Phosphodiester amidates of unsaturated nucleoside analogues: Synthesis and Anti-HIV activity

Article abstract of DOI:10.1021/jm970069q

The effect of introduction of a lipophilic phosphodiester amidate moiety on the HIV activity of inactive unsaturated nucleoside analogues was investigated. Phesphodiester alaninates 5a, 5b, and 6 derived from unsaturated nucleoside analogues 3b, 3c, and 4

Full text of DOI:10.1021/jm970069q

J . Med. Chem. 1997, 40, 2191-2195
2191
P h osp h od iester Am id a tes of Un sa tu r a ted Nu cleosid e An a logu es: Syn th esis a n d
An ti-HIV Activity1
Holger Winter,† Yosuke Maeda,‡ Hiroyuki Uchida,‡ Hiroaki Mitsuya,‡ and J iri Zemlicka*,†
Department of Chemistry, Experimental and Clinical Chemotherapy Program, Barbara Ann Karmanos Cancer Institute,
Wayne State University School of Medicine, Detroit, Michigan 48201-1379, and The Experimental Retrovirology Section,
Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received February 4, 1997X
The effect of introduction of a lipophilic phosphodiester amidate moiety on the HIV activity of
inactive unsaturated nucleoside analogues was investigated. Phosphodiester alaninates 5a ,
5b, and 6 derived from unsaturated nucleoside analogues 3b, 3c, and 4a were synthesized
and investigated as inhibitors of cytopathic effect and replication of HIV-1 in ATH-8 cells.
Compound 5a is an inhibitor of HIV-1 whereas analogue 6 is inactive with cytotoxicity appearing
above 10 µM and 5b is both inactive and nontoxic. Alkaline or enzymic hydrolysis of 5a gave
phosphomonoester alaninate 14, a putative product of intracellular metabolism. Compound
14 as well as adenallene derivative 15c were devoid of anti-HIV activity, and they also failed
to inhibit HIV reverse transcriptase. A new regioselective method for preparation of (Z)-4-
(benzoyloxy)-1-hydroxy-2-butene, 7, a key intermediate for the synthesis of unsaturated
nucleoside analogues of cis configuration such as 3a , 3b, and 3c, is also described.
Ch a r t 1
Unsaturated nucleosides 1 are important anti-HIV
agents (Chart 1). A recently approved2 drug for AIDS,
d4T (1a , stavudine, Zerit), belongs to this class of
analogues. The corresponding cytosine analogue 1b is
also a strong inhibitor of replication3 of HIV. In the
acyclic series, two groups of analogues were studied as
potential antiviral agents. Compounds of the type 2 (R
) H), formally derived from 1 by deletion of the
ribofuranose oxygen, did not exhibit antiviral (anti-HIV)
activity4 per se, but phosphonate analogue 2a was an
anti-HIV agent (EC50 9 µM). The corresponding diphos-
phate phosphonates 2b inhibited viral DNA polymeras-
es and HIV-1 reverse transcriptase.4 Foreshortened
acyclic analogues of unsaturated nucleosides 1 such as
3a and 3b were also investigated as potential antiviral
agents. Compound 3b can be regarded5 as an acyclic
analogue of antibiotic neplanocin A. Analogues of type
3 as well as the respective E-isomers 4 were devoid of
antiviral activity,5,6 and only guanine analogue 3a
exhibited a moderate antiherpetic effect.5,7-9
Recent results have indicated10,11 that lipophilic phos-
phodiester amidates of anti-HIV agents such as AZT
and d4T can be successfully employed as delivery forms
of the corresponding 5′-phosphorylated intermediates to
overcome kinase deficiency of particular host cells.
Similar derivatives have also been shown to impart anti-
HIV activity on inactive nucleoside analogues.12 In
addition, anti-HIV activity of allenic nucleoside ana-
logues such as adenallene6,13 was significantly enhanced
by introduction of a lipophilic phenylphosphoralaninate
moiety.14 It was therefore of interest to study additional
phosphoramidates derived from other inactive acyclic
and unsaturated nucleoside analogues. In this paper
we describe results obtained with phenylphosphoralani-
nates 5a , 5b, and 6.
