Phosphodiester amidates of allenic nucleoside analogues: Anti-HIV activity and possible mechanism of action

Article abstract of DOI:10.1021/jm960330n

Lipophilic phosphodiester amidates 2a, 2b, 4a, 4b, and 6 derived from anti-HIV agent adenallene 1a, 3a, inactive hypoxallene 1b, 3b, and 9-(4- hydroxy-2-butyn-1-yl)adenine (5) were synthesized and studied as inhibitors of HIV-1 in ATH8 cell system. All phosphodiester amidates were more biologically active than their parent nonphosphorylated compounds. Analogues 2a and 4a derived from (±)-adenallene 1a and (R)-enantiomer 3a are effective anti-HIV agents with EC₅₀ approximately 0.88 and 0.21 μM, respectively. Both analogues are 16 and 28 times more effective than parent compounds 1a and 3a, respectively. Some anti-HIV activity of hypoxallene derivatives 2b and 4b was noted in the range of 0.1-10 μM but the dose- response relationship was poor. Phosphodiester amidate analogue 6 also exhibited anti-HIV activity in the range of 0.1-100 μM, but this effect was accompanied by cytotoxicity. Hydrolytic studies performed at pH 9.8 and with pig liver esterase at pH 7.4 have shown that analogue 2a gives adenallene 4'-phosphoralaninate (10a) as the major product. These results can be interpreted in terms of initial hydrolysis of phosphodiester amidates 2a, 2b, 4a, 4b, and 6 catalyzed by intracellular esterase(s) to give stable phosphomonoester amidate intermediates with a free carboxyl group. The results obtained with hypoxallene phosphoramidates 2b and 4b indicate that the aminosuccinate-fumarate enzyme system responsible for activation of AIDS drug ddIno (didanosine, Videx) can also, albeit less efficiently, activate hypoxallene 4'-phosphate (9b) and the respective (R)-enantiomer released inside the HIV-infected cells.

Full text of DOI:10.1021/jm960330n

3300
J . Med. Chem. 1996, 39, 3300-3306
P h osp h od iester Am id a tes of Allen ic Nu cleosid e An a logu es: An ti-HIV Activity
a n d P ossible Mech a n ism of Action 1
Holger Winter,† Yosuke Maeda,‡ 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 May 2, 1996X
Lipophilic phosphodiester amidates 2a , 2b, 4a , 4b, and 6 derived from anti-HIV agent
adenallene 1a , 3a , inactive hypoxallene 1b, 3b, and 9-(4-hydroxy-2-butyn-1-yl)adenine (5) were
synthesized and studied as inhibitors of HIV-1 in ATH8 cell system. All phosphodiester
amidates were more biologically active than their parent nonphosphorylated compounds.
Analogues 2a and 4a derived from (()-adenallene 1a and (R)-enantiomer 3a are effective anti-
HIV agents with EC50 approximately 0.88 and 0.21 µM, respectively. Both analogues are 16
and 28 times more effective than parent compounds 1a and 3a , respectively. Some anti-HIV
activity of hypoxallene derivatives 2b and 4b was noted in the range of 0.1-10 µM but the
dose-response relationship was poor. Phosphodiester amidate analogue 6 also exhibited anti-
HIV activity in the range of 0.1-100 µM, but this effect was accompanied by cytotoxicity.
Hydrolytic studies performed at pH 9.8 and with pig liver esterase at pH 7.4 have shown that
analogue 2a gives adenallene 4′-phosphoralaninate (10a ) as the major product. These results
can be interpreted in terms of initial hydrolysis of phosphodiester amidates 2a , 2b, 4a , 4b,
and 6 catalyzed by intracellular esterase(s) to give stable phosphomonoester amidate intermedi-
ates with a free carboxyl group. The results obtained with hypoxallene phosphoramidates 2b
and 4b indicate that the aminosuccinate-fumarate enzyme system responsible for activation
of AIDS drug ddIno (didanosine, Videx) can also, albeit less efficiently, activate hypoxallene
4′-phosphate (9b) and the respective (R)-enantiomer released inside the HIV-infected cells.
Synthesis and biological evaluation of phosphate
forms of nucleoside analogues capable of being delivered
inside the cells are important facets of current medicinal
chemistry.2-6 Several studies have established that
such phosphates can overcome the activating-enzyme
(kinase) deficiency3,6 in cells resistant to antiviral agents
such as AZT. Of the reported phosphate delivery forms,
phosphodiesters carrying a lipophilic ester moiety and
an amino acid as an amidate are particularly promising
and have undergone much scrutiny.3,7 Nevertheless,
most of the work in this area has centered on active
antiviral agents such as AZT and, very recently8 d4T,
but reports on activation of less active or inactive
nucleoside analogues are scant.9
For initial experiments, we have selected the phenyl
phosphoralaninate 2a derived from an active anti-HIV
agent adenallene 1a and a similar analogue 2b obtained
from hypoxallene (1b) which lacks anti-HIV activity.
