Synthesis of quaternary 1-[2-(phosphonomethoxy)ethyl] derivatives of 2,4-diaminopyrimidine and related acyclic nucleotide analogs

Article abstract of DOI:10.1135/cccc19990242

Quaternization of 2,4-diaminopyrimidine (2) by diisopropyl 2-chloroethoxymethane-phosphonate (3) followed by bromotrimethylsilane treatment and subsequent hydrolysis gave zwitterionic N1-[2-(phosphonomethoxy)ethyl] derivative, hydrogen ([2-(2,4-diaminopyrimidin-1-io)ethoxy]methyl)phosphonate (5). Its structure was confirmed by X-ray crystallography. The same product was obtained from 2-amino-4-[(dimethylaminomethylene)amino)pyrimidine (6) by an analogous reaction sequence followed by an aqueous ammonia treatment after the transsilylation reaction. Also the quaternizations of 4,6-diaminopyrimidine (7) and 2,4,6-triaminopyrimidine (8) with the halo derivative 3 afforded the zwitterionic N1-substituted compounds 9 and 10, respectively. In contrast to this regiospecific reaction, 2-aminopyrimidine (11) gave on treatment with compound 3 and following deprotection the exo-N2-isomer 13. This compound was also obtained by the reaction starting from 2-[(dimethylaminomethylene)amino]pyrimidine (12) which was prepared by treatment of compound 11 with dimethylformamide dineopentyl acetal. Also 2,3-diaminopyridine (14) gave by the above reaction a mixture of 2-amino-3-([2-(phosphonomethoxy)ethyl]amino)pyridine (15) and quaternary N1-[2-(phosphonomethoxy)ethyl] derivative (16). None of these analogs of the antiviral PMEDAP exhibited any antiviral activity against DNA viruses or retroviruses, nor any cytostatic activity.

Full text of DOI:10.1135/cccc19990242

242
Holý, Buděšínský, Podlaha, Císařová:
SYNTHESIS OF QUATERNARY 1-[2-(PHOSPHONOMETHOXY)ETHYL]
DERIVATIVES OF 2,4-DIAMINOPYRIMIDINE AND RELATED ACYCLIC
NUCLEOTIDE ANALOGS
b1
Antonín HOLÝa1,*, Miloš BUDĚŠÍNSKÝa2, Jaroslav PODLAHA
b2
and Ivana CÍSAŘOVÁ
a
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10 Prague 6, Czech Republic; e-mail: uochb@uochb.cas.cz, budesinsky@uochb.cas.cz
Department of Inorganic Chemistry, Charles University, 128 40 Prague 2, Czech Republic;
1
2
b
1
2
e-mail: podlaha@prfdec.natur.cuni.cz, cisarova@prfdec.natur.cuni.cz
Received Decem ber 15, 1998
Accepted Jan uary 12, 1999
Dedicated to the memory of Dr Miroslav Protiva.
Quatern ization of 2,4-diam in opyrim idin e (2) by diisopropyl 2-ch loroeth oxym eth an e-
ph osph on ate (3) followed by brom otrim eth ylsilan e treatm en t an d subsequen t h ydrolysis
gave zwitterion ic N1-[2-(ph osph on om eth oxy)eth yl] derivative, h ydrogen {[2-(2,4-diam in o-
pyrim idin -1-io)eth oxy]m eth yl}ph osph on ate (5). Its structure was con firm ed by X-ray crys-
tallography. The same product was obtained from 2-amino-4-[(dimethylaminomethylene)amino]py-
rimidine (6) by an an alogous reaction sequen ce followed by an aqueous am m on ia treatm en t
after th e tran ssilylation reaction . Also th e quatern ization s of 4,6-diam in opyrim idin e (7) an d
2,4,6-triam in opyrim idin e (8) with th e h alo derivative 3 afforded th e zwitterion ic N1-substi-
tuted com poun ds
9 an d 10, respectively. In con trast to th is regiospecific reaction ,
2-am in opyrim idin e (11) gave on treatm en t with com poun d 3 an d followin g deprotection
th e exo-N2-isom er 13. Th is com poun d was also obtain ed by th e reaction startin g from
2-[(dim eth ylam in om eth ylen e)am in o]pyrim idin e (12) wh ich was prepared by treatm en t of
com poun d 11 with dim eth ylform am ide din eopen tyl acetal. Also 2,3-diam in opyridin e (14) gave
by th e above reaction a m ixture of 2-am in o-3-{[2-(ph osph on om eth oxy)eth yl]am in o}pyridin e
(15) an d quatern ary N1-[2-(ph osph on om eth oxy)eth yl] derivative (16). Non e of th ese an alogs
of th e an tiviral PMEDAP exh ibited an y an tiviral activity again st DNA viruses or retroviruses,
n or an y cytostatic activity.
Key w ord s: Acyclic n ucleotide an alogs; Pyrim idin es; Ph osph on ates; An tivirals; PMEDAP;
NMR spectroscopy, 1H an d 13C; Crystal structure.
9-[2-(Ph osph on om eth oxy)eth yl]-2,6-diam in opurin e (1, PMEDAP) is a pow-
erful an tiviral drug active both again st DNA viruses1 an d retroviruses2
(HIV); in vivo treatm en t of m ice in fected with eith er Molon ey m urin e sar-
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2,4-Diaminopyrimidine Acyclic Phosphonates
243
com a virus (MSV), Frien d leukem ia virus (FLV), or m urin e cytom egalovirus
(MCMV) revealed th e h igh th erapeutic effect of th is com poun d3,4. Also its
h igh activity again st h epatitis B virus (HBV) h as been firm ly establish ed5. It
was recen tly sh own th at PMEDAP also h as a sign ifican t cytostatic activity6.
Modification s of th is lead structure wh ich con cen trated m ain ly on th e sub-
stitution of its N6-am in o fun ction , resulted in n um erous biologically active
com poun ds with an tiviral7,8 an d cytostatic9 properties.
Th e m ost con spicuous structural feature of th e purin e system in th e
PMEDAP m olecule is its pyrim idin e part bearin g two am in o fun ction s.
2-Am in opurin e substituted at th e position 6 was postulated as a
ph arm acoph or of th e acyclic n ucleoside ph osph on ates active again st MSV
(ref.10). Th us, it seem ed of in terest to syn th esize an d in vestigate com poun ds
bearin g th e 2-(ph osph on om eth oxy)eth yl (PME) ch ain in 2-am in o-
pyrim idin e or 2,4-diam in opyrim idin e base, or th eir deaza an alogs.