Syn th esis. The 2-butenol 3b, a starting material for
synthesis of 5a , was obtained by the known procedure.9
The previous method15 for preparation of 1-(benzoyloxy)-
4-hydroxy-2-butene, 7, a key intermediate in this syn-
thesis, suffers from a lack of regioselectivity. This
drawback was removed by transformation of butene-
1,4-diol 8 to the cyclic orthobenzoate 9 using trimethyl
orthobenzoate and p-toluenesulfonic acid in DMF
(Scheme 1) in 68% yield. Hydrolysis of 9 with 1% acetic
acid in THF afforded smoothly compound 7 (66% yield).
The latter was converted as described15 to the respective
1-(benzoyloxy)-4-bromo-2-butene, 10.
Although E-olefin 4a was readily deaminated with
adenosine deaminase5 on a preparative scale to give the
corresponding hypoxanthine derivative 4b in 79% yield,
the reaction of the Z-isomer 3b was sluggish. Therefore,
6-chloropurine was alkylated with 1-(benzoyloxy)-4-
bromo-2-butene, 10, to furnish intermediate 11 (51%
yield). Hydrolysis with 80% formic acid16 followed by
debenzoylation with methanolic ammonia afforded the
requisite hypoxanthine derivative 3c in 60% yield.
* Send correspondence to this author at the Barbara Ann Karmanos
Cancer Institute, E. Warren Ave., Detroit, MI 48201-1379. Tele-
phone: (313) 833-0715, ext 2452 or 2266. Fax: (313) 832-7294.
E-mail: zemlicka@kci.wayne.edu.
† Wayne State University School of Medicine.
‡ National Institutes of Health.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(97)00069-1 CCC: $14.00 © 1997 American Chemical Society

2192 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Winter et al.
Sch em e 1
Sch em e 3
Sch em e 2
F igu r e 1. Inhibition of infectivity and cytopathic effect of
HIV-1 in ATH-8 cells by analogue 5a . For details, see the
Experimental Section.
singlets, but both diastereoisomers of the E-amidate 6
gave only a single peak.
Biologica l Activity. Analogues 5a , 5b, and 6 were
tested as inhibitors of replication and cytopathic effect
of HIV-1 in ATH-8 cells. The results are given in Figure
1. Phosphoramidate 5a derived from Z-alkene 3b was
the most potent compound. The protective effect against
HIV infection was seen in the range of 1-10 µM with a
relatively low cytotoxicity. The corresponding hypox-
anthine derivative 5b was noncytotoxic in the tested
concentration region with a negligible anti-HIV effect.
Analogue 6 derived from E-alkene 4a was devoid of anti-
HIV activity, but a significant cytotoxicity was apparent
at a concentration of 1 µM and higher (data not shown).
Results with compound 5a provide additional evidence
that inactive unsaturated acyclic nucleoside analogues
can be activated by a transformation to the correspond-
ing lipophilic phosphoralaninates. In this respect, the
present findings extend our previous observations with
allenic phosphoralaninates14 to structurally more sim-
plified analogues such as 5a , 5b, and 6.
Hydrolysis of analogue 5a with aqueous triethy-
lamine11 afforded phosphomonoester alaninate 14 in
77% yield. Pig liver esterase (PLE)-catalyzed reaction
at pH 7.4 led to the same product. A similar transfor-
mation was observed with d4T and allenic phosphodi-
ester alaninates.11,14 Kinetics of PLE-catalyzed hydrol-
ysis of phosphoralaninates 5a , 5b, and 6 indicated a
significant retardation relative to allenic derivatives 15a
Analogues 5a , 5b, and 6 were obtained by phospho-
rylation of the corresponding 2-butenols 3b, 3c, and 4a
with phenyl chlorophosphoralaninate 12 and N-meth-
ylimidazole17 in yields ranging from 53 to 75% (Schemes
2 and 3). In the case of hypoxanthine derivative 3c the
crude product contained, in addition to 5b, a significant
amount of a faster moving component, presumably
intermediate 13. A similar byproduct was observed
earlier14 during reaction of hypoxallene with 12. The
workup of the reaction mixture with 80% acetic acid led
to a disappearance of 13, and phosphodiester amidate
5b was obtained in 53% yield.
