Synthesis and antiviral activity of acyclic nucleotide analogues derived from 6-(aminomethyl)purines and purine-6-carboxamidines

Article abstract of DOI:10.1135/cccc19961525

The synthesis of a series of 9-(2-phosphonomethoxyalkyl) derivatives of 6-(aminomethyl)purine 11, 2-amino-6-(aminomethyl)purine 12 and purine-6-carboxamidine 14 is reported. The 6-cyanopurines 1 and 2 were selectively alkylated with 2-[bis(isopropyloxy)phosphonylmethoxy]alkyl synthons 3 and 4 at the 9-position. Catalytic hydrogenation of the obtained 9-{2-[bis(isopropyloxy)phosphonylmethoxy]-alkyl}-6-cyanopurines 9 and 10 followed by treatment with bromotrimethylsilane afforded the title compounds 11 and 12. Analogous acyclic nucleotides derived from purine-6-carboxamidines 14 were prepared from the cyanopurines 9a and 10a by treatment with sodium methoxide and ammonium chloride followed by deprotection. Compounds 11 and 12 exhibited moderate activity (MIC₅₀ = 3-50 μg/ml) against herpes simplex virus type 1, varicella-zoster virus and Moloney murine sarcoma virus in vitro.

Full text of DOI:10.1135/cccc19961525

Acyclic Nucleotide Analogues
1525
SYNTHESIS AND ANTIVIRAL ACTIVITY OF ACYCLIC NUCLEOTIDE
ANALOGUES DERIVED FROM 6-(AMINOMETHYL)PURINES AND
PURINE-6-CARBOXAMIDINES
a1
a
a
b
Michal HOCEK , Milena MASOJIDKOVA , Antonin HOLY , Graciela ANDREI ,
b
b
b
Robert SNOECK , Jan BALZARINI and Erik DE CLERCQ
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10 Prague 6, Czech Republic; e-mail: 1 hocek@uochb.cas.cz
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received May 31, 1996
Accepted July 9, 1996
Dedicated to the memory of Dr Zdenek Arnold.
The synthesis of a series of 9-(2-phosphonomethoxyalkyl) derivatives of 6-(aminomethyl)purine 11,
2-amino-6-(aminomethyl)purine 12 and purine-6-carboxamidine 14 is reported. The 6-cyanopurines 1
and 2 were selectively alkylated with 2-[bis(isopropyloxy)phosphonylmethoxy]alkyl synthons 3 and 4 at
the 9-position. Catalytic hydrogenation of the obtained 9-{2-[bis(isopropyloxy)phosphonylmethoxy]-
alkyl}-6-cyanopurines 9 and 10 followed by treatment with bromotrimethylsilane afforded the title
compounds 11 and 12. Analogous acyclic nucleotides derived from purine-6-carboxamidines 14 were
prepared from the cyanopurines 9a and 10a by treatment with sodium methoxide and ammonium
chloride followed by deprotection. Compounds 11 and 12 exhibited moderate activity (MIC50 = 3–50 µg/ml)
against herpes simplex virus type 1, varicella-zoster virus and Moloney murine sarcoma virus in vitro.
Key words: Acyclic nucleoside phosphonates; Phosphonomethoxyalkylpurine derivatives; 6-Cyano-
purines; 6-(Aminomethyl)purines; Purine-6-carboxamidines; Antivirals.
In the structure–activity relationship study of a series of antiviral phosphonomethoxy-
alkyl derivatives of pyrimidine and purine bases (for reviews see ref.1) it was found that
the presence of an amino group at the heterocyclic moiety is necessary for the antiviral
activity. Recently, several N(6)-monosubstituted and N(6)-disubstituted derivatives of
adenine and 2,6-diaminopurine were also found to be active2.
To study the role of the amino group at the purine base in the biological activity of
these compounds a series of acyclic nucleotide analogues bearing the strongly basic
2-(aminomethyl) function on the purine ring was prepared3. All compounds of that
series were found to be inactive. As a continuation of this study we describe here the
synthesis of 9-[2-(phosphonomethoxy)alkyl] derivatives of 6-(aminomethyl)purines
and purine-6-carboxamidines. The 6-(aminomethyl)purines are reported4 to be unstable.
They can be prepared4,5 by catalytic hydrogenation of 6-cyanopurines and were usually
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1526
Hocek et al.:
isolated5 as stable N-acyl derivatives. On the contrary, the afore-mentioned phosphono-
methoxyalkyl derivatives of 2-(aminomethyl)purines are stable, probably due to
zwitterion formation.
A facile method of preparation of 6-cyanopurines 1 and 2 was recently reported6.
Synthons 3 and 4 were used for the introduction of the phosphonomethoxyalkyl group.
While the 2-phosphonomethoxyethyl (PME) synthon 3 was already known7, the
racemic 3-hydroxy-2-phosphonomethoxypropyl (HPMP) synthon 4 containing a hydro-
genolytically cleavable O-benzyl protecting function8 was prepared from tritylglycidol 5.
Sodium benzoxide was used for oxirane ring opening of the glycidol 5 that proceeded
regioselectively to afford 3-O-benzyl-1-O-tritylglycerol 6. The 2-hydroxy group was
alkylated with diisopropyl tosyloxymethylphosphonate9 7 and the trityl function was
cleaved10 using wet Dowex 50 (H+) in methanol to give the protected 2-O-phosphono-
methylglycerol 8. The target HPMP-synthon 4 was prepared by tosylation of this inter-
mediate with tosyl chloride in pyridine (Scheme 1).
BnONa
OTr
a
b
BnO
OTr
BnO
OH
BnO
OTs
OH
O
O
O
5
6
P(O)(OiPr)
P(O)(OiPr)
2
2
8
4
+
a) 1. TsOCH P(O)(OiPr) (7), NaH, 2. Dowex 50 (H ); b) TsCl, Py
2
2
SCHEME 1
The alkylation of 6-cyanopurines 1 and 2 with the synthons 3 and 4 under standard
conditions using sodium hydride in dimethylformamide at 100 °C gave only traces of
the alkylated products. With cesium carbonate as a base the 9-substituted 6-cyano-
purine phosphonates 9 and 10 were obtained selectively with only trace impurities of
other regioisomers. Preparative TLC chromatography afforded pure oily phosphonates 9
and 10 in the yields of 48–60% that were used in further steps (Scheme 2).