Th e syn th esis of pyrim idin e an d/or purin e acyclic n ucleoside ph os-
ph on ates from th e h eterocyclic bases con tain in g acid NH fun ction is
m ain ly based on an N-alkylation of th e base with an alkylation agen t bear-
in g th e structural features of th e PME ch ain an d protected at th e
ph osph on ate residue by ester fun ction s11. Dieth yl esters of [2-(tosyl-
oxy)ethoxy]methylphosphonic acid12 and/or [(2-chloroethoxy)m eth yl]ph ospho-
nic acid were originally utilized for this purpose13. However, th is approach was
con siderably im proved by th e use of th e diisopropyl ester 3 (ref.10) wh ich
elim in ates th e form ation of N-eth yl derivatives as side products of th e
N-alkylation .
All th ese alkylation s take place in th e presen ce of a base: th e h eterocyclic
purin e or pyrim idin e bases are tran sform ed to th eir sodium salts by th e re-
action with NaH or, th e alkylation is perform ed in th e presen ce of cesium
carbon ate13 or, 1,7-diazabicyclo[5.4.0]un dec-7-en e11 (DBU). However, in
2,4-diam in opyrim idin e an d related com poun ds of pyrim idin e an d pyridin e
series lackin g NH-groupin gs, th e key-step in th e reaction sequen ce for th e
syn th esis of th e desired ph osph on ate an alogs is a quatern ization reaction .
Such a reaction with com poun d 3 was also applied to th e quatern ization of
n icotin am ide14.
Reaction of 2,4-diam in opyrim idin e (2) with excess com poun d 3 used as a
solven t takes place at m oderately in creased tem perature. It affords an ion ic
product bearin g a pron oun ced electropositive ch arge as witn essed by its
electroph oretic m obility at sligh tly alkalin e pH. Th is in term ediate was n ot
furth er ch aracterized; after rem oval of excess com poun d 3 it was directly
subm itted to tran ssilylation with BrSiMe3 followed by h ydrolysis. Th e reac-
tion m ixture con tain ed, in addition to un reacted 2, a sin gle product with
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

244
Holý, Buděšínský, Podlaha, Císařová:
sligh tly acidic ch aracter (Sch em e 1). It was deion ized by Dowex 50
ch rom atograph y an d afforded com poun d 5 by furth er purification on
Dowex 1 X 2. No oth er regioisom er of com poun d 5 was detected in th e re-
action m ixture15. Th e assign m en t of th is structure is based on 1H an d 13C
spectra (vide infra). Also th e UV spectrum (λm ax 270 n m ) of th e product cor-
NH
2
N
N
N
O
P(O)(Oi-Pr)
2
Cl
H N
2
N
O
P(O)(OH)
2
3
1
NH
N
NH
2
2
N
3
N
H N
2
H N
2
N
O
P(O)(Oi-Pr)
2
Cl
2
4
3
NH
NH
N
2
2
N
N
O
(CH ) N HC
3 2
N
N
H N
2
O
O
P
6
OH
SCHEME 1
5
respon ds rath er to th e value publish ed for th e quatern ary N1-m eth yl
derivative (λm ax 271 n m ) th an to th e UV spectrum of th e isom eric 4-am in o-
2-m eth ylam in opyrim idin e16 (λm ax 276 n m ). An iden tical product 5 was also
obtain ed from th e form am idin e 6 wh ich was prepared from 2,4-diam in o-
pyrim idin e (2) by th e action of dim eth ylform am ide din eopen tyl acetal17 or
dim eth yl acetal. Th e am idin e form ation proceeds solely at th e 2-NH2 fun c-
tion even wh en th e reaction is perform ed at elevated tem perature; th e
N4-m on o- an d N2,N4-bis(am idin e)-protected derivatives were detected as
im purities in th e crude reaction product on ly in spite of a careful workup18
(as sh own by NMR an alysis of th e crude 6).
Clearly th ese data can n ot un equivocally distin guish between th e zwitter-
ion ic quatern ary structure 5a, zwitterion ic structure with proton ated (2- or
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

2,4-Diaminopyrimidine Acyclic Phosphonates
245
4-) im m on ium fun ction (5b) or th e ph osph on ic acid structure 5c. However,
th e electroph oretic m obility wh ich is m uch lower th an th at expected for
th e m on oan ion derived from cytosin e or 2,6-diam in opurin e acyclic
ph osph on ate, speaks in favor of th e quatern ary structure, i.e. h ydrogen
2-{[2-(2,4-diam in opyrim idin -1-io)eth oxy]m eth yl}ph osph on ate (5a).
Th e fin al structural proof of com poun d 5 was provided by low-
tem perature X-ray structure determ in ation . All h ydrogen atom s were un -
am biguously located an d refin ed. Th e structure is zwitterion ic with sin gly
deproton ated ph osph on ate group (Fig. 1). Th e bon d len gth s in th e pyrim i-
din e rin g, wh ich is plan ar with in ±0.017 Å in cludin g N2 an d N4 atom s, in -
dicate certain degree of electron delocalization over all its n itrogen atom s.
Wh ile th e geom etry aroun d N1 is typical of a quatern ary N-alkylpyridin ium
derivative, th e distan ces to th e exocyclic am in o groups are som ewh at sh ort-
en ed com pared to sim ple arom atic am in es; m ore sign ifican tly, th e NH2 h y-
drogen s lie alm ost exactly with in th e h eteroarom atic plan e (less so for N4
but even th ese N3–C–N–H torsion an gles are below 9(1)° ). On th e oth er
h an d, th e electron delocalization in th e C4 to C6 part is in com plete an d, al-
th ough th e h ybridization at C5 an d C6 is strictly sp2, th e C4–C5 bon d is by
0.088(2) Å lon ger th an C5–C6. Th e overall geom etry of th e pyrim idin e base
is alm ost iden tical with th at of bis(2,4-diam in opyrim idin ium ) sulfate
m on oh ydrate19, th e on ly closely related structure foun d in Cam bridge
Structural Database. Th ough th is com poun d is th erein form ulated19 with
th e ch arge localized at N1, such a presen tation is purely form al: n o direct
eviden ce of ch arge localization can be obtain ed from a stan dard electron
den sity determ in ation .
