Pyrimidine acyclic nucleotide analogues with aromatic substituents in C-5 position

Article abstract of DOI:10.1135/cccc20070927

NH₂-protected 5-phenylcytoslne and its derivatives 2a-2d were treated with (2S)-2-[(trityloxy)methyl]oxirane (3) followed by etheriflcation with diisopropyl [(tosyloxy)methyl]phosphonate (5) in the presence of sodium hydride. The intermediary phosphonate esters 6 were debenzoylated and subsequently transformed to free phosphonic acids, i.e. (S)-1-[3-hydroxy-2- (phosphonomethoxy)propyl]-5-phenylcytosine (5-phenyl-HPMPC) derivatives (8a-8d) by the action of bromotrimethylsilane and subsequent hydrolysis. Deamination of these compounds with 3-methylbutyl nitrite afforded corresponding (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-phenyluracil (5-phenyl-HPMPU) derivatives (9a-9d). R-Enantiomers 14 and 15 were prepared analogously starting from (2R)-2-[(trityloxy)methyl]oxirane. 5-Benzyl-, 5-[(1-naphthyl)methyl]- and 5-[(2-naphthyl)methyl]HPMPU (24a-24c) and -HPMPC (25a-25c) were synthesized from appropriate 5-arylmethyl-4-methoxypyrimidin-2(lH)-ones similarly as described for 5-phenyl derivatives. Antiviral activity was found for (S)-l-[3-hydroxy-2- (phosphonomethoxy)propyl]-5-phenyluracil (9a) (HSV-1 and HSV-2) and (R)-1-[3-hydroxy2-(phosphonomethoxy)propyl]-5-phenylcytosine (14) (cytomegalovirus and varicella-zoster virus), both tested in cell cultures. Some of the 5-phenyluracil derivatives possessed inhibitory activity against thymidine Phosphorylase from SD-lymphoma.

Full text of DOI:10.1135/cccc20070927

Pyrimidine Acyclic Nucleotide Analogues
927
PYRIMIDINE ACYCLIC NUCLEOTIDE ANALOGUES WITH AROMATIC
SUBSTITUENTS IN C-5 POSITION
1,
2
3
Marcela KREČMEROVÁ *, Antonín HOLÝ and Milena MASOJÍDKOVÁ
Gilead Sciences & IOCB Research Centre, Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6,
1
2
3
Czech Republic; e-mail: marcela@uochb.cas.cz, holy@uochb.cas.cz, masojid@uochb.cas.cz
Received February 27, 2007
Accepted March 27, 2007
NH2-protected 5-ph en ylcytosin e an d its derivatives 2a–2d were treated with (2S)-2-[(trityl-
oxy)methyl]oxirane (3) followed by etherification with diisopropyl [(tosyloxy)methyl]ph osph on ate
(5) in th e presen ce of sodium h ydride. Th e in term ediary p h osph on ate esters 6 were
debenzoylated and subsequently transform ed to free phosphonic acids, i.e. (S)-1-[3-h ydroxy-
2-(ph osph on om eth oxy)propyl]-5-ph en ylcytosin e (5-ph en yl-HPMPC) derivatives (8a–8d ) by
th e action of brom otrim eth ylsilan e an d subsequen t h ydrolysis. Deam in ation of th ese com -
poun ds with 3-m eth ylbutyl n itrite afforded correspon din g (S)-1-[3-h ydroxy-2-(ph osph on o-
m eth oxy)propyl]-5-ph en yluracil (5-ph en yl-HPMPU) derivatives (9a–9d ). R-En an tiom ers 14
an d 15 were prepared an alogously startin g from (2R)-2-[(trityloxy)m ethyl]oxirane. 5-Benzyl-,
5-[(1-naphthyl)m ethyl]- and 5-[(2-naphthyl)m ethyl]HPMPU (24a–24c) an d -HPMPC (25a–25c)
were syn th esized from appropriate 5-arylm eth yl-4-m eth oxypyrim idin -2(1H)-on es sim ilarly as
described for 5-ph en yl derivatives. An tiviral activity was foun d for (S)-1-[3-h ydroxy-2-(ph os-
ph on om eth oxy)propyl]-5-ph en yluracil (9a) (HSV-1 an d HSV-2) an d (R)-1-[3-h ydroxy-
2-(ph osph on om eth oxy)propyl]-5-ph en ylcytosin e (14) (cytom egalovirus an d varicella-zoster
virus), both tested in cell cultures. Som e of th e 5-ph en yluracil derivatives possessed in h ibi-
tory activity again st th ym idin e ph osph orylase from SD-lym ph om a.
Keyw ord s: Acyclic n ucleotide an alogues; Acyclic n ucleoside ph osph on ates; Pyrim idin es;
An tivirals; Th ym idin e ph osph orylase in h ibitors; Cidofovir; Cytosin e.
Acyclic n ucleotide an alogues (ANPs) are com poun ds of con siderable im por-
tan ce due to th eir broad spectrum of an tiviral activities1. An im portan t
group of th ese com poun ds are (S)-[3-h ydroxy-2-(ph osph on om eth oxy)propyl]
(HPMP) derivatives of purin e an d pyrim idin e bases: aden in e an d
diam in opurin e (HPMPA, HPMPDAP) an d, in particular, th e cytosin e deriva-
tive (HPMPC, cidofovir2), an active con stituen t of Vistide™. Th is drug was
approved for th e treatm en t of cytom egalovirus retin itis in AIDS patien ts3.
At th e presen t tim e, it is im portan t particularly for th e treatm en t of severe
cases of (m align izin g) papillom atoses (an ogen ital, laryn geal)4, progressive
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 7, pp. 927–951
© 2007 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20070927

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Krečmerová, Holý, Masojídková:
m ultifocal leukoen ceph alopath y (caused by JC virus)5 aden ovirus in fec-
tion s6 an d som e rath er obscure severe in fection s caused by poxviruses
(vaccinia7, orf, molluscum contagiosum). The attractivity of cidofovir is drama-
tically en h an ced by its suprem e activity again st sm allpox virus8 an d related
m on keypox virus9; both of th ese h igh ly in fectious viruses can be easily cul-
tivated an d purposely used in a bioterrorist attack.
Th ese facts support furth er in vestigation of variously m odified cidofovir
derivatives. An in ten sive atten tion is paid to th e developm en t of n eutral
ester prodrugs10 an d, at presen t, we focus our atten tion also to preparation
of an alogues m odified in th e cytosin e m oiety (an alogues substituted in po-
sition s C-5 an d N4, refs11,12).
In th is paper, we describe preparation of HPMPC substituted in position 5
with various arom atic system s as com poun ds for an tiviral screen in g. Sim ul-
tan eously, we prepared also th eir uracil coun terparts (HPMPU derivatives).
ANPs derived from uracil an d th ym in e are usually an tivirally in active1;
th eir in vestigation is directed to study of oth er biological properties, in par-
ticular in h ibitory effects on th ym idin e ph osph orylase13, an en zym e catalyz-
in g ph osph orolysis of th ym idin e to th ym in e an d 2-deoxy-D-ribose
1-ph osph ate.
