Acyclic nucleotide analogs derived from 8-azapurines: Synthesis and antiviral activity

Article abstract of DOI:10.1021/jm960314q

Reaction of phosphoroorganic synthons with 8-azaadenine, 8-aza-2,6- diaminopurine, and 8-azaguanine using cesium carbonate yielded regioisomeric 8-azapurine N⁷-, N⁸-, and N⁹-(2(phosphonomethoxy)alkyl) derivatives. This rea

Full text of DOI:10.1021/jm960314q

J . Med. Chem. 1996, 39, 4073-4088
4073
Acyclic Nu cleotid e An a logs Der ived fr om 8-Aza p u r in es: Syn th esis a n d An tivir a l
Activity
Anton´ın Holy´,*,† Hana Dvorˇa´kova´,† J indrˇich J indrˇich,†,‡ Milena Masoj´ıdkova´,† Milosˇ Budeˇsˇ´ınsky´,† J an Balzarini,§
Graciella Andrei,§ and Erik De Clercq§
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Praha 6, Czech Republic,
and Rega Institute for Medical Research, Catholic University Leuven, B-3000 Leuven, Belgium
Received April 26, 1996X
Reaction of phosphoroorganic synthons with 8-azaadenine, 8-aza-2,6-diaminopurine, and
8-azaguanine using cesium carbonate yielded regioisomeric 8-azapurine N7-, N8-, and N9-(2-
(phosphonomethoxy)alkyl) derivatives. This reaction followed by deprotection afforded isomeric
2-(phosphonomethoxy)ethyl (PME), (S)-(3-hydroxy-2-(phosphonomethoxy)propyl) [(S)-HPMP],
(S)-(3-fluoro-2-(phosphonomethoxy)propyl) [(S)-FPMP], (S)-(2-(phosphonomethoxy)propyl) [(S)-
PMP], and (R)-(2-(phosphonomethoxy)propyl) [(R)-PMP] derivatives. 13C NMR spectra were
used for structural assignment of the regioisomers. None of the 8-isomers exhibited any
antiviral activity against herpesviruses, Moloney murine sarcoma virus (MSV), and/or HIV.
9-(S)-HPMP-8-azaadenine (23) and PME-8-azaguanine (65) were active against HSV-1, HSV-
2, and CMV at 0.2-7 µg/mL, VZV at 0.04-0.4 µg/mL, and MSV (at 0.3-0.6 µg/mL). PME-8-
azaguanine (65) and (R)-PMP-8-azaguanine (71a ) protected MT-4 and CEM cells against HIV-
1- and HIV-2-induced cytopathicity at a concentration of ∼2 µg/mL.
In tr od u ction
Retroviruses are the exclusive target for compounds of
the PMP group,12 and the (R)-enantiomers of adenine
(PMPA, 5) and 2,6-diaminopurine (PMPDAP, 6) display
the highest known antiretroviral effect among nucle-
otide analogues.13 (R)-PMPA is the first compound
reported to completely protect macaques against SIV
infection.14
In the series of acyclic nucleotide analogues that
contain a phosphonomethyl ether function replacing the
phosphoric acid ester group four structural types of
these analogues possessing high activity against DNA
viruses and retroviruses have emerged: N-(2-(phospho-
nomethoxy)ethyl) (PME) derivatives and their 2-sub-
stituted analogues, HPMP [N-(3-hydroxy-2-(phospho-
nomethoxy)propyl)] derivatives, FPMP [N-(3-fluoro-2-
(phosphonomethoxy)propyl)] derivatives, and PMP [N-(2-
(phosphonomethoxy)propyl)] derivatives. The antiviral
activity is mostly connected with the presence of certain
purine heterocyclic bases: in the above cases, the
activity was always encountered in adenine, 2,6-diami-
nopurine and guanine derivatives.1
Adenine and 2,6-diaminopurine derivatives of the (S)-
HPMP series, (S)-HPMPA (1) and (S)-HPMPDAP (2),
exert a marked antiviral effect against DNA viruses.2
The guanine derivatives (S)-HPMPG (9) and its race-
mate are also active;3 however, they exhibit higher
cytotoxicity to the host cells. PME derivatives PMEA
(3) and PMEDAP (4) show potent in vitro and in vivo
antiviral activity against DNA viruses and retroviruses.4
The latter effect was verified in animal models for
retroviral infection (i.e., FIV, MLV, Visna virus).5
PMEA (Adefovir, 3)6 and its oral diester prodrug [bis-
(POM)-PMEA]7 are pursued in clinical trials against
AIDS and hepatitis B.8 The guanine derivative PMEG
(8) is also toxic to most cell lines in vitro; however, it
has a remarkable activity against papillomaviruses.9
The anticancer activity of purine PME derivatives was
reported in the literature.10 Similar activity against
animal retroviruses in vivo and HIV in vitro was found
for the fluoromethyl derivative of PMEA (FPMPA, 7).11
Detailed studies have further shown that the biologi-
cal effect has an enantiospecific character in all adenine
derivatives, while no absolute distinction between the
enantiomers was observed in the 2,6-diaminopurine and
guanine series.13 This effect can be interpreted by the
involvement of different nucleotide kinases in the
activation of these compounds.15 Guanine derivatives
are always the most active and simultaneously the most
* To whom correspondence should be addressed.
† Academy of Sciences of the Czech Republic.
‡ Present address: Institute of Organic Chemistry, Faculty of
Sciences, Charles University, Prague, Czech Republic.
§ Catholic University Leuven.
X Abstract published in Advance ACS Abstracts, September 1, 1996.
S0022-2623(96)00314-7 CCC: $12.00 © 1996 American Chemical Society

4074 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Holy´ et al.
Sch em e 1
toxic of the triad, the toxicity of the adenine derivatives
being the weakest. The inhibition of purine nucleoside
phosphorylase by the guanine phosphonates and their
metabolites (mono- and diphosphates) that perturbs the
general purine nucleotide pools in the cell could explain
the cellular toxicity in these cases.16
In numerous structure-activity studies we have
demonstrated that no activity in any of the usual targets
ever occurs with the phosphonate derivatives derived
from bases that are devoid of amino groups (hypoxan-
thine, xanthine) and that an additional substitution of
the purine ring at the position 2, 6, and/or 8 typically
suppresses the biological activity.1 However, in some
cases the nitrogen atom of the pyrimidine ring in purine
moiety can be replaced by the -CHd grouping without
substantial loss of antiviral activity. In particular, the
3-deazaadenine analogue of 9-(S)-(3-hydroxy-2-(phospho-
nomethoxy)propyl)adenine (1)17 showed not only a po-
tent antiviral effect18 but also a notable activity against
Plasmodium sp. surpassing that of the parent adenine
derivative 1.19 8-Azapurine (ν-triazolo[4,5-d]pyrimi-
dine) derivatives are subject of continuous interest in
medicinal chemistry. Thus, 9-isomers of diverse 8-azaad-
enine and 8-azahypoxanthine nucleosides exhibit anti-
viral or cytostatic activity.20 An antileishmanial or
antitrypanosomal activity was also reported for 8-aza-
inosine.21
As a continuation of our studies on base modification
in the series of acyclic nucleotide analogues, we have
already investigated the effects of the replacement of
-Nd for -CHd grouping at position 2 of the purine
ring.22 In this communication, we report the synthesis
of several types of nucleotide analogues derived from
8-azapurines. Our results were published in prelimi-
nary form.23 Several (phosphonylmethoxy)alkyl deriva-
tives of 8-azapurines were also reported by other
authors.24
8-azaadenine series, we have examined the alkylation
of 8-azaadenine (11) by (R)-2,2-dimethyl-4-[[(p-tolylsul-
fonyl)oxy]methyl]-1,3-dioxolane (14) (Scheme 1) in the
presence of cesium carbonate. The protected 1,3-diox-
olane derivatives 15-17 were separated by silica gel
chromatography, and their structure was unequivocally
assigned by 13C NMR and IR spectra. Contrary to the
N9 (15) and N8 (16) isomers that are in IR spectra
characterized by the frequencies of free and associated
Ch em istr y
6-amino function [ν(NH2)free, 3523, 3410 cm-1; νassoc
,
Although the formation of 9-isomers predominates in
the glycosylation reactions of 8-azapurines whether by
acid fusion, by reaction of halogenoses with chloromer-
curi or silyl derivative of the bases,25 or by the phase
transfer glycosylation,26 other regioisomers are regularly
formed during such reactions. In order to avoid the low
regiospecificity of 8-azapurine alkylation, the acyclic
analogues of nucleosides derived from 8-azapurines
[9-(2,3-dihydroxypropyl) derivatives of 8-azahypoxan-
thine and 8-azaadenine] were prepared by ring closure
reactions from 4-(azidomethyl)oxirane derivatives.24,27,28
We have been interested in evaluating the biological
activity of all regioisomers in the 8-azapurine series.
Therefore, in our synthetic approach, we have employed
alkylations of alkali metal salts of the commercially
available 8-azapurine bases. As alkylating agents, we
have mostly used synthons and procedures described
earlier for the preparation of other nucleotide analogues.
As the base, cesium carbonate29 was frequently found
to be useful for such alkylations. The individual major
regioisomers were in most cases isolated as phosphonate
diesters and characterized by NMR and UV spectra
before bromotrimethylsilane deprotection to the free
phosphonic acids.
3474, 3327 cm-1] the amino group in the N7-isomer 17
participates in the hydrogen bond formation with the
adjacent oxygen atom of the 1,3-dioxolane ring that is
reflected by the presence of ν(NH2)intramol.bridge, 3467,
3347 cm-1
. The ratio of regioisomers (estimated by
HPLC) was in this case N9:N8:N7 ) 47:46:7. This
structural assignment was used for other cases. 9-(S)-
(2,3-Dihydroxypropyl)-8-azaadenine (18) and its N8-
isomer 19 were prepared from these intermediates by
acid treatment. These compounds are 8-aza analogues
of 9-(S)-(2,3-dihydroxypropyl)adeninesan established
methylation inhibitor and antiviral agent30sand were
synthesized in the optically active form for the first time.
The synthesis of the 8-aza analogue of the antiviral
HPMPA (23) (Scheme 2) made use of the cesium
carbonate-mediated alkylation of 8-azaadenine (11) with
2(R)-[(diisopropylphosphono)methoxy]-3-(trimethyl-
acetoxy)propyl p-toluenesulfonate (20).31 This reaction
8-Aza a d en in e Der iva tives. In order to make un-
equivocal assignment of individual regioisomers in the

Acyclic Nucleotide Analogs Derived from 8-Azapurines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4075
Sch em e 2
Sch em e 3
afforded, after methanolysis, an equimolar mixture of
the protected isomeric intermediates 21 and 22. Their
treatment with bromotrimethylsilane followed by hy-
drolysis gave ultimately (S)-9-(3-hydroxy-2-(phospho-
nomethoxy)propyl)-8-azaadenine (23) and its 8-isomer
(24). The formation of 7-isomer was not detected in this
case.
Sch em e 4
A similar procedure consisting of the alkylation of
8-azaadenine (11) by a synthon bearing structural
features of the required side chain was applied to the
synthesis of 9-(2-(phosphonomethoxy)ethyl)-8-azaad-
enine (8-aza analogue of PMEA) (28) and its regioiso-
mers. 8-Azaadenine (11) afforded on treatment with
bis(2-propyl) [(2-chloroethoxy)methyl]phosphonate (25)32
protected intermediate diesters: the 9-isomer 26 slightly
prevailed over the 8-isomer 27 (ratio, 1.5:1). Additional
regioisomers were not detected. Free 8-aza analogues
of PMEA (28) and its 8-isomer 29 were obtained by
transsilylation reaction of individual diesters and sub-
sequent hydrolysis (Scheme 3).
An approximately equimolar ratio in the formation
of the N9- and N8-isomers was observed also in the
cesium carbonate-promoted alkylation of 8-azaadenine
with synthon 30.33 The intermediary diesters 31 and
32 were separated by silica gel chromatography, and
their structures were proven by 13C NMR. Treatment
with bromotrimethylsilane afforded the isomers of
N-(S)-(3-fluoro-2-(phosphonomethoxy)propyl)-8-azaad-
enine (33, 34) related to the antiretroviral compound
FPMPA [9-(S)-(3-fluoro-2-(phosphonomethoxy)propyl)-
adenine (7) (Scheme 4).
The synthon 35 was applied for the preparation of (R)-
PMP derivatives.11 Alkylation of 8-azaadenine in a
cesium carbonate-promoted reaction afforded the usual
mixture of regioisomeric diesters 36 and 37 with the
8-isomer 37 prevailing. This observation is in agree-
ment with recently published21 results of other authors
who made use of our previously described procedure.34
The 9- and 8-isomeric PMP-derivatives 38 and 39
were obtained from the protected intermediates (sepa-