Th e con form ation of th e side ch ain is obviously dictated by
in term olecular h ydrogen bon din g. Th e ph osph on ate groups are lin ked by a
N4
C4
C5
C6
N3
C2
O4
O1
P1
N1
O3
N2
C7
O9
C8
O2
FIG. 1
Perspective view of 5·H2O with atom label-
lin g (50% probability ellipsoids)
C10
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246
Holý, Buděšínský, Podlaha, Císařová:
sh ort, cen trosym m etric double h ydrogen bon d (O2···O3 2.538 Å) supported
by lon ger NH2–ph osph on ate an d NH2–N3 in teraction s, eith er direct or m e-
diated by th e water m olecules. In th is bon din g, all th e am in o an d water h y-
drogen s participate as don ors an d th e ph osph on ate an d water oxygen s an d
N3 as acceptors. Th e eth er oxygen O9 an d th e h eterocyclic n itrogen N1 are
n ot in volved in h ydrogen bon din g. In th e crystal, th e zwitterion s are lin ked
in to two-dim en sion al layers propagatin g approxim ately parallel to th e crys-
tallograph ic ac-plan e wh ich is also th e direction of th e preferen tial growth
of th e crystals.
Sim ilarly, 4,6-diam in opyrim idin e (7) an d 2,4,6-triam in opyrim idin e (8)
gave by th e reaction with th e ph osph on ate 3 quatern ary N1-[2-(ph osph o-
n om eth oxy)eth yl] derivatives 9 an d 10, respectively (Sch em e 2), with out
an y detectable form ation of oth er regioisom ers. Th eir structure was also as-
sign ed on th e basis of NMR data (vide infra) an d corroborated by com parin g
th eir UV spectra with th ose of correspon din g m eth yl derivatives: th e un -
equivocal course of th e reaction correspon ds to a sim ilar reaction of th e
base 7 with m eth yl iodide wh ich affords th e quatern ary N1-m eth yl deriva-
tive as th e on ly reaction product20. Its UV ch aracteristics (λm ax 265 n m )
NH
N
NH
N
NH
N
2
2
2
N
N
N
O
P
O
P
O
P
H N
2
H N
2
HN
O
O
OH
OH
O
O
O
OH
OH
5a
5b
5c
NH
N
2
NH
N
2
N
N
3
R
NH
2
O
R
NH
O
2
O
P
OH
7, R = H
8, R = NH
9, R = H
10, R = NH
2
2
N
N
N
3
O
P(O)(OH)
2
N
NH
2
N
H
3
11
13
N
N
N
CH N(CH )
3 2 12
SCHEME 2
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2,4-Diaminopyrimidine Acyclic Phosphonates
247
agrees with th e data for com poun d 9 (λm ax 267 n m ) wh ile th e λm ax value of
th e isom eric 4-am in o-2-m eth ylam in opyrim idin e as a m odel for th e
regioisom er of com poun d 9 (λm ax 277 n m at pH 1)21 sign ifican tly differs
from our experim en tal value. Sim ilarly, th e UV spectra of th e com poun d 10
(λm ax 275 n m at pH 1 an d 7) are in accord with th e N1-isom er an d differ
substan tially from th ose of 2,4-diam in o-6-m eth ylam in opyrim idin e ((λm ax
280 n m at pH 1 an d 270 n m at pH 7)22. Th e low electroph oretic m obility of
com poun ds 9 an d 10 is com parable with th e value for com poun d 5. All
th ese argum en ts corroborate th e quatern ary structures bearin g th e side
ch ain at N1-position .
A differen t situation arose in th e application of th e above reaction with
com poun d 3 to 2-am in opyrim idin e (11) an d/or its N2-dim eth ylam in o-
m eth ylen e derivative 12 (prepared by th e reaction of 11 with dim eth yl-
form am ide dim eth yl acetal): th e cleavage of th e in term ediate with BrSiMe3
an d subsequen t h ydrolysis of th e silyl ester groups (in th e case of 12 fol-
lowed by aqueous am m on ia treatm en t) gave 2-{[2-(ph osph on o-
m eth oxy)eth yl]am in o}pyrim idin e (13) as th e on ly product. Its structural
assign m en t is based on NMR spectra (vide infra); also th e UV spectrum (λm ax
232 an d 314 n m ) correspon ds to th at structure. Furth erm ore, th e electro-
ph oretic m obility of com poun d 13 is substan tially h igh er com pared to
com poun ds 5, 9 an d 10 as expected for th e structure lackin g th e positive
ch arge wh ich equilibrates th e n egative ch arges of th e ph osph on ate residue.
Th e form ation of exo-N2 derivative could be in terpreted by direct alkylation
of th e im in o form in th e 2-am in o derivative 11. In th e case of am idin e 12,
th e m ech an ism im plies quatern ization of th e am idin e α-n itrogen atom fol-
lowed by h ydrolysis of th e am idin ium salt. Altern atively, th e form ation of
th e n on -quatern ized exo-N2 derivative 13 both from 11 an d 12 could be ex-
plain ed by quatern ization at N1 followed by Dim roth type rearran gem en t
(rin g open in g–rin g closure reaction at alkalin e pH).
Gen erally, com poun ds with zwitterion ic ch aracter (5, 9, 10, 16) elute
from the strongly basic anion exchanger already with very diluted (≈0.02 M)
acetic acid solution s wh ile th e derivatives with th e com m on ph osph on o-
m eth yl ch aracter (13, 15) are eluted on ly by th e solution s of a h igh er ion ic
stren gth of acetic acid23
.