As to n ucleobases th em selves, som e atten tion in th e recen t years, was
paid to substituted 5-ph en ylcytosin es an d in term ediates form ed in th eir
preparation as an tiviral agen ts: 2-[(carboxym eth yl)sulfan yl-5-(4-ch loro-
ph en yl)pyrim idin ]-4-am in e is active in vitro again st vaccin ia virus14, som e
activity was also foun d for its o- an d m-isom er. 5-Ph en ylcytosin e an d its de-
rivatives (p-am in o, p-ch loro an d m-ch loro) were studied as in h ibitors of
WEE (Western equin e en ceph alom yelitis) virus. In in vivo experim en ts all
th ese com poun d were in active, probably due to th eir in solubility in wa-
ter14. Nucleosides prepared from 5-ph en ylcytosin e an d 5-ph en yluracil are
also in m ost cases an tivirally in active15,16. 5-Ben zyluracil n ucleosides an d
th eir acyclic an alogues were foun d to be in h ibitors of uridin e ph osph orylase,
an en zym e respon sible for un desirable ph osph orolysis of pyrim idin e
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Pyrimidine Acyclic Nucleotide Analogues
929
n ucleoside an alogues used in can cer ch em oth erapy17,18. Pyrim idin e bases
an d th eir n ucleoside an d/or n ucleotide an alogues substituted in position 5
with bulky n aph th ylm eth yl group h ave n ot been described yet.
RESULTS AND DISCUSSION
Our approach to 5-aryl HPMPC an d HPMPU an alogues is based on utiliza-
tion of appropriately substituted 5-phenylcytosines (1). These bases are accessible
from corresponding phenylacetonitriles by the process involving form ylation,
tran sform ation of form yl derivatives to en ol eth ers an d th eir cyclization with
th iourea to 5-ph en yl-2-th iocytosin es19,20. Th eir reaction with ch loroacetic
acid followed by h ydrolysis with h ydroch loric acid affords correspon din g
5-ph en ylcytosin es. 5-(4-Nitroph en yl)cytosin e (1b) was prepared by n itra-
tion of 5-ph en ylcytosin e (1a)20.
Th e am in o groups in 5-ph en ylcytosin es 1 were protected by ben zoylation
with ben zoic an h ydride in DMF (Sch em e 1). Th is step is useful due to a very
(i) Ben zoic an h ydride, DMF, r.t.; (ii) DMF, Cs2CO3, 100 °C; (iii) NaH, DMF, r.t.; (iv)
NH3/CH3OH, 4 °C; (v) (CH3)3SiBr, CH3CN, r.t; (vi) 3-m eth ylbutyl n itrite, 80% acetic acid,
r.t.
SCHEME 1
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Krečmerová, Holý, Masojídková:
pure solubility of free ph en ylcytosin es. Th e followin g step, base-catalyzed
reaction with (2S)-2-[(trityloxy)m eth yl]oxiran e (3) th us proceeds with h igh
yields in con trast to reaction s perform ed with un protected cytosin e com po-
n en ts wh ich are n ot quan titative. Th e n ucleoph ilic open in g of an oxiran e
rin g proceeded regiospecifically an d afforded in all cases N1-substituted
intermediates 4 which gave, on treatment with diisopropyl [(tosyloxy)methyl]-
ph osph on ate (5) in th e presen ce of excess sodium h ydride in dim eth yl-
form am ide, fully protected ph osph on ates 6. Th eir partial deprotection to
ph osph on ates 7 with free am in o group, followed by sim ultan eous rem oval
of trityl an d diisopropyl ester groups with brom otrim eth ylsilan e an d subse-
quen t h ydrolysis afforded th e free ph osph on ic acids: 5-ph en yl-HPMPC (8a)
an d its substituted derivatives (8b–8d ) as fin al products. Th ese com poun ds
can be also tran sform ed to th eir uracil coun terparts: (S)-1-[3-h ydroxy-
2-(ph osph on om eth oxy)propyl]-5-ph en yluracil (5-ph en yl-HPMPU, 9a) an d
its 4-n itro, 4-ch loro an d 2-ch loro derivatives (9b–9d ); deam in ation reaction
was perform ed with 3-m eth ylbutyl n itrite (isoam yl n itrite) in acetic acid.
Th e an ti-h erpes virus activity foun d for (S)-1-[3-h ydroxy-2-(ph osph on o-
m eth oxy)propyl]-5-ph en yluracil (9a) substan tiates our effort to prepare for
com parison also th e correspon din g (R)-en an tiom er (15) an d 5-ph en yluracil
n ucleotide an alogues with m odified aliph atic m oiety, in particular
5-ph en yl-1-[(R)-2-(ph osph on om eth oxy)propyl]uracil, a com poun d belon g-
in g to th e PMP-series1. Th e (R)-en an tiom er 15 was prepared from
(2R)-2-[(trityloxy)m eth yl]oxiran e (10) an d N4-ben zoyl-5-ph en ylcytosin e
(2a) via in term ediates 11–14 by th e sam e procedure as described for (S)-en -
an tiom er 9a.
ANPs of th e (R)-2-(ph osph on om eth oxy)propyl series are usually prepared
by condensation of an appropriate base com ponent with (R)-2-[(diisopropoxy-
ph osph oryl)m eth oxy]propyl tosylate (16) un der basic con dition s21. Th e re-
action of un protected 5-ph en ylcytosin e with 16 in DMF was n ot successful,
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Pyrimidine Acyclic Nucleotide Analogues
931
probably due to very low solubility of 5-ph en ylcytosin e. Th e h eatin g of
N4-ben zoyl-5-ph en ylcytosin e with a large excess of 16 in th e presen ce of ce-
sium carbon ate gave th e desired com poun d 17 accom pan ied by several
oth er products, partly deprotected ph osph on ates in cludin g m on oisopropyl
esters (as determ in ed by paper electroph oresis). Th is m ixture of products af-
forded, after treatm en t with m eth an olic am m on ia an d deprotection of ester
groups with brom otrim eth ylsilan e, 5-ph en yl-1-[(R)-2-(ph osph on om eth oxy)-
propyl]cytosin e (18) as a sin gle reaction product. Its deam in ation with
isoam yl nitrite afforded 5-ph en yl-1-[(R)-2-(ph osph on om eth oxy)propyl]uracil
(19) (Sch em e 2).
(i) DMF, Cs2CO3, 120 °C; (ii) NH3/CH3OH,
3-m eth ylbutyl n itrite, 80% acetic acid, r.t.