Phosphodiester amidates 5a , 5b, and 6 are each a
mixture of two diastereoisomers which is reflected in
their HPLC profiles and NMR spectra. A similar
differentiation was observed previously in the case of
phosphodiester amidates derived from AZT10 and allenic
analogues.14 Thus, adenine derivatives 5a and 6 were
resolved into two peaks whereas hypoxanthine amidate
1
5b gave only a single one. The CH3O signals in the H
NMR and phosphorus in 31P NMR appeared in all cases
as two singlets. It is also interesting that the H8 signals
1
of H NMR of the Z-derivatives 5a and 5b formed two

Phosphodiester Amidates of Nucleoside Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2193
Ta ble 1. Kinetics of Hydrolysis of Phenylphosphoralaninate
Analogues 5a , 5b, and 6 Catalyzed by Pig Liver Esterase at 38
°C and pH 7.4a
77 (phenyl, 40.2); HRMS calcd M 206.09428, found 206.0946.
Anal. (C12H14O3) C, H.
(Z)-4-(Ben zoyloxy)-1-h yd r oxy-2-bu ten e (7). 1,3-Dioxa-
2-methoxy-2-phenyl-5-cycloheptene (9) (600 mg, 2.91 mmol)
was dissolved in THF (50 mL), and aqueous acetic acid (1%,
100 mL) was added. The resultant mixture was stirred at
room temperature for 5 h, whereupon it was extracted with
CH2Cl2 (2 × 100 mL). The organic phase was dried (Na2SO4),
and it was evaporated to yield 7 as a colorless oil (370 mg,
66.2%): 1H NMR ∂ 7.99 and 7.45 (2 m, 5, benzoyl), 5.85 and
5.70 (2m, 2, H2 and H3), 4.87 and 4.30 (2d, J ) 6.9 and 6.3 Hz,
H1 and H4); 13C NMR 166.72 (CO), 133.68, 133.14, 129.60,
128.41, 125.27 (phenyl, C2 and C3), 60.72 (C4), 58.30 (C1). This
product was used for the preparation of (Z)-4-(benzoyloxy)-1-
bromo-2-butene15 (10).
compd
half-life (t1/2, h)
compd
half-life (t1/2, h)
5a
5b
6
43.5
58.3
57.1
15a
15b
8.5b
7.3b
a
b
For details, see the Experimental Section. Reference 14.
and 15b (Table 1). It is then possible that differences
9-[(Z)-4-(Ben zoyloxy)-2-bu ten -1-yl]-6-ch lor opu r in e (11).
6-Chloropurine (2.00 g, 12.9 mmol) was dissolved in DMF (100
mL), and NaH (60%, 600 mg, 0.016 mol) was added in portions.
The mixture was stirred at room temperature for 30 min.
1-(Benzoyloxy)-4-bromo-2-butene15 (10) (4.0 g, 0.016 mol) was
added over a period of 20 min. The solution was stirred at
room temperature for 3 h. The solvent was evaporated, and
the residue was chromatographed on a silica gel column using
petroleum ether-ethyl acetate (1:1) to give 11 as a colorless
solid (2.18 g, 51.4%), which was recrystallized from metha-
nol: mp 105-106 °C; Rf (petroleum ether-ethyl acetate, 2:3)
0.5; UV max (EtOH) 265 nm (9800), 230 (15 200), 209 (18 700);
1H NMR (CDCl3) ∂ 8.73 (s, 1, H8), 8.28 (s, 1, H2), 8.03 (dd),
7.58 (split t) and 7.44 (t, total 5 protons, benzoyl), 6.05 and
5.90 (2 m, 2, H2′ and H3′), 5.14 and 5.07 (2d, 4, J ) 6.9 and J
) 7.2 Hz, H1′ and H4′); FAB MS 437 (M + thioglycerol + H,
7.8), 329.0 (M + H, 53.1). Anal. (C16H13N4O2Cl) C, H, N, Cl.