The reaction of the com pound 3 with 2,3-diam in opyridin e (14) afforded
two reaction products wh ich were separated by silica gel ch rom atograph y
an d obtain ed as ligh t-sen sitive oils. Th eir treatm en t with BrSiMe3 an d h y-
drolysis gave isom eric 2-(ph osph on om eth oxy)eth yl derivatives wh ich were
purified by an ion exch an ge ch rom atograph y: th e N3-isom er, 2-am in o-
3-{[2-ph osph on om eth oxy)eth yl]am in o}pyridin e 15 an d 1-[2-(ph osph on o-
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

248
Holý, Buděšínský, Podlaha, Císařová:
m eth oxy)eth yl] derivative 16 (Sch em e 3). Th eir structures follow from th e
NMR spectra (vide infra). Th e argum en ts are sustain ed by th eir electroph o-
retic beh aviour: th e quatern ary ch aracter of com poun d 16 is supported by
its low electroph oretic m obility wh ich is com parable with com poun ds 5
an d 10, wh ile th e m obility of com poun d 15 wh ich correspon ds to th e
structurally related com poun d 13 clearly reflects th e double dissociation of
th e ph osph on ate group at weakly alkalin e pH an d excludes th e quatern ary
structure bearin g an expressed positive ch arge wh ich partly n eutralizes th e
ph osph on ate residue. Th e reaction of th e base 14 with m eth yl iodide was
reported to proceed also with th e form ation of two products:
N1-substituted (quatern ary) m eth yl derivative an d 2-am in o-3-(m eth yl-
am in o)pyridin e24. Th e presen ce of a regioisom er 17 was n ot detected.
NH
NH
2
2
NH
2
2
N
O
P
N
NH
O
O
14
OH
16
H
N
NH
2
O
P(O)(OH)
2
O
P(O)(OH)
2
N
N
H
N
NH
2
15
17
SCHEME 3
NMR Discussion
To obtain addition al argum en ts for th e assign m en t of sign als an d structural
in terpretation of NMR spectra of th e isom eric products, we h ave also in ves-
tigated th e NMR spectra of th e correspon din g h eterocyclic bases (Table I).
Th e presen ce of th e ph osph on om eth oxy group in th e 1-[2-(ph osph on o-
m eth oxy)eth yl] (PME) derivatives is m an ifested by addition al spin –spin
2
1
1
3
couplin gs J(P,H) in H NMR or J(P,C) an d J(P,C) in 13C NMR spectra. Th e
ch aracter of sign als (doublets of CH2–P an d C-2′ carbon s in proton -
decoupled 13C NMR spectra) and different splitting (150–165 and 10–13 Hz,
respectively) allow un equivocal assign m en t of carbon sign als CH2–P an d
C-2′ an d th eir distin guish in g from C-1′.
Main structural problem in th e series of com poun ds 5, 9, 10, 13, 15 an d
16 con sists in th e determ in ation of th e PME-group location at th e
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

2,4-Diaminopyrimidine Acyclic Phosphonates
249
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

250
Holý, Buděšínský, Podlaha, Císařová:
h eteroarom atic rin g. Th eir poor solubility m akes im possible to apply th e
m eth od based on vicin al couplin gs J(C,H) of α-m eth ylen e proton s of
substituen t observable in proton -coupled 13C NMR spectra (e.g. ref.25). Th e
structural argum en ts are th erefore based on ch em ical sh ifts, differen t sym -
m etry of altern ative structures, NOE an d/or spin –spin couplin g in 1H NMR
spectra.
Th e N1-position of th e PME-group in com poun d 5 was derived from dif-
feren ce 1D NOE spectra. Saturation of C(1′)H2 sign al at δ 4.13 resulted in
th e 8% NOE en h an cem en t of doublet at δ 7.67 belon gin g to proton H-6.
Sim ilar approach h as been applied in case of com poun d 9, wh ere saturation
of C(1′)H2 sign al at δ 4.28 resulted in th e 12% NOE en h an cem en t of sin glet
at δ 8.23 belon gin g to proton H-2 in accordan ce with proposed structure 9.
Th e argum en ts followin g from th e sym m etry of th e m olecule h ave been
used for com poun ds 10 an d 13. Two-fold sym m etry axis (m an ifested by th e
ch em ical sh ift equivalen ce of proton s H-4, H-6 as well as carbon s C-4, C-6)
an d spin –spin couplin g between H-1′ an d NH (observed in 1H NMR spec-
trum of com poun d 13 taken in DMSO) in dicate th e substitution by th e
PME-ch ain at th e am in o group in position 2. On th e oth er h an d, four n on -
equivalen t carbon sign als of th e substituted pyrim idin e rin g in com poun d
10 exclude th e substitution at th e exocyclic N2-am in o group. Th e absen ce
of th e NOE at sin glet of H-5 (at δ 5.15) un der irradiation of N-CH2 sign al at
δ 4.18 an d close sim ilarity of th e 13C ch em ical sh ift of C-1′ (δ 49.29) with
above discussed com poun ds 5 an d 9 stron gly support th e structure 10 with
th e 2-(ph osph on om eth oxy)eth yl group in position 1.
Reaction of 2,3-diam in opyridin e (14) provided two isom eric products:
lowfield position of carbon C-1′ (δ 57.04) an d correspon din g m eth ylen e
proton s (δ 4.46) in com poun d 16 an d, particularly, 9% NOE en h an cem en t
of th e H-6 sign al (at δ 7.46) observed at irradiation of H-1′ proton s (at δ
4.46) in dicate th at th e PME-group is located at th e position N1. Th e iso-
m eric com poun d 15 sh owed an upfield sh ift of C-1′ (δ 45.96) an d H-1′ (δ
3.21) an d 8% NOE at H-4 (δ 6.98) un der irradiation of H-1′ proton s in ac-
cordan ce with th e substitution at C(3)–NH2 group.
Biological Activity
Th e free ph osph on ates 5, 9, 10, 13, 15 an d 16 were screen ed for th eir an ti-
viral activity again st DNA viruses an d retroviruses un der stan dard in vitro
con dition s (cf. ref.10). Also th eir cytostatic activity un der in vitro con dition s
was exam in ed in L-1210, P388 an d CEM cell lin es. No activity was en coun -
tered in th ese system s with an y of th e com poun ds tested26,27
.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

2,4-Diaminopyrimidine Acyclic Phosphonates
251
EXPERIMENTAL
Un less oth erwise stated, th e solven ts were evaporated at 40 °C/2 kPa an d th e com poun ds
were dried overn igh t at 2 kPa over P2O5. Meltin g poin ts were determ in ed on a Kofler block
an d are un corrected. TLC was perform ed on Silufol UV254 plates (Kavalier Votice, Czech
Republic) in system S1, ch loroform –eth an ol (4 : 1). Paper electroph oresis was perform ed on
a Wh atm an No. 3 MM paper at 40 V/cm for 1 h in 0.05 M trieth ylam m on ium h ydrogen -
carbon ate (TEAB) at pH 7.5; th e electroph oretical m obilities were referen ced to uridin e
3′-ph osph ate.