The protected 6-cyanopurines 9 or 10 on treatment with bromotrimethylsilane
(TMSBr) gave a complicated pattern of reaction products. Therefore, the compounds 9
and 10 were first hydrogenated. After two days the TLC detection showed that the
cyano group was completely reduced. In the case of the O-benzyl protected compounds 9b
and 10b, however, the removal of the benzyl group was not quantitative. The sluggish
reaction could be explained by a catalyst poisoning by the aminomethyl group; a simi-
lar effect was recently described11 for selective inhibition of hydrogenolytic cleavage of
the O-benzyl function in the presence of ammonia.
Without isolation or characterization, the crude products were treated immediately
with TMSBr to cleave the isopropyl ester groups. This reaction was not quantitative
either and, due to lower solubility of the intermediates in acetonitrile, in some cases
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Acyclic Nucleotide Analogues
1527
dimethylformamide had to be used as a solvent. Standard purification by a combination
of cation and anion exchange chromatography did not afford pure products; in the case
of compounds 11b, 12a and 12b preparative reversed phase HPLC was used to remove
the phosphonate monoester and the O-benzyl protected side-products. In one case, the
pure O-benzyl byproduct 12c was isolated and characterized (Scheme 2).
The 9-substituted 6-cyanopurines 9a and 10a were converted to the 6-carboxamidino
derivatives 14 with the use of the recently published method12 for the preparation of
hetarylcarboxamidines. On treatment with a catalytic amount of sodium methoxide in
methanol the compounds 9a and 10a formed imidate intermediates that were without
isolation treated with ammonium chloride to afford the protected amidine derivatives 13.
While the reaction of compound 9a with methanol proceeded at room temperature, the
reaction of the compound 10a had to be performed at reflux temperature to reach com-
CN
X
O
P(O)(OiPr)
2
N
N
R
N
H
Y
N
X
R
H
1,2
Cl
3
OTs CH OCH Ph
4
2
2
a
NH
2
CN
N
N
N
N
N
N
N
b
Y
Y
N
O
P(O(OiPr)
O
P(O(OH)
2
2
R
R
9,10
11,12
Y
R
a) 3 or 4, Cs CO , DMF;
H
1
2
3
b) 1. H , Pd-C; 2. TMSBr, CH CN
H N
2
2
2
3
H
H
H
9a
9b
CH OBn
2
H N
2
H
10a
10b
11a
11b
12a
12b
12c
H N CH OBn
2
2
H
H
H
CH OH
2
H N
2
H
H N CH OH
2
2
H N CH OBn
2
2
SCHEME 2
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1528
Hocek et al.:
plete conversion. The amidines 13 were without isolation deprotected using TMSBr in
dimethylformamide to afford the desired 9-(2-phosphonomethoxyethyl) derivatives of
purine-6-carboxamidine 14a and of 2-aminopurine-6-carboxamidine 14b in the yields
of 44% and 51%, respectively, that were easily isolated by ion exchange chromato-
graphy (Scheme 3).
Cl
H N
2
NH
2
CN
N
N
N
N
N
N
N
Y
a
Y
N
O
P(O(OiPr)
O
P(O)(OiPr)
2
2
13
Y = H
9a
10a
Y = H N
2
b
H N
2
NH
2
Y
H
13,14
N
N
a
b
N
Y
H N
2
O
N
O
P
O
OH
a) 1. MeONa/MeOH, 2. NH Cl; b) TMSBr
14
4
SCHEME 3
Although the method does not offer high yields and the purification of products is
quite complicated, the acyclic nucleotide analogues derived from 6-(amino-
methyl)purines 11a, 11b and 12a, 12b were prepared in amounts and purity sufficient
for the biological activity tests. The compounds were much less stable than the analo-
gous phosphonate derivatives of 2-(aminomethyl)purines3 and were characterized as
hygroscopic lyophilizates that quickly turned violet (compounds 11) or green (com-
pounds 12) on exposure to air. The amidine compounds 14, on the other hand, were
stable.
Similarly to the 2-(aminomethyl)purine analogues3, the electrophoretical mobilities
of compounds 11, 12 and 14 are substantially lower than those of adenine derivatives.
UV spectra of the compounds 11 exhibited maxima at 265 nm, while the fluorescent
compounds 12 displayed maxima at 306 nm with significant bathochromic shift to 317 nm
in an acidic medium. The UV spectrum of the compound 14a exhibited a maximum at
296 nm with hypsochromic shift to 279 nm in an alkaline medium and compound 14b
absorbed at 361 nm with hypsochromic shift to 334 nm in alkalies. The alkali-induced
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Acyclic Nucleotide Analogues
1529
hypsochromic shifts of the amidine compounds indicate that the amidine function is
fully protonated even at pH 7.
Structure of the compounds was proved by 1H and 13C NMR spectra. Chemical shifts
of the base carbon atoms (Table I) are in good accord with the reported values for
6-mono- and 2,6-disubstituted purines13 considering the shielding of the substituents
CN, CH2NH2, C(=NH)NH2 and NH2. While the C-4 and C-5 signals of the free bases 1
and 2 are broad, the corresponding signals of the 9-substituted derivatives 9–14 are
sharp due to the excluded 7H–9H tautomerism.
The acyclic nucleotide analogues 11, 12 and 14 were tested in vitro for cytostatic
activity (L-929, L-1210 and HeLa cells) and for inhibitory effect on the DNA viruses:
herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), cytomegalovirus (CMV), vari-
cella-zoster virus (VZV) and vaccinia virus, and retroviruses: Moloney murine sarcoma
virus (MSV) and human immunodeficiency virus type 1 (HIV-1/IIIb) and type 2 (HIV-
2/ROD). Compound 11a exhibited a moderate inhibitory effect (MIC50 = 20 µg/ml)
against HSV-1 (F); compounds 11b and 12a were active against both the TK+ and TK–
VZV strains at concentrations varying between 3 and 33 µg/ml and against MSV at 30
and 18 µg/ml, respectively. Also compound 12b showed a moderate antiviral activity
against VZV (MIC50 = 30–48 µg/ml). Other antiviral activity assays as well as cyto-
static activity tests were negative. None of these compounds showed any considerable
cell toxicity.