in affinity toward intracellular esterase(s) can also
influence the biological activity of prodrugs containing
a carboxylic ester group. Phosphoralaninates 14 and
15c are inactive against HIV, and they are not inhibi-
tors of HIV reverse transcriptase. Interestingly, less
polar amino acid phosphoramidate esters derived from
AZT were found to exhibit antiviral activity surpassing
that of parent compound, but they were inactive toward
reverse transcriptase.18 It is recognized that the less
polar phosphoramidate esters may penetrate cellular
membrane easier than phosphoramidate acids. At any
rate, our results show that intermediates 14 and 15c
cannot be directly responsible for antiviral effect of
phosphoralaninates 5a and 15a . Additional intracel-
lular activation appears to be necessary. Previously, we
suggested that intermediates such as 15c were further
processed to monophosphates by an intracellular phos-
phoramidase.14 Therefore, a similar transformation of
14 to phosphate 16 is also likely. This reaction course
was also postulated for phosphomonoester amidates
derived from 2′-deoxy-5-fluorouridine.19 Nevertheless,
a detailed mechanism of intracellular transformation
of P f N-amino acid amidates to phosphates remains
to be elucidated. Our results with compound 5a and
previous findings14 indicate that lipophilic nucleotide
analogues lacking an intact ribofuranose ring can be
activated by such a mechanism.
9-[(Z)-4-Hyd r oxy-2-bu ten -1-yl]h yp oxa n th in e (3c).
A
solution of 9-[(Z)-4-(benzoyloxy)-2-buten-1-yl]-6-chloropurine
(11, 1.00 g, 3.05 mmol) in 80% formic acid (100 mL) was stirred
at 80 °C for 5 h. After cooling, the volatile components were
removed in vacuo and water (20 mL) was evaporated from the
residue, which was then dissolved in methanolic ammonia
(20%, 100 mL). The solution was stirred at room temperature
for 36 h. The solvent was evaporated, and the residue was
chromatographed on a silica gel column using CH2Cl2-MeOH
(9:1) to give a colorless solid (380 mg, 60.3%), which was
recrystallized from methanol: mp 215-216 °C; Rf (CH2Cl2-
MeOH, 9:1) 0.1; UV max (EtOH) 207 nm (18 100), 250 (10 800);
1H NMR (CD3SOCD3, 500 MHz) ∂ 12.26 (s, 1, NH), 8.04 and
8.02 (2s, 2, H8 and H2), 5.70 and 5.58 (2m, 2, H2′ and H3′), 4.80
(m, 3, H1′ and OH), 4.16 (m, 2, H4′); 13C NMR (125 MHz) 134.72,
124.00 (C2′, C3′), 57.01 (C4′), C1′ is overlapped with solvent
signal; purine 156.61 (C6), 148.12 (C2), 145.50 (C4), 139.89 (C8),
124.25 (C5); FAB MS 207 (M + H, 61.4), 137 (hypoxanthine +
H, 24.4). Anal. (C9H10N4O3) C, H, N.