4076 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Holy´ et al.
Sch em e 5
Sch em e 6
The synthesis of 9-(S)-(3-fluoro-2-(phosphonomethoxy)-
propyl)-8-aza-2,6-diaminopurine (55) followed the route
described for the 8-azaadenine analogue 31, i.e., treat-
ment of the base 12 with the synthon 30. The isomeric
diester intermediates 53 and 54 were separated on silica
gel and deprotected to afford the 9-isomer 55 and its
8-congener 56 (Scheme 4).
Contrary to the reaction with compound 25, 8-aza-
2,6-diaminopurine (12) gave on treatment with the
tosylate 35a in the presence of cesium carbonate the
(R)-9- (57) and (R)-8-isomer (58) only, in an approxi-
mately equimolar (1.25:1) ratio. The deprotection of the
phosphonate diester grouping gave the regioisomers of
the 8-aza analogues of (R)-PMP-series 59 and 60.
(Scheme 5).
8-Aza gu a n in e Der iva tives. Extremely low solubil-
ity of 8-azaguanine in the reaction medium unfavorably
influences the process of its alkylation. To circumvent
the problem of low solubility, 8-azaguanine (13) was
transformed by treatment with dimethylformamide
dineopentylacetal to its N2-[(dimethylamino)methylene]
derivative 61 which is well soluble in dimethylforma-
mide (the use of dimethylformamide dimethyl acetal led
to undesirable formation of N-methyl derivatives as side
products). Compound 61 gave on treatment with syn-
thon 25 and following methanolysis a mixture of 9- (62),
8- (63), and 7-isomers (64) in the ratio of 2:2:1. Their
chromatographic separation on silica gel is difficult.
However, the 9-isomer 62 could successfully be sepa-
rated from the other isomers by ion exchange chroma-
tography on Dowex 50 × 8 in the acid form. Elution
with water afforded chromatographically pure 9-isomer
62 while the mixture of the remaining 7- and 8-isomers
was eluted subsequently with diluted ammonia in
aqueous methanol. The diesters were deprotected by
transsilylation: compound 62 gave the pure 9-isomer
65 while the mixture of isomers 66 and 67 obtained by
rated by silica gel chromatography) by standard depro-
tection method (vide supra). No significant formation
of any additional regioisomer was observed under the
reaction conditions (Scheme 5).
8-Aza -2,6-d ia m in op u r in e Der iva tives. The syn-
thesis of (S)-9-(3-hydroxy-2-(phosphonomethoxy)propyl)-
8-aza-2,6-diaminopurine (47) makes use of a general
procedure for stepwise synthesis of HPMP compounds:
35
8-Aza-2,6-diaminopurine (12) was treated with [(tri-
tyloxy)methyl]-(R)-oxirane (40), and the resulting tri-
tylated intermediates 41 and 42 that were formed in
an approximately equimolar ratio were separated by
silica gel chromatography. The trityl derivative 41 was
transformed to the N2,N6-bis((dimethylamino)methyl-
ene) derivative 44 by the reaction with dimethylforma-
mide dimethyl acetal.36 Condensation with bis(2-propyl)
[[(p-tolylsulfonyl)oxy]methyl]phosphonate (46)31 in the
presence of excess sodium hydride followed by acid
hydrolysis gave the diester 45 that was cleaved to the
ultimate (S)-HPMP derivative 47 by transsilylation
reaction. The structure of the 9-isomer was also proven
by detritylation of compound 41 to 9-(S)-(2,3-dihydroxy-
propyl)-8-aza-2,6-diaminopurine (43) (Scheme 6).
Cesium carbonate mediated reaction of 8-aza-2,6-
diaminopurine (12) with bis(2-propyl) [(2-chloroethoxy)-
methyl]phosphonate (25) gave 9-, 7-, and 8-isomeric
diesters 48-50 in the ratio 10:1:8. We have not suc-
ceeded in the complete separation of the 7- (50) and
8-isomer (49); however, the 1H-NMR signals of com-
pound 50 in the 1:10 mixture with compound 49 were
unequivocally separated and assigned. The diesters 48
and 49 were transformed into the free phosphonates 51
and 52 (Scheme 3).

Acyclic Nucleotide Analogs Derived from 8-Azapurines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4077
Sch em e 7
the deprotection of their diesters was separated by
chromatography on Sephadex A-25 in triethylammo-
nium hydrogen carbonate (Scheme 7).
classes of 8-azapurine derivatives under study. Al-
though the 13C NMR data of some 8-azapurine nucleo-
sides were described by Seela25 and of 8-azapurine
acyclic phosphonates by Franchetti et al.,27 there is some
doubt left on the assignment of signals.25,27,37 Therefore,
we have examined this assignment and determined
experimentally the effects of substitution at the position
N7, N8, or N9.
A similar procedure was applied in the synthesis of
the enantiomeric PMP derivatives of 8-azaguanine:
reaction of N2-[(dimethylamino)methylene] derivative
61 with the synthons 35a or 35b followed by am-
monolysis gave the mixture of 9-, 7-, and 8-isomer in
the 9:1:8 ratio. This mixture was deprotected by trans-
silylation, and the resulting free phosphonates were
separated on Dowex 50 column (vide supra) to afford
pure 9-isomers 71a and 71b by elution with water. The
mixtures of the corresponding 8- (72a , 72b) and 7-iso-
mers (73a ,73b), obtained by elution of the column with
aqueous ammonia, were separated on Sephadex A-25
in triethylammonium hydrogen carbonate (Scheme 7).
NMR Sp ectr a . The structure of acyclic nucleosides
and nucleotides has been determined from their NMR
spectra. The acyclic moieties were unequivocally char-
Detailed NMR study was performed with three iso-
meric nucleosides derived from 8-azaadenine (15, 16,
and 17). Proton-decoupled “attached proton test” 13C
NMR spectra of 8-azaadenine derivatives allow a signal
of the methine carbon C-2 to be distinguished from
signals of the quaternary carbon atoms C-4, C-5, and
C-6. Proton-coupled 13C NMR spectra confirm the
assignment of C-2 (1J (C,H) ∼ 200 Hz) and distinguish
the signals of carbons C-4 and C-6 (with large trans-
3
3
coupling constants J (C4,H2) respectively J (C6,H2) ∼
12 Hz) from those of carbon C-5 which is not coupled to
proton H-2. The tentative assignment of signals be-
longing to C-4 and C-6 has been verified as follows: (a)
In a proton-coupled 13C NMR spectrum of compound 17
the signal at δ 151.91 (with J (C,H) ) 11.7 Hz) shows
additional splitting to triplet of doublets by interaction
with the NH2 protons (this splitting disappears after
addition of D2O) and therefore belongs to carbon C-6.
(b) Another evidence is based on the deuterium isotopic
effects on carbon chemical shifts: The presence of small
amount of D2O in a DMSO solution results in a partial
exchange of labile NH2 protons with deuterium that is
indicated by known isotopic upfield shifts of carbon
signals in the vicinity of the deuterated amino group.
Slow exchange between molecules containing ND2 and
NH2 in DMSO solution results in “doubling” of the
corresponding signals of two isotopomers. The observed
isotopic shifts in 17 (N7-isomer) (-0.066 ppm for signal
at δ 151.91, -0.031 ppm for signal at δ 114.23, and ∼ 0
ppm for signals at δ 160.35 and 154.57) together with
the expected decrease of the isotopic shifts in the â, γ,
and δ position led to the assignment of carbon signals
1
acterized by H NMR (see Experimental Section) and
13C NMR spectra (Tables 1-3). The presence of a
phosphonate group is manifested in 1H and 13C NMR
spectra by additional splitting of signals of the corre-
sponding hydrogen and carbon atoms in the vicinity of
phosphorus due to J (H,P) and J (C,P), respectively.
Determination of the position of the acyclic substitu-
ent at the heterocyclic base is more complicated. In the
8-azapurine series, solely 8-azaadenine contains hydro-
gen atom at C-2 while both 8-azaguanine and 8-aza-
2,6-diaminopurine possess exchangeable amine or amide
protons only. Therefore, their 1H NMR spectra are
useless for the purpose and the required structural
evidence has to be deduced mainly from the 13C NMR
spectra. Correct structural assignment of carbon sig-
nals is thus essential for precise determination of the
position of the acyclic side chain. Structural modifica-
tions of the substituent have little influence on the
chemical shifts of the carbon atoms of the heteroaro-
matic base. Hence, similar substitution effects of the
regioisomers (N7, N8, or N9) can be expected in all three

4078 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Ta ble 1. Carbon-13 NMR Spectral Data of 8-Azaadenine Derivatives
Holy´ et al.
comp
solvent
DMSO
C-2
C-4
C-5
C-6
C-1′ (J (C,F))
C-2′ (J (C,P); J (C,F))
C-3′ (J (C,F))
OCH2P (J (P,CH2))
11
156.20
151.35
123.28
156.23
N-9 Isomers
49.01
15a
18
DMSO
DMSO
D2O
156.97
156.97
157.84
156.77
156.89
156.84
149.44
149.87
147.50
148.29
149.47
149.50
123.83
124.37
125.50
124.41
123.86
123.84
156.41
156.72
158.10
156.57
156.39
156.43
73.53
(-)
66.48
64.09
61.74
50.49
47.89
47.01
70.64
(-)
23
81.30
(10.3)
69.85
(10.0)
77.45
(12.5; 19.0)
75.16
(13.7)
69.20
(150.2)
69.19
(150.5)
63.55
(164.1)
62.60
(164.8)
28
D2O
31b
36c
DMSO
DMSO
45.64
(8.8)
50.25
81.90
(169.2)
17.22
N-8 Isomers
59.17
16d
19
DMSO
DMSO
D2O
157.01
156.87
157.53
156.82
157.00
156.86
157.52
158.01
158.14
156.30
157.18
158.07
158.01
157.66
125.73
125.73
126.69
125.87
125.77
125.63
126.47
156.77
156.83
157.78
156.69
156.78
156.80
157.42
73.98
(-)
66.37
63.75
61.57
60.57
57.86
56.85
71.11
(-)
24
81.43
(12.4)
70.23
(10.0)
78.03
(13.2; 19.1)
75.71
(12.2)
77.01
(12.2)
67.30
(156.8)
69.21
(150.2)
63.75
(164.8)
62.67
(164.8)
66.52
29
D2O
32e
37f
39
DMSO
DMSO
D2O
56.21
(8.1)
60.70
81.80
(169.9)
16.91
61.60
17.54
(155.7)
N-7 Isomer
52.35
17g
DMSO
154.57
160.35
114.43
151.91
74.25
65.92
(-)
Other carbons are as follows: Di-O-isopropylidene: a109.30, 26.76, 25.73; d109.49, 26.84, 25.40; g109.36, 26.13, 25.08. P(OCH(CH3)2)2:
c
b70.42 d and 70.03 d (J (C,P) ) 6.6), 23.84 d and 23.79 d (J (C,P) ) 3.7), 23.63 d and 23.57 d (J (C,P) ) 4.4); 70.34 d and 70.21 d (J (C,P)
e
) 6.1), 23.88 d and 23.86 d (J (C,P) ) 3.1), 23.69 d and 23.65 d (J (C,P) ) 4.6); 70.48 d and 70.37 d (J (C,P) ) 5.9), 24.23 d and 23.82 d
(J (C,P) ) 3.7), 23.66 d and 23.58 d (J (C,P) ) 3.6); f70.38 d and 70.26 d (J (C,P) ) 6.1), 23.87 d and 23.85 d (J (C,P) ) 3.1), 23.68 d and 23.64
d (J (C,P) ) 4.6).
in the order of C-6, C-5 and C-2, C-4 which agrees with
the assignment derived above. Similarly, the isotopic
shifts for compound 16 (N8-isomer) are -0.074 ppm for
signal at δ 156.77 and -0.037 ppm at δ 125.73, and for
compound 15 (N9-isomer) -0.070 ppm at δ 156.41 and
-0.039 ppm at δ 123.83 always for carbon atoms C-6
and C-5, respectively. Analogous isotopic effects, in-
duced by deuteration of hydroxyl protons, were de-
scribed in 13C NMR spectra of saccharides.38 The above
results are in contrast with the earlier described as-
signment of C-4 and C-6 signals in the N7- isomer of
8-aza-2′-deoxyadenosine36 and in the N8-isomers of
8-aza-2′-deoxyguanosine25 and 2-amino-8-aza-2′-deoxy-
adenosine,25b respectively.
signal by vicinal coupling with hydrogens H-1′ of the
substituent can be observed which is absent in the N8-
isomer.
To determine the effect of substitution in individual
positions of 8-azapurines we have recorded also 13C
NMR spectra of the free bases in DMSO (Tables 1-3).
Average values of the substituent effects of 8-azaad-
enine, 8-azaguanine, and 8-aza-2,6-diaminopurine and
their N9-, N8-, and N7-substituted derivatives are pre-
sented in Table 4. It is evident that the substituent
effects for regioisomeric derivatives of all three 8-azapu-
rine bases are similar and can be used to distinguish
between N9-, N8-, and N7-isomers. In fact, each isomer
should be referred to the appropriate tautomeric form
of the unsubstituted base. Unfortunately, neither the
13C chemical shifts of individual tautomers nor their
population in solution have been published. Signifi-
cantly larger substituent effects on carbon atoms C-4
and C-5 which were observed in the N8-isomers, and
particularly the N7-isomers compared to the N9-isomers
(Table 4) may be due to a lower population of the
corresponding N8- and N7-tautomers of the free bases
in solution. Due to the limited solubility of the free
phosphonates in DMSO, their NMR spectra were mea-
sured in D2O. The data in Tables 1-3 show that the
13C chemical shifts of the bases in water are rather
similar to those in DMSO. However, a different situ-
ation is encountered in an alkaline aqueous solution.
The 8-azaguanine derivatives 72a (N-8) and 73a (N-7)
measured in D2O + NaOD show significant downfield
On the basis of the structural assignment of the
carbon signals, the experimental NMR evidence for the
assignment of different regioisomers was made possible
by the analysis of vicinal J (C,H) couplings between the
R-hydrogens of the N-substituent and the corresponding
carbon atom of the base in proton-coupled 13C NMR
spectra. In our previous paper39 we have used this
method for providing evidence for the N9- and N2-
isomers in 2-azaadenine derivatives. Different vicinal
couplings J (C,H) for the N9-, N8-, and N7-isomers of
8-aza-2′-deoxyadenosine were described in the litera-
ture.36 This method is applicable for distinguishing the
N9-, N8-, and N7-isomers in compounds 15, 16, and 17.
We have found that in proton-coupled 13C NMR spectra
of the N9- and/or N7-isomer a characteristic splitting or
at least significant line broadening of the C-4 and/or C-5