NMR spectra were recorded on an FT NMR spectrom eter Varian UNITY-500. 1H NMR spec-
tra (at 500 MHz) were taken in D2O (with 3-(trim eth ylsilyl)-1-propan esulfon ic acid, sodium
salt as in tern al referen ce) an d CD3SOCD3 solution s (referen ced to th e sign al of solven t at δ
2.50). Ch em ical sh ifts an d couplin g con stan ts were obtain ed by th e first order an alysis of
spectra. Proton 1D NOE spectra were obtain ed by selective irradiation durin g 5 s before data
acquisition an d subtraction of two spectra taken with irradiation frequen cy placed on - an d
off-reson an ce. 13C NMR spectra (at 125.7 MHz) were m easured in D2O (referen ced to TSP)
an d/or CD3SOCD3 (referen ced to th e solven t sign al at δ 39.7) usin g eith er “attach ed proton
test” or stan dard broadban d proton decouplin g pulse sequen ce.
Mass spectra were m easured on a ZAB-EQ (VG An alytical) spectrom eter usin g FAB (ion iza-
tion by Xe, acceleratin g voltage 8 kV, glycerol m atrix).
UV absorption spectra were m easured on a Beckm an DU-65 spectrom eter in aqueous solu-
tion s.
Materials
Brom otrim eth ylsilan e, 2-am in opyrim idin e, 2,4-diam in opyrim idin e, 2,4,6-triam inopyrim idine,
2,4-diamino-6-hydroxypyrimidine and 2,3-diaminopyridin e were purch ased from Sigm a–Aldrich
(Prague, Czech Republic), 4,6-diam in opyrim idin e from Lach em a (Brn o, Czech Republic).
Com poun d 3 was syn th esized accordin g to th e literature10. Dim eth ylform am ide din eopen tyl
acetal was prepared accordin g to ref.15. Dim eth ylform am ide was distilled from ph osph orus
pen toxide in vacuo an d stored over m olecular sieves.
Purification of th e Ph osph on ates by Dowex 1 X 2 Colum n Ch rom atograph y.
Gen eral Procedure
Un less stated oth erwise, 100 m l colum n s of Dowex 1 X 2 (100–200 m esh , acetate form ,
prewash ed with water) were used. Th e sam ple was dissolved in water (20–25 m l), alkalized
with con cen trated aqueous am m on ia to pH 9–9.5 an d applied on th e colum n . Elution with
water (3 m l/m in ) was con tin ued till th e UV absorption (254 n m ) of th e eluate dropped to
th e in itial value. Th e colum n was th en eluted eith er with water to afford th e (zwitterion ic)
product with reten tion , with 0.02 M acetic acid (3 m l/m in , fraction s 30 m l), or with a lin ear
gradien t of acetic acid (0–0.5 m ol/l, 1 l each ) as in dicated.
2-[(Dim eth ylam in om eth ylen e)am in o]pyrim idin e (12)
2-Am in opyrim idin e (11; 10.5 g, 0.1 m ol) in dim eth ylform am ide dim eth yl acetal (80 m l) was
stirred at 100 °C till dissolution (15 m in ) an d left to stan d overn igh t at room tem perature.
Th e m ixture was taken down to dryn ess in vacuo, codistilled with toluen e (2 × 25 m l) an d
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Holý, Buděšínský, Podlaha, Císařová:
th e residue was crystallized from toluen e (eth er added to turbidity). Yield 13.6 g (90.5%) of
com poun d 12, m .p. 105 °C. For C7H10N4 (150.2) calculated: 55.98% C, 6.71% H, 37.31% N;
foun d: 56.13% C, 6.75% H, 37.10% N. 1H NMR (CD3SOCD3): 3.02 d, 3 H, J(CH3,CH) = 0.6
(CH3); 3.11 s, 3 H (CH3); 6.92 t, 1 H, J(5,4) = J(5,6) = 4.7 (H-5); 8.47 d, 2 H, J(4,5) = 4.7 (H-4
an d H-6); 8.60 bs, 1 H (=CH-). 13C NMR (CD3SOCD3): 34.55 an d 40.45 (2 × CH3); 114.80
(C-5); 158.21 (C-4 an d C-6); 158.25 (=CH-); 166.69 (C-2).
4-Am in o-2-[(dim eth ylam in om eth ylen e)am in o]pyrim idin e (6)
2,4-Diam in opyrim idin e (2; 2.75 g, 25 m m ol) an d dim eth ylform am ide din eopen tyl acetal (15
m l) were stirred in DMF (30 m l) for 16 h at 80 °C an d taken down to dryn ess at 40 °C/14 Pa.
Th e residue was treated with eth an ol (25 m l) an d a few pieces of dry ice for 20 m in an d
evaporated. Th e residue was stirred with eth an ol (25 m l), eth er (50 m l) added, an d th e
product was filtered, wash ed with eth er an d dried in vacuo. Yield 4.6 g (83.5%), m .p.
197–198 °C. For C10H16N6 (220.3) calculated: 54.53% C, 7.32% H, 38.15% N; foun d: 54.88% C,
7.55% H, 38.40% N. Mass spectrum : MH+ 221.1. 1H NMR (CD3SOCD3): 2.97 bs, 3 H (CH3);
3.07 s, 3 H (CH3); 6.02 d, 1 H, J(5,6) = 5.5 (H-5); 6.70 bs, 2 H (NH2); 7.88 d, 1 H, J(6,5) = 5.5
(H-6); 8.48 bs, 1 H (N=CH-N). 13C NMR (CD3SOCD3): 34.47 an d 40.48 (2 × CH3); 104.06
(C-5); 156.18 (C-6); 157.85 (N=CH-N); 163.65 (C-4); 167.99 (C-2).