SCHEME 2
4 °C; (iii) (CH3)3SiBr, CH3CN, r.t.; (iv)
For syn th eses of 5-ben zyl- an d both 5-(n aph th ylm eth yl)-HPMPU an d
-HPMPC derivatives, we selected an approach , wh ich is in prin ciple an alo-
gous to preparation of 5-ph en yl derivatives: n ucleoph ilic open in g of
(2S)-2-[(trityloxy)m eth yl]oxiran e (3) with appropriate base com pon en t, fol-
lowed by th e treatm en t with diisopropyl [(tosyloxy)m eth yl]ph osph on ate
(5) to give fully protected ph osph on ate esters 23 (Sch em e 3). In th is case
5-substituted uracils were used as startin g bases. Th e preparation of
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932
Krečmerová, Holý, Masojídková:
5-ben zyluracil (20a), 5-(1-n aph th ylm eth yl)uracil (20b) an d 5-(2-n aph th yl-
m eth yl)uracil (20c) was perform ed from eth yl 3-ph en ylpropan oate or eth yl
3-n aph th ylpropan oates accordin g to refs22,23. Th eir tran sform ation to
5-(arylm eth yl)-4-m eth oxypyrim idin -2(1H)-on es 21 con sists in th e treat-
m en t with POCl3 givin g 2,4-dich loropyrim idin es, reaction with sodium
m eth oxide leadin g to 2,4-dim eth oxypyrim idin es an d th eir fin al tran sfor-
m ation to 4-m eth oxypyrim idin -2(1H)-on es 21 by th e action of acetyl ch lo-
ride followed by sodium m eth oxide. Th is procedure, developed origin ally
for uracil an d th ym in e protection 24 was foun d useful for all types of
5-substituted uracils; recen tly we used th is process for preparation of
(i) POCl3, N,N-dim eth ylan ilin e; (ii) CH3ONa; (iii) CH3COCl, followed by CH3ONa; (iv) DMF,
Cs2CO3, 80 °C; (v) NaH, THF, r.t.; (vi) (CH3)3SiBr, CH3CN, r.t.; (vii) NH3/CH3OH, 110 °C, au-
toclave
SCHEME 3
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Pyrimidine Acyclic Nucleotide Analogues
933
5-eth yl-4-m eth oxypyrim idin -2(1H)-on e11
.
Reaction s of 5-substituted
4-m eth oxypyrim idin -2(1H)-on es with oxiran e 3 were perform ed in DMF
un der Cs2CO3 catalysis at 80 °C. An in crease in tem perature (up to 120 °C)
led to deprotection of 4-m eth oxy group (com poun d 27), sim ultan eously
with th e form ation of un desired disubstituted derivative 28. Ph osph on ate
diesters 23 were deprotected in on e step with brom otrim eth ylsilan e in
aceton itrile an d subsequen t h ydrolysis to 5-ben zyl-1-[(S)-3-h ydroxy-
2-(ph osph on om eth oxy)propyl]uracil (24a) an d its 5-(n aph th ylm eth yl) an a-
logues (24b, 24c) as fin al products for biological testin g. Th eir cytosin e
coun terparts 25a–25c were prepared from 4-m eth oxypyrim idin -2(1H)-on e
in term ediates 23a–23c by h igh -pressure reaction with am m on ia followed
by deprotection with brom otrim eth ylsilan e.
Biological Activity
All target com poun ds were tested for an tiviral activity in vitro in cell cul-
tures (E6SM cell culture for h erpesviruses, h um an em bryon ic lun g cells for
cytom egalovirus an d varicella-zoster virus). A selective an ti-h erpes virus
activity was foun d for (S)-1-[3-h ydroxy-2-(ph osph on om eth oxy)propyl]-
5-ph en yluracil (9a) wh ile th e correspon din g 5-ph en ylcytosin e derivative 8a
was com pletely in active. In terestin gly, an an tiviral activity was foun d also
for (R)-con figurated HPMP-5-ph en ylcytosin e 14, in spite of th e fact th at
(R)-HPMP derivatives are usually in active. Activity data of th ese com poun ds
are sh own in Table I. All oth er com poun ds described in th is paper were
an tivirally in active.
All 5-ph en yluracil derivatives were tested for in h ibitory activity again st
th ym idin e ph osph orylase (E.C.2.4.2.4.) from SD-lym ph om a. Th is in vestiga-
tion was perform ed un der con dition s described recen tly in th e study of TP
in h ibition with diverse th ym in e (ph osph on om eth oxy)alkyl derivatives:
100 µM [3H]-2′-deoxyth ym idin e, 250 µM Pi, tested com poun d 10 µm ol l–1,
pH 6.7; an appropriate am oun t of en zym e an d 10 m in in cubation at 37 °C
(ref.13). In h ibitory activity was foun d at 1-[(R)-3-h ydroxy-2-(ph osph on o-
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 7, pp. 927–951

934
Krečmerová, Holý, Masojídková:
m eth oxy)propyl]-5-ph en yluracil (15, Vi/V0 = 0.25), th e activity of oth er
com poun ds was on ly m argin al: Vi/V0 = 0.53 at 5-ph en yl-1-[(R)-2-(ph os-
ph on om eth oxy)propyl]uracil (19) an d Vi/V0 = 0.72 at 1-(S)-[3-h ydroxy-
2-(ph osph on om eth oxy)propyl]-5-(4-n itroph en yl)uracil (9b).
TABLE I
An tiviral activity of com poun ds 9a an d 14 again st cytom egalovirus (CMV), varicella-zoster
virus (VZV) an d h erpes sim plex viruses (HSV-1, HSV-2) com pared to cidofovir (HPMPC) an d
gan ciclovir
An tiviral activitya EC50, µg/m l
MCCb
µg/m l
Com poun d
CMV
AD 169
strain
CMV
Davis
strain
TK+ VZV TK– VZV
HSV-1
(KOS)
HSV-2
(G)
OKA
07/01
strain
strain
9a
76
55
>100
4.4
>100
1.5
9.6
240
9.6
>400
>400
>400
>400
14
2.2
0.8
0.35
1.7
48
Cidofovir
Gan ciclovir
0.13
0.64
0.20
–
0.05
–
0.16
0.54
0.0768
0.0064
a
b
Effective con cen tration required to reduce virus plaque form ation by 50%.
Min im um
cytotoxic con cen tration th at causes a m icroscopically detectable alteration of cell m orph ology.
Non e of th e com poun ds presen ted in th is study exh ibited cytostatic
activity in vitro tested in m ouse lym ph ocytic leukem ia L1210 cells,
CCRF-CEM T lym ph oblastoid cells, h um an prom yelocytic leukem ia HL-60
cells an d h um an cervix carcin om a HeLa cells.
EXPERIMENTAL
Un less stated oth erwise, solven ts were evaporated at 40 °C/2 kPa an d com poun ds were dried
at 13 Pa. Meltin g poin ts were determ in ed on a Kofler block an d are un corrected. An alytical
TLC were perform ed on silica gel 60 F254 plates (Merck KGaA, Darm stadt, Germ an y); ch ro-
m atograph ic system s are described in text. Colum n ch rom atograph y was perform ed on silica
gel 60 µm (Fluka) or alum in um oxide (50–150 µ, pH 7.0 ± 0.5, Fluka). Reverse ph ase HPLC
separation s were perform ed on a Waters Delta 600 in strum en t with a Waters 2487 Dual λ
Absorban ce Detector usin g colum n s XTerra® RP18 (3.9 × 150 m m , an alytical colum n ) an d
Lun a Ph en om en ex® C-18 (21 × 250 m m , preparative colum n ). 1H NMR spectra were m ea-
sured on a Varian Un ity 500 in strum en t (at 500 MHz) in DMSO-d6 solution s (referen ced to
th e solven t sign al at
δ 2.50) or in D2O solution s with in tern al stan dard sodium
3-(trim eth ylsilyl)propan e-1-sulfon ate (DSS). 1H NMR ch em ical sh ifts (δ, ppm ) an d couplin g
con stan ts (J, Hz) were obtain ed by first-order an alysis of th e spectra. 13C NMR spectra were
recorded on th e sam e in strum en t (at 125.7 MHz) usin g APT pulse sequen ce in DMSO-d6 (ref-
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 7, pp. 927–951

Pyrimidine Acyclic Nucleotide Analogues
935
eren ced to th e solven t sign al δ 39.70). Th e n um berin g system for assign m en t of NMR sign als
is outlin ed in Fig. 1. Mass spectra were m easured on a ZAB-EQ (VG An alytical) spectrom eter
usin g FAB (ion ization with xen on , acceleratin g voltage 8 kV, glycerol m atrix) or ESI. Optical
rotation s were m easured on Autopol IV polarim eter (Rudolph Research An alytical, U.S.A.) at
20 °C, [α]D values are given in 10–1 deg cm 2 g–1
.