Because our previous studies12,13 showed that (R)-
adenallene (3a ) and (R)-cytallene were responsible for
biological effects of the parent racemic compounds, we
have included the phosphoralaninates 4a and 4b in our
investigation. Likewise, analogue 6 derived from 9-(4-
hydroxy-2-butyn-1-yl)adenine (5), a synthetic precur-
sor14 of adenallene (1a ) which is devoid11 of anti-HIV
effect, was also of interest. In this paper, we report on
the synthesis and biological evaluation of analogues 2a ,
2b, 4a , 4b, and 6 as well as chemical and enzymatic
hydrolysis of 2a , 2b.
In recent years, we have investigated a new class of
anti-HIV agents, allenic nucleoside analogues.10 The
antiretroviral effect of two of these analogues, adenal-
lene (1a ) and cytallene, is similar to the corresponding
2′,3′-dideoxyribonucleosides,11 2′,3′-dideoxyadenosine,
and 2′,3′-dideoxycytidine, whereas the allene derivatives
of hypoxanthine 1b, guanine, and thymine were inac-
tive. It is therefore of interest to examine lipophilic
phosphate derivatives of active and inactive allenic
analogues as potential anti-HIV agents. Two questions
are of particular importance: (i) Will a lipophilic moiety
increase the anti-HIV efficacy of active allenic analogues
and (ii) will such a function activate inactive analogues?
Syn th esis. Analogues 2a , 2b, 4a , 4b, and 6 were
prepared by a method used for synthesis3 of phen-
ylphosphoralaninates of AZT (Schemes 1-3). Com-
pounds 1a , 1b, 3a , 3b, and 5 which were described
previously12,14,15 served as starting materials and phos-
phorochloridate16 7 as a phosphorylating agent. The
butynol derivative 5 was prepared by alkylation of
adenine with 1-bromo-4-(benzoyloxy)-2-butyne17 as de-
scribed.15 The 1,4-butynediol monobenzoate which is a
starting material for preparation of the latter alkylating
agent was obtained by the known procedure17 or by
reesterification of 2-butyne-1,4-diol dibenzoate with
2-butyne-1,4-diol according to a method described for
ethylene glycol monobenzoate.18 The advantage of this
procedure is the utilization of 2-butyne-1,4-diol diben-
zoate, a side-product which forms in substantial amounts
during preparation of the corresponding monoben-
zoate.17 The (R)-hypoxallene (3b) was obtained by
deamination of (R)-adenallene (3a ) with adenosine
* Send correspondence to this author at the Barbara Ann Karmanos
Cancer Institute, 110 E. Warren Ave., Detroit, MI 48201-1379. Tele-
phone: (313) 833-0715, ext 312 or 262. Fax: (313) 832-7294. e-mail:
zemlicka@kci.wayne.edu.
† Wayne State University School of Medicine.
‡ National Cancer Institute.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(96)00330-5 CCC: $12.00 © 1996 American Chemical Society

Phosphodiester Amidates of Allenic Nucleoside Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3301
Sch em e 1a
a
Schemes 1 and 2: Series a: B ) adenin-9-yl, series b: B )
hypoxanthin-9-yl. Also see legend for Scheme 3.
Sch em e 2a
a
See legend for Schemes 1 and 3.
Sch em e 3
a
F igu r e 1. Inhibition of infectivity and cytopathic effect of
HIV-1 in ATH8 cells by phosphodiester alaninates 2a and 4a .
For details see Experimental Section.
Schemes 1-3: (i) 7, N-methylimidazole, THF.
deaminase.12,14 The chromatography was avoided by
performing the reaction in water instead of a phosphate
buffer, enzyme was denatured by a brief heating, and
product 3b was obtained in 71% yield.
atom. Consequently, analogues 2a and 2b derived from
racemic allenes 1a and 1b are the mixtures of four
diastereoisomers whereas each of the phosphates 4a ,
4b derived from (R)-allenes 3a , 3b and optically inactive
butynol 5 contains only two diastereoisomers. This was
corroborated by NMR spectra although not all the
signals were resolved. The 31P spectra of phosphodi-
ester amidates 2a , 2b, 4a , 4b, and 6 showed the
requisite number of peaks expected for a given diaste-
reoisomeric composition. The HPLC was also indicative
of a partial resolution of diastereoisomers. Thus, all
phosphodiester amidates examined gave two poorly
resolved peaks. A similar behavior was noted in case
of the corresponding analogues of AZT3 which are
mixtures of two diastereoisomers.