Acyclic Nucleotide Analogs Derived from 8-Azapurines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4079
Ta ble 2. Carbon-13 NMR Spectral Data of 8-Azaguanine Derivatives
compd
solvent
DMSO
C-2
C-4
C-5
C-6
C-1′
C-2′ (J (P,2′))
C-3′
OCH2P (J (P,CH2))
13
155.45
153.45
123.76
155.88
N-9 Isomers
155.98
62b
65
DMSO
D2O
155.63
157.15
155.64
151.77
152.17
151.91
124.34
125.26
124.22
45.43
47.30
49.86
69.94
(12.2)
70.68
(10.7)
75.30
(12.7)
64.81
(164.8)
69.75
(151.1)
62.79
159.54
156.00
68a a
DMSO
17.25
(165.0)
N-8 Isomers
156.71
63d
66
DMSO
D2O
154.01
155.54
154.34
165.35
159.83
159.51
159.77
161.08
126.66
126.84
126.71
128.74
55.52
56.95
59.74
60.82
70.18
(12.2)
71.13
(10.7)
75.59
(12.7)
77.02
(10.7)
64.74
(163.3)
68.75
(154.1)
62.70
(165.0)
68.25
(151.1)
156.51
156.75
169.73
69a c
72a
DMSO
NaOD
17.02
17.16
N-7 Isomers
154.28
64d
67
DMSO
D2O
153.82
155.35
161.14
160.82
113.78
114.81
49.62
51.43
70.51
(12.2)
71.26
(10.7)
64.82
(164.8)
68.86
155.54
(152.6)
70a e
73a
DMSO
NaOD
153.96
165.71
160.99
162.63
113.91
116.74
154.16
164.54
53.87
55.25
75.75
(12.7)
77.32
(11.4)
17.00
17.63
62.70
(165.0)
68.48
(151.1)
Other carbons are as follows. P(OCH(CH3)2)2: a70.44 d and 70.32 d (J (C,P) ) 5.9), 23.92 d and 23.90 d (J (C,P) ) 2.9), 23.76 d and 23.71
d (J (C,P) ) 4.9); b70.46 d (J (C,P) ) 6.1), 23.93 d (J (C,P) ) 3.1), 23.74 d (J (C,P) ) 4.6); c70.42 d and 70.32 d (J (C,P) ) 5.9), 23.92 d (J (C,P)
) 2.9), 23.76 d (J (C,P) ) 4.9); d70.49 d and 70.47 d (J (C,P) ) 6.1), 23.95 d and 23.92 d (J (C,P) ) 3.1), 23.75 d and 23.74 d (J (C,P) ) 4.6);
e70.42 d and 70.26 d (J (C,P) ) 5.9), 23.92 d (J (C,P) ) 2.9), 23.73 d (J (C,P) ) 4.9).
shifts of carbons C-2 and C-6 (10-13 ppm) and much
smaller downfield shifts of carbons C-4 and C-5 (1-3
ppm). Similar effects in the alkaline aqueous solutions
were described by Seela25b and explained by the oc-
curence of 8-aza-2′-deoxyguanosine monoanion.
MSV-induced transformation (IC50: ∼2 µg/mL). Inter-
estingly, several other 8-azapurine derivatives proved
markedly more active against MSV than HIV. For
example, compounds 51, 47, 39, and 59 were almost 2
orders of magnitude less inhibitory to HIV than to MSV
(Table 6). The reason for this phenomenon is unclear
so far, but could be related to a differential uptake and/
or metabolism in the murine versus human cells.
Alternatively, MSV reverse transcriptase (RT) might be
more susceptible to the inhibitory effects of these test
compounds (in their diphosphate form) than HIV RT.
Further experiments are needed to resolve these issues.
Biologica l Activity. Several 8-azapurine deriva-
tives within the PME, (S)-HPMP, (S)- and (R)-PMP, and
(S)-FPMP series were evaluated for their inhibitory
effect on the DNA viruses herpes simplex virus type 1
(HSV-1), HSV-2, cytomegalovirus (CMV), varicella-
zoster virus (VZV), and vaccinia virus, and against the
retroviruses Moloney murine sarcoma virus (MSV) and
human immunodeficiency virus type 1 (HIV-1) and HIV-
2. Among the PME derivatives, only PME-8-azagua-
nine (65) was inhibitory to HSV-1, the thymidine ki-
nase (TK-) deficient HSV-1 strains B2006 and
VMW1837, HSV-2, CMV, both TK+ and TK- VZV
strains and vaccinia virus at concentrations varying
between 0.04 and 0.4 µg/mL for VZV to 2 and 7 µg/mL
for HSV-1, HSV-2, and CMV. (S)-HPMP-8-azaadenine
(23) proved markedly inhibitory to all DNA viruses
tested. In particular, 23 was exquisitely inhibitory to
VZV (MIC: 0.2-2 µg/mL) (Table 5). In fact, compound
23 was endowed with an antiviral potency that was
comparable to that of the prototype compound (S)-
HPMPA (data not shown). (S)-HPMP-8-aza-2,6-diami-
nopurine (47) was approximately 2 orders of magnitude
less potent an inhibitor of these viruses than compound
23. None of the 8-aza-PMP or 8-aza-(S)-FPMP deriva-
tives proved markedly inhibitory to any of the DNA
viruses tested, except for compounds 38 and 39 that
showed a moderate antiviral activity against VZV
(MIC: 5-10 µg/mL).
Exp er im en ta l Section
Unless otherwise stated, solvents were evaporated at 40 °C/2
kPa and compounds were dried at 2 kPa over P2O5. Melting
points were determined on a Kofler block and are uncorrected.
TLC was performed on Silufol UV254 plates (Kavalier Votice,
Czech Republic) in systems S1, chloroform-ethanol (95:5); S2,
chloroform-ethanol (9:1); S3, chloroform-ethanol (4:1); S4,
ethyl acetate-acetone-ethanol-water (4:1:1:1). HPLC was
performed on Separon SGX RPS columns (200 × 4 mm) in 0.05
M triethylammonium hydrogen carbonate, pH 7.5 containing
1% acetonitrile (S5), 2% acetonitrile (S6), 4% acetonitrile (S7),
16% acetonitrile (S8), and 20% acetonitrile (S9). Preparative
TLC was carried out on 40 × 17 × 0.4 cm loose layer plates of
silica gel containing UV indicator. Paper electrophoresis was
performed on a Whatman No. 3 MM paper at 40 V/cm for 1 h
in 0.05 M triethylammonium hydrogen carbonate (TEAB) at
pH 7.5; the electrophoretical mobilities are referenced to
uridine 3′-phosphate.
Proton NMR spectra were taken on Varian UNITY-200 (at
200 MHz) and/or Varian UNITY-500 (at 500 MHz) instru-
ments in CD3SOCD3, D2O, or D2O + NaOD solutions with
tetramethylsilane (TMS) or sodium disilapentanesulfonate
(DSS) as the respective internal standards. Proton chemical
shifts and coupling constants were obtained by the first-order
analysis of the spectra. Carbon-13 NMR spectra were mea-
sured on a Varian UNITY-500 (at 125.7 MHz) instrument and
With respect to the antiretroviral activity of the test
compounds, PME-8-azaguanine (65) and (R)-PMP-aza-
guanine (71a ) had a substantial inhibitory effect against

4080 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Holy´ et al.
Ta ble 3. Carbon-13 NMR Spectral Data of 8-Aza-2,6-diaminopurine Derivatives
compd
solvent
DMSO
C-2
C-4
C-5
C-6
C-1′ (J (1′,F))
C-2′ (J (2′,P); J (2′,F))
C-3′ (J (3′,F))
OCH2P (J (P,CH2))
12
162.58
153.22
119.85
156.31
N-9 Isomers
49.22
43
DMSO
D2O
162.83
163.71
162.92
163.69
163.00
162.99
163.51
151.95
152.20
151.90
151.90
152.13
152.05
151.85
120.40
121.61
120.40
121.84
120.32
120.26
121.49
156.42
157.74
156.36
157.83
156.38
156.37
157.62
69.85
(-)
63.78
61.82
47
47.27
45.05
47.22
80.90
(10.0)
69.91
(11.4)
70.87
(12.2)
77.60
(13.0;18.3)
75.15
(12.7)
76.29
(10.8)
69.33
(150.3)
64.74
(163.3)
70.22
(150.0)
63.68
(164.0)
62.72
48c
51
DMSO
D2O
53a
57b
59
DMSO
DMSO
D2O
44.75
(8.4)
49.57
82.09
(169.4)
17.31
(165.0)
68.10
(150.4)
51.26
17.68
N-8 Isomers
56.23
41d
49g
54e
58f
60
DMSO
DMSO
DMSO
DMSO
D2O
162.52
162.49
162.55
162.54
163.25
160.54
160.51
160.51
160.57
159.70
122.40
122.43
122.62
122.44
123.58
156.57
156.56
156.58
156.58
158.41
68.95
(-)
65.85
55.45
70.37
(11.4)
78.07
(13.0;19.1)
75.75
(12.7)
76.77
(10.8)
64.86
(164.0)
63.78
(164.0)
62.77
(164.1)
68.12
(150.4)
55.12
(7.6)
59.83
82.01
(169.4)
17.11
60.94
17.62
N-7 Isomer
49.86
50h
DMSO
161.43
163.73
109.78
151.79
71.35
(10.0)
64.98
(162.5)
Other carbons are as follows: P (OCH(CH3)2)2: a70.54 d and 70.40 d (J (C,P) ) 6.1), 23.89 d and 23.85 d (J (C,P) ) 3.8), 23.69 d and
b
c
23.62 d (J (C,P) ) 4.6); 70.37 d and 70.23 d (J (C,P) ) 5.9), 23.91 d and 23.88 d (J (C,P) ) 3.9), 23.73 d and 23.68 d (J (C,P) ) 4.9); 70.38
e
d (J (C,P) ) 6.9), 23.92 d (J (C,P) ) 3.8), 23.72 d (J (C,P) ) 4.6); 70.59 d and 70.50 d (J (C,P) ) 6.1), 23.87 d and 23.84 d (J (C,P) ) 3.8),
23.67 d and 23.62 d (J (C,P) ) 4.6); f70.36 d and 70.26 d (J (C,P) ) 5.9), 23.92 d and 23.89 d (J (C,P) ) 3.9), 23.73 d and 23.70 d (J (C,P) )
3.9); g70.39 d (J (C,P) ) 6.1), 23.91 d (J (C,P) ) 3.8), 23.71 d (J (C,P) ) 4.6); h70.39 d (J (C,P) ) 6.1), 23.88 d (J (C,P) ) 3.8), 23.71 d (J (C,P)
) 4.6). OC(C6H5)3: d86.20, 143.84 (3 × C), 128.45 (6 × C), 128.05 (6 × C), 127.19 (3 × C).
Ta ble 4. Substituent Effects on Carbon-13 Chemical Shifts in
8-Azaadenine, 8-Azaguanine and 8-Aza-2,6-diaminopurine
Derivatives
8-aza-2,6-diaminopurine hemisulfate, and 8-azaguanine were
obtained from Sigma (Germany). Dimethylformamide di-
methyl acetal was purchased from BASF (Germany), di-
methylformamide dineopentyl acetal was prepared according
to ref 41. Dimethylformamide was distilled from P2O5 and
stored over molecular sieves (4 Å). Acetonitrile was refluxed
with CaH2 and distilled over molecular sieves (4 Å).
substituent effect (ppm)
compounds
base
8-azaadenine
8-azaguanine
8-aza-2,6-diaminopurine
C-2
C-4
C-5
C-6
examined
N-9 Isomer
0.72 -1.78
0.21 -1.50
0.36 -1.21
0.70
0.62
0.50
0.26
0.15
0.07
4
4
1
9-(S)-[(2,2-Dim eth yl-1,3-d ioxola n -4-yl)m eth yl]-8-a za a d -
en in e(15), 8-(S)-[(2,2-Dim eth yl-1,3-d ioxola n -4-yl)m eth yl]-
8-a za a d en in e (16), a n d 7-(S)-[(2,2-Dim eth yl-1,3-d ioxola n -
4-yl)m eth yl]-8-a za a d en in e (17). A mixture of 8-azaadenine
(11) (3.0 g, 22 mmol), cesium carbonate (3.6 g, 11 mmol), and
(R)-2,2-dimethyl-4-[(tosyloxy)methyl]-1,3-dioxolane42 (14) (6.9
g, 24 mmol) in dimethylformamide (100 mL) was heated at
100 °C under a calcium chloride protecting tube for 20 h until
the starting base disappeared (monitored by TLC in S2).
According to HPLC analysis (S8) the mixture contains three
regioisomers in the ratio of 47:46:7, k1 ) 5.73, k2 ) 4.66, k3 )
3.97. The mixture was taken down in vacuo, and codistilled
twice with toluene (50 mL portions), and the residue was
extracted with boiling chloroform (total, 250 mL). The extract
was evaporated to dryness and the residue adsorbed from
methanol on silica gel (30 g). This material was applied onto
a column of silica gel (120 g) and chromatographed with
chloroform-ethanol mixture (98:2) to afford, after crystalliza-
tion from methanol, 1.1 g (20%) of the N9-isomer (15). Mp:
192-193 °C. Rf ) 0.4 (S2). k ) 5.73 (S8). Anal. (C10H14N6O2)
C, H, N. 1H NMR ((CD3)2SO): δ 8.30 s, 1H (H-2 arom); 8.40
and 8.09 2 × br, 2H (NH2); 4.62 br s, 3H (H-1′ and H-2′); 4.08
dd, 1H (H-3′, J (3′,2′) ) 6.3, J (gem) ) 8.8); 3.93 dd, 1H (H-3′′,
J (3′′,2′) ) 4.4, J (gem) ) 8.8); 1.22 s, 6H (2 × CH3). UV
Spectrum (λmax (ꢀmax)): (pH 2) 265.0 nm (11 600); (pH 7) 278.0
nm (12 200); (pH 12) 278.0 nm (11 700). IR spectrum (chlo-
roform, cm-1): ν(NH2) free, 3523 w, 3410 m; assoc, 3474 w,
3327 w; sciss(NH2) free,1638 vs; assoc,1655 m, sh; (ring) 1585
m; s(CH3) 1384 w, 1374 w; (CH3) 2992 m, 2890 w; (C-O) 1083
w, sh, 1064 w.
N-8 Isomer
8-azaadenine
8-azaguanine
8-aza-2,6-diaminopurine -0.06
0.74
-1.21
6.71
6.37
7.31
2.44
3.02
2.62
0.55
0.82
0.26
4
3
2
N-7 Isomer
8-azaadenine
8-azaguanine
-1.63
9.00 -8.85 -4.32
4
4
2
-1.56
7.62 -9.92 -1.66
8-aza-2,6-diaminopurine -1.24 10.42 -10.06 -4.50
referenced either internally with the solvent signal (δ (CD3-
SOCD3) ) 39.7 ppm) or externally (using chemical shift of
dioxane in D2O: δ (dioxane) ) 66.86 ppm). Carbon chemical
shifts and 13C-31P coupling constants were obtained from
“normal” proton-decoupled spectra or “attached proton test”
spectra.40 Proton-coupled 13C NMR spectra with NOE
enhancement were used to obtain J (C,H) coupling constants
in some compounds. Deuterium isotopic shifts of 13C signals
were extracted from partially exchanged proton-decoupled 13C
NMR spectra in CD3SOCD3 solution with a small amount (ca.
3 drops) of D2O added.
Mass spectra were measured on a ZAB-EQ (VG Analytical)
spectrometer using FAB (ionization by Xe, accelerating voltage
8 kV, glycerol matrix). UV absorption spectra were measured
on a Beckman DU-65 spectrometer in aqueous solutions.
Materials. Bromotrimethylsilane and cesium carbonate
were purchased from Fluka (Switzerland); 8-azaadenine,