FIG. 1
Gen eral n um berin g sch em e for assign m en t of NMR sign als
Materials an d Solven ts
Most ch em icals an d ion -exch an ge resin s (Dowex 50WX8-200 an d Dowex 1X2-400) were
purch ased from Sigm a–Aldrich (Czech Republic). Dim eth ylform am ide an d aceton itrile were
dried by distillation from CaH2 (DMF in vacuo) an d stored over m olecular sieves (4Å).
Diisopropyl [(tosyloxy)m eth yl]ph osph on ate was prepared by th e described procedure25
.
(2S)-2-[(Trityloxy)m eth yl]oxiran e was purch ased from DAISO Co. Ltd (Japan ).
Preparation of 5-Aryl-N4-ben zoylcytosin es 2a–2d . Gen eral Procedure
Ben zoic an h ydride (6.8 g, 30 m m ol) an d 4-(dim eth ylam in o)pyridin e (50 m g) were added to
a suspen sion of an appropriate 5-ph en ylcytosin e 1 (20 m m ol) in DMF (250 m l). Th e m ixture
was stirred at room tem perature for 3 days, th en diluted with eth an ol (50 m l) an d evapo-
rated. Th e residue was coevaporated with toluen e (2 × 50 m l) an d subsequen tly refluxed
with eth an ol (150 m l) for 15 m in . After coolin g to room tem perature, eth er (50 m l) was
added, th e crystallin e product collected by suction , wash ed with eth er an d dried in vacuo.
N4-Benzoyl-5-phenylcytosine (2a ). Yield 4.1 g (70%), wh ite crystals. M.p. 237 °C. For
C
17H13N3O2 (291.3) calculated: 70.09% C, 4.50% H, 14.42% N; foun d: 69.68% C, 4.38% H,
14.29% N. FAB MS, m/z (%): 292 (100) [M + H]. 1H NMR (DMSO-d6): 13.35 brs, 1 H an d
12.03 brs, 1 H (NH); 8.0 d, 2 H (H-arom .); 7.60–7.30 m , 8 H (H-arom .); 7.86 s, 1 H (H-6).
13C NMR (DMSO-d6): 172.54 (Bz); 159.88 (C-4); 148.07 (C-2); 143.42 (C-6); 137.25 (Bz);
133.50 (Ph ); 132.55 (Bz); 129.67, 2 C an d 129.44, 2 C (Ph ); 128.44, 2 C an d 128.02 (Bz);
127.56 (Ph ); 113.44 (C-5).
N4-Benzoyl-5-(4-nitrophenyl)cytosine (2b). Yield 5.0 g (74%), yellowish crystals. M.p. 284–288 °C.
For C17H12N4O4 (336.3) calculated: 60.71% C, 3.60% H, 16.66% N; foun d: 60.37% C, 3.72% H,
16.68% N. FAB MS, m/z (%): 337 (4) [M + H], 233 (16) [M – benzoyl + 2 H]. 1H NMR (DMSO-d6):
broad sign als due to in sufficien t solubility of com poun d.
N4-Benzoyl-5-(4-chlorophenyl)cytosine (2c). Yield 5.8 g (89%), wh ite crystals. M.p. 308 °C.
For C17H12ClN3O2 (325.8) calculated: 62.68% C, 3.71% H, 10.88% Cl, 12.90% N; foun d:
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Krečmerová, Holý, Masojídková:
62.78% C, 3.70% H, 10.64% Cl, 13.00% N. FAB MS, m/z (%): 326 (18) [M + H], 105 (28)
[ben zoyl]. 1H NMR (DMSO-d6): 13.30 brs, 1 H an d 12.10 brs, 1 H (NH); 8.03 brd, 2 H
(H-arom .); 7.91 s, 1 H (H-6); 7.66 brd, 2 H, 7.55 t, 1 H, 7.49 d, 2 H an d 7.45 t, 2 H
(H-arom .). 13C NMR (DMSO-d6): 178.41 (Bz); 159.71 (C-4); 148.91 (C-6); 143.91 (C-2);
137.05 (Ph ); 132.59 (Bz); 132.19, 131.36, 2 C an d 129.42, 2 C (Ph ); 128.50, 2 C an d 128.02,
2 C (Bz); 112.13 (C-5).
N4-Benzoyl-5-(2-chlorophenyl)cytosine (2d ). Th e crude product was ch rom atograph ed in
toluen e–eth yl acetate (1:1). Yield 900 m g (14%), wh ite crystals. M.p. 236–238 °C. FAB MS,
m/z (%): 326 (100) [M + H], 105 (74) [ben zoyl]. 1H NMR (DMSO-d6): 13.04 brs, 1 H an d
11.99 brs, 1 H (NH); 7.87 m , 2 H (H-arom .); 7.85 s, 1 H (H-6); 7.60–7.36 m , 7 H (H-arom .).
Reaction of (2S)-2-[(Trityloxy)m eth yl]oxiran e (3) with 5-Aryl-N4-ben zoylcytosin es 2a–2d .
Gen eral Procedure
Com poun d 3 (2.72 g, 8.6 m m ol) an d cesium carbon ate (250 m g, 0.76 m m ol) were added to
a stirred suspen sion of ben zoylated 5-arylcytosin e 2 (8 m m ol) in DMF (50 m l). Th e m ixture
was h eated at 100 °C un til th e con version was com plete (6–12 h , TLC con trol). Th e m ixture
was cooled to room tem perature, evaporated an d th e residue coevaporated with toluen e (2 ×
50 m l). Th e crude product was purified by ch rom atograph y on silica gel in system toluen e–
eth yl acetate (3:1) con tain in g 1% trieth ylam in e.