Hydrogen {[2-(2,4-Diam in opyrim idin -1-io)eth oxy]m eth yl}ph osph on ate (5)
Method A. Th e m ixture of 2,4-diam in opyrim idin e (2; 5.0 g, 45.4 m m ol) an d com poun d 3
(20 m l) was stirred in Ar atm osph ere for 16 h at 80 °C. Eth er (150 m l) was slowly added un -
der stirrin g an d th e solven t decan ted. Th e residue was stirred in eth er (100 m l), filtered,
wash ed with eth er an d dried in vacuo for 20 m in at am bien t tem perature. Th e h ygroscopic
residue (hom ogeneous by paper electrophoresis, EUp –0.48) was dissolved in acetonitrile (100 m l),
BrSiMe3 (10 m l) was added an d th e m ixture was left to stan d overn igh t at room tem pera-
ture. Th e solven ts were stripped down in vacuo, th e residue codistilled with aceton itrile
(2 × 25 m l) an d water (50 m l) was added to th e residue. After 10 m in , th e solution was
sligh tly alkalized with con cen trated aqueous am m on ia an d evaporated in vacuo. Th e residue
was applied in con cen trated aqueous solution on to a colum n (150 m l) of Dowex 50 X 8
(H+-form ) an d th e colum n eluted with water (3 m l/m in ); th e elution was followed by con -
tin uous m easurem en t of UV absorption of th e eluate at 254 n m . After rem oval of th e n eu-
tral UV-absorbin g fraction th e colum n was eluted with 2.5% aqueous am m on ia an d th e
UV-absorbin g fraction collected. It was taken down in vacuo, redissolved in water (20 m l),
brough t to pH 9–10 by con cen trated aqueous am m on ia an d applied on a colum n (100 m l)
Dowex 1 X 2 (acetate form ) th orougly prewash ed with water. Th e colum n was processed as
described in Gen eral Procedure an d th e product was eluted with 0.02 M acetic acid. Th e
UV-absorbin g eluate was taken down in vacuo, th e residue was codistilled with water (2 × 25 m l)
an d th e residue crystallized from water. Yield 5.10 g (42.4%) of com poun d 5, m .p. >280 °C
(water). EUp 0.63. For C7H13N4O4P·H2O (266.2) calculated: 31.59% C, 5.68% H, 21.05% N,
11.64% P; foun d: 31.73% C, 5.76% H, 21.27% N, 11.88% P. UV [λm ax (εm ax)] (pH 2, pH 7):
270 (6 200). Mass spectrum : MH+ 249.1. 1H NMR (CD3SOCD3): 3.37 d, 2 H, J(CH2,P) = 6.4
(CH2P); 3.77 t, 2 H, J(2′,1′) = 6.5 (H-2′); 4.20 t, 2 H, J(1′,2′) = 6.5 (H-1′); 6.02 d, 1 H, J(5,6) =
7.2 (H-5); 7.83 d, 1 H, J(6,5) = 7.2 (H-6); 7.90 bs, 1 H (NH); 7.98 bs, 1 H (NH). 1H NMR
(D2O): 3.65 d, 2 H, J(CH2,P) = 8.7 (CH2P); 3.93 t, 2 H, J(2′,1′) = 5.0 (H-2′); 4.13 t, 2 H, J(1′,2′) =
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

2,4-Diaminopyrimidine Acyclic Phosphonates
253
5.0 (H-1′); 6.23 d, 1 H, J(5,6) = 7.3 (H-5); 7.67 d, 1 H, J(6,5) = 7.3 (H-6). 13C NMR (D2O):
54.93 (C-1′); 70.57 d, J(CH2,P) = 156.6 (CH2P); 72.42 d, J(2′,P) = 11.9 (C-2′); 102.08 (C-5);
149.48 (C-6); 158.55 (C-4); 167.12 (C-2).
Method B. Com poun d 6 (3.3 g, 15 m m ol) was stirred with com poun d 3 (20 m l) for 16 h at
100 °C an d precipitated with eth er (100 m l). Th e product was filtered, wash ed with eth er
an d dried in vacuo. Aceton itrile (70 m l) an d BrSiMe3 (10 m l) were added an d th e m ixture
was left to stan d overn igh t at room tem perature. Th e volatiles were taken down in vacuo,
th e residue was dissolved in water (50 m l) an d th e solution was left to stan d for 1 h at am bi-
en t tem perature. Th e m ixture was alkalized with con cen trated aqueous am m on ia an d evapo-
rated. Th e ch rom atograph y on Dowex 50 was perform ed as described in m eth od A (th e
am m on ia eluate was left to stan d overn igh t prior to evaporation ). Furth er work-up an d
ch rom atograph y on Dowex 1 colum n was perform ed as described in m eth od A. Yield 1.9 g
(47.3%) of com poun d 5, iden tical with th e m aterial prepared by m eth od A.
Hydrogen {2-[(2,6-Diam in opyrim idin -1-io]eth oxy]m eth yl}ph osph on ate (9)
Com poun d 9 was prepared from 2.5 g (19.8 m m ol) 2,6-diam in opyrim idin e (7) as described
for com poun d 5 (m eth od A). Yield 1.70 g (32.3%), m .p. 187 °C (80% aqueous eth an ol).
EUp 0.60. For C7H13N4O4P·H2O (266.2) calculated: 31.59% C, 5.68% H, 21.05% N, 11.64% P;
foun d: 31.80% C, 5.67% H, 21.23% N, 11.54% P. UV [λm ax (εm ax)] (pH 2, pH 7): 267
(10 200). 1H NMR (CD3SOCD3): 3.38 d, 2 H, J(CH2,P) = 6.3 (CH2P); 3.78 t, 2 H, J(2′,1′) = 6.7
(H-2′); 4.31 t, 2 H, J(1′,2′) = 6.7 (H-1′); 5.60 s, 1 H (H-5); 7.46 bs, 1 H an d 7.64 bs, 1 H (NH2);
8.32 s, 1 H (H-2); 9.00 vbs, 2 H (NH2). 1H NMR (D2O): 3.66 d, 2 H, J(CH2,P) = 8.6 (CH2P);
3.93 t, 2 H, J(2′,1′) = 5.0 (H-2′); 4.28 t, 2 H, J(1′,2′) = 5.0 (H-1′); 5.86 s, 1 H (H-5); 8.23 s, 1 H
(H-2). 13C NMR (D2O): 52.60 (C-1′); 70.61 d, J(CH2,P) = 156.7 (CH2P); 72.28 d, J(2′,P) = 11.7
(C-2′); 85.86 (C-5); 155.70 (C-2); 158.02 (C-6); 165.31 (C-4).
Hydrogen {2-[(2,4,6-Triam in opyrim idin -1-io)eth oxy]m eth yl}ph osph on ate (10)
Th e m ixture of 2,4,6-triam in opyrim idin e (8; 3.0 g, 24 m m ol), com poun d 3 (7 m l) an d DMF
(7 m l) was stirred for 14 h at 100 °C, taken down in vacuo and codistilled with DMF (2 × 20 m l).