Acyclic Nucleotide Analogs Derived from 8-Azapurines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4081
a
Ta ble 5. Anti-DNA-viral Activity (IC50
)
of Acyclic 8-Azapurine Nucleotide Analogues
HSV-1 TK-
HSV-2
196
CMV
VZV TK+
OKA YS
VZV TK-
07/1 YS/R
HSV-1
KOS
Vaccinia
virus
compd
B2006 VMW1837
G
Lyons AD169 Davis
PME Derivatives
28
29
51
65
66
>200
>200
>400
150
>200
>400
150
2
>200
>400
20
>200
>400
150
>200 >100
>100
>100
>40
2.3
>40
>100
>100
>100
>40
>100
>100
>40
0.1
>40
>100
>400
>400
400
0.700
150
>400
70
>400 >100
>100
>40
>100
>40
150
2
>40
2.3
2
7
2
7
0.4
40
0.04
40
0.4
40
>400
>400
300
>400
>400
>300 >100
(S)-HPMP Derivatives
0.2
23
24
47
0.4
0.9
150
20
2
>400
40
40
>400
20
2
2
3.5
0.06
0.04
0.02
0.01
0.7-2
>400
>400
>400
>400 >200
>200
>100
>100
>100
>100
>100
20
20
40 >100
9
6
6
7
20
PMP Derivatives
22
38
39
59
>400
70
150
300
>100
20
>400
70
150
300
200
>100
>400
>400
>400
100
100
>400
200
>400
>400
100
70
>400
>400
>400
70
150
300
200
>100
400
>400
>400
>50
>40
>40
>50
>50
35
>50
>40
>40
>50
>50
50
>50
8
10
>50
>50
40
>50
8
>50
8
>400
>400
>400
>400
>100
>100
300
100
5
5
>50
>50
20
100
300
5
5
300
>50
>50
28
4
>20
>50
>50
26
>40
>20
60
>200
>200
71a
71b
72a
20
20
300
>100
300
>400
300
>400
>40
>50
>40
>50
10
>20
>40
>400
(S)-FPMP Derivatives
33
34
55
56
>400
>40
>400
>400
>400
>40
>400
>400
>400
>400
>40
>400
>50
>40
>400
>50
>40
>10
>50
>50
>40
>10
>50
>50
>40
>10
>50
>50
>40
>10
>50
>50
>400
>40
>400
>400
>40
>400
>400
>400
>400
>50
>50
Other Compounds
18
19
>200
>200
>100
>200
>200
>100
>100
>100
>50
13
>40
>40
>50
>40
>50
>40
37
43
>50
11
>50
22
ACVb
0.015
0.02
0.18
0.35
a
50% inhibitory concentration (µg/mL), or concentration required to reduce virus plaque formation by 50% under experimental conditions
b
(see Methods). ACV ) acyclovir.
a
Ta ble 6. Antiretroviral Activity (IC50
)
of Acyclic 8-Azapurine Nucleotide Analogues
HIV-1
HIV-2
compd
MSV
MT-4
CEM
MT-4
CEM
PME Derivatives
28
29
51
65
66b
12.1 ( 1.72
>100
>100
>100
>100
>100
68
0.750
0.32 ( 0.1
>40
45.8
2
2
>100
2
2
>100
>100
>100
(S)-HPMP Derivatives
23
24
47
0.57 ( 0.03
>0.8
>100
>100
>0.8
>100
>100
>100
1.0 ( 0.22
>100
>100
PMP Derivatives
38
39
59
60
71a
72a
73a
71b
72b
0.85 ( 0.14
2.90 ( 1.68
1.58 ( 0.73
28.4 ( 0.81
0.43 ( 0.24
5.200
7
6.700
>100
45 ( 5
>100
10
>100
40
>100
>100
44.4 ( 10
30 ( 14
>100
>100
2
20
>100
>100
2.42 ( 0.91
20
1.75 ( 0.2
2
20
>40
>40
20
>100
>100
>100
27 ( 0.7
>40
52.7 ( 43.4
40 ( 28
>100
62 ( 34
85 ( 21
>100
>100
>100
(S)-FPMP Derivatives
>100
33
34
55
56
>200
>100
13.1+7.5
14.9 ( 5.9
>200
15
16
50
50
50
55
Other Compounds
18
19
>100
>100
>40
>100
>100
>100
>100
>100
>100
43
AZTc
0.020
0.005
0.008
a
IC50 is the minimum concentration (µg/mL) of the test compound which protects 50% of the cells against virus-induced transformation.
Contains 9% of compound 67. c AZT ) azidothymidine.
b
The following fraction contained a mixture of two com-
pounds that were separated by preparative HPLC (water-
methanol gradient, 1%/min, up to 50% methanol). The first
fraction afforded by crystallization from methanol 1.2 g (22%)

4082 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Holy´ et al.
of the N8-isomer 16. Mp: 122-124°C. Rf ) 0.3 (S2). k ) 4.66
(S8). Anal. (C10H14N6O2) C, H, N. 1H NMR ((CD3)2SO): δ 8.28
s, 1H (H-2 arom); 8.25 and 8.07 2 × br, 2H (NH2); 4.66-4.88
m, 3H (H-1′ and H-2′); 4.15 dd, 1H (H-3′, J (3′,2′) ) 5.8, J (gem)
) 8.8); 3.93 dd, 1H (H-3′′, J (3′′,2′) ) 4.6, J (gem) ) 8.8); 1.29 s
and 1.24 s, 6H (2 × CH3). UV spectrum (λmax (ꢀmax): (pH 2)
284.0 nm (12 300); (pH 7) 293.0 nm (11 000), 262.0 nm (4800);
(pH 12) 292.0 nm (10 800), 263.0 nm (4800). IR spectrum
(chloroform): ν(NH2) free, 3523 w, 3409 m; assoc, 3472 w, 3338
m, br; νsciss(NH2) free, 1635 vs; assoc., 1650 s, sh; (ring) 1596
m, 1563 s; s(CH3) 1385 mw, 1374 sw; (CH3) 2992 s, 2891 w;
(C-O) 1073 m, 1054 m, sh.
These compounds were treated with bromotrimethylsilane (2.5
mL) in acetonitrile (25 mL) at room temperature overnight
and evaporated in vacuo and the residues dissolved in water
(50 mL). The solutions were alkalized by ammonia and after
20 min evaporated in vacuo. The residues were deionized on
columns (100 mL) of Dowex 50 × 8 (H+-form) and the crude
residues purified by chromatography on Dowex 1 × 2 column
in acetate form. The products were eluted by linear gradient
of acetic acid (0-0.5 M, 1 L each). The UV-absorbing fraction
of the product was pooled and evaporated in vacuo, and the
residue was codistilled with water (3 × 20 mL) and finally
crystallized from 80% aqueous ethanol (ether added to turbid-
ity). Yield, 0.21 g (34%) of compound 23. Mp: 125-128 °C. k
) 2.5 (S5). EUp ) 0.85. Anal. (C8H13N6O5P‚H2O) C, H, N, P.
1H NMR (D2O + NaOD): δ 8.31 s, 1H (H-2 arom); 4.49 d, 2H
(H-1′, J (1′,2′) ) 5.6); 4.13 m, 1H (H-2′, ∑J ) 20.0); 3.84 dd,
1H (H-3′, J (3′,2′) ) 3.4, J (gem) ) 12.45); 3.57 dd, 1H (H-3′′,
J (3′′,2′) ) 5.4, J (gem) ) 12.4); 3.95 dd, 1H (PCH2, J (P-CH) )
8.5, J (gem) ) 12.2); 3.49 dd, 1H (PCH2, J (P-CH) ) 10.0,
J (gem) ) 12.2). UV spectrum (λmax (ꢀmax)): (pH 2) 265 nm
(11 700); (pH 7) 279 nm (12 000); (pH 13) 279 nm (11 700).
8-(S )-(3-H yd r oxy-2-(p h osp h on om e t h oxy)p r op yl)-8-
a za a d en in e (24) was obtained in a similar manner from the
bis(2-propyl) ester 22 (0.76 g, 0.19 mmol) in the yield of
0.16 g (27%). Mp: >250 °C. k ) 2.4 (S5). EUp ) 0.85. Anal.
(C8H13N6O5P‚H2O) C, H, N, P. 1H NMR (D2O + NaOD): δ 8.24
s, 1H (H-2 arom); 4.98 dd, 1H (H-1′, J (1′,2′) ) 4.6, J (gem) )
14.2); 4.91 dd, 1H (H-1′′, J (1′′,2′) ) 6.6, J (gem) ) 14.2); 4.19
m, 1H (H-2′, ∑J ) 20.5); 3.88 dd, 1H (H-3′, J (3′,2′) ) 3.9,
J (gem) ) 12.45); 3.68 dd, 1H (H-3′′, J (3′′,2′) ) 5.4, J (gem) )
12.45); 3.70 dd, 1H (PCH2, J (P-CH) ) 9.8, J (gem) ) 12.9);
3.65 dd, 1H (PCH2, J (P-CH) ) 9.5, J (gem) ) 12.9). UV
spectrum (λmax (ꢀmax)): (pH 2) 284 nm (12 000); (pH 7) 292 nm
(11 100), 256 nm (5100); (pH 13) 294 nm (10 500), 264 nm
(5200).
The next (minor) fraction from the HPLC gave (after
crystallization from methanol 0.22 g (4%) N7-isomer 17. Mp:
182 -183 °C. Rf ) 0.3 (S2). k ) 3.97 (S8). Anal. (C10H14N4O2)
C, H, N. 1H NMR ((CD3)2SO): δ 8.30 s, 1H (H-2 arom); 7.70
brs, 2H (NH2); 5.09 dd, 1H (H-1′, J (1′,2′) ) 3.4, J (gem) ) 14.7);
4.92 dd, 1H (H-1′′, J (1′′,2′) ) 6.3, J (gem) ) 14.7); 4.47 qd, 1H
(H-2′, ∑J ) 22.5); 4.12 dd, 1H (H-3′, J (3′,2′) ) 6.8, J (gem) )
8.8); 3.78 dd, 1H (H-3′′, J (3′′,2′) ) 5.9, J (gem) ) 8.8); 1.19 s
and 1.08 s, 6H (2 × CH3). UV spectrum (λmax (ꢀmax): (pH 2)
288.0 nm (9000); (pH 7) 290.0 nm (8000); (pH 12) 289.5 nm
(7500). IR spectrum (chloroform): ν(NH2)intramol.bridge 3467 m,
3347 m; sciss(NH2) 1635 vs; (ring) 1592 m, 1566 s; s(CH3) 1385
m, 1376 m; (CH3) 2994 m, 2899 w; (C-O) 1057 m, 1042 m, sh.
9-(S)-(2,3-Dih yd r oxyp r op yl)-8-a za a d en in e (18) a n d 8-
(S)-(2,3-d ih yd r oxyp r op yl)-8-a za a d en in e (19). The solution
of compound 15 or 16 (4 mmol) in 0.25 M sulfuric acid (50
mL) was left to stand overnight at the ambient temperature.
The mixtures were diluted with water (50 mL) and neutralized
with saturated barium hydroxide solution. The resulting
suspensions were heated to boil and filtered through Celite,
and the filter was washed with boiling water (500 mL). The
filtrates were taken down in vacuo, and the residues were
codistilled with ethanol (100 mL) and crystallized from 80%
aqueous ethanol (ether added to turbidity). Yield: 0.76 g (84%)
N9-isomer 18. Mp: 228-230 °C. k ) 4.02 (S7). Anal.
(C7H10N6O2‚H2O) C, H, N. 1H NMR ((CD3)2SO): δ 8.28 s, 1H
(H-2 arom); 8.35 and 8.05 2 × br, 2H (NH2); 5.02 d, 1H (OH-
2′, J ) 5.6); 4.84 t, 1H (OH-3′, J ) 5.6); 4.59 dd, 1H (H-1′,
J (1′,2′) ) 4.1, J (gem) ) 13.9); 4.43 dd, 1H (H-1′, J (1′′,2′) )
8.1, J (gem) ) 13.9); 4.07 m, 1H (H-2′, ∑J ) 29.0); 3.44 m, 2H
(H-3′). UV spectrum (λmax (ꢀmax): (pH 2) 265.0 nm (11 700);
(pH 7) 278.0 nm (12 000); (pH 12) 278.0 nm (11 700). N8-
isom er 19, yield 0.72 g (86%). Mp: 225-227 °C. k ) 3.37
(S7). Anal. (C7H10N6O2) C, H, N. 1H NMR ((CD3)2SO): δ 8.27
s, 1H (H-2 arom); 8.19 and 8.02 2 × br, 2H (NH2); 5.13 d, 1H
(OH-2′, J ) 5.6); 4.89 t, 1H (OH-3′, J ) 5.6); 4.79 dd, 1H (H-
1′, J (1′,2′) ) 3.4, J (gem) ) 13.4); 4.50 dd, 1H (H-1′, J (1′′,2′) )
8.6, J (gem) ) 13.4); 4.14 m, 1H (H-2′, ∑J ) 29.0); 4.46 2 × dt,
2H (H-3′). UV spectrum (λmax (ꢀmax)): (pH 2) 284.0 nm (12 400);
(pH 7) 293.0 nm (11 500), 262.0 nm (4900); (pH 12) 292.0 nm
(10 800), 263.0 nm (4700).
9-(2-((Diisop r op ylp h osp h on o)m eth oxy)eth yl)-8-a za a d -
en in e (26) a n d 8-(2-((Diisop r op ylp h osp h on o)m eth oxy)-
eth yl)-8-a za a d en in e (27). A mixture of 8-azaadenine (11)
(0.95 g,7.0 mmol), dimethylformamide (40 mL), cesium carbon-
ate (1.3 g, 4.0 mmol), and bis(2-propyl) [(2-chloroethoxy)-
methyl]phosphonate (25) (2.1 g, 8.0 mmol) was heated at 100
°C under stirring and exclusion of moisture for 4 h until the
starting compound disappeared (TLC in S3). After evaporation
of the solvent in vacuo and codistillation with toluene (3 × 50
mL), the residue was extracted with boiling chloroform and
the residue of the extract chromatographed on the column (50
g) of silica gel in chloroform. The N9-isomer was eluted by
the system S1; yield 0.65 g (1.8 mmol, 26%) of an amorphous
compound 26.
Further elution by system S2 gave 0.96 g (2.7 mmol) (38%)
of the N8-isomer 27 (amorphous foam). The products were
directly used for the deprotection.
9-(2-(P h osp h on om eth oxy)eth yl)-8-a za a d en in e (28) a n d
8-(2-(P h osp h on om eth oxy)eth yl)-8-a za a d en in e (29). Bro-
motrimethylsilane (1.8 or 2.7 mL, respectively) was added to
the above residue of the bis(2-propyl) ester 26 (1.8 mmol) in
acetonitrile (20 or 25 mL, respectively), and the mixtures were
stirred in stoppered flasks for 24 h at room temperature. The
mixtures were evaporated in vacuo, water (20 mL) was added,
and after 30 min of standing at room temperature, the mixture
was alkalized by aqueous ammonia and evaporated. The
residue in water (20 mL) was applied onto a column (100 mL)
of Dowex 50 × 8 (H+-form). The column was washed with
water to the drop of UV absorption, and the column was then
eluted with diluted (1:10) ammonia. The UV-absorbing frac-
tions were chromatographed on the column (100 mL) of Dowex
1 × 2 (acetate form). The product was eluted with a linear
gradient of acetic acid (0.05-1 M, 1 L each). After crystalli-
sation from water-ethanol mixture (1:4) (ether added to
turbidity), there was obtained 0.31 g (63%) of the N9-isomer
28. Mp: >250 °C. k ) 2.3 (S5). EUp ) 0.89. Anal.
(C7H11N6O4P) C, H, N, P. 1H NMR (D2O + NaOD): δ 8.22 s,
1H (H-2 arom); 4.79 t, 2H (H-1′, J (1′,2′) ) 5.4); 4.10 t, 2H (H-
2′, J (1′,2′) ) 5.4); 3.50 d, 2H (PCH2, J (P-CH) ) 8.5). UV
spectrum (λmax (ꢀmax)): (pH 2) 264.0 nm (8700); (pH 13) 277.5
nm (8700).
9-(S )-(2-(P h osp h on om e t h oxy)-3-h yd r oxyp r op yl)-8-
a za a d en in e (23) a n d 8-(S)-(2-(P h osp h on om et h oxy)-3-
h ydr oxypr opyl)-8-azaaden in e (24). The mixture of 8-azaad-
enine (11) (0.95 g, 7.0 mmol) (2R)-2-[bis(2-propyl)phos-
phonylmethoxy]-3-(trimethylacetoxy)propyl p-toluenesulfonate
(20) (4.1 g, 8.0 mmol) and cesium carbonate (1.1 g, 3.5 mmol)
in 45 mL of dimethylformamide was heated at 100 °C for 8 h
until the starting compound disappeared (TLC S3). The
mixture was evaporated in vacuo and the residue codistilled
with toluene (3 × 50 mL). The residue was extracted by
boiling chloroform (250 mL) and filtered, the filtrate was
evaporated, and the residue was left to stand overnight at
ambient temperature with 0.1 M sodium methoxide in metha-
nol (100 mL). The resulting mixture was neutralized by
addition of Dowex 50 × 8 (H+-form), the suspension was
alkalized with triethylamine and filtered, the resin was
washed with methanol (200 mL), and the filtrate was evapo-
rated. The residue was separated by chromatography on the
column of silica gel (100 g) in chloroform; the N9-isomer 21
was eluted by system S2. There was obtained 0.8 g (2.1 mmol)
of 9-(S)-(2-((diisopropylphosphono)methoxy)-3-hydroxypropyl)-
8-azaadenine (21) or 0.76 g (0.19 mmol) respectively of the N8-
isomer (22) (that was eluted by the system S3) as thick oils.