N4-Benzoyl-1-[(S)-2-hydroxy-3-(trityloxy)propyl]-5-phenylcytosine (4a). Yield 4.0 g (82%) of a
wh ite foam . [α]D –28.9 (c 0.802, CHCl3). For C39H33N3O4 (607.7) calculated: 77.08% C,
5.47% H, 6.91% N; foun d: 77.10% C, 5.53% H, 6.54% N. FAB MS, m/z (%): 608 (58) [M +
H], 366 (10) [M – trityl + 2 H], 243 (100) [trityl], 105 (38) [ben zoyl]. 1H NMR (DMSO-d6):
13.55 s, 1 H (NH); 8.08 d, 2 H (H-arom .); 8.04 s, 1 H (H-6); 7.59 d, 2 H, 7.56 t, 1 H, 7.46 t, 3 H,
7.39 d, 6 H, 7.31 t, 6 H an d 7.24 m , 5 H (H-arom .); 5.44 d, 1 H, J(OH,2′) = 5.5 (OH); 4.11 m ,
2 H (H-1′a, H-2′); 3.82 dd, 1 H, J(1′b,2′) = 8.1, J(gem ) = 13.2 (H-1′b); 3.08 br dd, 1 H, J(3′a,2′)
= 4.4, J(gem ) = 9.7 (H-3′a); 2.94 dd, 1 H, J(3′b,2′) = 5.5 (H-3′b). 13C NMR (DMSO-d6): 178.475
(C=O); 158.98 (C-4); 147.945 (C-6); 147.84 (C-2); 143.82, 3 C (Tr), 137.15 (Bz); 133.11 (Ph );
132.59, 129.47, 2 C an d 129.27, 2 C (Bz); 129.06, 2 C (Ph ); 128.38, 8 C (Ph , Tr); 128.06, 6 C
(Tr); 127.18, 3 C (Tr); 125.47 (Ph ); 113.16 (C-5); 86.185 (Tr); 66.99 (C-2′); 66.18 (C-3′); 52.72
(C-1′).
N4-Benzoyl-1-[(S)-2-hydroxy-3-(trityloxy)propyl]-5-(4-nitrophenyl)cytosine (4b ). Yield 2.9 g
(56%), yellowish crystals. M.p. 233 °C. [α]D –19.3 (c 0.427, CHCl3). For C39H32N4O6 (652.7)
calculated: 71.77% C, 4.94% H, 8.58% N; foun d: 71.72% C, 4.93% H, 8.49% N. FAB MS, m/z
(%): 653 (17) [M + H], 243 (100) [trityl], 105 (72) [ben zoyl]. 1H NMR (DMSO-d6): 13.50 brs,
1 H (NH); 8.29 m , 2 H (H-arom .); 8.24 s, 1 H (H-arom .); 8.03 m , 2 H, 7.87 m , 2 H, 7.57 t, 1 H,
7.47 t, 2 H, 7.38 d, 6 H, 7.31 t, 6 H an d 7.24 t, 3 H (H-arom .); 5.46 d, 1 H, J(OH,2′) = 5.7
(OH); 4.17 dd, 1 H, J(1′a,2′) = 3.2, J(gem ) = 12.7 (H-1′a); 4.11 m , 1 H (H-2′); 3.83 dd, 1 H,
J(1′b,2′) = 8.4 (H-1′b); 3.07 dd, 1 H, J(3′a,2′) = 4.5, J(gem ) = 9.4 (H-3′a); 2.94 dd, 1 H, J(3′b,2′) =
5.8 (H-3′b). 13C NMR (DMSO-d6): 167.50 (Bz); 156.80 br (C-4); 147.60 br (C-6); 147.20 br
(C-2); 146.46, 2 C (NO2-Ph ); 143.81, 3 C (Tr); 132.74 (Bz); 129.37, 2 C (NO2-Ph ); 128.59, 4 C
(Bz); 128.38, 6 C, 128.09, 6 C an d 127.23, 3 C (Tr); 123.40, 2 C (NO2-Ph ); 110.98 (C-5);
86.20 (Tr); 66.87 (C-2′); 66.225 (C-3′); 53.34 br (C-1′).
N4-Benzoyl-5-(4-chlorophenyl)-1-[(S)-2-hydroxy-3-(trityloxy)propyl]cytosine (4c). Yield 3.8 g
(74%), wh ite foam . [α]D –26.3 (c 0.426, CHCl3). For C39H32ClN3O4 (642.2) calculated:
72.95% C, 5.02% H, 5.52% Cl, 6.54% N; foun d: 73.06% C, 5.29% H, 5.49% Cl, 6.38% N.
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Pyrimidine Acyclic Nucleotide Analogues
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FAB MS, m/z (%): 642 (1) [M+], 400 (30) [M – trityl + H], 243 (20) [trityl], 105 (100) [ben zoyl].
1H NMR (DMSO-d6): 13.50 br s, 1 H (NH); 8.07 s, 1 H (H-6); 8.03 m , 2 H, 7.57 t, 1 H, 7.50 m ,
2 H, 7.47 t, 2 H, 7.37 d, 6 H, 7.30 t, 6 H an d 7.23 m , 5 H (H-arom .); 5.44 d, 1 H, J(OH,2′) =
5.6 (OH); 4.14 dd, 1 H, J(1′a,2′) = 4.0, J(gem ) = 13.2 (H-1′a); 4.09 m , 1 H (H-2′); 3.81 dd, 1 H,
J(1′b,2′) = 8.1 (H-1′b); 3.06 dd, 1 H, J(3′a,2′) = 4.6, J(gem ) = 9.4 (H-3′a); 2.93 dd, 1 H, J(3′b, 2′) =
5.7 (H-3′b). 13C NMR (DMSO-d6): 167.0 (Bz); 158.0 br (C-4); 148.0 br (C-6); 147.96 (C-2);
144.30 (Cl-Ph ); 143.84,
3 C (Tr); 132.68 (Bz); 132.29 br an d 129.40, 4 C (Cl-Ph );
128.57–126.83 (arom . C); 111.89 (C-5); 86.20 (Tr); 66.93 (C-2′); 66.24 (C-3′); 53.0 br
(C-1′).
N4-Benzoyl-5-(2-chlorophenyl)-1-[(S)-2-hydroxy-3-(trityloxy)propyl]cytosine (4d ). Yield 4.1 g
(80%), wh ite foam . [α]D –30.3 (c 0.321, CHCl3). For C39H32ClN3O4 (642.2) calculated:
72.95% C, 5.02% H, 5.52% Cl, 6.54% N; foun d: 72.43% C, 4.89% H, 5.76% Cl, 6.31% N.
1H NMR (DMSO-d6): 13.50 br s, 1 H (NH); 8.00 br d, 2 H (H-arom ., H-6); 7.50–7.10 m , 23 H
(H-arom .); 4.16 dd, 1 H, J(1′a,2′) = 2.0, J(gem ) = 13.8 (H-1′a); 4.14 m , 1 H (H-2′); 3.73 dd, 1 H,
J(1′b,2′) = 7.2 (H-2′b); 3.30 dd, 1 H, J(3′a,2′) = 4.6, J(gem ) = 9.8 (H-3′a); 3.20 dd, 1 H, J(3′b,2′) =
6.0 (H-3′b), 2.80 br, 1 H (OH). 13C NMR (DMSO-d6): 167.0 br (Bz); 158.79 (C-2); 150.79
(C-2); 146.27 (C-2); 143.33, 3 C ((Tr); 134.50 an d 132.63 (Cl-Ph ); 132.36 (Bz); 131.50 an d
129.89 (Cl-Ph ); 129.66, 2 C an d 129.60, 2 C (Bz); 128.47, 6 C, 128.03, 6 C an d 127.32, 3 C
(Tr); 126.46 (Cl-Ph ); 112.0 br (C-5); 87.16 (Tr); 69.25 (C-2′); 64.78 (C-3′); 52.45 (C-1′).