9-[(E)-4-Hydr oxy-2-bu ten -1-yl]h ypoxan th in e (4b). 9-[(E)-
4-Hydroxy-2-buten-1-yl]adenine20 (4a ) (200 mg, 0.98 mmol)
was dissolved in 0.02 M Na2HPO4 (30 mL, pH 7.5). Adenosine
deaminase (Sigma Chemical Co., St. Louis, MO, type II, 60
mg) was added, and the solution was stirred at room temper-
ature for 22 h. The solvent was evaporated, and the residue
was extracted with CH2Cl2-MeOH (1:1, 2 × 200 mL). The
organic phase was evaporated, and the residue was chromato-
graphed on a silica gel column using CH2Cl2-MeOH (9:1) to
give the title compound 4b as a colorless solid (158 mg, 79.1%),
which was recrystallized from methanol: mp 216-218 °C; Rf
(CH2Cl2-MeOH, 9:1) 0.1; UV max (EtOH) 209 nm (13 400),
250 (12 000); 1H NMR (CD3SOCD3) ∂ 12.28 (s, 1, NH), 8.04
and 8.01 (2s, 2, H8 and H2), 5.75 and 5.65 (2 m, 2, H2′ and H3′),
4.74 (m, 3, H1′ and OH), 3.89 (bs, 2, H4′); 13C NMR 134.95,
123.99 (C2′, C3′), 60.86 (C4′), 44.84 (C1′); purine 157.10 (C6),
148.63 (C4), 146.03 (C2), 140.50 (C8), 124.32 (C5); FAB MS 413
(2M + H, 4.8), 315 (M + thioglycerol + H, 5.0), 207 (M + H,
61.4), 137 (hypoxanthine + H, 24.4). Anal. (C9H10N4O3) C,
H, N.
Exp er im en ta l Section
Gen er a l Met h od s. See ref 14. The NMR spectra were
determined in CDCl3 at 300.095 (1H), 74.47 (13C), and 121.47
MHz (31P) unless stated otherwise.
1,3-Dioxa -2-m eth oxy-2-p h en yl-5-cycloh ep ten e (9).
A
mixture of 2-butene-1,4-diol (8) (2 mL, 0.024 mol), trimethyl
orthobenzoate (5 mL, 0.30 mol), and p-toluenesulfonic acid (200
mg, 1.1 mmol) in DMF (15 mL) was stirred at room temper-
ature for 8 h. Triethylamine (1 mL) was added, followed by
water (150 mL), and the solution was extracted with petroleum
ether (5 × 150 mL). The organic phase was dried (Na2SO4)
and evaporated. The crude product was chromatographed on
a silica gel column using petroleum ether-triethylamine (98:
2) to give compound 9 as a colorless oil (3.34 g, 68.0%): 1H
NMR (CDCl3) ∂ 7.65 and 7.36 (2 m, 5, phenyl), 5.70 (apparent
t, 2, H5 and H6), 4.45 and 4.07 (2d, 4, J ) 15.6 Hz, H4 and H7),
3.05 (s, 3, OCH3); 13C NMR 136.63, 128.87, 127.97, 127.40
(phenyl, C5 and C6), 116.01 (C2), 61.41 (C4 and C7), 50.87
(OCH3); EI-MS 206 (M, 1.9), 175 (50.6, M - CH3O), 105 (100.0),
9-[(Z)-4-Hyd r oxy-2-bu ten -1-yl]a d en in e (R,S)-4′-(Meth yl
p h en yl p h osp h or yl-/P fN/-L-a la n in a te) (5a ). 9-[(Z)-4-Hy-
droxy-2-buten-1-yl]adenine20 (3b, 120 mg, 0.58 mmol) was
added to a solution of phenyl methoxyalaninylphosphorochlo-
ridate17 (12, 400 mg, 1.35 mmol) in THF (10 mL). After

2194 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Winter et al.