Acyclic Nucleotide Analogs Derived from 8-Azapurines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4083
The N8-isomer 29 was obtained in a similar manner from
compound 27 (2.7 mmol) in the yield of 70%. Mp: >250 °C. k
) 1.9 (S5). EUp ) 0.89. Anal. (for C7H11N6O4P) C, H, N, P.
1H NMR (D2O + NaOD): δ 8.20 s, 1H (H-2 arom); 4.95 t, 2H
(H-1′, J (1′,2′) ) 5.2); 4.20 t, 2H (H-2′, J (1′,2′) ) 5.1); 3.50 d,
2H (PCH2, J (P-CH) ) 8.3). UV spectrum (λmax (ꢀmax)): (pH
2) 288.0 nm (10 500); (pH 13) 290.5 nm (10 900).
(S)-9-[2-((Diisop r op ylp h osp h on o)m et h oxy)-3-flu or o-
p r op yl]-8-a za a d en in e (31) a n d (S)-8-[2-((d iisop r op yl-
p h osp h on o)m eth oxy)-3-flu or op r op yl]-8-a za a d en in e (32).
A mixture of 8-azaadenine (11) (531 mg, 3.9 mmol), cesium
carbonate (611 mg, 1.9 mmol), and compound 30 (1.3 g, 3.7
mmol) in dimethylformamide (15 mL) was stirred for 24 h at
100 °C under exclusion of moisture and taken to dryness. The
residue was extracted with boiling chloroform and filtered and
the filtrate evaporated. The residue was chromatographed on
a column of silica gel (100 mL) in chloroform-methanol
mixture, 20:1. Compound 31 (320 mg, 22%) and 32 (510 mg,
35%) were obtained as thick oils.
one portion. The mixture was then heated for 6 h at 110 °C
under exclusion of moisture and taken to dryness. The residue
was extracted with boiling chloroform (total 300 mL), filtered
and the filtrate evaporated. The residue was chromatographed
on a column of silica gel (300 mL). Elution with system S1
afforded 9-(R)-(2-((diisopropylphosphono)methoxy)propyl)-8-
azaadenine (36) as a thick oil. Yield: 0.90 g (37%). UV
spectrum (pH 2): λmax 265.5 (ꢀmax 14 000). Rf ) 0.50 (S2).
Further elution with the same solvent afforded 8-(R)-(2-
((diisopropylphosphono)methoxy)propyl)-8-azaadenine (37) as
semisolid material in the yield of 0.75 g (22%). UV spectrum
(pH 2): λmax 284 nm. Rf 0.40 (S2).
Each fraction was separately treated with acetonitrile (25
mL) and bromotrimethylsilane (2.5 mL) overnight, and the
mixtures were worked up as described for the compound 26.
Chromatography on Dowex 1 × 2 column (50 mL) afforded
9-(R)-(2-(phosphonomethoxy)propyl)-8-azaadenine (38) on elu-
tion with 2 M acetic acid. Yield (after crystallization from
water-ethanol): 79%. UV spectrum (λmax (ꢀmax)): (pH 2): 265
nm (14 000). Anal. (C8H14N6O4P) C, H, N, P.
Com p ou n d 31. Anal. (C14H24FN6O4P) C, H, N, F, P. 1H-
NMR ((CD3)2SO): δ 1.04 d + 1.08 d + 1.10 d + 1.14 d, 4 × 3 H
(CH3); 3.75 dd, 1 H (PCH, J (H,P) ) 9.5, J (gem) ) 13.7); 3.89
dd, 1 H (PCH, J (H,P) ) 9.5, J (gem) ) 13.7); 4.41 m, 2 H (OCH);
4.33 dm, 1 H (H-2′, J (2′,F) ) 23); 4.54 ddd, 1 H (H-3′, J (3′,2′)
) 4.2, J (gem) ) 10.5, J (3′,F) ) 46.2); 4.78 ddd, 1 H (H-3′′,
J (3′′,2′) ) 3.2, J (gem) ) 10.5, J (3′′,F) ) 47.6); 4.68 dd, 1 H
(H-1′, J (1′,2′) ) 7.1, J (gem) ) 14.6); 4.78 dd, 1 H (H-1′′, J (1′′,2′)
) 5.1, J (gem) ) 14.6); 8.30 s, 1 H (H-2); 8.10 + 8.43, 2 × bs,
2 H (NH2).
8-(R)-(2-(Phosphonomethoxy)propyl)-8-azaadenine (39) was
obtained similarly (final elution with 1 M acetic acid). Yield:
72%. UV spectrum (pH 2): λmax 284 nm. Anal. (C8H14N6O4P)
C, H, N, P.
8-Aza -2,6-d ia m in op u r in e (12) was obtained by stirring a
suspension of its hemisulfate (25 mmol) in water (100 mL)
under addition of Dowex 50 × 8 (H+-form) until dissolution,
the suspension was poured onto a column of the same cation
exchanger (100 mL), and the column was washed with water
until neutral. The resin was then treated in aqueous suspen-
sion under stirring with concentrated aqueous ammonia until
alkaline and filtered, and the resin was washed with boiling
water (total, 1 L). The filtrate and washings were evaporated
to dryness, the residue was codistilled with ethanol (2 × 50
mL), and the product was filtered from ether, washed with
the same solvent, and dried over phosphorus pentoxide in
vacuo.
9-(S)-(2-Hyd r oxy-3-(t r ip h en ylm et h oxy)p r op yl)-8-a za -
2,6-d ia m in op u r in e (41) a n d 8-(S)-(2-H yd r oxy-3-(t r i-
p h en ylm eth oxy)p r op yl)-8-a za -2,6-d ia m in op u r in e (42). A
mixture of 8-aza-2,6-diaminopurine (12) (3 g, 20 mmol), (R)-
tritylglycidol (40) (7.6 g, 24 mmol), and cesium carbonate (0.2
g, 0.6 mmol) in dimethylformamide (100 mL) was heated for
20 h at 120 °C under stirring with exclusion of air moisture
until the starting compound disappeared (TLC in S3). The
solvent was taken to dryness in vacuo and the residue
codistilled with toluene (three 100-mL portions). The chro-
matography of the residue on silica gel column (300 g) in
chloroform followed by elution with S1 mixture gave (on
crystallization from ether, petroleum ether added to turbidity)
3.2 g (15%) of the 9-isomer 41. Mp: 225 °C. Rf ) 0.80 (S3).
Anal. (C26H25N7O2) C, H, N. 1H NMR ((CD3)2SO): δ 7.70 br
(1H, NH), 6.35 br s (2H, NH2); 7.20-7.45 m (16 H arom +
NH); 5.42 δ (OH), J ) 5.6; 4.35 dd (H-1′), J (1′,2′) ) 5.1, J (gem)
) 13.2; 4.29 dd, (H-1′′), J (1′′,2′) ) 7.1, J (gem) ) 13.2; 4.25 m,
(H-2′), ∑J ) 27.9; 3.03 dd (H-3′), J (3′,2′) ) 5.1, J (gem) ) 9.5;
2.95 dd, (H-3′′), J (3′′,2′) ) 5.1, J (gem) ) 9.5. MS (M + H):
468.2.
Further elution afforded 1.3 g (14%, ethanol-ether) of the
8-isomer 42. Mp: 139 °C. Rf ) 0.76 (S3). Anal. (C26H25N7O2)
C, H, N. 1H NMR ((CD3)2SO): δ 7.50 br (1H, NH2), 6.08 br s
(2H, NH2); 7.20-7.40 m (15 H arom + NH); 5.36 δ (OH), J )
6.1; 4.61 dd (H-1′), J (1′,2′) ) 4.6, J (gem) ) 13.4; 4.41 dd, (H-
1′′), J (1′′,2′) ) 8.1, J (gem) ) 13.4; 4.31 m, (H-2′), ∑J ) 29.1;
3.04 dd (H-3′), J (3′,2′) ) 4.9, J (gem) ) 9.5; 3.00 dd, (H-3′′),
J (3′′,2′) ) 5.4, J (gem) ) 9.5.
9-(S)-(2,3-Dih yd r oxyp r op yl)-8-a za -2,6-d ia m in op u r in e
(43). A solution of the trityl derivative 41 (0.75 g, 1.6 mmol)
in the mixture of 0.25 M sulfuric acid and dioxane (1:1, 50
mL) was set aside overnight at ambient temperature. The
mixture was alkalized with aqueous ammonia to pH 7-8 and
taken to dryness. The residue was treated with water and
the mixture extracted with ether (three 100-mL portions). The
aqueous phase afforded by evaporation a crude product that
gave upon recrystallization from water-ethanol mixture (1:
1) compound 43 in 0.35 g (97%) yield. Mp: >250 °C. Rf )
Com p ou n d 32. Anal. (C14H24FN6O4P) C, H, N, F, P. 1H-
NMR((CD3)2SO): δ 1.04 d + 1.08 d + 1.10 d + 1.14 d, 4 × 3 H
(CH3); 3.75 dd, 1 H (PCH, J (H,P) ) 9.5, J (gem) ) 13.7); 3.89
dd, 1 H (PCH, J (H,P) ) 9.5, J (gem) ) 13.7); 4.42 ddd, 1 H
(H-3′, J (3′,2′) ) 4.2, J (gem) ) 10.5, J (3′,F) ) 46.2); 4.78 ddd,
1 H (H-3′′, J (3′′,2′) ) 3.2, J (gem) ) 10.5, J (3′′,F) ) 47.6); 4.81
dd, 1 H (H-1′, J (1′,2′) ) 7.6, J (gem) ) 14.2); 4.99 dd, 1 H (H-
1′′, J (1′′,2′) ) 4.4, J (gem) ) 14.6); 8.29 s, 1 H (H-2); 8.05 +
8.25, 2 × bs, 2 H (NH2).
(S)-9-(3-Flu or o-2-(ph osph on om eth oxy)pr opyl)-8-azaad-
en in e (33). Bromotrimethylsilane (437 µL, 3.31 mmol) was
added to a solution of compound 21 (323 mg, 0.83 mmol) in
acetonitrile (1.6 mL), and the reaction mixture was stirred at
ambient temperature for 24 h. The solution was concentrated
in vacuo and codistilled with toluene. Aqueous ammonia
(2,5%, 5 mL) was added and the solution taken to dryness.
The residue in water (5 mL) was applied onto a Dowex 50 ×
8 column (20 mL, H+-form) and the column washed with water.
Subsequent elution of the column with diluted (2.5%) aqueous
ammonia afforded a UV-absorbing eluate that was evaporated
in vacuo, the residue in water (5 mL) was applied on a column
(20 mL) of Dowex 1 × 2 (acetate), and the column was washed
with water and then with 1 M acetic acid. The product was
then obtained by elution with 4 M acetic acid. The eluate was
taken to dryness and the residue codistilled with water to
afford compound 33 (200 mg, 79%). Anal. (C8H12FN6O4P) C,
H, N, F, P. UV spectrum (λmax (ꢀmax): (pH 2) 264.0 nm (10 700);
(pH 7) 278.0 nm (10 000); (pH 13) 278.0 nm (11 600).
(S)-8-(3-Flu or o-2-(ph osph on om eth oxy)pr opyl)-8-azaad-
en in e (34). A solution of compound 32 (390 mg, 1.0 mmol) in
acetonitrile (2.0 mL) was treated with bromotrimethylsilane
(515 µL, 3.9 mmol), and the reaction mixture was stirred for
24 h at ambient temperature. The further workup was
performed as described for compound 33. The ultimate
purification was performed by chromatography on Dowex 1 ×
2 column and the product eluted with 2 M acetic acid. Yield:
230 mg (75%) of compound 34. Anal. (For C8H12FN6O4P) C,
H, N, F, P. UV spectrum (λmax (ꢀmax)): (pH 2) 284.0 nm (10 100);
(pH 7) 278.0 nm (11 600), 256.0 nm (4200); (pH 13) 293 nm
(9000), 255 nm (4100). MS (FAB): 307 (M + H), 171, 157.
9-(R)-(2-(P h osph on om eth oxy)pr opyl)-8-azaaden in e (38)
a n d 8-(R)-(2-(P h osp h on om eth oxy)p r op yl)-8-a za a d en in e
(39). A mixture of 8-azaadenine (11) (1.45 g, 10.8 mmol),
cesium carbonate (1.75 g, 5.4 mmol), and dimethylformamide
(25 mL) was preheated to 100 °C, and a solution of (R)-2-[(bis-
(2-propyl)phosphono)methoxy]propyl p-toluenesulfonate (35a )
(3.67 g, 9 mmol) in dimethylformamide (10 mL) was added in