Reaction of Com poun ds 4 with Diisopropyl [(Tosyloxy)m eth yl]ph osph on ate (5)
A solution of ph osph on ate 5 (2.0 g, 5.7 m m ol) an d appropriate trityl derivative 4a–4d
(3.9 m m ol) in DMF (100 m l) was cooled to –20 °C an d 60% sodium h ydride dispersion
(470 m g, 11.8 m m ol) was added un der stirrin g. Th e m ixture was warm ed slowly to room
tem perature, stirred for addition al 20 h an d th en n eutralized with acetic acid to pH 7.0 an d
evaporated. Th e residue was coevaporated with toluen e (100 m l) an d ch rom atograph ed on
silica gel (400 m l) in th e system described below.
N4-Benzoyl-1-{(S)-2-[(diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-5-phenylcytosine
(6a). Ch rom atograph ed in system eth yl acetate–toluen e (2:1). Yield 2.4 g (78%), wh ite foam .
[α]D –53.8 (c 0.614, CHCl3). For C46H48N3O7P (785.9) calculated: 70.30% C, 6.16% H, 5.35% N,
3.94% P; foun d: 70.10% C, 6.20% H, 5.18% N, 4.00% P. FAB MS, m/z (%): 786.5 (7) [M + H],
544 (10) [M – trityl + 2 H], 243 (100) [trityl]. 1H NMR (DMSO-d6): 13.55 s, 1 H (NH); 8.07 d,
2 H (H-arom .); 8.03 s, 1 H (H-6); 7.64 d, 2 H, 7.55 t, 1 H an d 7.45–7.24 m , 20 H (H-arom .);
4.52 m , 2 H (P-OCH); 4.10–4.00 m , 3 H (H-1′, H-2′); 3.83 dd, 1 H, J(P,CHa) = 9.0, J(gem ) =
13.8 (PCHa); 3.76 dd, 1 H, J(P,CHb) = 9.5 (PCHb); 3.27 dd, 1 H, J(3′a,2′) = 3.0, J(gem ) = 10.6
(H-3′a); 3.04 dd, 1 H, J(3′b,2′) = 3.5 (H-3′b); 1.15 d, 3 H, 1.13 d, 3 H, 1.12 d, 3 H an d 1.10 d,
3 H, J(CH3,CH) = 6.2 (CH3). 13C NMR (DMSO-d6): 178.545 (C=O); 158.93 (C-4); 147.81
(C-2); 147.58 (C-6); 143.63, 3 C (Tr); 137.09 (Bz); 133.08 (Ph ); 132.66 (Bz); 129.60, 2 C an d
129.49, 2 C (Bz); 128.51, 2 C (Ph ); 128.39, 6 C an d 128.12, 6 C (Tr); 128.02, 2 C an d 127.72
(Ph ); 127.29, 3 C (Tr); 113.51 (C-5); 86.47 (Tr); 78.03 d, J(P,C) = 11.7 (C-2′); 70.32 d, 2 C,
J(P,C) = 6.3 (P-O-C); 63.97 d, J(P,C) = 164.6 (P-C); 62.81 (C-3′); 50.11 (C-1′); 23.85 d, 2 C,
J(P,C) = 3.4 an d 23.70 d, 2 C, J(P,C) = 4.4 (CH3).
N4-Benzoyl-1-{(S)-2-[(diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-5-(4-nitrophenyl)cyto-
sine (6b). Ch rom atograph ed in system eth yl acetate–toluen e (2:1). Yield 2.6 g (80%), yellow
foam . [α]D –39.2 (c 0.172, CHCl3). For C46H47N4O9P (830.9) calculated: 66.50% C, 5.70% H,
6.74% N, 3.73% P; foun d: 66.65% C, 5.64% H, 6.40% N, 3.87% P. FAB MS, m/z (%): 831 (4)
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Krečmerová, Holý, Masojídková:
[M + H], 243 (100) [trityl]. 1H NMR (DMSO-d6): 13.50 brs, 1 H (NH); 8.32 d, 2 H (H-arom .);
8.25 s, 1 H (H-6); 8.07 d, 2 H, 7.98 d, 2 H, 7.57 t, 1 H, 7.48 t, 2 H, 7.39 d, 6 H, 7.32 t, 6 H
an d 7.26 t, 3 H (H-arom .); 4.51 m , 2 H (P-OCH); 4.11 m , 2 H an d 4.02 m , 1 H (H-1′, H-2′);
3.84 dd, 1 H an d 3.78 dd, 1 H, J(P,CH) = 8.8, J(gem ) = 13.9 (PCH2); 3.28 dd, 1 H, J(3′a,2′) =
3.8, J(gem ) = 10.6 (H-3′a); 3.03 dd, 1 H, J(3′b,2′) = 3.2 (H-3′b); 1.14 d, 6 H an d 1.12 d, 6 H,
J(CH3,CH) = 6.1 (CH3). 13C NMR (DMSO-d6): 167.35 (Bz); 158.22 (C-4); 149.05 (C-6); 147.68
(C-2); 146.58, 2 C (NO2-Ph ); 143.60, 3 C (Tr); 132.78 (Bz); 130.51, 2 C (NO2-Ph ); 128.60, 4 C
(Bz); 128.37, 6 C, 128.12, 6 C an d 127.29, 3 C (Tr); 123.19, 2 C (NO2-Ph ); 111.24 (C-5);
86.46 (Tr); 77.93 d, J(P,C) = 10.2 (C-2′); 70.35 d an d 70.30 d, J(P,C) = 6.3 (P-O-C); 63.97 d,
J(P,C) = 164.6 (P-C); 62.82 (C-3′); 50.28 (C-1′); 23.85 d, 2 C, J(P,C) = 3.4, 23.80 d an d 23.69 d,
J(P,C) = 4.4 (CH3).
N4-Benzoyl-5-(4-chlorophenyl)-1-{(S)-2-[(diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-
cytosine (6c). Ch rom atograph ed in system eth yl acetate–toluen e (1:2). Yield 2.4 g (75%),
am orph ous solid. [α]D –40.5 (c 0.380, CHCl3). For C46H47ClN3O7P.3H2O (874.4) calculated:
63.19% C, 6.11% H, 4.05% Cl, 4.81% N, 3.54% P; foun d: 63.01% C, 6.15% H, 3.98% Cl,
4.82% N, 3.43% P. FAB MS, m/z (%): 820 (1) [M + H], 578 (2) [M – trityl + H], 243 (68)
[trityl], 105 (42) [ben zoyl]. 1H NMR (DMSO-d6): 13.50 brs, 1 H (NH); 8.06 s, 1 H (H-6); 8.04 m ,
2 H, 7.65 m , 2 H, 7.56 t, 1 H, 7.50 d, 2 H, 7.46 t, 2 H, 7.39 d, 6 H, 7.32 t, 6 H an d 7.25 t, 3 H
(H-arom .); 4.50 m , 2 H (P-OCH); 4.09 m , 2 H an d 4.01 m , 1 H (H-1′, H-2′); 3.81 dd, 1 H an d
3.76 dd, 1 H, J(P,CH) = 8.8, J(gem ) = 13.9 (PCH2); 3.26 dd, 1 H, J(3′a,2′) = 3.9, J(gem ) = 10.5
(H-3′a); 3.02 dd, 1 H, J(3′b,2′) = 3.3 (H-3′b); 1.14 d, 3 H, 1.13 d, 3 H, 1.12 d, 3 H an d 1.10 d,
3 H, J(CH3,CH) = 6.2 (CH3). 13C NMR (DMSO-d6): 145.70 (C-2); 143.70, 3 C (Tr); 137.54
(Cl-Ph ); 132.72 (Bz); 132.32 br an d 129.44 br, 4 C (Cl-Ph ); 128.59, 4 C (Bz); 128.39, 6 C,
128.13, 6 C an d 127.31, 3 C (Tr); 112.19 (C-5); 86.47 (Tr); 77.92 d, J(P,C) = 12.7 (C-2′);
70.37 d an d 70.32 d, J(P,C) = 6.3 (P-O-C); 63.95 d, J(P,C) = 165.0 (P-C); 62.81 (C-3′); 23.87 d
an d 23.82 d, J(P,C) = 3.4 (CH3); 23.70 d an d 23.64 d, J(P,C) = 4.4 (CH3).