Th e residue was treated with aceton itrile (60 m l) an d BrSiMe3 (7 m l) overn igh t an d worked
up as described for com poun d 5 (m eth od A). Elution with 0.02 M acetic acid afforded 1.85 g
(29%) of com poun d 10, m .p. 260 °C (decom p.) (80% aqueous eth an ol–eth er). EUp 0.48. For
C7H13N5O4P (263.2) calculated: 31.95% C, 5.36% H, 26.61% N, 11.77% P; foun d: 32.20% C,
5.62% H, 26.81% N, 11.85% P. UV [λm ax (εm ax)] (pH 2, pH 7): 275 (15 500). Mass spectrum :
MH+ 264.0. 1H NMR (CD3SOCD3): 3.39 d, 2 H, J(CH2,P) = 6.4 (CH2P); 3.76 t, 2 H, J(1′,2′) =
6.2 (H-1′); 4.18 t, 2 H, J(2′,1′) = 6.2 (H-2′); 5.15 s, 1 H (H-5); 6.90 vbs, 2 H (NH2); 7.90 bs, 4 H
(2 × NH2). 1H NMR (D2O): 3.62 d, 2 H, J(CH2,P) = 8.5 (CH2P); 3.93 t, 2 H, J(2′,1′) = 4.8
(H-2′); 4.16 t, 2 H, J(1′,2′) = 4.8 (H-1′); 5.41 s, 1 H (H-5).13C NMR (D2O): 49.29 (C-1′); 70.95 d,
J(CH2,P) = 155.3 (CH2P); 73.01 d, J(2′,P) = 11.7 (C-2′); 79.53 (C-5); 158.66 an d 159.34 (C-2
an d C-6); 165.84 (C-4).
2-{[2-(Ph osph on om eth oxy)eth yl]am in o}pyrim idin e (13)
a) Com poun d 12 (3.0 g, 20 m m ol) an d com poun d 3 (15 m l) were stirred for 2 h at 100 °C
an d th e m ixture was worked up as described for com poun d
5 (m eth od A). After
deion ization , th e product was eluted from Dowex 1 colum n by 0.5 M acetic acid. Yield 2.15 g
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254
Holý, Buděšínský, Podlaha, Císařová:
(42.8%) of com poun d 13, m .p. 199 °C (eth an ol–eth er). EUp 1.00. For C7H12N3O4P·H2O
(251.2) calculated: 33.47% C, 5.62% H, 16.73% N, 12.33% P; foun d: 33.60% C, 5.56% H,
17.01% N, 12.22% P. UV [λm ax (εm ax)] (pH 2, pH 7): 232, 310 (3 200). Mass spectrum : MH+
234.0. 1H NMR (CD3SOCD3): 3.43 q, 2 H, J(1′,2′) = J(1′,NH) = 6.1 (H-1′); 3.56 d, 2 H,
J(CH2,P) = 8.7 (CH2P); 3.61 t, 2 H, J(2′,1′) = 6.1 (H-2′); 6.57 t, 1 H, J(5,4) = J(5,6) = 4.7 (H-5);
7.02 bt, 1 H, J(1′,NH) = 6.1 (NH); 8.26 d, 2 H, J(4,5) = J(6,5) = 4.7 (H-4, H-6). 1H NMR (D2O):
3.68 d, 2 H, J(CH2,P) = 8.7 (CH2P); 3.71 m , 2 H (H-1′); 3.82 m , 2 H (H-2′); 6.99 t, 1 H, J(5,4) =
J(5,6) = 5.4 (H-5); 8.52 d, 2 H, J(4,5) = 5.4 (H-4, H-6). 13C NMR (D2O): 43.64 (C-1′); 69.88 d,
J(CH2,P) = 156.8 (CH2P); 73.92 d, J(2′,P) = 11.2 (C-2′); 113.58 (C-5); 161.12 (C-4 an d C-6);
162.95 (C-2).
b) 2-Am in opyrim idin e (11; 3.0 g, 31.6 m m ol) was treated for 24 h at 100 °C with com -
poun d 3 (15 m l). Th e stan dard work-up gave 1.6 g (20.3%) of com poun d 13 wh ich was
eluted with 0.35–0.40 M acetic acid (lin ear gradien t, 1 l water, 1 l 0.5 M acetic acid). Th e
product was iden tical by NMR an d UV spectra with th e m aterial prepared by previous proce-
dure.
2-Am in o-3-{[2-(ph osph on om eth oxy)eth yl]am in o}pyridin e (15) an d
Hydrogen {2-[(2,3-Diam in opyridin -1-io)eth oxy]m eth yl}ph osph on ate (16)
Th e m ixture of 2,3-diam in opyridin e (14; 5.0 g, 45.9 m m ol) an d com poun d 3 (25 m l) was
stirred for 24 h at 80 °C an d precipitated with eth er as described for com poun d 5. Th e pre-
cipitate was ch rom atograph ed on silica gel colum n (300 m l) in ch loroform to give two frac-
tion s (RF 0.37 an d 0.16, respectively, S1) as red ligh t-sen sitive oils. Th e products were dried
in vacuo over P2O5 an d treated each with BrSiMe3 (5 m l) in aceton itrile (50 m l) overn igh t.