Biologica l Activity. Analogues 2a , 2b, 4a , 4b, and
6 were tested as inhibitors of replication and cytopathic
effect of HIV-1 in ATH8 cells.11,12 Results with phos-
phoramidates of adenallene 2a and 4a are given in
Figure 1. Analogue 2a (EC50 0.88 µM) is 16 times more
effective than the parent racemic analogue12 1a (EC50
14 µM). Phosphoramidate 4a is more active than 2a
(EC50 0.21 µM), and the results then follow the pattern12
observed in case of adenallene (1a ) and (R)-enantiomer
3a . The presence of a monophosphate generated from
(S)-enantiomer of adenallene does not seem to influence
the activity profile. A higher activity of 2a and 4a was
accompanied by an increase of cytotoxicity relative to
(()-adenallene11 (1a ) and (R)-enantiomer12 3a (IC50 2.1
and 3 µM, respectively). This indicates that an increase
of intracellular concentration of the phosphorylated
intermediate(s) does not necessarily lead to an improve-
ment of the selectivity index of a given analogue. A
toxicity effect related to an intracellular generation of
the free 5′-nucleotide of an antiviral agent was reported
earlier.20 Possibly, the higher levels of intracellular
phosphorylated intermediates can increase their sub-
strate activity toward mammalian enzymes and, con-
sequently, inhibit the DNA polymerase. It is likely that
The yields of phosphorylation were dependent on the
extent of precipitation of a syrupy material, presumably
a complex between phosphorochloridate 7 and N-meth-
ylimidazole, containing also the starting material as
shown by TLC. Thus, allenic phosphates 2a and 4a
were obtained in 71 and 65% yield, respectively, whereas
yields of 2b, 4b, and 6 were lower (33-52%). No
significant N-phosphorylation was observed in case of
adenine derivatives 2a and 4a . With hypoxanthine
derivatives 1b and 3b, formation of an intermediate less
polar than 2b or 4b was observed. The column chro-
matography or treatment of the crude reaction product
with silica gel and methanol led to disappearance of this
compound for which a tentative structure 8 is proposed.
It is known that the O6 of purine nucleosides can be
attacked by phosphorylating agents.19
Analogues 2a , 4a and 2b, 4b are hygroscopic and of
limited stability in solid form and, therefore, microana-
lytical data were not obtained. Their frozen solutions
in DMSO stored at -70 °C were stable for several
months without change in the biological activity. All
compounds were obtained as amorphous foams, and
they were characterized by 1H, 13C, 31P NMR, mass, and
quantitative UV spectra as well as TLC and HPLC.
Phosphodiester amidates 2a , 2b, 4a , 4b, and 6 contain
an L-alanine moiety and an asymmetric phosphorus

3302 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Winter et al.
all diastereoisomers of the examined phosphodiester
amidates are capable of penetrating the cellular mem-
brane by a nonfacilitated transport, but their intrinsic
cytotoxicities, if any, may differ.
The data obtained with phosphodiester amidates 2a
and 4a are also of importance from the viewpoint of
mechanism of action of adenallene 1a and 3a . It was
established that cytallene was phosphorylated by deoxy-
cytidine kinase from leukemic spleen cells21 although
with a decreased efficiency. By contrast, enzyme that
phosphorylates adenallene (1a ) or 3a remains to be
identified22 although the fact that both analogues
inhibit11 HIV-1 and HIV-2 strongly suggests23 involve-
ment of phosphate intermediates. Anti-HIV activity of
2a and 4a clearly indicates involvement of phosphory-
lated species in the mechanism of action of adenallene
1a and 3a . The results with butyne phosphoramidate
6 derived from an inactive analogue11 5 are shown in
Figure 2. Although the former analogue was biologi-
cally active, no clear separation of antiviral activity and
cytotoxicity was observed.
Another inactive analogue, hypoxallene11 (1b) and
(R)-enantiomer 3b, became also activated after conver-
sion to phosphodiester amidates 2b and 4b (Figure 2).
The results indicated in both cases some anti-HIV
activity in the range of 0.1-10 µM. However, no clear
dose-response pattern was seen with either analogue
and cytotoxicity of 2b was noted at 10 µM and above.
Amidate 4b derived from (R)-enantiomer 3b was sig-
nificantly less toxic. These results also show that
hypoxallene 4′-phosphate (9b) (generated from 2b or 4b
inside the cells) is able to participate in further enzy-
matic activation by the adenylate succinate-fumarate
synthetase/lyase system operating in case of AIDS drug
2′,3′-dideoxyinosine (ddI, didanosine, Videx).24 The
lower potency of 2b and 4b relative to 2a and 4a reflects
probably the less efficient generation of free phosphate
9b and/or the latter metabolite is not an effective
substrate for synthetase/lyase enzyme system necessary
to provide adenallene 4′-phosphate (9a ). Also, the
stereochemical (R vs S) requirements of phosphate 9b
toward synthetase/lyase are not known. They may not
be necessarily the same as those for anti-HIV activity.
The difference in enantioselectivities between the anti-
HIV effect and action of adenosine deaminase12 toward
adenallene 1a may be seen as a precedent.
F igu r e 2. Inhibition of infectivity and cytopathic effect of
HIV-1 in ATH8 cells by phosphodiester alaninates 2b, 4b, and
6. For details see Experimental Section.
phosphodiesterase to give AZT and the corresponding
5′-phosphate. Other lipophilic but structurally unre-
lated (non-amidate) phosphodiester analogues were
shown to be converted to free phosphate monoesters by
esterase4 or combined esterase-phosphodiesterase ac-
tion.6 It was therefore of interest to undertake model
experiments which could shed some light on this
important problem.