addition of N-methylimidazole (0.25 mL, 3 mmol), the mixture
was stirred at room temperature for 2.5 h. The solvent was
removed and the residue dissolved in CH2Cl2 (50 mL). The
solution was washed with water (3 × 15 mL), dried (Na2SO4),
and evaporated. The residue was chromatographed on a silica
gel column using CH2Cl2-MeOH (95:5) to yield the title
compound 5a as a colorless foam (195 mg, 75.3%): Rf (CH2-
Cl2-MeOH, 9:1) 0.4; HPLC, retention time 10.25 and 10.85
min; purity 97.8%; UV max (EtOH) 209 nm (25 700), 261
(14 900 nm); 1H NMR (CDCl3) ∂ 8.31, 8.23 (2s, 1, H8), 7.85 (s,
1, H2), 7.21 (m, 5, phenyl), 6.33 (s, 2, NH2), 5.80 (m, 2, H2′ and
H3′), 5.02 - 4.73 (m, 5, H1′, H4′ and NH of Ala), 4.10 (m, 1, CH
of Ala), 3.68, 3.65 (2s, 3, OCH3 of Ala), 1.37 (t, 3, CH3 of Ala);
31P NMR 3.73, 3.41; 13C NMR 128.53, 128.44, 128.09, 127.94
(C3′, C2′), 61.76 (C4′), 40.38, 40.28 (C1′); purine 155.61 (C6),
152.87, 152.77 (C2), 149.67 (C4), 140.18 (C8), 119.45, 119.40
(C5); alanine 174.06 (split peak, CO, ester), 52.36, 52.32
(OMe); 50.25, 50.16 (CH); 20.79, 20.82 (Me); phenyl 150.61 (C-
ipso), 129.68 (C-para), 124.93 (C-ortho); 120.17 (C-meta); FAB
MS 555 (M + thioglycerol + H, 2.5), 447 (M + H, 17.2), 296.0
(33.1), 189 (97.1), 137 (hypoxanthine + H, 35.0). Anal.
(C19H22N5O6P) C, H, N.
9-[(Z)-4-Hyd r oxy-2-bu ten -1-yl]a d en in e 4′-(P h osp h or yl-
/P fN/-L-a la n in a te) (14). Analogue 5a (180 mg, 0.41 mmol)
was dissolved in H2O-Et3N (1:1, 15 mL), and the reaction
mixture was stirred at room temperature for 2 h. The
triethylamine phase was removed and the aqueous phase
evaporated in vacuo at room temperature. The resulting crude
product was chromatographed on silica gel column using CH2-
Cl2-MeOH-H2O-NH3 (120:70:10:1) to give compound 14 as
a colorless foam (121.5 mg, 77.4%): Rf 0.35; HPLC, retention
time 4.6 min; purity 100%; UV max (EtOH) 262 nm (ꢀ 11 800),
211 (ꢀ 15 500); 1H NMR (D2O + 1 drop of Et3N) ∂ 8.06 and
8.04 (2s, 2, H8 and H2), 5.80 and 5.70 (2m, 2, H2′ and H3′), 4.82
and 4.44 (m and t, 4, H4′ and H1′), 3.52 (m, 1, CH-Ala), 1.18 (d,
3, CH3-Ala); 31P NMR 7.65.
En zym e Stu d ies. A. P ig Liver Ester a se. Analogues 5a ,
5b, and 6 were dissolved in 0.02 M Na2HPO4 (pH 7.4) at a
concentration of 2.2 × 10-3 M. To each solution (1 mL) was
added pig liver esterase (PLE, 200 units), and the mixtures
were stirred at 38 °C. At selected time intervals aliquots (20
µL) were analyzed by HPLC as described above using water
-CH3CN (9:1, 0-3 min) and water -CH3CN (7:3, 3-15 min).
The half-lives t1/2 are listed in Table 1.
(17.8), 188 (100), 136 (adenine + H, 24.5). Anal. (C19H23
-
N6O5P) C, H, N.