In conclusion, the replacement of the 6-amino or (di)alkylamino group of the
strongly active purine nucleotide analogues by the aminomethyl function (derivatives
11 and 12) caused substantial decrease of activity in most antiviral assays but still some
activity against HSV-1, VZV and MSV was preserved. Replacement of the amino
group by the carboxamidine function (derivatives 14) led to complete loss of antiviral
activity.
EXPERIMENTAL
Unless otherwise stated, solvents were evaporated at 40 °C/2 kPa and compounds were dried at 60 °C/2 kPa
over P2O5. Melting points were determined on a Kofler block and are uncorrected. TLC was per-
formed on Silufol UV254 plates (Kavalier Votice, Czech Republic) (A) in CHCl3–MeOH (80 : 20)
mixture or (B) in ethyl acetate. Preparative TLC was carried out on 40 × 17 × 0.4 cm plates of silica
gel containing a UV indicator. Reversed phase preparative HPLC was carried out on a Separon SGX
column (300 × 16 mm) in water for 20 min, followed by a linear gradient of methanol (0–10% in 30 min).
Paper electrophoresis was performed on a Whatman No. 3 MM paper at 40 V/cm for 1 h in 0.05 M tri-
ethylammonium hydrogen carbonate at pH 7.5 and the electrophoretical mobilities are referenced to
uridine 3′-phosphate. NMR spectra were measured on a Varian Unity 500 spectrometer (500 MHz for
1H and 125.7 MHz for 13C NMR) in hexadeuteriodimethyl sulfoxide referenced to the solvent signals
2.5 ppm for 1H and 39.7 ppm for 13C NMR, or in deuterium oxide containing sodium deuteroxide
with sodium disilapentanesulfonate as internal standard for 1H and dioxane as external standard for
13C NMR (δ(dioxane) = 66.86). Mass spectra were measured on a ZAB-EQ (VG Analytical) spec-
trometer using FAB technique (ionization by Xe, accelerating voltage 8 kV, glycerol matrix). UV
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1530
Hocek et al.:
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Acyclic Nucleotide Analogues
1531
absorption spectra (λmax, nm; (ε)) were measured on a Beckman DU-65 spectrometer in aqueous sol-
utions.
Dimethylformamide was distilled from P2O5 and stored over molecular sieves. Acetonitrile was
refluxed with calcium hydride and distilled. The 6-cyanopurines6 1 and 2 as well as the phosphonate
synthon7 3 were prepared according to the previously published methods.
1-Benzyloxy-3-(triphenylmethoxy)-2-propanol (6)
To a stirred solution of NaH (60% dispersion in mineral oil; 10.8 g, 289 mmol) in DMF (100 ml) a
solution of benzyl alcohol (33.4 ml, 323 mmol) in DMF (50 ml) was added and the stirring at room
temperature was continued for 15 min. Then a solution of trityloxymethyloxirane14 (5; 17 g, 54
mmol) in DMF (50 ml) was added dropwise and the mixture was stirred at 100 °C for 2.5 h. After
cooling, the mixture was diluted with water (150 ml) and extracted with ether (3 × 250 ml). The
combined organic layers were dried with MgSO4 and evaporated. Excess of benzyl alcohol was dis-
tilled off (bath temperature 100 °C, 130 Pa) and the residue was purified by column chromatography
on silica gel (200 g, ethyl acetate–light petroleum 1 : 2) to afford compound 6 as an amorphous
solid, m.p. 71–73 °C. Yield 19 g (83.3%). For C29H28O3 (424.5) calculated : 82.05% C, 6.65% H;
found: 81.57% C, 6.61% H. Mass spectrum (FAB), m/z (rel.%): 243 (100) [Tr], 165 (22) [M – Tr],
1
91 (38). H NMR spectrum (CDCl3): 3.20 dd, 1 H, J(1b,2) = 5.5, J(gem) = 9.5 (Hb-1); 3.26 dd, 1 H,
J(1a,2) = 5.5, J(gem) = 9.5 (Ha-1); 3.56 dd, 1 H, J(3b,2) = 6.1, J(gem) = 9.5 (Hb-3); 3.62 dd, 1 H,
J(3a,2) = 4.6, J(gem) = 9.5 (Ha-3); 4.00 m, 1 H (H-2); 4.54 s, 2 H (CH2Ph); 7.20–7.35 m, 14 H
(H-arom); 7.40–7.50 m, 6 H (H-arom).
Diisopropyl {1-[(Benzyloxy)methyl]-2-hydroxyethoxy}methylphosphonate (8)
To a stirred suspension of NaH (60% dispersion; 2.0 g, 50 mmol) in dry THF (50 ml) a solution of 6
(19 g, 44.8 mmol) in THF (50 ml) was slowly added and the suspension was stirred at room tem-
perature for 1 h. Then a solution of diisopropyl tosyloxymethylphosphonate9 (7; 18.2 g, 52 mmol) in
THF (20 ml) was added dropwise and the stirring at room temperature was continued for 36 h. Meth-
anol (50 ml) followed by ethyl acetate (250 ml) was added and the solution was washed with water
(3 × 100 ml). The combined organic phases were dried over MgSO4 and evaporated. The residue was
refluxed with Dowex 50 (H+) (40 ml) in methanol (240 ml) and water (60 ml) for 3 h, filtered, the
Dowex was washed with methanol (100 ml), and the combined filtrates were evaporated. The residue
was dissolved in ethyl acetate (350 ml) and washed with water (3 × 200 ml). The organic layer was
dried over MgSO4 and evaporated. Column chromatography of the residue (150 g silica gel, toluene–
ethyl acetate gradient 0–100%) afforded compound 8 as a yellowish oil, RF (B) 0.17. Yield 7.68 g
(48%). Mass spectrum (FAB), m/z (rel.%): 361 (55) [M + H], 91 (100). 1H NMR spectrum
((CD3)2SO): 1.21–1.24 m, 12 H (CH3); 3.46–3.63 m, 5 H (H-1, H-2, H-3); 3.86 dd, 1 H, J(P,CHb)
= 8.8, J(gem) = 13.9 (PC-Hb); 3.90 dd, 1 H, J(P,CHa) = 8.8, J(gem) = 13.9 (PC-Ha); 4.48 and 4.51
2 × d, 2 × 1 H, J(gem) = 12.2 (CH2Ph); 4.59 m, 2 H (POCH); 4.69 t, 1 H, J(OH,CH2) = 5.4 (OH);
7.30–7.40 m, 5 H (H-arom).