Phosphodiester amidates 2a and 2b were found stable
at pH 7.5 for at least 24 h at room temperature. Some
hydrolysis (ca. 15% after 24 h) was noted at 37 °C.
Hydrolysis of both analogues was studied at pH 9.8 and
1.2. At pH 9.8 hypoxallene phosphoramidate 2b was
hydrolyzed significantly slower (t1/2 308 min) than
adenallene derivative 2a (t1/2 151 min). This may be
explained by ionization of the hypoxanthine residue (pK
∼ 9.5) which has a retardation influence on the rate of
hydrolysis. As expected, both amidates were hydrolyzed
at pH 1.2 with almost equal half-lives t1/2 24.4 h (2a )
and 23.4 h (2b), respectively.
Analogues 2a and 2b were inactive in a clonogenic
assay with murine leukemia L1210 cells (ID50 > 300
µM). They were less active than adenallene25 (1a )
(ID50 150 µM). It is possible that the metabolism in
these cells does not provide for an effective conver-
sion of 2a and 2b to the respective phosphorylated
intermediates.
Ch em ica l a n d En zym a tic Hyd r olysis of P h os-
p h od iester Am id a tes 2a a n d 2b. Although various
aspects of biological activity of phosphodiester amino
acid amidates of antiviral nucleosides, mostly AZT,
were studied earlier3,7,9,26,27 the mechanism of their
intracellular conversion to active phosphorylated spe-
cies has not been clarified. Hypotheses were advanced
that these analogues may be substrates for HIV pro-
teinase26,27 or an unspecified intracellular enzyme ca-
pable of cleaving the amino acid moiety.26 It was
surmised28 that phosphodiester of AZT resultant from
HIV proteinase cleavage may be further digested by
Pig liver esterase at pH 7.4 was used as a model4,8
for hydrolysis of the phosphodiester amidates 2a and

Phosphodiester Amidates of Allenic Nucleoside Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3303
12a . Hydrolysis of the latter product then gives phos-
phoramidate 10a . A similar mechanism was proposed
for hydrolysis of simple phosphodiester amino acid
amidates.30 The 5′-phosphoramidates similar to 10a
derived from nucleosides were a subject of much scru-
tiny.31,32 A similar course of chemical and enzymatic
hydrolysis was recently observed8 for phenyl phospho-
ralaninate of d4T 13a where phosphoramidate 13b was
identified as a major metabolite.
F igu r e 3. Kinetics of hydrolysis of phosphodiester alaninates
2a and 2b catalyzed by pig liver esterase at pH 7.4 and 37 °C.
For details see Experimental Section.
It is possible that phosphoramidate monoesters 10a
and 10b are intracellular metabolic products of phos-
phodiester amidates 2a and 2b. As already mentioned,
product 13b, similar to phosphoramidate monoester
10a , was found8 among the metabolites of phospho-
ralaninate 13a . It was suggested that 13b can contrib-
ute to the overall antiviral activity by intracellular
release of d4TMP but no details were given. Very
recently, it was shown that phosphoralaninate of anti-
HIV analogue isoddA 14a is metabolized in CEM cell
extract to a similar phosphomonoester amidate33 14b.
A hypothesis was proffered that further metabolic
disposition of 14b includes phosphodiesterase-catalyzed
hydrolysis to isoddAMP. However, it is known34 that
the P-N bond of nucleotide phosphomonoester amidates
derived from amino acids is resistant to phosphodi-
esterases. Allenic amidate 10a was also not hydrolyzed
by phosphodiesterase from snake venom. The question
of metabolic fate of 10a , 10b, 13b, and 14b thus remains
open. It cannot be excluded that such intermediates can
directly interfere with reverse transcriptase. To our
knowledge, there is no indication that nucleotide amino
acid amidates themselves can participate in a phospho-
rylation pathway catalyzed by nucleotide kinases to
obtain triphosphates necessary for inhibition of HIV
reverse transcriptase. Hydrolysis of benzylphosphora-
midate in dioxane led to a formation of the correspond-
ing symmetrical pyrophosphate,35 but it is doubtful
whether such a transformation can mimic some process
inside the cells. Alternately, an enzyme different from
phosphodiesterase may be involved in releasing free
phosphate monoesters 9a , 9b from phosphomonoester
amidates 10a , 10b. In this context, it is worthwhile to
note that a ribonucleoside phosphoamidase capable of
hydrolyzing ribonucleoside 5′-phosphoramidates was
isolated from bacterial and mammalian sources.31 The
substrates for this enzyme include phosphoramidates
with a free amino group, L- or D-amino acid residue.
However, 2′-deoxyribonucleoside 5′-phosphoramidates
are either weak substrates or they are inactive. It is
then not clear whether structurally more distant phos-
phoramidates such as 10a and 10b can be effectively
converted to phosphates 9a or 9b with this enzyme.
Intermediates such as 10a and 10b may also, at least
in some cases, represent a potential source of cytotox-
icity. Additional studies are needed to clarify all these
points.