9-[(E)-4-Hyd r oxy-2-bu ten -1-yl]a d en in e (R,S)-4′-(Meth yl
p h en yl p h osp h or yl-/P fN/-L-a la n in a te) (6). 9-[(E)-4-Hy-
droxy-2-buten-1-yl]adenine20 (4a , 100 mg, 0.486 mmol) was
added to a solution of phenyl methoxyalaninylphosphorochlo-
ridate (12, 340 mg, 1.13 mmol) in dry THF (10 mL). After
addition of N-methylimidazole (0.213 mL, 2.6 mmol), the
reaction mixture was stirred at room temperature for 3 h. The
solvent was removed and the residue dissolved in CH2Cl2 (50
mL). The solution was washed with water (3 × 15 mL), dried
(Na2SO4), and evaporated. The residue was purified by
chromatography on a silica gel column using CH2Cl2-MeOH
(95:5) to yield compound 6 as a colorless foam (131 mg,
60.5%): Rf (CH2Cl2-MeOH, 9:1) 0.4; HPLC, retention time
9.58 and 10.53 min; purity 98.7%; UV max (EtOH) 210 nm
B. HIV R ever se Tr a n scr ip t a se. Inhibitory activity of
phosphoramidates 14 and 15c against reverse transcriptase
(RT) was determined as previously described.21,22 Briefly,
various concentrations of tested analogues were added to
reaction mixtures (50 µL) containing poly(rA)‚(dT)12-18, Tris‚-
HCl (50 mM, pH 7.8), MgCl2 (6 mM), Triton X-100 (0.01%),
[3H]TTP, and RT (2 nM) at 0 °C. Reaction was initiated by
raising the temperature of the reaction mixtures from 0 to 37
°C. The mixtures were incubated for 30 min at 37 °C, and
they were quenched by the addition of EDTA (0.5 M, 25 µL).
Products were then examined by a DE81 filter binding assay.22
A portion of the reaction mixture (40 µL) was spotted on DE81
filters, and in order to remove unincorporated [3H]TTP, filters
were washed twice with sodium citrate (0.3 M, pH 7.0), twice
with ethanol, and once with acetone before they were air-dried.
The amount of incorporated [3H] nucleotides was determined
by a scintillation counter. The results were expressed as
counts per minute of [3H]TMP (40-60 Ci/mmol; 1 Ci ) 37 GBq)
incorporated per 40 µL of the reaction mixture. Analogues 14
and 15c did not inhibit RT at 20 µM. In a control experiment,
AZTTP blocked 90% of the activity of RT at 1 µM.
1
(24 500), 261 (14 700 nm); H NMR (CDCl3) ∂ 8.32 (s, 1, H8),
7.78 (s, 1, H2), 7.26 and 7.14 (t and m, 5, phenyl), 6.34 (s, 2,
NH2), 5.90 and 5.70 (m, 2, H2′ and H3′), 4.78 (d, 2, J ) 5.4 Hz,
H1′), 4.57 and 4.46 (2 m, 3 H, H4′ and NH of Ala); 4.00 (m, 1,
CH of Ala), 3.66, 3.64 (2s, 3, OCH3 of Ala), 1.33 (t, 3, CH3 of
Ala); 31P NMR 3.03, 2.92; 13C NMR 129.01, 128.91, 127.32,
127.22 (C3′, C2′), 65.77 (split peak, C4′), 44.38 (C1′); purine
155.65 (C6), 153.03 (C2), 149.78 (C4), 140.10 (C8), 119.40 (C5);
alanine 173.93 (CO, ester), 52.36 (OMe); 50.07 (CH); 20.84,
20.77 (Me); phenyl 150.66 (C-ipso), 129.56 (C-para), 124.79 (C-
ortho); 120.04 (split peak, C-meta); FAB MS 555 (M +
thioglycerol + H, 16.4), 447 (M + H, 72.0), 296 (42.1), 188
(100.0), 136 (adenine + H, 51.9). Anal. (C19H23N6O5P) C, H,
N.