Diisopropyl {1-[(Benzyloxy)methyl]-2-(p-toluenesulfonyloxy)ethoxy}methylphosphonate (4)
To a stirred solution of 8 (7.52 g, 20.9 mmol) and 4-(N,N-dimethylamino)pyridine (200 mg) in py-
ridine (70 ml) TsCl (4.58 g, 24 mmol) was added portionswise at –20 °C. The stirring was continued
for 30 min at –20 °C and for 16 h at room temperature. Water (25 ml) was added and the mixture
was concentrated in vacuo to about 50 ml and codistilled with water (20 ml). Ethyl acetate (250 ml)
was added and the solution was washed successively with 0.1 M HCl (2 × 200 ml), 10% NaHCO3
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1532
Hocek et al.:
(2 × 200 ml) and water (8 × 200 ml). The organic layer was dried with MgSO4 and eva-
porated to give compound 4 as yellowish oil, RF (B) 0.49. Yield 8.6 g (80%). For C24H35O8PS
(514.6) calculated: 56.02% C, 6.86% H, 6.02% P, 6.23% S; found: 55.57% C, 6.98% H, 6.29% P,
6.58% S. Mass spectrum (FAB), m/z (rel.%): 515 (55) [M + H], 431 (100). 1H NMR spectrum
((CD3)2SO): 1.19–1.22 4 × d, 4 × 3 H, J(CH3,CH) = 6.1 (CH3); 2.40 s, 3 H (CH3-Ts); 3.46 dd, 1 H,
J(1b,2) = 5.1, J(gem) = 10.5 (Hb-1); 3.50 dd, 1 H, J(1a,2) = 5.1, J(gem) = 10.5 (Ha-1); 3.77 dd, 1 H,
J(P,CHb) = 8.8, J(gem) = 13.7 (PC-Hb); 3.83 dd, 1 H, J(P,CHa) = 9.0, J(gem) = 13.7 (PC-Ha); 3.84 m,
1 H (H-2); 4.03 dd, 1 H, J(3b,2) = 5.4, J(gem) = 10.5 (Hb-3); 4.17 dd, 1 H, J(3a,2) = 3.4, J(gem) = 10.5
(Ha-3); 4.42 s, 2 H (CH2Ph); 4.56 dsept, 2 H, J(CH,CH3) = 6.1, J(P,OCH) = 7.8 (POCH); 7.22–7.35 m,
5 H (H-Ph); 7.79 d, 2 H and 7.46 d, 2 H, J = 8.3 (CH-Ts).
Alkylation of 6-Cyanopurines 1 and 2. General Procedure
Cesium carbonate (490 mg, 1.5 mmol) was added to a solution of the 6-cyanopurine 1 or 2 (3 mmol)
in DMF (30 ml) and the mixture was stirred at 100 °C for 1 h, then cooled to 50 °C and a solution
of the synthon 3 or 4 (3.2 mmol) in DMF (5 ml) was added dropwise. The mixture was then stirred
at 120 °C for 16 h (TLC detection showed that most of the starting material had disappeared). The
solvent was evaporated, water (30 ml) was added and the mixture was extracted with ethyl acetate
(3 × 50 ml). The combined organic layers were dried over MgSO4 and evaporated. The residue was
chromatographed on a preparative layer of silica gel. The obtained chromatographically homogeneous
(TLC) oily compounds 9 and 10 were used in further steps.
9-{2-[Bis(isopropyloxy)phosphonylmethoxy]ethyl}-6-cyanopurine (9a), yield 48%, RF (A) 0.50.
For C15H22N5O4P . 1/2 H2O (376.4) calculated: 47.86% C, 6.16% H, 18.61% N, 8.23% P; found:
1
47.98% C, 6.15% H, 18.55% N, 8.15% P. Mass spectrum (FAB), m/z (rel.%): 368 (55) [M + H]. H NMR
spectrum ((CD3)2SO): 1.08 d, 6 H and 1.14 d, 6 H, J(CH3,CH) = 6.1 (4 × CH3); 3.78 d, 2 H, J(P,CH2) = 8.1
(PCH2); 3.96 t, 2 H, J(2′,1′) = 5.1 (H-2′); 4.44 dsept, 2 H, J(CH,CH3) = 6.1, J(P,OCH) = 7.8 (2 × POCH);
4.54 t, 2 H, J(1′,2′) = 5.1 (H-1′); 8.89 s, 1 H (H-8); 9.14 s, 1 H (H-2).
9-{3-Benzyloxy-2-[bis(isopropyloxy)phosphonylmethoxy]propyl}-6-cyanopurine (9b), yield 53%, RF
(A) 0.73. Exact mass (EI HRMS) for C23H30N5O5P calculated: 487.1985; found: 487.1918. Mass
spectrum (FAB), m/z (rel.%): 488 (10) [M + H], 91 (100). 1H NMR spectrum ((CD3)2SO): 1.06 d,
3 H, 1.115 d, 3 H, 1.12 d, 3 H and 1.16 d, 3 H, J(CH3,CH) = 6.1 (4 × CH3); 3.58 dd, 1 H, J(3′b,2′) =
4.6, J(gem) = 10.7 (Hb-3′); 3.62 dd, 1 H, J(3′a,2′) = 4.6, J(gem) = 10.7 (Ha-3′); 3.78 dd, 1 H, J(P,CHb) =
9.3, J(gem) = 13.9 (PC-Hb); 3.89 dd, 1 H, J(P,CHa) = 8.8, J(gem) = 13.9 (PC-Ha); 4.11 m, 1 H
(H-2′); 4.42 dsept, 1 H and 4.47 dsept, 1 H, J(CH,CH3) = 6.1, J(P,OCH) = 7.6 (2 × POCH); 4.47 d,
1 H and 4.49 d, 1 H, J(gem) = 12.0 (CH2Ph); 4.51 dd, 1 H, J(1′b,2′) = 7.3, J(gem) = 14.6 (Hb-1′); 4.57 dd,
1 H, J(1′a,2′) = 3.9, J(gem) = 14.6 (Ha-1′); 7.30–7.40 m, 5 H (H-arom.); 8.83 s, 1 H (H-8); 9.12 s, 1 H
(H-2).