Th e reaction m ixtures were worked up as described for com poun d 5 (m eth od A). Purification
of th e desalted m ixture from th e diester RF 0.37 on Dowex 1 colum n (elution with 0.02 M ace-
tic acid) gave 1.8 g (15.9%) of th e N3-isom er 15, EUp 0.82 (blue fluorescen ce), m .p. 259 °C
(water). For C8H14N3O4P (247.2) calculated: 38.87% C, 5.71% H, 17.00% N, 12.53% P;
foun d: 38.80% C, 5.77% H, 16.83% N, 12.58% P. Mass spectrum : MH+ 248.1. 1H NMR
(CD3SOCD3): 3.12 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.59 d, 2 H, J(CH2,P) = 7.0 (CH2P); 3.85 t, 2 H,
J(1′,2′) = 5.0 (H-1′); 6.63 dd, 1 H, J(5,4) ≈ 7.5, J(5,6) ≈ 6.0 (H-5); 6.68 dd, 1 H, J(4,5) ≈ 7.5,
J(4,6) ≈ 1.0 (H-4); 7.14 dd, 1 H, J(6,5) ≈ 6.0, J(6,4) ≈ 1.0 (H-6); 8.42 bs, 2 H (NH2). 1H NMR
(D2O): 3.67 d, 2 H, J(CH2,P) = 10.6 (CH2P); 3.21 m , 2 H (H-1′); 3.86 m , 2 H (H-2′); 6.82 dd,
1 H, J(5,4) = 8.0, J(5,6) = 6.3 (H-5); 6.98 dd, 1 H, J(4,5) = 8.0, J(4,6) = 1.3 (H-4); 7.27 dd, 1 H,
J(6,5) = 6.3, J(6,4) = 1.3 (H-6). 13C NMR (D2O): 45.96 (C-1′); 70.45 d, J(CH2,P) = 151.0
(CH2P); 73.20 d, J(2′,P) = 12.7 (C-2′); 116.73, 121.51 an d 126.00 (C-4, C-5 an d C-6); 135.90
an d 148.89 (C-2 an d C-3).
Th e secon d fraction from th e silica gel ch rom atograph y (RF 0.16) gave after th e purifica-
tion by Dowex 1 ch rom atograph y (elution with 0.02 M acetic acid) product wh ich was re-
peatedly coevaporated with water an d treated with aceton e an d eth er to afford am orph ous
quatern ary com poun d 16 (1.9 g, 16.8%), sligh tly con tam in ated with th e N3-isom er 15.
Rech rom atograph y on Dowex 1 X 2 un der th e sam e con dition s rem oved th is im purity (EUp
0.43). For C8H14N3O4P (247.2) calculated: 38.87% C, 5.71% H, 17.00% N, 12.53% P; foun d:
39.17% C, 5.48% H, 16.78% N, 12.86% P. Mass spectrum : MH+ 248.1. 1H NMR (CD3SOCD3):
3.44 d, 2 H, J(CH2,P) = 7.0 (CH2P); 3.90 t, 2 H, J(2′,1′) = 6.0 (H-2′); 4.48 t, 2 H, J(1′,2′) = 6.0
(H-1′); 6.04 bs, 2 H (NH2); 6.64 dd, 1 H, J(5,4) = 7.7, J(5,6) = 6.6 (H-5); 6.88 dd, 1 H, J(4,5) =
7.7, J(4,6) = 1.3 (H-4); 7.32 dd, 1 H, J(6,5) = 6.6, J(6,4) = 1.3 (H-6); 8.96 bs (NH2). 1H NMR
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

2,4-Diaminopyrimidine Acyclic Phosphonates
255
(D2O): 3.63 d, 2 H, J(CH2,P) = 8.7 (CH2P); 4.04 m , 2 H (H-1′); 4.46 m , 2 H (H-2′); 6.83 dd,
1 H, J(5,4) = 7.8, J(5,6) = 6.7 (H-5); 7.28 dd, 1 H, J(4,5) = 7.8, J(4,6) = 1.4 (H-4); 7.46 dd, 1 H,
J(6,5) = 6.7, J(6,4) = 1.4 (H-6). 13C NMR (D2O): 57.04 (C-1′); 70.59 d, J(CH2,P) = 156.3
(CH2P); 72.54 d, J(2′,P) = 11.7 (C-2′); 116.96, 126.74 an d 133.44 (C-4, C-5 an d C-6); 135.87
an d 150.11 (C-2 an d C-3).
X-Ray An alysis of Com poun d 5
Com poun d 5·H2O, C8H15N4O4P, M = 266.20, triclin ic, space group P1 (No. 2), a = 7.476(1),
b = 7.562(1), c = 11.217(1) Å, α = 90.86(1), β = 92.36(1), γ = 117.19(1)°, V = 563.2(1) Å3,
F(000) = 280, Dc = 1.570 g/cm 3 for Z = 2. A colourless plate of th e dim en sion s 0.6 × 0.4 ×
0.15 m m (from h ot water) was m easured on a CAD4 diffractom eter at 150.0(1) K (MoKα ra-
diation , λ = 0.71073 Å). From a total of 2 480 in depen den t reflection s m easured in th e ran ge
h = –9 to 8, k = 0 to 9, l = –14 to 14, 2 249 were regarded as observed accordin g to th e I >
2σ(I) criterion . Th ree stan dard reflection s m on itored every 1 h sh owed 1.9% total decay. Ab-
sorption was n eglected (µ = 0.263 m m –1). Th e structure was solved by direct m eth ods
(SIR97, ref.30) an d refin ed by full-m atrix least squares based on F2 (SHELXL93, ref.31) with
h ydrogen atom s isotropic, all oth er atom s an isotropic. Th e refin em en t con verged to R =
0.0290, wR = 0.0792, GOF = 1.073 for 214 param eters. Th e fin al differen ce m ap displayed n o
peaks of ch em ical sign ifican ce. Stan dard SHELX output files are available from th e auth or
(J. P.) on request. Crystallograph ic data for th e structure 5 reported in th is paper h ave been
deposited with th e Cam bridge Crystallograph ic Data Cen tre as supplem en tary publication
n um ber CCDC-112393. Copies of th e data can be obtain ed free of ch arge on application to
CCDC, e-m ail: deposit@ccdc.ca.ac.uk.
This work was supported by grants of the Grant Agency of the Czech Republic (No. 203/96/K001,
No. 203/96/0497 and No. 203/97/0242), by Gilead Sciences (Foster City, CA, U.S.A.) and by the
General Health Insurance Agency of the Czech Republic. The cytostatic activity was examined by Dr I.
Votruba of This Institute, the antiviral effect by Drs R. Snoeck, G. Andrei, J. Balzarini and E. De
Clercq at the Rega Institute, Katholic University, Leuven (Belgium). The UV spectra were measured by
Dr E. Tloušová, mass spectra by Dr J. Kohoutová, both from This Institute. The excellent technical
assistance of Ms B. Nováková is gratefully acknowledged.
REFERENCES AND NOTES
1. De Clercq E., Sakuma T., Baba M., Pauwels R., Balzarini J., Rosenberg I., Holý A.: Antiviral
Res. 1987, 8, 261.
2. Balzarini J., Perno C. F., Schols D., De Clercq E.: Biochem. Biophys. Res. Commun. 1991,
178, 329.
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