4084 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Holy´ et al.
0.64 (S4). k ) 1.66 (S7). Anal. (C7H11N7O2) C, H, N. 1H NMR
((CD3)2SO): δ 7.70 and 7.40 2 × br, 2H (NH2); 6.40 br s, 2H
(NH2); 5.05 d, 1H (OH-2′), J ) 4.9; 4.82 t, 1H (OH-3′), J ) 5.6;
4.35 dd, 1H (H-1′), J (1′,2′) ) 4.4, J (gem) ) 13.9; 4.32 dd, 1H
(H-1′), J (1′′,2′) ) 8.0, J (gem) ) 13.9; 4.00 m, 1H (H-2′), ∑J )
28.2; 3.41 pent and 3.35 pent, 2H (H-3′).
) 0.64 (S3). k ) 2.7 (S9). Anal. (C13H24N7O4P) C, H, N, P.
1H NMR ((CD3)2SO): δ 7.55 br and 6.40 br s, 2H (2 × NH2);
4.46 dsept (POCH), J (CH-CH3) ) 6.1 and 6.3, J (P-OCH) )
7.6; 4.45 t (NCH2), J (CH2-CH2) ) 5.2; 3.98 t, 2H (OCH2),
J (CH2-CH2) ) 5.2; 3.74 d (PCH2), J (P-CH) ) 8.5; 1.15 and
1.09 2 × d, (2 × CH3). UV spectrum (λmax (ꢀmax)): (pH 2) 253.0
nm (11 600); (pH 7, 13) 259.5 nm (5700), 287.0 nm (9500).
Continued elution of the column with a chloroform-ethanol
mixture (96:5) gave 1.0 g (18%) of the N8- (49) and N7-isomer
(50) in the ratio 10:1, respectively; Rf ) 0.52 and 0.43,
respectively (S3). k ) 2.2 and 1.6 (S9). N8-isom er (49). 1H
NMR ((CD3)2SO): δ 7.90 br, 2H and 6.35 br s, 2H (2 × NH2);
4.67 t (NCH2), J (CH2-CH2) ) 5.1; 4.46 dsept (POCH), J (CH-
CH3) ) 6.1 and 6.3, J (P-OCH) ) 7.9; 4.06 t (OCH2), J (CH2-
CH2) ) 5.1; 3.74 d (PCH2), J (P-CH) ) 8.2; 1.15 and 1.08 2 ×
d, (2 × CH3). N7-isom er (50). 1H NMR ((CD3)2SO): d 7.20
br and 6.05 br s (2 × NH2); 4.89 t (NCH2), J (CH2-CH2) ) 4.9;
4.46 dsept (POCH), J (CH-CH3) ) 6.1 and 6.3, J (P-OCH) )
4.9; 3.91 t (OCH2), J (CH2-CH2) ) 4.9; 3.72 d (PCH2),
J (P-CH) ) 7.9; 1.15 and 1.08 2 × d, (2 × CH3).
9-(S)-(2-(P h osp h on om eth oxy)-3-h yd r oxyp r op yl)-8-a za -
2,6-d ia m in op u r in e (47). A solution of trityl derivative 41
(2.3 g, 5.0 mmol) and N,N-dimethylformamide dimethyl acetal
(5 mL, 38 mmol) in dimethylformamide (25 mL) was left to
stand overnight at room temperature in a closed flask. The
reaction mixture was then evaporated at 40 °C/13 Pa and the
residue codistilled with dimethylformamide (three 50-mL
portions). The residue was then treated with water-pyridine
mixture (1:1) in the presence of solid carbon dioxide. The
mixture was stirred to reach the room temperature (ca. 30 min)
and evaporated in vacuo under the above conditions. The
residue of compound 44 was then dried by repeated codistil-
lation with pyridine (3 × 50 mL) and dimethylformamide (3
× 50 mL). Bis(2-propyl) [[(p-tolylsulfonyl)oxy]methyl]phos-
phonate (46) (2.1 g, 6 mmol) was added to the residue followed
by dimethylformamide (25 mL), the solution was cooled down
to -20 °C and 60% sodium hydride dispersion in paraffin (0.6
g, 15 mmol) was added under stirring. The mixture was
stirred at ambient temperature under exclusion of moisture
for 24 h. Methanol-concentrated aqueous ammonia mixture
(100 mL, 1:1) was added and the mixture set aside overnight.
The reaction mixture was taken to dryness in vacuo and the
residue refluxed for 2 h with 80% acetic acid (100 mL). The
resulting solution was evaporated, the residue codistilled with
water (3 × 50 mL) and the residue was deionized on a column
of Dowex 50 × 8 (H+-form, 100 mL); the ammonia eluate
containing crude compound 45 was dried over phosphorus
pentoxide in vacuo. Acetonitrile (30 mL) was added to the
residue followed by bromotrimethylsilane (3 mL) and the
mixture stirred in a closed flask overnight. The residue was
evaporated in vacuo and codistilled with acetonitrile (3 × 50
mL), and the residue was dissolved in water (50 mL). Tri-
ethylamine was added to pH 8, and after 1 h of standing, the
mixture was evaporated in vacuo. The residue was deionized
on a column (100 mL) of Dowex 50 × 8 (H+-form, 100 mL),
the ammonia eluate was evaporated in vacuo, and the crude
product was purified by chromatography on a column of Dowex
1 × 2 (acetate form, 100 mL). The column was eluted with
linear gradient of acetic acid (0-0.5M, 1 L each) to afford, after
evaporation of the relevant fractions, codistillation with water,
and crystallization from ethanol (ether added to turbidity) 0.83
9-(2-(P h osp h on om eth oxy)eth yl)-8-a za -2,6-d ia m in op u -
r in e (51). Bromotrimethylsilane (3 mL) was added to a
suspension of the bis(2-propyl) ester 48 (0.8 g, 2.1 mmol) in
acetonitrile (30 mL), and the mixture was stirred in a stop-
pered flask at ambient temperature overnight. The mixture
was further worked up as described above for compound 28
and deionized on a column (100 mL) of Dowex 50 × 8 (H+-
form). The crude product was purified by chromatography on
the column (100 mL) of Dowex 1 × 2 (acetate). The product
was eluted with a linear gradient of acetic acid (0.5-1 M, 1 L
each). The crystallization of the residue from 80% aqueous
ethanol (ether added to turbidity) gave 0.2 g (31%) of compound
51. Mp: >250 °C. k ) 1.45 (S6). EUp ) 0.70. Anal.
(C7H12N7O4P‚H2O) C, H, N, P. 1H NMR (D2O + NaOD): δ 4.59
t (NCH2,J (CH2-CH2) ) 5.1); 4.03 t, 2H (OCH2, J (CH2-CH2) )
5.4); 3.48 d J (P-CH) ) 8.3 (PCH2).
(S)-2,6-Dia m in o-8-a za -9-[2-((d iisop r op ylp h osp h on o)-
m eth oxy)-3-flu or opr opyl]pu r in e (53) an d (S)-2,6-Diam in o-
8-a za -8-[2-((d iisop r op ylp h osp h on o)m et h oxy)-3-flu or o-
p r op yl]p u r in e (54). A mixture of 2,6-diamino-8-azapurine
(12) (581 mg, 3.85 mmol), cesium carbonate (576 mg, 1.8
mmol), and compound 30 (1.225 g, 3.5 mmol) in dimethylfor-
mamide (18 mL) was stirred at 120 °C for 6 h under exclusion
of moisture and taken to dryness. The residue was extracted
with boiling chloroform and filtered and the filtrate evapo-
rated. The residue was chromatographed on a column of silica
gel (100 mL) in chloroform-methanol mixtures, 30:1 and 20:
1, respectively. Compound 53 (350 mg, 24%) and 54 (100 mg,
7%) were obtained as thick oils.
Com p ou n d 53. Anal. (C14H25FN7O4P) C, H, N, F, P. 1H-
NMR ((CD3)2SO): δ 1.04 d + 1.08 d + 1.10 d + 1.14 d, 4 × 3 H
(CH3); 3.75 dd, 1 H (PCH, J (P,CH) ) 9.5, J (gem) ) 13.7); 3.89
dd, 1 H (PCH, J (P,CH) ) 9.5, J (gem) ) 13.7); 4.41 m, 2 H
(OCH); 4.33 dm, 1 H (H-2′, J (2′,F) ) 23); 4.54 ddd, 1 H (H-3′,
J (3′,2′) ) 4.2, J (gem) ) 10.5, J (3′,F) ) 46.2); 4.78 ddd, 1 H
(H-3′′, J (3′′,2′) ) 3.2, J (gem) ) 10.5, J (3′′,F) ) 47.6); 4.68 dd,
1 H (H-1′, J (1′,2′) ) 7.1, J (gem) ) 14.6); 4.78 dd, 1 H (H-1′′,
J (1′′,2′) ) 5.1, J (gem) ) 14.6); 8.30 s, 1 H (H-2); 8.10 + 8.43,
2 × bs, 2 H (NH2). Com p ou n d 54. Anal. (C14H25FN7O4P)
C, H, N, F, P. 1H-NMR (D2O + NaOD): δ 1.04 d + 1.08 d +
1.10 d + 1.14 d, 4 × 3 H (CH3); 3.75 dd, 1 H (PCH, J (P,CH) )
9.5, J (gem) ) 13.7); 3.89 dd, 1H (PCH, J (H,P) ) 9.5, J (gem)
) 13.7); 4.42 m, 1 H (H-3′, J (3′,2′) ) 4.2, J (gem) ) 10.5, J (3′,F)
) 46.2); 4.78 ddd, 1 H (H-3′′, J (3′′,2′) ) 3.2, J (gem) ) 10.5,
J (3′′,F) ) 47.6); 4.81 dd, 1 H (H-1′, J (1′,2′) ) 7.6, J (gem) )
14.2); 4.99 dd, 1 H (H-1′′, J (1′′,2′) ) 4.4, J (gem) ) 14.6); 8.29
s, 1 H (H-2); 8.05 + 8.25, 2 × bs, 2 H (NH2).
g (50%) of compound 47. Mp: 160 °C. k ) 3.5 (S6). EUp
)
0.75. Anal. (C8H14N7O5P) C, H, N, P. 1H NMR (D2O +
NaOD): δ 4.57 dd, 1H (H-1′), J (1′,2′) ) 6.4, J (gem) ) 14.6; 4.53
dd, 1H (H-1′′), J (1′′,2′) ) 5.6, J (gem) ) 14.6; 3.97 qd, 1H (H-
2′), ∑J ) 20.8; 3.78 dd, 1H (H-3′), J (3′,2′) ) 3.4, J (gem) ) 12.4;
3.54 dd, 1H (H-3′′), J (3′′,2′) ) 5.4, J (gem) ) 12.4; 3.58 dd, 1H
(P-CH2), J (P-CH) ) 9.5, J (gem) ) 12.4; 3.47 dd, 1H (P-CH,
J (P-CH) ) 9.5, J (gem) ) 12.4).
9-(2-((Diisop r op ylp h osp h on o)m et h oxy)et h yl)-8-a za -
2,6-d ia m in op u r in e (48), 8-(2-((Diisop r op ylp h osp h o-
n o)m eth oxy)eth yl)-8-aza-2,6-diam in opu r in e (49), an d 7-(2-
((D iis o p r o p y lp h o s p h o n o )m e t h o x y )e t h y l)-8-a za -2,6-
d ia m in op u r in e (50). 8-Aza-2,6-diaminopurine (12) (2.26 g,
15.0 mmol), cesium carbonate (2.4 g,7.5 mmol), and bis(2-
propyl) [(2-chloroethoxy)methyl]phosphonate (25) (4.5 g, 17.4
mmol) in dimethylformamide (75 mL) were heated for 20 h at
100 °C under stirring and exclusion of moisture until the
starting base disappeared (TLC in S3). After evaporation of
the solvent in vacuo and codistillation of the residue with
toluene (3 × 50 mL) the residue was adsorbed from methanol
on silica gel (50 mL) and the material transferred to a column
of the same sorbent (150 g) in chloroform. The N9-isomer 48
was eluted with the system S1, and the pertinent fractions
were pooled and evaporated in vacuo. Yield: 0.85 g (15%)
(methanol-ether) of the 9-isomer 48. Mp: 185-187 °C. Rf
9-(S)-(3-F lu or o-2-(p h osp h on om et h oxy)p r op yl)-8-a za -
2,6-d ia m in op u r in e (55) a n d 8-(S)-(3-F lu or o-2-(p h osp h o-
n om eth oxy)p r op yl)-8-a za -2,6-d ia m in op u r in e (56). A sus-
pension of compound 53 (80 mg) in acetonitrile (1 mL) was
treated with bromotrimethylsilane (26 µL) overnight and
worked up as described for compound 51. Yield: 21 mg (33%)
of compound 55, not melting below 260 °C. UV spectrum (λmax
(ꢀmax)): (pH 2) 253.0 nm (8700), 275.0 nm (6700); (pH 7,13)
259.0 nm (4400), 287.0 nm (8000). For C8H13FN7O4P (321.2):