2-Amino-9-{2-[bis(isopropyloxy)phosphonylmethoxy]ethyl}-6-cyanopurine (10a), yield 60%, RF
(A) 0.35. Exact mass (EI HRMS) for C15H23N6O4P calculated: 382.1518; found: 382.1528. Mass
spectrum (FAB), m/z (rel.%): 383 (40) [M + H], 299 (100). 1H NMR spectrum ((CD3)2SO): 1.11 d,
6 H and 1.16 d, 6 H, J(CH3,CH) = 6.1 (4 × CH3); 3.77 d, 2 H, J(P,CH2) = 8.3 (PCH2); 3.88 t, 2 H,
J(2′,1′) = 5.1 (H-2′); 4.26 t, 2 H, J(1′,2′) = 5.1 (H-1′); 4.47 dsept, 2 H, J(CH,CH3) = 6.1, J(P,OCH) = 7.6
(2 × POCH); 7.08 bs, 2 H (NH2); 8.29 s, 1 H (H-8).
2-Amino-9-{3-benzyloxy-2-[bis(isopropyloxy)phosphonylmethoxy]propyl}-6-cyanopurine (10b),
yield 54%, RF (A) 0.65. Exact mass (EI HRMS) for C23H31N6O5P calculated: 502.2094; found:
502.2048. Mass spectrum (FAB), m/z (rel.%): 503 (10) [M + H], 91 (100). 1H NMR spectrum ((CD3)2SO):
1.08 d, 3 H; 1.13 d, 6 H; 1.17 d, 3 H, J(CH3,CH) = 6.1 (4 × CH3); 3.54 dd, 1 H, J(3′b,2′) = 4.6,
J(gem) = 10.5 (Hb-3′); 3.60 dd, 1 H, J(3′a,2′) = 4.6, J(gem) = 10.5 (Ha-3′); 3.74 dd, 1 H, J(P,CHb) = 9.3,
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Acyclic Nucleotide Analogues
1533
J(gem) = 13.9 (PC-Hb); 3.87 dd, 1 H, J(P,CHa) = 8.8, J(gem) = 13.9 (PC-Ha); 4.04 m, 1 H (H-2′);
4.23 dd, 1 H, J(1′b,2′) = 7.3, J(gem) = 14.4 (Hb-1′); 4.27 dd, 1 H, J(1′a,2′) = 4.4, J(gem) = 14.4 (Ha-1′);
4.44 dsept, 1 H and 4.47 dsept, 1 H, J(CH,CH3) = 6.1, J(P,OCH) = 7.6 (2 × POCH); 4.50 d, 1 H and
4.52 d, 1 H, J(gem) = 12.0 (CH2Ph); 7.08 bs, 2 H (NH2); 7.27–7.37 m, 5 H (H-arom.); 8.26 s, 1 H
(H-8).
6-(Aminomethyl)-9-(2-phosphonomethoxyethyl)purine (11a)
A solution of compound 9a (380 mg, 1.04 mmol) in absolute ethanol (50 ml) was hydrogenated at
25 °C/101 kPa in the presence of 5% Pd-C (500 mg) for 48 h. The mixture was then filtered through
a Celite pad and the solvent was evaporated. The amorphous residue was dried, dissolved in aceto-
nitrile (30 ml) and, while stirring under argon atmosphere, TMSBr (1 ml, 7.6 mmol) was added and
the resulting solution was allowed to stand overnight at 25 °C. The mixture was taken down in vacuo
and codistilled with toluene (2 × 50 ml). The residue was dissolved in water (30 ml), aqueous am-
monia (35%; 1 ml) was added, the solution was washed with ether (2 × 30 ml) and applied onto a
column of Dowex 1X2 (acetate form, 50 ml). The column was washed with water and eluted with
0.01 M aqueous acetic acid. Evaporation of the appropriate fractions afforded the compound 11a (130 mg, 44%)
as amorphous glass that crystallized from aqueous ethanol on addition of ether. M.p. 161–164 °C
(dec.), EUp 0.67. For C9H14N5O4P . H2O (305.2) calculated: 35.41% C, 5.28% H, 22.94% N, 10.14% P;
found: 35.28% C, 5.28% H, 23.06% N, 9.95% P. Mass spectrum (FAB), m/z (rel.%): 288 (100) [M + H],
1
272 (21) [M + H – NH2]. H NMR spectrum (D2O): 3.64 d, 2 H, J(P,CH) = 8.55 (PCH2); 4.04 t, 2 H,
J(2′,1′) = 4.9 (H-2′); 4.61 t, 2 H, J(1′,2′) = 4.9 (H-1′); 4.81 s, 2 H (NCH2); 8.65 s, 1 H and 8.98 s,
1 H (H-2 and H-8). UV spectrum (water): pH 7, 265 (6 700); pH 12, 264 (6 700); pH 2, 264 (5 900).
6-(Aminomethyl)-9-(3-hydroxy-2-phosphonomethoxypropyl)purine (11b)
A solution of compound 9b (800 mg, 1.64 mmol) in ethanol (150 ml) was hydrogenated in the same
manner as described for the compound 9a and the reaction with TMSBr (2 ml, 15.2 mmol) was per-
formed in acetonitrile (70 ml). Anion exchange chromatography followed by preparative HPLC and
freeze-drying afforded pure compound 11b as an extremely hygroscopic violet lyophilizate (60 mg,
12%). EUp 0.80. For C10H16N5O5P . x H2O N/P found: 5.15; calculated: 5.00. Mass spectrum (FAB),
1
m/z (rel.%): 318 (31) [M + H]. H NMR spectrum (D2O): 3.52 dd, 1 H, J(P,CHb) = 9.5, J(gem) = 12.9
(PC-Hb); 3.62 dd, 1 H, J(3′b,2′) = 4.6, J(gem) = 12.5 (Hb-3′); 3.70 dd, 1 H, J(P,CHa) = 9.3, J(gem) = 12.9
(PC-Ha); 3.84 dd, 1 H, J(3′a,2′) = 3.9, J(gem) = 12.5 (Ha-3′); 3.95 m, 1 H (H-2′); 4.54 dd, 1 H,
J(1′b,2′) = 7.3, J(gem) = 14.9 (Hb-1′); 4.64 dd, 1 H, J(1′a,2′) = 3.9, J(gem) = 14.9 (Ha-1′); 4.82 s, 2 H
(NCH2); 8.65 s, 1 H and 8.98 s, 1 H (H-2 and H-8). UV spectrum (water): pH 7, 265; pH 12, 264; pH 2,
263 nm.