Sch em e 4
2b by intracellular esterase(s). At room temperature,
the cleavage was slow (t1/2 ∼ 3 days). The reaction
became faster at 37 °C, but a relatively high concentra-
tion of enzyme was required. Both analogues were
smoothly hydrolyzed at pH 7.4 by pig liver esterase with
t1/2 440 min (2b) and 510 min (2a ), respectively (Figure
3). These model experiments have indicated that hy-
drolysis of phosphodiester amidates 2a , 2b, 4a , 4b, and
6 catalyzed by intracellular esterase(s) is plausible.
Peptidases capable of hydrolyzing the amino acid (pep-
tide) esters29 may also be involved.
Analysis of the hydrolysis products of phosphodiester
amidate 2a showed the following. Phosphomonoester
amidate 10a was identified as a major component after
hydrolysis of 2a at pH 9.8 and with pig liver esterase.
It is then likely that chemical or enzymatic hydrolysis
of the carboxylic ester function of 2a to give intermedi-
ate 11a is the first step of the process in both cases
(Scheme 4). A nucleophilic attack of the phosphorus
atom by the carboxylate function of 11a leads to an
expulsion of phenolate and formation of cyclic anhydride

3304 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Winter et al.
reaction with (R)-(-)-adenallene12 (3a ) was performed as in
the case of racemic compound 1a on 0.5 mmol scale using 4.26
mmol of N-methylimidazole. Yield 156 mg (65.1%) of 4a as a
mixture of two diastereoisomers. Rf (S2) 0.5; HPLC, retention
time: 24.62 and 23.62 min, purity 99.5%. UV (EtOH) λmax 261
nm (ꢀ 14 100 nm), 218 (ꢀ 24 800). 1H NMR δ 8.35, 8.34 and
8.02 (3s, 2H, H8 and H2), 7.4-7.0 (m, 6H, phenyl and H1′), 6.19
(m, 3H, H3′ and NH2); 4.78 (m, 2H, H4′), 4.55 (t), 4.44 (t) and
4.05 (m, 2H, CHNH of Ala), 3.71, 3.66 (2s, 3H, OCH3), 1.37
(apparent t, 3H, Ala-CH3). 31P NMR 3.28 and 3.20. 13C NMR
197.53, 197.23 (C2′), 101.57, 101.48, 101.38 (C3′), 95.09, 94.96
(C1′), 63.51, 63.28, 63.24 (C4′); alanine: 174.17, 174.09 (CO,
ester), 52.47 (OCH3), 50.23, 50.13 (CH); 20.79, 20.73 (CH3);
phenyl: 150.42 (C-ipso), 129.60, 129.48 (C-para), 124.94 (C-
ortho); 120.15, 119.48 (C-meta); adenine: 155.79 (C6), 153.30
(C2), 148.70 (C4), 138.47, 138.35 (C8), 119.48 (C5). FAB MS
445 (M + H, 49.3), 186 (100.0), 136 (adenine + H, 44.9).
Meth yl (R,S)-9-(4-Hyd r oxy-1,2-bu ta d ien -1-yl)h yp oxa n -
th in e (R,S)-4′-P h en ylph osph or yl-/P fN/-(S)-alan in ate (2b).
The procedure described for compound 2a was followed on 0.83
mmol scale of (R,S)-hypoxallene14 (1b). The crude product was
purified by chromatography on a silica gel column using
solvent system S2 to afford colorless foam 2b (192 mg, 51.9%)
as a mixture of four diastereoisomers. Rf (S2) 0.45; HPLC,
retention time: 19.17 and 18.48 min, purity 98.0%. UV
(EtOH) λmax 222 nm (ꢀ 24 700). 1H NMR δ 8.23, 7.96 and 7.94
(3s, 2H, H8 and H2), 7.4-7.0 (m, 6H, phenyl and H1′), 6.19 (qt,
1H, H3′), 4.77 (m, 2H, H4′), 4.33 (q), 4.16 (t) and 4.02 (m, 2H,
CHNH of Ala), 3.68 and 3.64 (2s, 3H, OCH3), 1.36 (apparent
t, 3H, Ala-CH3). 13P NMR 2.94, 3.01, 3.07 and 3.09. 13C NMR
197.69, 197.48 (C2′), 101.78, 101.66 (C3′), 95.04 (C1′), 63.30 (C4′);
alanine: 173.93 (CO, ester), 52.53, 52.51 (OCH3), 50.27, 50.18
(CH), 20.78 (CH3); phenyl: 150.50 (C-ipso), 129.63, 129.58 (C-
para), 125.02 (C-ortho), 120.21, 120.17 (C-meta); hypoxan-
thine: 158.54 (C6), 147.79 (C2), 145.93 (C4), 137.92 (C8), 124.70
(C5). FAB MS 446 (M + H, 28.4), 260 (55.8), 200 (86.0), 137
(hypoxanthine + H, 43.7).
Meth yl (R)-9-(4-Hyd r oxy-1,2-bu ta d ien -1-yl)h yp oxa n -
th in e (R,S)-4′-P h en ylph osph or yl-/P fN/-(S)-alan in ate (4b).