Preparation of Com poun ds 7a–7c. Gen eral Procedure
A solution of ben zoylated ph osph on ate 6a–6c (3.1 m m ol) in 30% m eth an olic am m on ia
(40 m l) was kept at 4 °C for 24 h an d th en evaporated. Th e residue was ch rom atograph ed
on silica gel (300 m l) in th e system described below.
1-{(S)-2-[(Diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-5-phenylcytosine (7a). Ch rom ato-
graph ed in system eth yl acetate–aceton e–eth an ol–water (18:3:2:2). Yield 1.9 g (90%), wh ite
foam . [α]D –34.8 (c 0.480, CHCl3). For C39H44N3O6P (681.8) calculated: 68.71% C, 6.50% H,
6.16% N, 4.54% P; foun d: 68.30% C, 6.51% H, 6.01% N, 4.83% P. FAB MS, m/z (%): 682 (7)
[M + H], 440 (12) [M – trityl + 2 H], 243 (100) [trityl]. 1H NMR (DMSO-d6): 7.44 s, 1 H (H-6);
7.40–7.30 m , 20 H (H-arom .); 6.30 brs, 2 H (NH2); 4.56 m , 2 H (P-OCH); 3.95 dd, 1 H,
J(1′a,2′) = 4.0, J(gem ) = 12.6 (H-1′a); 3.92 m , 1 H (H-2′); 3.87 dd, 1 H, J(1′b,2′) = 5.9 (H-1′b);
3.78 dd, 1 H, J(P,CHa) = 8.9, J(gem ) = 13.6 (PCHa); 3.70 dd, 1 H, J(P,CHb) = 9.8 (PCHb); 3.21 dd,
1 H, J(3′a,2′) = 2.9, J(gem ) = 10.6 (H-3′a); 2.93 dd, 1 H, J(3′b,2′) = 3.9 (H-3′b); 1.17 d, 3 H,
1.165 d, 3 H, 1.135 d, 3 H an d 1.13 d, 3 H, J(CH3,CH) = 6.2 (CH3). 13C NMR (DMSO-d6):
163.795 (C-4); 155.28 (C-2); 146.36 (C-6); 143.71, 3 C (Tr); 133.96 (Ph ); 129.00, 2 C an d
128.83, 2 C (Ph ); 128.40, 6 C an d 128.09, 6 C (Tr); 127.57 (Ph ); 127.24, 3 C (Tr); 106.76
(C-5); 86.255 (Tr); 78.57 d, J(P,C) = 13.2 (C-2′); 70.34 d, 2 C, J(P,C) = 6.4 (P-O-C); 64.05 d,
J(P,C) = 166.0 (P-C); 62.86 (C-3′); 50.18 (C-1′); 23.94 d, J(P,C) = 3.9, 23.91 d, J(P,C) = 3.9,
23.83 d, J(P,C) = 4.4 an d 23.75 d, J(P,C) = 4.9 (CH3).
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Pyrimidine Acyclic Nucleotide Analogues
939
1-{(S)-2-[(Diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-5-(4-nitrophenyl)cytosine (7b ).
Ch rom atograph ed in eth yl acetate–aceton e–eth an ol–water (15:3:4:3). Yield 2.15 g (95%),
yellow foam . [α]D –7.7 (c 0.764, CHCl3). For C39H43N4O8P (726.8) calculated: 64.45% C,
5.96% H, 7.71% N, 4.26% P; foun d: 63.88% C, 6.02% H, 7.52% N, 4.06% P. FAB MS, m/z (%):
749 (8) [M + Na], 243 (100) [trityl]. 1H NMR (DMSO-d6): 8.24 d, 2 H (H-arom .); 7.65 s, 1 H
(H-6); 7.56 d, 2 H, 7.38 d, 6 H, 7.32 t, 6 H an d 7.26 t, 3 H (H-arom ); 4.52 m , 2 H (P-OCH);
3.97 dd, 1 H, J(1′a,2′) = 3.3, J(gem ) = 11.8 (H-1′a); 3.93 m , 1 H (H-2′); 3.88 dd, 1 H, J(1′b,2′) =
7.0 (H-1′b); 3.78 dd, 1 H, J(P,CHa) = 8.7, J(gem ) = 13.7 (PCHa); 3.70 dd, 1 H, J(P,CHb) = 9.9
(PCHb); 3.22 dd, 1 H, J(3′a,2′) = 2.7, J(gem ) = 10.5 (H-3′a); 2.92 dd, 1 H, J(3′b,2′) = 3.9
(H-3′b); 1.17 d, 3 H, 1.16 d, 3 H, 1.135 d, 3 H, 1.13 d, 3 H, J(CH3,CH) = 6.1 (CH3). 13C NMR
(DMSO-d6): 163.27 (C-4); 155.02 (C-2); 147.77 (C-6); 146.44 (NO2-Ph ); 143.70, 3 C (Tr);
141.44 an d 129.88, 2 C (NO2-Ph ); 128.39, 6 C, 128.11, 6 C an d 127.27, 3 C (Tr); 124.05, 2 C
(NO2-Ph ); 104.94 (C-5); 86.29 (Tr). 78.49 d, J(P,C) = 12.7 (C-2′); 70.38 d an d 70.34 d, J(P,C) =
6.3 (P-O-C); 64.03 d, J(P,C) = 165.5 (P-C); 62.83 (C-3′); 50.32 (C-1′); 24.93 d an d 23.90 d,
J(P,C) = 3.4, 23.83 d an d 23.77 d, J(P,C) = 4.4 (CH3).
5-(4-Chlorophenyl)-1-{(S)-2-[(diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}cytosine (7c).
