTI boom. Antiviral Chem. Chemoth-
er. 1999, 10, 285-314.
(4) Miyasaka, T.; Tanaka, H.; Baba, M.; Hayakawa, H.; Walker, R.
T.; Balzarini, J .; De Clercq, E. A novel lead for specific anti-
HIV-1 agents: 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thym-
ine. J . Med. Chem. 1989, 32, 2507-2509.
(5) Tanaka, H.; Takashima, H.; Ubasawa, M.; Sekiya, K.; Inouye,
N.; Baba, M.; Shigeta, S.; Walker, R. T.; De Clercq, E.; Miyasaka,
T. Synthesis and antiviral activity of 6-benzyl analogues of 1-[(2-
hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent
and selective anti-HIV-1 agents. J . Med. Chem. 1995, 38, 2860-
2865.
(6) Hopkins, A. L.; Ren, J .; Esnouf, R. M.; Willcox, B. E.; J ones, E.
Y.; Ross, C.; Miyasaka, T.; Walker, R. T.; Tanaka, H.; Stammers,
D. K.; Stuart, D. I. Complexes of HIV-1 reverse transcriptase
with inhibitors of the HEPT series reveal conformational
changes relevant to the design of potent non-nucleoside inhibi-
tors. J . Med. Chem. 1996, 39, 1589-1600.
(7) Baba, M.; Tanaka, H.; Miyasaka, T.; Yuasa, S.; Ubasawa, M.;
Walker, R. T.; De Clercq, E. HEPT derivatives: 6-Benzyl-1-
(ethoxymethyl)-5-isopropyluracil (MKC-442). Nucleosides Nucle-
otides 1995, 14, 575-583.
(8) Press statement from Triangle Pharmaceuticals Inc. 17 J anuary
2002, http://www.tripharm.com/static/news/drug.html.
(9) Hopkins, A. L.; Ren, J .; Tanaka, H.; Baba, M.; Okamato, M.;
Stuart, D. I.; Stammers, D. K. Design of MKC-442 (emivirine)
analogues with improved activity against drug-resistant HIV
mutants. J . Med. Chem. 1999, 42, 4500-4505.
(23) (a) Richman, D.; Shih, C. K.; Lowy, I.; Rose, J .; Prodanovich, P.;
Goff, S.; Griffin, J . Human immunodeficiency virus type 1
mutants resistant to nonnucleoside inhibitors of reverse tran-
scriptase arise in tissue culture. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 11241-11245. (b) Hara, H.; Fujihashi, T.; Sakata, T.;
Kaji, A.; Kaji, H. Tetrahydronaphthalene lignan compounds as
potent anti-HIV type 1 agents. AIDS Res. Hum. Retroviruses
1997, 13, 6695-705.
(24) Nunberg, J . H.; Schleif, W. A.; Boots, E. J .; O’Brien, J . A.;
Quintero, J . C.; Hoffman, J . M.; Emini, E, A.; Goldman, M. E.
Viral resistance to human immunodeficiency virus type 1-specific
pyridinone reverse transcriptase inhibitors. J . Virol. 1991, 65,
4887-4892.
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