N-Methylimidazole (0.35 mL, 4.25 mmol) was added to a
solution of phosphorochloridate 7 (600 mg, 1.98 mmol) in dry
THF (6 mL). After 5 min, (R)-(-)-hypoxallene (3b) (100 mg,
0.49 mmol) was added in portions, and the reaction mixture
was stirred for 24 h at room temperature. The solvent was
removed, and the residue was dissolved in CH2Cl2 (50 mL).
The solution was washed with 5% aqueous HCl (2 × 15 mL)
and saturated NaHCO3 (2 × 15 mL), dried (Na2SO4), and
evaporated. The residue was dissolved in MeOH (10 mL), and
silica gel (1 g) was added. The suspension was stirred for 5 h
at room temperature, it was evaporated to dryness, and the
residue was put on the top of a silica gel column which was
eluted with solvent S2 to give a colorless foam 4b (71 mg,
32.5%) as a mixture of two diastereoisomers. Rf (S2) 0.45;
HPLC, retention time: 17.76 and 20.21 min, purity 97.2%. UV
(EtOH) λmax 213 nm (ꢀ 24 600). 1H NMR δ 8.20, 7.97 and 7.96
(3s, 2H, H8 and H2), 7.4-7.0 (m, 6H, phenyl and H1′), 6.20 (qt,
1H, H3′), 4.79 (m, 2H, H4′), 4.12-3.87 (m, 2H, CHNH of Ala),
3.70 and 3.67 (2s, 3H, OCH3), 1.40 (s, 3H, Ala-CH3). 13P NMR
3.06 and 3.03. 13C NMR 197.70, 197.46 (C2′), 101.78, 101.66
(C3′), 95.04, 94.97 (C1′), 63.40, 63.23, 63.18 (C4′); alanine: 173.89
(CO, ester), 52.52 (OCH3), 50.16 (CH), 20.79, 20.73 (CH3);
phenyl: 150.49 (C-ipso), 129.58 (C-para), 125.04 (C-ortho),
120.17 (C-meta); hypoxanthine: 158.66 (C6), 147.82 (C2),
145.79 (C4), 137.99 (C8), 124.83 (C5). FAB MS 446 (M + H,
27.6), 260 (28.4), 200.0 (100.0), 137 (hypoxanthine + H, 35.1).
Met h yl 9-(4-H yd r oxy-2-b u t yn -1-yl)a d en in e 4′-P h en -
ylp h osp h or yl-/P fN/-(S)-a la n in a te (6). The procedure de-
scribed for analogue 4b was applied for phosphorylation of
butynol15 5 on a 0.99 mmol scale. The reaction mixture was
stirred at room temperature for 3 h. The solvent was
evaporated, and the residue was dissolved in CH2Cl2 (50 mL).
The solution was washed three times with water (3 × 10 mL),
dried (Na2SO4), and evaporated. The residue was purified by
chromatography on silica gel column using solvent S2 to give
two diastereoisomers of 6 as a colorless foam (188 mg, 43.2%).
Rf (S2) 0.4; HPLC, retention time: 22.20 and 22.77 min, purity
Exp er im en ta l Section
Gen er a l Meth od s. See ref 13. High-performance liquid
chromatography (HPLC) was performed using Synchropak
RP-P column (C18, 6.6 µm, 2.1 × 250 mm, SynChrom, Inc.,
Lafayette, IN) in water-CH3CN (9:1, 0-10 min; 85:15, 10-
15 min and 4:1, 15-30 min, flow-rate 1 mL/min, detection at
260 nm). The following solvents were used for TLC and
column chromatography: S1 (CH2Cl2-MeOH, 95:5) and S2
(CH2Cl2-MeOH, 9:1). The NMR spectra were determined in
CDCl3 at 300.095 (1H), 74.57 (13C), and 121.57 (31P) unless
stated otherwise. Adenosine deaminase (EC 3.5.4.4, Type II,
1.5 units/mg solid) and pig liver esterase (esterase from porcine
liver, EC 3.1.1.1, 210 units/mg protein) were obtained from
Sigma Chemical Co., St. Louis, MO. Phosphodiesterase from
Crotalus durissus (EC 3.1.4.1, 1.5 units/mg) was the product
of Boehringer Mannheim Biochemicals, Indianapolis, IN.
2-Bu tyn e-1,4-d iol Mon oben zoa te. A mixture of crude
2-butyne-1,4-diol dibenzoate (recovered from benzoylation of
2-butyne-1,4-diol,17 604.1 g, 2.05 mol), 2-butyne-1,4-diol (176.5
g, 2.05 mol), and chloroform (1 L) was refluxed for 7 h. After
cooling, the dark solution was washed with water (2 × 1 L),
the organic phase was dried (Na2SO4) and it was evaporated.
The crude product was distilled in vacuo and then redistilled
to give a yellow oil, bp 160-185 °C/0.08 mmHg, 324.8 g (41.7
%), containing, according to TLC (CH2Cl2), a trace of diben-
zoate. This product was suitable for a preparation of 1-bromo-
4-(benzoyloxy)-2-butyne.