Acyclic Nucleotide Analogs Derived from 8-Azapurines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4085
mass spectrum (CAD) 322 [(M + H)]+, 298 (MH - 2N), 276
(MH - 2N - H2O).
mamide (30 mL), and dimethylformamide dineopentyl acetal
(15 mL) was stirred under exclusion of moisture overnight at
ambient temperature. The mixture was taken to dryness at
40 °C/13 Pa, and the residue was codistilled with dimethyl-
formamide (30 mL) under the same conditions and stirred with
70% aqueous ethanol (70 mL) under simultaneous additions
of dry ice to neutralize alkaline reaction. Acetone (70 mL) was
added, and the precipitate was filtered, washed with acetone
and ether, and dried to afford 2.0 g of the HPLC-homogeneous
product; k ) 3.9 (S7). The mother liquor gave on evaporation
and trituration with acetone an additional 2.0 g (yield, total
4.0 g, 98%) of compound 61, slightly contaminated with
8-azaguanine; k ) 0.8 (4% acetonitrile in 0.05 M TEAB). Anal.
(C7H9N7O) C, H, N.
Compound 54 (80 mg) was treated with bromotrimethylsi-
lane in a similar manner to afford compound 56 (36 mg, 57%)
not melting below 260 °C. UV spectrum (λmax (ꢀmax)): (pH 2)
259.0 nm (10 200), 286.0 nm (9500); (pH 7) 259.0 nm (7900),
289.0 nm (6700); (pH 13) 258.0 nm (4400), 310 nm (6200). For
C8H13FN7O4P (321.2): mass spectrum (CAD) 322 [(M + H)]+,
304 (MH - H2O).
9-(R)-(2-((Diisop r op ylp h osp h on o)m et h oxy)p r op yl)-8-
a za -2,6-d ia m in op u r in e (57) a n d 8-(R)-(2-((Diisop r op yl-
p h osp h on o)m et h oxy)p r op yl)-8-a za -2,6-d ia m in op u r in e
(58). A suspension of 8-aza-2,6-diaminopurine (12) (3.02 g,
20 mmol) and cesium carbonate (3.3 g, 10 mmol) in dimeth-
ylformamide (60 mL) was heated at 100 °C for 1 h, and a
solution of compound 35a (8.6 g, 21 mmol) in dimethylforma-
mide (30 mL) was added over 15 min under stirring. The
heating and stirring was then continued for an additional 16
h, the mixture stripped of the solvent in vacuo, and the resi-
due extracted with boiling chloroform (total, 300 mL). The
extract was chromatographed on a column (250 mL) of silica
gel in chloroform and the column eluted with system S1 to
afford 1.75 g (22.5%) of crystalline compound 57 (Rf 0.70, S3).
9-(2-((Diisop r op ylp h osp h on o)m et h oxy)et h yl)-8-a za -
gu a n in e (62), 8-(2-((Diisop r op ylp h osp h on o)m eth oxy)-
eth yl)-8-a za gu a n in e (63), a n d 7-(2-((Diisop r op ylp h osp h o-
n o)m eth oxy)eth yl)-8-a za gu a n in e (64). A mixture of N2-
[(dimethylamino)methylene]-8-azaguanine (61) (4.2 g, 20 mmol),
compound 25 (6.5 g, 25 mmol), and cesium carbonate (3.5 g,
10 mmol) in dimethylformamide (60 mL) was stirred at 100
°C for 8 h under exclusion of moisture. The mixture was taken
to dryness at 40 °C/13 Pa and the residue codistilled with
toluene (30 mL) under the same conditions. The residue was
dissolved in methanol (70 mL), concentrated aqueous ammonia
(70 mL) was added, and the mixture was left to stand
overnight at room temperature. The solution was evaporated
in vacuo, and the residue was dissolved in water (100 mL) by
addition of Dowex 50 × 8 (H+-form, ca. 60 mL). This
suspension was applied onto a column (200 mL) of the same
ion exchanger and the column eluted with water. The elution
afforded first the salts followed by an UV-absorbing eluate.
This material was taken to dryness in vacuo to afford 1.42 g
(19%) of the compound 62. Rf ) 0.30 (S2). Anal. (C13H23N6O5P)
C, H, N, P. 1H NMR ((CD3)2SO): δ 10.93 br, 1H (NH); 6.89 br
s, 2H (NH2); 4.46 m, 2H (POCH), J (P-OCH) ) 7.8, J (CH-
CH3) ) 6.1; 4.45 t, 2H (NCH2), J ) 5.2; 3.97 t, 1H (OCH2), J
) 5.2; 3.73 d, 2H (PCH2, J (P-CH) ) 8.6); 1.15 and 1.10 2 × d
(2 × CH3), J ) 6.1.
Mp: 120-122 °C (ethyl acetate-petroleum ether). [R]D
)
-4.7° (c ) 0.5, 0.1 M HCl). Anal. (C14H26N7O4P) C, H, N, P.
1H-NMR spectrum (D2O + NaOD): δ 7.35 + 7.70, 2 × br, 2 ×
1H (NH2); 6.37 br s, 2H (NH2); 4.34 dd, 1H, J (1′a,2′) ) 7.1,
J (gem) ) 14.4 (H-1′a); 4.28 2 × dd, 1H, J (1′a,2′) ) 4.90, J (gem)
) 14.4 (H-1′a); 4.47 dq, 1H, J ) 6.1, J (P,OCH′′a) ) (P-OCH′′a);
4.43, dq, 1H, J ) 6.3, J (P,OCH′′b) ) 7.8 (POCH′′b); 4.11 pent
d, 1H, J ) 29.5 (H-2′); 3.74 dd, 1H, J (P,CHa) ) 9.5, J (gem) )
12.7 (P-CHa); 3.65 dd, 1H, J (P,CHb) ) 9.0, J (gem) ) 12.7
(PCHb); 1.10 + 1.12 + 1.14 + 1.16 + 1.17, 5 × d (5 × 3H), J )
6.1 (CH3).
Further elution of the column gave 1.40 g (18%) of 8-(R)-
(2-((Diisopropylphosphono)methoxy)propyl)-8-aza-2,6-diami-
nopurine (58) (Rf 0.50, S3). Mp: 148-150 °C (ethyl acetate-
petroleum ether). [R]D ) -43.7° (c ) 0.5, 0.1 M HCl). Anal.
(C14H26N7O4P) C, H, N, P. 1H-NMR spectrum (D2O + NaOD):
δ 7.50 br, 2H + 6.08 br, 2H (NH2); 4.53 dd, 1H J (1′a,2′) ) 5.4,
J (gem) ) 13.9 (H-1′a); 4.47 dq, 1H and 4.43 dq, 1H, J (P,OCH)
) 7.6, J (CH,CH3) ) 6.1 (POCH); 4.50 dd, 1H, J (1′b,2′) ) 6.60,
J (gem) ) 13.9 (H-1′b); 4.17 pent d, J ) 31.0 (H-2′); 3.75 dd,
1H, J (P,CHa) ) 9.5, J (gem) ) 12.7 (P,CHb); 3.63 dd, 1H, J (P,-
CHb) ) 9.0, J (gem) ) 12.7 (PCHb); 1.10 + 1.11 + 1.14 + 1.16
+ 1.18, 5 × d (5 × 3H), J ) 6.1 (CH3).
Further elution of the Dowex 50 column with 2.5% aqueous
ammonia gave a UV-absorbing eluate that afforded after
evaporation and chromatography of the residue on a silica gel
column (200 mL) with methanol-chloroform mixture (5:95)
5.77 g (15.4 mmol, 77%) of an unseparable mixture of the 7-
(64) and 8-isomer (63). Rf ) 0.30 (S2), in the ratio 1:10,
respectively (as estimated from the relative area of H-1
signals). 1H-NMR (D2O + NaOD) (63): δ 10.93 br, 1H (NH);
6.51 br s, 2H (NH2); 4.64 t, 2H (1′-CH2), J ) 5.0; 4.46 sept, 1H
(POCH), J (P-OCH) ) 7.6, J (CH,CH3) ) 6.1; 4.04 t, 2H (2′-
CH2), J ) 5.0; 3.73 d, 2H (PCH2), J (P,CH) ) 8.3; 1.10 and
1.105, 2 × d, 2 × 3H (2 × CH3), J ) 6.1. (64): δ 11.25 br, 1H
(NH); 6.46 br s, 2H (NH2); 4.75 t, 2H (1′-CH2), J ) 5.1; 4.46
sept, 1H (POCH), J (P-OCH) ) 7.6, J (CH,CH3) ) 6.1; 4.01 t,
2H (2′-CH2), J ) 5.1; 3.75 d, 2H (PCH2), J (P,CH) ) 8.3; 1.15
and 1.145, 2 × d, 2 × 3H (2 × CH3), J ) 6.1.
9-(R)-(2-(P h osp h on om eth oxy)p r op yl)-8-a za -2,6-d ia m i-
n op u r in e (59) a n d 8-(R)-(2-(P h osp h on om eth oxy)p r op yl)-
8-a za -2,6-d ia m in op u r in e (60). Compound 57 (1.75 g, 4.5
mmol) was treated with acetonitrile (25 mL) and bromotri-
methylsilane (2.5 mL) overnight at room temperature and the
solution evaporated in vacuo. Water (50 mL) was added, the
mixture was alkalized by addition of concentrated aqueous
ammonia, and the solution was evaporated. Further workup
and purification was performed essentially as described for
compound 28. Yield: 0.90 g (65.5%) of 9-(R)-(2-(phospho-
nomethoxy)propyl)-2,6-diamino-8-azaadenine (59). Mp: 238-
242 °C. [R]D ) +5.6° (c ) 0.5, 0.1 M HCl). Anal. (C8H14N7O4P)
C, H, N, P. 1H-NMR spectrum (D2O + NaOD): δ 4.49 dd, 1H,
J (1′a,2′) ) 5.6, J (gem) ) 14.9 (H-1′a); 4.45 dd, 1H, J (1′b,2′) )
5.4, J (gem) ) 14.9 (H-1′b); 4.08 m, 1H, J ) 30.0 (H-2′); 3.59
dd, 1H, J (P,CHa) ) 9.3, J (gem) ) 12.2 (PCHa); 3.50 dd, 1H,
J (P,CHb) ) 9.1, J (gem) ) 12.2 (PCHb); 1.17 d, 3H, J (3′,2′) )
6.3 (H-3′). EUp (pH 7.5) ) 0.85.
9-(2-(P h osp h on om et h oxy)et h yl)-8-a za gu a n in e (65).
Compound 62 (1.42 g, 3.8 mmol) in acetonitrile (25 mL) was
treated with bromotrimethylsilane (2.5 mL) overnight, and the
volatiles were taken to dryness in vacuo. The residue in water
(50 mL) was alkalized by ammonia and evaporated. The
residue was applied on a column (100 mL) of Dowex 50 × 8
(H+-form) and eluted with water. UV-absorbing fraction was
alkalized by ammonia and evaporated in vacuo. The residue
was applied on a column (150 mL) of Dowex 1 × 2 (acetate)
and washed with water till the drop of the absorption. The
resin was then stirred batchwise with 1 M formic acid (400
mL) for 30 min, filtered by suction and washed with boiling
water (1 L). The filtrates were evaporated, and the residue
was codistilled with water (5 × 50 mL) and dissolved in boiling
water. Ethanol (2 volumes) was added to the solution and the
mixture left to crystallize at 0 °C. Yield: 0.70 g (63%) of
compound 65, not melting below 250 °C. Anal. (C7H11N6O5P)
C, H, N, P. 1H-NMR spectrum (D2O + NaOD): δ 4.57 t, 2H,
(H-1′) J (1′,2′) ) 5.1; 4.09 t, 2H, (H-2′) J ) 5.1; 3.60 d, 2H,
(PCH2) J (P,CH) ) 8.8. EUp(pH 7.5) ) 0.87.
The reaction of compound 58 (1.40 g, 3.6 mmol) with
bromotrimethylsilane was performed essentially as de-
scribed for compound 59. Yield: 0.80 g (72.5%) of 8-(R)-(2-
(phosphonomethoxy)propyl)-8-aza-2,6-diaminopurine
(60).
Mp: 238-240 °C. [R]D ) -23.5° (c ) 0.5, 0.1 M HCl). Anal.
(C8H14N7O4P) C, H, N, P. 1H-NMR spectrum (D2O + NaOD):
δ 4.70 d, 2H, J (1′,2′) ) 5.2; (H-1′); 4.17 m, ∑J ) 29.3 (H-2′);
3.58 dd, 1H, J (P,CHa) ) 9.4, J (gem) ) 12.2 (PCHa); 3.50 dd,
1H, J (P,CHb) ) 9.2, J (gem) ) 12.2 (PCHb); 1.21 d, 3H, J (3′,2′)
) 6.3 (CH3). EUp (pH 7.5) ) 0.85.
N2-[(Dim eth yla m in o)m eth ylen e]-8-a za gu a n in e (61). A
mixture of 8-azaguanine (13) (3.0 g, 20 mmol), dimethylfor-