In h ibition of HIV-1 Cytop a th ic Effect. The assay was
+
performed with CD4 ATH8 cells as described.14 The ATH-8
cells (2 × 105) were exposed to HIV-1/HIVLAI (500 50% tissue
culture infectious dose) for 45 min, and they were cultured in
the presence of compounds 5a , 5b, and 6. Total viable cells
were counted on day 7. Percent protective effect of a compound
on survival and growth of ATH-8 cells exposed to the virus
was determined by the following formula: 100 × [(number of
viable cells exposed to HIV-1 and cultured in the presence of
the compound) - (number of viable cells exposed exposed to
HIV-1 and cultured in the absence of of the compound)]/
[(number of viable cells cultured alone) - (number of viable
cells exposed to HIV-1 and cultured in the absence of the
compound)]. Percent cytotoxicity of a compound was deter-
mined as follows: 100 × [1 - (number of total viable cells
cultured in the presence of the compound)/(number of total
viable cells cultured alone)]. The data obtained for 5a are
given in Figure 1. Positive controls performed with ddI as well
as results with less effective compounds 5b and 6 are not
shown.
9-[(Z)-4-Hyd r oxy-2-b u t en -1-yl]h yp oxa n t h in e (R,S)-4′-
(Met h yl p h en yl p h osp h or yl-/P fN/-L-a la n in a t e) (5b ).
9-[(Z)-4-Hydroxy-2-buten-1-yl]hypoxanthine (3c, 100 mg, 0.486
mmol) was added to a solution of phenyl methoxyalani-
nylphosphorochloridate (12, 340 mg, 1.23 mmol) in dry THF
(10 mL). After addition of N-methylimidazole (0.213 mL, 2.59
mmol), the reaction mixture was stirred at room temperature
for 4 h. The solvent was evaporated, the residue was dissolved
in 80% acetic acid (40 mL), and the mixture was stirred at
room temperature for 16 h. The solvent was evaporated and
the residue purified by chromatography on a silica gel column
using CH2Cl2-MeOH (9:1) to give compound 5b as a colorless
foam (115 mg, 53%): Rf (CH2Cl2-MeOH, 9:1) 0.3; HPLC,
retention time 9.96 min; purity 98.7%; UV max (EtOH) 209
nm (17 300), 250 (11 100 nm); 1H NMR (CDCl3) ∂ 8.16 and 8.12
(2s, 1, H8), 7.86 (s, 1, H2), 7.34-7.12 (m, 5, phenyl), 5.90 and
5.80 (2m, 2, H2′ and H3′), 4.90 (m, 4, H1′ and H4′), 4.26 and
4.12 (2m, 2, NH of Ala and CH of Ala), 3.71, 3.68 (2s, 3, OCH3
of Ala), 1.39 (t, 3, CH3 of Ala); 31P NMR 3.45, 3.25; 13C NMR
128.93, 128.84, 127.71, 127.59 (C3′, C2′), 61.95 (split peak, C4′),
40.69 (C1′); purine 158.71 (C6), 148.77 (C2), 145.18 (C4), 139.81
(C8), 124.43 (C5); alanine 173.90 (CO, ester), 52.44 (OMe); 50.23
(split peak, CH); 20.76 (Me); phenyl 150.64 (C-ipso), 129.62
(C-para), 124.93 (C-ortho); 120.10 (split peak, C-meta); FAB
MS 556 (M + thioglycerol + H, 6.9), 448 (M + H, 52.0), 297
Ack n ow led gm en t. We thank the Central Instru-
mentation Facility (Department of Chemistry, Wayne
State University, Dr. Robin J . Hood, Director) and,
particularly, Drs. M. Ksebati and C. Tronche for NMR
spectra and Dr. M. Kempff for mass spectra. The work
at the Barbara Ann Karmanos Cancer Institute was
supported in part by U.S. Public Health Service Re-
search Grant CA32779 from the National Cancer In-
stitute, Bethesda, MD, and in part by an institutional

Phosphodiester Amidates of Nucleoside Analogues
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grant to the Barbara Ann Karmanos Cancer Institute
from the United Way of Southeastern Michigan.
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