2-Amino-6-(aminomethyl)-9-(2-phosphonomethoxyethyl)purine (12a)
A solution of compound 10a (350 mg, 0.92 mmol) in absolute ethanol (100 ml) was hydrogenated in
the presence of 5% Pd-C (200 mg) at 25 °C/101 kPa for 48 h. The mixture was then filtered through
a Celite pad and the solvent was evaporated. The residue was dissolved in water (50 ml) and filtered
again. The filtrate was evaporated and dried. The residue was then dissolved in a mixture of DMF
(10 ml) and acetonitrile (30 ml) and TMSBr (3 ml, 22.8 mmol) was added under stirring in an argon
atmosphere. The stirring was continued for 2 h and the solution was allowed to stand overnight at 25 °C.
The mixture was taken down in vacuo and codistilled with toluene (2 × 50 ml). The residue was
dissolved in water (30 ml), aqueous ammonia (35%; 1 ml) was added, the solution was washed with
ether (2 × 50 ml) and applied onto a column of Dowex 50X8 (H+ form, 50 ml), washed with water and
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1534
Hocek et al.:
eluted with 5% aqueous ammonia. After evaporation of the appropriate UV absorbing fraction the
residue was applied to a column of Dowex 1X2 (acetate form, 50 ml), the column was washed with
water and the compound was eluted with 0.01 M aqueous acetic acid. Preparative HPLC of the
residue (110 mg) after evaporation of the appropriate fractions followed by freeze-drying afforded the
compound 12a as a greenish hygroscopic lyophilizate (80 mg, 30%). EUp 0.66. For C9H15N6O4P . H2O
(320.3) calculated: 33.75% C, 5.35% H, 26.24% N, 9.67% P; found: 34.12% C, 5.41% H, 26.04% N,
9.35% P. Mass spectrum (FAB), m/z (rel.%): 303 (32) [M + H], 185 (100). 1H NMR spectrum
(D2O): 3.62 d, 2 H, J(P,CH) = 8.6 (PCH2); 3.96 t, 2 H, J(2′,1′) = 4.9 (H-2′); 4.37 t, 2 H, J(1′,2′) = 4.9
(H-1′); 4.56 s, 2 H (NCH2); 8.21 s, 1 H (H-8). UV spectrum (water): pH 7, 306 (6 000), 245 (3 200); pH 12,
304 (6 200), 244 (3 400); pH 2, 317 (5 000), 237 sh (3 900).
2-Amino-6-(aminomethyl)-9-(3-hydroxy-2-phosphonomethoxypropyl)purine (12b)
and 2-Amino-6-(aminomethyl)-9-(3-benzyloxy-2-phosphonomethoxypropyl)purine (12c)
Compound 10b (850 mg, 1.7 mmol) was hydrogenated in ethanol (150 ml) over 5% Pd-C (500 mg)
as described for compound 10a and the intermediate was treated with TMSBr (5 ml, 38 mmol) in
acetonitrile (50 ml). The ion exchange chromatography followed by preparative HPLC and freeze
drying afforded compound 12b as a greenish hygroscopic lyophilizate (120 mg, 21%). EUp 0.67. For
C10H17N6O5P . 2 H2O (368.3) calculated: 32.61% C, 5.75% H, 22.82% N, 8.41% P; found: 32.87%
C, 5.09% H, 22.54% N, 8.27% P. Mass spectrum (FAB), m/z (rel.%): 333 (18) [M + H], 243 (100).
1H NMR spectrum (D2O): 3.38 dd, 1 H, J(P,CHb) = 9.5, J(gem) = 12.9 (PC-Hb); 3.44 dd, 1 H,
J(3′b,2′) = 4.9, J(gem) = 12.5 (Hb-3′); 3.53 dd, 1 H, J(P,CHa) = 9.3, J(gem) = 12.9 (PC-Ha); 3.64 dd,
1 H, J(3′a,2′) = 3.9, J(gem) = 12.5 (Ha-3′); 3.74 m, 1 H (H-2′); 4.15 dd, 1 H, J(1′b,2′) = 7.1, J(gem) = 14.9
(Hb-1′); 4.25 dd, 1 H, J(1′a,2′) = 3.9, J(gem) = 14.9 (Ha-1′); 4.42 s, 2 H (NCH2); 8.04 s, 1 H (H-8). UV
spectrum (water): pH 7, 307 (4 800), 243 (1 800); pH 12, 303 (5 100), 243 (2 300); pH 2, 317 (3 600),
236 sh (2 200). Further elution of the HPLC column afforded the 3-O-benzyl derivative 12c (50 mg, 7%)
as amorphous solid. EUp 0.61. Exact mass (FAB HRMS) for C17H24N6O5P [M + H] calculated:
423.1546, found: 423.1541. 1H NMR spectrum (D2O): 3.43 dd, 1 H, J(3′b,2′) = 4.2, J(gem) = 11.0
(Hb-3′); 3.48 dd, 1 H, J(P,CHb) = 10.0, J(gem) = 12.9 (PC-Hb); 3.54 dd, 1 H, J(3′a,2′) = 4.6, J(gem) = 11.0
(Ha-3′); 3.61 dd, 1 H, J(P,CHa) = 9.0, J(gem) = 12.9 (PC-Ha); 3.84 m, 1 H (H-2′); 4.18 dd, 1 H,
J(1′b,2′) = 5.9, J(gem) = 14.9 (Hb-1′); 4.22 dd, 1 H, J(1′a,2′) = 4.6, J(gem) = 14.9 (Ha-1′); 4.30 d, 1 H
and 4.34 d, 1 H, J(gem) = 12.0 (CH2Ph); 4.31 d, 1 H and 4.37 d, 1 H, J(gem) = 16.4 (NCH2);
7.11–7.21 m, 5 H (H-arom); 7.95 s, 1 H (H-8).