(R)-(-)-9-(4-Hyd r oxy-1,2-bu ta d ien -1-yl)h yp oxa n th in e
(3b). A mixture of (R)-(-)-adenallene12 (3a , 310 mg, 1.51
mmol) and adenosine deaminase (Type II, 50 mg, 90 units)
was stirred in water (32 mL) at room temperature for 36 h.
The solution was then refluxed for 5 min to denature the
enzyme which was then filtered off. The solvent was evapo-
rated, and the solid residue was washed several times with
warm CH2Cl2-MeOH (4:1, 700 mL total). The combined
organic phase was evaporated, and the product was recrystal-
lized from 90% EtOH to give (R)-(-)-hypoxallene (3b) as
colorless crystals (229 mg, 71.4%), mp 228-229 °C after
another recrystallization from EtOH. UV (pH 7.0) λmax 224
nm (ꢀ 24 300). [R]25D -178° (MeOH, c 0.021), lit.12 for (S)-(+)-
enantiomer [R]25D 175° (MeOH, c 0.04). 1H NMR (CD3SOCD3)
δ 12.41 (s, 1H, NH), 8.12 and 8.07 (2s, 2H, H8 and H2), 7.3 (m,
1H, H1′), 6.20 (m, 1H, H3′), 5.15 (t, 1H, OH), 4.10 (m, 2H, H4′).
13C NMR 196.26 (C2′), 156.50 (C6), 147.60 and 146.68 (C4 and
C2), 138.37 (C8), 124.78 (C5), 106.63 (C3′), 94.27 (C1′), 59.18 (C4′).
Anal. C9H8N4O2 (C, H, N).
Meth yl (R,S)-9-(4-Hyd r oxy-1,2-bu ta d ien -1-yl)a d en in e
(R,S)-4′-P h en ylph osph or yl-/P fN/-(S)-alan in ate (2a). (R,S)-
Adenallene14 (1a ) (200 mg, 0.99 mmol) was added to a solution
of methyl chlorophenylphosphoryl-/PfN/-(L)-alaninate16 (7,
600 mg, 2.15 mmol) in dry THF (12 mL). After addition of
N-methylimidazole (0.34 mL, 4.15 mmol), the reaction mixture
was stirred at room temperature for 3 h. The solvent was
evaporated, and the residue was dissolved in CH2Cl2 (50 mL).
The solution was washed with water (3 × 15 mL), and it was
dried (Na2SO4) and evaporated. The crude product was
purified by chromatography on a silica gel column using
solvent system S1 to yield a colorless foam 2a (312 mg, 70.8%)
as a mixture of four diastereoisomers. Rf (S2) 0.5; HPLC,
retention time: 23.26 and 22.46 min, purity 98.5%. UV
(EtOH) λmax 261 nm (ꢀ 14 000), 211 (ꢀ 27 400). 1H NMR δ
8.343, 8.341, 8.336 and 8.00, 7.99, 7.98 (6s, 2H, H8 and H2),
7.4-7.0 (m, 6H, phenyl and H1′), 6.21-6.15 (m, 3H, H3′ and
NH2), 4.75 (m, 2H, H4′), 4.40 (q), 4.25 (q), and 4.05 (m, 2H,
CHNH of Ala), 3.71, 3.69, 3.67 and 3.66 (4s, 3H, OCH3), 1.37
(m, 3H, Ala-CH3). 31P NMR 2.84, 2.89, 3.00 and 3.03. 13C
NMR 197.57 (C2′), 101.54 (C3′), 95.9, 94.95 (C1′), 63.58, 63.29
(C4′); alanine: 173.98 (CO, ester), 52.51 (OCH3), 50.25, 50.16
(CH), 20.90 (CH3); phenyl: 150.48 (C-ipso), 129.64, 129.62,
129.51 (C-para), 125.00 (C-ortho), 120.20, 120.17 (C-meta);
adenine: 155.53 (C6), 153.28 (C2), 148.83 (C4), 138.56, 138.47
(C8), 119.60 (C5). FAB-MS 445 (M + H, 6.6), 260 (19.6), 200
(75.7), 136 (adenine + H, 100.0).
Met h yl (R)-9-(4-H yd r oxy-1,2-b u t a d ien -1-yl)a d en in e
(R,S)-4′-P h en ylp h osp h or yl-/P fN/-(S)-a la n in a te (4a ). The

Phosphodiester Amidates of Allenic Nucleoside Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3305
1
99.6%. UV (EtOH) λmax 261 nm (ꢀ 14 700), 209 (ꢀ 24 700). H
NMR δ 8.370, 8.365 and 8.00 (3s, 2H, H8 and H2), 7.4-7.0 (2m,
5H, phenyl), 5.88 (s, 2H, NH2), 5.01 (q, 2H, H1′), 4.81 (m, 2H,
H4′), 4.07 (m, 2H, CHNH of Ala), 3.71 and 3.69 (2s, 3H, OCH3),
1.38 (apparent t, 3H, Ala-CH3). 31P NMR 2.97 and 3.06. 13C
NMR 80.63, 80.52, 80.06, 79.98 (C2′ and C3′), 54.45, 54.38,
54.32 (C4′), 33.11 (C1′); alanine: 173.97 (CO, ester), 52.34
(OCH3), 50.04 (CH), 20.70 (CH3); phenyl: 150.47, 150.37
(C-ipso), 129.51 (C-para), 124.83 (C-ortho), 120.03, 120.01
(C-meta); adenine: 155.75 (C6), 153.06 (C2), 149.28 (C4),
139.54 (C8), 119.16 (C5). FAB MS 445 (M + H, 100.0), 186
(68.3), 136 (adenine + H, 46.3). Anal. C19H21N6O5P × 0.2 H2O
(C, H, N).