4086 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Holy´ et al.
8-(2-(P h osp h on om eth oxy)eth yl)-8-a za gu a n in e (66) a n d
7-(2-(P h osp h on om eth oxy)eth yl)-8-a za gu a n in e (67). Sus-
pension of crude mixture of compounds 63 and 64 (5.77 g, 15.4
mmol) in acetonitrile (40 mL) was treated overnight with
bromotrimethylsilane (4 mL), and the volatiles were evapo-
rated in vacuo. The residue in water (50 mL) was alkalized
with ammonia and reevaporated and the residue applied on a
column of Dowex 50 × 8 (150 mL). The product which is
sparingly soluble in water eluted with water with a consider-
able retention. The residue was filtered from ethanol, washed
with ether, and dried. Yield: 3.2 g (71.4%) of the mixture of
66 and 67. Anal. (C7H11N6O5P) C, H, N, P. EUp(pH 7.5) )
0.87 (fluorescent).
9-(S )-(2-(P h osp h on om e t h oxy)p r op yl)-8-a za gu a n in e
(71b ), 8-(S)-(2-(P h osp h on om et h oxy)p r op yl)-8-a za gu a -
n in e (72b), a n d 7-(S)-(2-(P h osp h on om eth oxy)p r op yl)-8-
a za gu a n in e (73b ). A mixture of N2-[(dimethylamino)-
methylene]-8-azaguanine (61) (2.15 g, 10 mmol), cesium
carbonate (1.75 g, 5.4 mmol), and dimethylformamide (40 mL)
was treated under stirring with 2(S)-[(bis(2-propyl)phospho-
nyl)methoxy]propyl p-toluenesulfonate (35b) (5 g, 12.2 mmol)
in dimethylformamide (10 mL) and heated at 100 °C for 4 h.
The mixture was filtered while hot, filtrate was evaporated to
dryness in vacuo, and the residue was left to stand overnight
at ambient temperature in a mixture of methanol and con-
centrated aqueous ammonia (1:1, 200 mL). The mixture was
evaporated to dryness and the residue adsorbed from metha-
nolic solution on silica gel (50 g). The sorbent was applied
onto the column (200 mL) of silica gel in chloroform and the
column eluted with chloroform followed by mixture S1. The
minor fractions were discarded, and the main fraction (1.82
g, 47%) of the mixture of isomeric (S)-(2-((diisopropylphospho-
no)methoxy)propyl)-8-azaguanines was dried overnight in
vacuo. Acetonitrile (25 mL) and bromotrimethylsilane (2.5
mL) were added, and the solution was left to stand overnight
at room temperature. The mixture was then evaporated to
dryness in vacuo, dissolved in water (20 mL), and alkalized
with concentrated aqueous ammonia. After 15 min the
solution was again evaporated and the residue applied in water
to Dowex 50 × 8 (H+-form) (100 mL). The elution of the
column with water afforded (after removal of salts) with
retention the UV-absorbing fraction that gave on evaporation
and trituration of the residue with ethanol 0.50 g of the
9-isomer 71b. Anal. (C7H11N6O5P) C, H, N, P.
The ammonia eluate afforded a gel-forming mixture that was
dissolved in water by addition of ammonia and applied onto a
column of Sephadex A-25 (150 mL) in 0.02 M triethylammo-
nium hydrogen carbonate. The column was eluted with a
linear gradient of the same buffer (0.02-0.20 M, 1 L each) to
give the main fraction consisting of the mixture of the 7- and
8-isomer (EUp ) 0.92, fluorescent spot). The residue after
codistillation with methanol was applied onto a column (20
mL) of Dowex 1 × 2 (acetate), the column was washed with
water (100 mL), and the product was eluted with 1 M acetic
acid. After evaporation in vacuo, codistillation with water, and
trituration with ethanol, the product was filtered, washed with
ethanol and ether, and dried to afford 0.50 g of the mixture of
7-isomer (73a ) and 8-isomer (72a ) in the ratio 1:4 (by 13C
NMR). Anal. (C7H11N6O5P) C, H, N, P. 1H-NMR (D2O +
NaOD): (8-isom er , 72a ): δ 4.65 2 × dd, 2H, J (1′,2′) ) 5.4,
J (1′′,2′) ) 5.1, J (gem) ) 14.0 (1′-CH2); 4.17 br sext, 1H, ∑J )
29.5 (2′-CH); 3.52 dd, 1H, J (P-CH) ) 9.7, J (gem) ) 12.2 +
3.47 dd, 1H, J (P-CH) ) 9.0, J (gem) ) 12.2 (PCH2); 1.18 d,
3H (J (3′,2′) ) 6.3 (CH3); (7-isom er , 73a ): δ 4.76 2 × dd, 2H,
J (1′,2′) ) J (1′′,2′) ) 5.2, J (gem) ) 14.0 (1′-CH2); 4.13 br sext,
1H, ∑J ) 29.5 (2′-CH); 3.50 m, 2H (PCH2); 1.16 d, 3H, J (3′,2′)
) 6.3 (CH3).
An tivir a l Assa ys. The antiviral assays, other than HIV-
1, were based on inhibition of virus-induced cytopathicity in
either E6SM (HSV-1, HSV-2, VV) or HEL (VZV, CMV) cell
cultures, following previously established procedure.2a-c,43,44
Briefly, confluent cell cultures in microtiter trays were inocu-
lated with 100 CCID50 of virus, 1 CCID50 being the virus dose
required to infect 50% of the cell cultures. After a 1-h virus
adsorption period, residual virus was removed, and the cell
cultures were incubated in the presence of varying concentra-
tions (400, 200, 100, ... µg/mL) of the test compounds. Viral
cytopathicity was recorded as soon as it reached completion
in the control virus-infected cell cultures that were not treated
with the test compounds.
In h ib it ion of H IV-1-In d u ced Cyt op a t h icit y in MT-4
Cells. The methodology of the anti-HIV assays has been
described previously.2b Briefly, human MT-4 (∼4 × 105cells‚
mL-1) or CEM (∼3 × 105 cells‚mL-1) cells were infected with
100 CCID50 HIV-1(IIIB) µL-1 and seeded in 200 µL wells
of a microtiter plate, containing appropriate dilutions of the
test compounds. After 5 days (MT-4) or 4 days (CEM) of
incubation at 37 °C, the number of viable (MT-4) cells was
determined in a blood cell counting chamber by trypan blue
dye exclusion or CEM giant cell formation was examined
microscopically.
In h ibition of MSV-In d u ced Tr a n sfor m a tion of Mu r in e
C3H/3T3 E m br yo F ib r obla st s. The anti-MSV assay was
performed as described previously.4a,5b Murine C3H/3T3
embryo fibroblast cells were seeded at 5 × 105 cells‚mL-1 into
1-cm2 wells of a 48-well microplate. Twenty-four hours later,
the cell cultures were infected with 80 focus-forming units of
MSV (prepared from tumours induced following intramuscular
inoculation of 3-day-old NMRI mice with MSV) for 90-120 min
at 37 °C. The medium was then replaced by 1 mL of fresh
medium containing various concentrations of the test com-
pounds. After 6 days, transformation of the cell culture was
examined microscopically.
Further elution of the column with 2.5% aqueous ammonia
gave in the same manner 0.45 g of a mixture of the 7- (73b)
and 8-isomer (72b) in the ratio of 1:4 (estimated by 13C NMR
spectra).
9-(R)-(2-(P h osp h on om et h oxy)p r op yl)-8-a za gu a n in e
(71a ),8-(R)-(2-(P h osp h on om eth oxy)p r op yl)-8-a za gu a n in e
(72a ), a n d 7-(R)-(2-(P h osp h on om et h oxy)p r op yl)-8-a za -
gu a n in e (73a ). The reaction was performed essentially as
described for the (S)-enantiomers. After the workup of the
condensation mixture with aqueous methanolic ammonia the
crude mixture of bis(2-propyl) esters was applied onto a column
of Dowex 50 × 8 (H+-form) (150 mL) and the column eluted
with 20% aqueous methanol. The UV-absorbing fraction was
taken to dryness in vacuo and dried, affording the 9-isomer
as an amorphous foam. The residue was treated with aceto-
nitrile (30 mL) and bromotrimethylsilane (3 mL) overnight
and evaporated in vacuo, and the residue was dissolved in
2.5% ammonia and reevaporated in vacuo. This residue
was applied onto a column (100 mL) of Dowex 1 × 2 (acetate
form) and washed with water (1 L) and with 1 M acetic acid
(500 mL). The eluates were discarded, and the resin was
extracted on filter with boiling water (500 mL). This eluate
was evaporated in vacuo and the residue crystallized from
water (poorly soluble) to afford the 9-(R)-isomer 71a (0.50 g).
Anal. (C7H11N6O5P) C, H, N, P.
Ack n ow led gm en t. This work was supported by
grants of the Czech State Grant Agency No. 203/93/
0118, Grant Agency of the Academy of Sciences No.
455407, PECO project no. ERBCIPDCT930194, Gilead
Sciences (Foster City, CA), the Biomedical Research
Programme of the European Commission, the Belgian
Fonds voor Geneeskundig Wetenschappelijk Onderzoek
No. 3.0180.95, the Belgian Genconcerteerde Onder-
zoeksacties No. 95/5, and the Belgian Nationaal Fonds
voor Wetenschappelijk Onderzoek No. 3.3010.91. The
excellent technical assistance of Mrs. Beˇla Nova´kova´,
Lizette van Berckelaer, Anita Van Lierde, Frieda De
Meyer, Ann Absillis, and Anita Camps is gratefully
acknowledged.
Futher elution of Dowex 50 column with 2.5% aqueous
ammonia gave a UV-absorbing fraction that was evaporated
in vacuo, dried, and treated with acetonitrile (20 mL) and
bromotrimethylsilane (2 mL). The reaction mixture formed a
thick gel that was dissolved by the addition of 5% aqueous
ammonia (100 mL), and the mixture was evaporated and
deionized on a column (100 mL) of Dowex 50 × 8 (H+-form).

Acyclic Nucleotide Analogs Derived from 8-Azapurines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4087
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