6-Carboxamidinio-9-(2-hydrogenphosphonatomethoxyethyl)purine (14a)
To a stirred solution of compound 9a (250 mg, 0.68 mmol) in absolute methanol (80 ml) 1 M sodium
methoxide solution (68 µl, 0.068 mmol) was added and the solution was allowed to stand for 2 days
at 20 °C (TLC showed almost quantitative conversion). Ammonium chloride (100 mg, 1.8 mmol)
was added and the solution was refluxed for 2 h. Evaporation of the solvent gave the crude amidi-
nium chloride 13a as yellow hygroscopic powder (containing some NH4Cl and NaCl). Exact mass
(FAB HRMS) found: 385.1780 (100); for C15H26N6O4P [M – Cl] calculated: 385.1753. 1H NMR
spectrum ((CD3)SO): 1.09 d, 6 H, and 1.15 d, 6 H, J(CH3,CH) = 6.1 (4 × CH3); 3.81 d, 2 H,
J(P,CH2) = 8.3 (PCH2); 4.01 t, 2 H, J(2′,1′) = 5.0 (H-2′); 4.46 dsept, 2 H, J(CH,CH3) = 6.1,
J(P,OCH) = 7.8 (2 × POCH); 4.62 t, 2 H, J(1′,2′) = 5.0 (H-1′); 7.90 bs, 15 H (NH + NH4Cl); 9.08 s, 1 H
and 9.14 s, 1 H (H-2 and H-8). This compound was without purification dissolved in a mixture of
acetonitrile (80 ml) and DMF (5 ml), filtered and the filtrate was treated with TMSBr (4 ml, 30 mmol)
under stirring. The solution was set aside for 2 days at 20 °C, the solvents were evaporated and the
residue dissolved in water (20 ml). Aqueous ammonia (35%; 1 ml) was added, the solution was
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Acyclic Nucleotide Analogues
1535
washed with ether (2 × 50 ml) and evaporated. The residue was dissolved in water (5 ml) and after
addition of 35% HCl (0.2 ml) applied onto a column of Dowex 50X8 (H+ form, 50 ml) which was
washed with water to drop of UV absorption and then with 1% ammonia. The UV-absorbing fraction
of the ammonia eluate was evaporated, the residue dissolved in water (5 ml) and after addition of
35% ammonia (1 ml) applied onto a column of Dowex 1X2 (acetate form, 50 ml). The column was
washed with water and the product eluted with 0.01 M acetic acid. Evaporation of the UV-absorbing
fraction afforded the amidine 14a as a brownish hygroscopic powder (90 mg, 44%), m.p. 239–242 °C
(dec.), EUp 0.62. For C9H13N6O4P . 1.75 H2O (331.7) calculated: 32.58%C, 5.01% H, 25.33% N;
found: 32.51% C, 4.40% H, 25.59% N. Mass spectrum (FAB), m/z (rel.%): 301 (28) [M + H], 57 (100).
1H NMR spectrum (D2O): 3.69 d, 2 H, J(P,CH) = 8.8 (PCH2); 4.07 t, 2 H, J(2′,1′) = 5.2 (H-2′); 4.66 t,
2 H, J(1′,2′) = 5.2 (H-1′); 8.84 s, 1 H and 9.10 s, 1 H (H-2 and H-8). UV spectrum (water): pH 7, 296
(8 100), 249 sh (3 600); pH 12, 279 (9 700); pH 2, 294 (7 700), 250 sh (3 900).
2-Amino-6-carboxamidinio-9-(2-hydrogenphosphonatomethoxyethyl)purine (14b)
To a solution of compound 10a in absolute methanol (120 ml) was added 1 M sodium methoxide
solution (150 µl, 0.15 mmol) and the solution was stirred for 2 days at 65 °C (TLC – almost quanti-
tative conversion). Ammonium chloride (250 mg, 4.5 mmol) was added and the stirring at 65 °C was
continued for 5 h. The solvent was evaporated to give the crude amidinium chloride 13b as a yellow
hygroscopic powder (contaminated with NH4Cl and NaCl). Exact mass (FAB HRMS) found:
400.1885 (100); for C15H27N7O4P [M – Cl]+ calculated: 400.1862. 1H NMR spectrum ((CD3)SO):
1.10 d, 6 H and 1.15 d, 6 H, J(CH3,CH) = 6.1 (4 × CH3); 3.78 d, 2 H, J(P,CH) = 8.3 (PCH2); 3.90 t, 2 H,
J(2′,1′)= 5.0 (H-2′); 4.32 t, 2 H, J(1′,2′) = 5.0 (H-1′); 4.47 dsept, 2 H, J(CH,CH3) = 6.1, J(P, OCH) = 7.6
(POCH); 6.95 bs, 2 H (NH2); 8.00 bs, 10 H (NH + NH4Cl); 8.44 s, 1 H (H-8). This material was dis-
solved without purification in a mixture of DMF (100 ml) and acetonitrile (50 ml), filtered and the
filtrate was treated with TMSBr (10 ml, 76 mmol) as described for compound 13a. Analogous isola-
tion by ion exchange chromatography afforded the amidine 14b as a yellow powder, m.p. 298–300 °C
(dec.), yield 240 mg (51%). EUp 0.54. For C9H14N7O4P (315.2) calculated: 34.29% C, 4.48% H,
31.10% N, 9.83% P; found: 34.07% C, 4.41% H, 30.86% N, 9.49% P. Mass spectrum (FAB), m/z
1
(rel.%): 316 (100) [M + H]. H NMR spectrum (D2O): 3.64 d, 2 H, J(P,CH) = 8.5 (PCH2); 4.04 t, 2 H,
J(2′,1′) = 4.9 (H-2′); 4.37 t, 2 H, J(1′,2′) = 4.9 (H-1′); 8.39 s, 1 H (H-8). UV spectrum (water): pH 7,
361 (6 200), 277 (1 000); pH 12, 334 (6 900), 266 (3 600); pH 2, 360 (6 300), 275 sh (1 100).