9-(4-Hyd r oxy-1,2-bu ta d ien -1-yl)a d en in e 4′-P h osp h or yl-
/P fN/-(S)-a la n in a te (10a ). Analogue 2a (270 mg, 0.60
mmol) was stirred in 0.08 M glycine buffer (pH 9.8, 100 mL)
at room temperature for 38 h. The solution was brought to
pH 7 with 80% acetic acid, and then it was applied on a DEAE
Sephadex A25 column (18 × 2 cm). The column was washed
with water (500 mL), and then it was eluted with a linear
gradient of water (500 mL) and 0.3 M NH4HCO3 (500 mL).
The major UV absorbing peak was lyophilized to give the title
compound 10a as an ammonium salt (132 mg, 59%), positive
with ninhydrin.36 Electrophoretic mobility of 10a at pH 7 was
1.04 of adenosine 5′-phosphate or adenallene 4′-phosphate37,38
(9a ). HPLC, water (0-9 min, 0.2 mL/min), and then water-
CH3CN, 9:1 (9-35 min, 1 mL/min), retention time: 5.9 min
(10a ), 7.9 min (9a ), and 16.6 min (1a ). UV (pH 7) λmax 260
nm (ꢀ 12 000), 218 (ꢀ 19 800) corresponds to that of adenallene
4′-phosphate37 9a . 1H NMR39 (D2O) δ 8.02 and 8.00 (2 split40
s, 2H, H8 and H2), 7.12 (m, 1H, H1′), 6.13 (m, 1H, H3′), 4.32
(bs, 2H, H4′), 3.39 (m, 1H, CH-Ala), 1.07 (d, 3H, CH3-Ala). 13P
NMR 7.78.
Hydrolysis of 10a with 0.05 M HCl gave adenallene 4′-
phosphate (9a ), adenallene (1a ), and alanine as the only
products. Product 10a was also formed during hydrolysis of
analogue 2a at pH 7.4 catalyzed by PLE as shown by HPLC
and paper electrophoresis.
Hyd r olysis Stu d ies. Aliquots of stock solutions of ana-
logues 2a and 2b in water (10-3 M) were diluted with
appropriate buffer: 0.08 M glycine buffer pH 9.8; aqueous
HCl, pH 1.2, and 0.02 M Na2HPO4, pH 7.0) to a final
concentration of 1.1 × 10-4 M. These solutions were stirred
at room temperature. At selected time intervals aliquots (20
µL) were removed and analyzed by HPLC using water-
CH3CN (9:1, 0-4 min) followed by water-CH3CN (7:3, 4-15
min). In case of acid hydrolysis (pH 1.2), the solution was
neutralized by adding saturated aqueous NaHCO3 (20 µL) and
then analyzed.
En zym e Stu d ies. A. P ig Liver Ester a se. Analogues 2a
and 2b 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 (200 units), and the mixtures were
stirred at 37 °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 results are shown in Figure 3.
B. P h osp h od iest er a se. Compound 10a was incubated
with phosphodiesterase from Crotalus durissus (55 µg, 0.082
units) in 0.1 M Tris-HCl (pH 8.6, 1 mL) at a concentration of
5.6 × 10-4 M for 72 h at 37 °C, and aliquots were analyzed by
HPLC as described above using water (0.2 mL/min) as eluent.
No degradation was observed. Adenylyl-/3′-5′/-uridine which
was used as a positive control was cleaved within 30 min.
In h ibition of HIV-1 Cytop a th ic Effect. The assay was
performed with CD4+ ATH8 cells as described.11,12,41 The ATH8
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 2a , 2b, 4a , 4b, and 6. Total viable
cells were counted on day 7. Percent protective effect of a
compound on survival and growth of ATH8 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
to HIV-1 and cultured in the absence 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 are summarized in
Figures 1 and 2. Positive controls performed with ddI and
adenallene 1a are not shown.
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, Dr. M. Ksebati and C. Tronche for NMR
spectra and Dr. M. Kempff for mass spectra. Our
thanks are also due to Dr. David Kessel, Department
of Pharmacology, Wayne State University School of
Medicine, for murine leukemia L1210 assays. The work
at the Barbara Ann Karmanos Cancer Institute was
supported in part by U. S. Public Health Service
Research Grant CA32779 from the National Cancer
Institute, Bethesda, MD, and in part by an institutional
grant to the Barbara Ann Karmanos Cancer Institute
from the United Way of Southeastern Michigan.
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