Antiviral Activity Assays
The antiviral activity assays other than for HIV-1 were based on inhibition of virus-induced cytopa-
thicity in either E6SM or HEL cell cultures, following the previously established procedures15,16
.
Briefly, confluent cell cultures in microtiter trays were inoculated with 100 CCID50 of the virus
(1 CCID50 being the virus dose required to infect 50% of the cell cultures). After a 1 h virus adsorp-
tion period, the residual virus was removed and the cell cultures were incubated in the presence of
varying concentrations (400, 200, 100, ... µg/ml) of the test compounds. Viral cytopathicity was re-
corded as soon as it reached completion in the control virus-infected cell cultures.
Inhibition of HIV-1-Induced Cytopathicity in MT-4 Cells
The methodology of the anti-HIV activity assays has been described previously17. Briefly, human
MT-4 cells (5 . 105 cell/ml) were infected with 100 CCID50 HIV-1 (strain HTLV-IIIB)/ml and seeded
in 200 µl-wells of a microtiter plate, containing the test compounds in appropriate dilutions. After
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1536
Hocek et al.:
5 days of incubation at 37 °C, the number of viable cells was determined in a blood cell counting
chamber by trypan blue dye exclusion.
Inhibition of MSV-Induced Transformation of Murine C3H/3T3 Embryo Fibroblasts
The anti-MSV activity assay was performed as described previously18. Murine C3H/3T3 embryo fi-
broblast cells were seeded at 5 . 105 cell/ml into 1 cm2 wells of a 48-well microplate. After 24 h, the
cell cultures were infected with 80 focus-forming units of MSV (prepared from tumours induced fol-
lowing intramuscular inoculation of 3 days old NMRI mice with MSV, as described previously19) for
60–90 min at 37 °C. The medium was then replaced by 1 ml of fresh medium containing various
concentrations of the test compounds. After 6 days, the transformation of the cell culture was exami-
ned microscopically.
This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic
(Grants No. 455402 and No. 455407), by the Grant Agency of the Czech Republic (Grant No.
203/93/0118), by the PECO project No. ERBCIPDCT 940202 of the European Commission, by the
Grants from the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek No. 3.3010.91, the Belgian
Genconcerteerde Onderzoeksacties No. 95/5 and by Gilead Sciences (Foster City, CA, U.S.A.). The
cytostatic activity was studied by Dr I. Votruba at the Institute of Organic Chemistry and Biochemistry.
The authors’ thanks are due to Mr J. Günter for performing the preparative HPLC, to the staff of mass
spectrometry and analytical departments and to Mrs A. Absillis, L. van Berckelaer, A. Van Lierde, F. De
Meyer and A. Camps for technical assistance with the antiviral activity assays.
REFERENCES
1. a) Holy A. in: Isopolar Phosphorus-Modified Nucleotide Analogues in Advances in Antiviral
Drug Design (E. De Clercq, Ed.), Vol. 1, p. 179. JAI Press Inc., Greenwich (U.S.A.)/London
1993. b) Holy A., Dvorakova H., Jindrich J. in: Antiviral Acyclic Nucleotide Analogues in
Antibiotics and Antiviral Compounds (K. Krohn, H. A. Kirst and H. Maag, Eds), p. 455. Verlag
Chemie, Berlin/Heidelberg 1993. c) Vesely J., Vlach J., Sediva K., Rosenberg I., Otmar M.,
Hrebabecky H., Travnicek M., Vonka V., Snoeck R., De Clercq E.: Antiviral Res. 13, 295
(1990).
2. Holy A., Snoeck R., Andrei G., Balzarini J., De Clercq E.: Antiviral Res. 26, A231 (1995).
3. Hocek M., Masojidkova M., Holy A.: Collect. Czech. Chem. Commun. 60, 875 (1995).
4. Giner-Sorolla A., Bendich A.: J. Am. Chem. Soc. 80, 3932 (1958).
5. Herdewijn P., Van Aerschot A., Pfleiderer W.: Synthesis 1989, 961.
6. Hocek M., Holy A.: Collect. Czech. Chem. Commun. 60, 1386 (1995).
7. Dvorakova H., Holy A.: Collect. Czech. Chem. Commun. 58, 1419 (1993).
8. Bronson J. J., Ghazzouli I., Hitchcock M. J. M., Webb R. R., Martin J. C.: J. Med. Chem. 32,
1457 (1989).
9. Holy A.: Collect. Czech. Chem. Commun. 58, 649 (1993).
10. Hockova D., Masojidkova M., Budesinsky M., Holy A.: Collect. Czech. Chem. Commun. 60, 224
(1995).
11. Sajiki H.: Tetrahedron Lett. 36, 3465 (1995).
12. Phelan M. J., Gabrielsen B., Kirsi J. J., Shannon W. M., Ussery M. A., Barthel-Rosa L., Schubert
E. M., Kini G. D., Robins R. K.: Nucleosides Nucleotides 14, 1315 (1995).
13. Thorpe M. C., Coburn W. C., Jr., Montgomery J. A.: J. Magn. Reson. 15, 98 (1974).
14. Lok C. M.: Chem. Phys. Lipids 22, 323 (1978).
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15. De Clercq E., Descamps J., Verhelst G., Walker R. T., Jones A. S., Torrence P. F., Shugar D.: J.
Infect. Dis. 141, 563 (1980).
16. De Clercq E., Holy A., Rosenberg I., Sakuma T., Balzarini J., Maudgal P. C.: Nature 323, 464
(1986).
17. Balzarini J., Naesens L., Slachmuylders J., Niphuis H., Rosenberg I., Holy A., Schellekens H.,
De Clercq E.: AIDS 5, 21 (1991).
18. Balzarini J., Naesens L., Herdewijn P., Rosenberg I., Holy A., Pauwels R., Baba M., Johns D.
G., De Clercq E.: Proc. Natl. Acad. Sci. U.S.A. 86, 332 (1989).
19. De Clercq E., Merigan T. C.: Proc. Soc. Exp. Biol. Med. 137, 590 (1971).
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