Ch rom atograph ed in eth yl acetate–aceton e–eth an ol–water (18:3:2:2). Yield 1.8 g (81%),
wh ite foam . [α]D –29.4 (c 0.445, CHCl3). For C39H43ClN3O6P (716.2) calculated: 65.40% C,
6.05% H, 4.95% Cl, 5.87% N, 4.32% P; foun d: 65.27% C, 5.97% H, 4.65% Cl, 6.18% N,
4.08% P. FAB MS, m/z (%): 738 (10) [M + Na], 243 (100) [trityl]. 1H NMR (DMSO-d6): 7.97 br,
1 H (NH2); 7.87 d, 2 H an d 7.51 t, 1 H (H-arom .); 7.46 s, 1 H (H-6); 7.45 m , 4 H, 7.38 d, 6 H,
7.31 t, 6 H an d 7.26 m , 5 H (H-arom .); 6.45 br, 1 H (NH2); 4.55 m , 2 H (P-OCH); 3.94 dd, 1 H,
J(1′a,2′) = 4.2, J(gem ) = 11.8 (H-1′a); 3.90 m , 1 H (H-2′); 3.85 dd, 1 H, J(1′b,2′) = 6.2 (H-1′b);
3.77 dd, 1 H, J(P,CHa) = 8.8, J(gem ) = 13.7 (PCHa); 3.69 dd, 1 H, J(P,CHb) = 9.8 (PCHb); 3.20 dd,
1 H, J(3′a,2′) = 3.2, J(gem ) = 10.5 (H-3′a); 2.92 dd, 1 H, J(3′b,2′) = 4.0 (H-3′b); 1.17 d, 3 H,
1.165 d, 3 H, 1.133 d, 3 H an d 1.131 d, 3 H, J(CH3,CH) = 6.1 (CH3). 13C NMR (DMSO-d6):
163.70 (C-4); 155.24 (C-2); 146.61 (C-6); 143.71, 3 C (Tr); 132.91, 132.25, 131.39, 2 C an d
130.75, 2 C (Cl-Ph ); 128.40, 6 C, 128.10, 6 C an d 127.63, 3 C (Tr); 105.60 (C-5); 86.27 (Tr);
78.55 d, J(P,C) = 12.2 (C-2′); 70.36 d, 2 C, J(P,C) = 5.4 (P-O-C); 64.05 d, J(P,C) = 165.5 (P-C);
62.86 (C-3′); 50.21 (C-1′); 23.96 d an d 23.90 d, J(P,C) = 3.9 (CH3); 23.85 d an d 23.76 d,
J(P,C) = 4.4 (CH3).
N4-Ben zoyl-1-[(R)-2-h ydroxy-3-(trityloxy)propyl]-5-ph en ylcytosin e (11)
Th e product was prepared from com poun ds 10 (2.72 g, 8.6 m m ol) an d 2a (2.5 g, 8.6 m m ol)
by th e sam e procedure as described for 4a. Yield 4.51 g (86%), wh ite foam . [α]D +30.0 (c 0.340,
CHCl3). 1H an d 13C NMR spectra are iden tical with th ose of com poun d 4a.
N4-Ben zoyl-1-{(R)-2-[(diisopropoxyph osph oryl)m eth oxy]-3-(trityloxy)propyl}-
5-ph en ylcytosin e (12)
Th e product was prepared from 11 (4.5 g, 7.4 m m ol) by th e sam e procedure as described for
6a. Yield 4.3 g (74%), wh ite foam . [α]D +57.2 (c 0.394, CHCl3). 1H an d 13C NMR spectra are
iden tical with th ose of com poun d 6a.
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1-{(R)-2-[(Diisopropoxyph osph oryl)m eth oxy]-3-(trityloxy)propyl}-5-ph en ylcytosin e (13)
Th e product was prepared from 12 (4.3 g, 5.5 m m ol) by th e sam e procedure as described for
7a. Yield 3.1 g (82%), wh ite foam . [α]D +31.8 (c 0.403, CHCl3). 1H an d 13C NMR spectra are
iden tical with th ose of com poun d 7a.
Free Ph osph on ic Acids Derived from 5-Substituted Cytosin es. Gen eral Procedure
Brom otrim eth ylsilan e (2 m l, 15 m m ol) was added to a solution of appropriate ester 7a–7c,
14 or 26a–26c (1.6 m m ol) in aceton itrile (10 m l), th e m ixture was set aside in th e dark at
room tem perature for 48 h an d evaporated. Th e residue was coevaporated with aceton itrile
(20 m l), dissolved in system eth an ol–water–trieth ylam in e (10:10:1, 45 m l) an d after 5 m in
stan din g evaporated. Th e residue was partition ed between water (100 m l) an d dieth yl eth er
(100 m l), th e aqueous layer con cen trated to ca. 10 m l an d applied on to a colum n of Dowex
50 (H+ form , 50 m l). Elution was perform ed with water (750 m l), followed by 2.5% aqueous
am m on ia. UV absorbin g am m on ia fraction s were evaporated, th e residue dissolved in water
(5 m l) an d applied on to a colum n of Dowex 1 (acetate form , 50 m l). Elution was perform ed
with water (100 m l), followed by gradien t of 0–1 M acetic acid (1 l) or 1 M form ic acid; con -
cen tration s n ecessary for product elution are given below. Product-con tain in g fraction s were
evaporated, th e residue coevaporated with water (4 × 20 m l) an d crystallized from water or
aqueous eth an ol.
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-phenylcytosine (8a). Eluted from Dowex 1
with 0.3 M acetic acid. Yield 431 m g (74%), wh ite crystals. M.p. 265–268 °C. [α ]D –55.0
(c 0.141, H2O). For C14H18N3O6P·0.5H2O (364.3) calculated: 46.16% C, 5.26% H, 11.53% N,
8.50% P; foun d: 46.22% C, 5.21% H, 11.30% N, 8.39% P. FAB MS, m/z (%): 356 (100) [M + H],
188 (12) [5-ph en ylcytosin e + H]. 1H NMR (D2O): 7.52 s, 1 H (H-6); 7.43 m , 3 H an d 7.35 d,
2 H (H-arom .); 3.87 dd, 1 H, J(1′a,2′) = 6.2, J(gem ) = 14.0 (H-1′a); 3.84 dd, 1 H, J(1′b,2′) = 5.8
(H-1′b); 3.71 m , 1 H (H-2′); 3.69 dd, 1 H, J(3′a,2′) = 3.2, J(gem ) = 13.2 (H-3′a); 3.55 dd, 1 H,
J(P,CHa) = 8.3, J(gem ) = 12.4 (PCHa); 3.46 dd, 1 H, J(3′b,2′) = 6.0, J(gem ) = 13.2 (H-3′b); 3.40 dd,
1 H, J(P,CHb) = 9.5 (PCHb). 13C NMR (D2O): 164.75 (C-4); 158.00 (C-2); 145.95 (C-6);
132.15, 129.36, 2 C, 129.24, 2 C an d 128.75 (C-arom .); 109.96 (C-5); 80.11 d, J(P,C) = 9.8
(C-2′); 68.49 d, J(P,C) = 150.4 (P-C); 60.81 (C-3′); 49.965 (C-1′).
1-[(R)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-phenylcytosine (14). Yield 414 m g (71%),
wh ite crystals. M.p. 266 °C. [α]D +54.5 (c 0.206, H2O). 1H an d 13C NMR spectra are iden tical
with th ose of com poun d 8a.
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-(4-nitrophenyl)cytosine (8b ). Eluted from
Dowex 1 with 1 M acetic acid. Yield 482 m g (72%), yellowish solid. M.p. 178 °C. [α]D –13.9
(c 0.512, H2O). For C14H17N4O8P·H2O (418.3) calculated: 40.20% C, 4.58% H, 13.39% N,
7.40% P; foun d: 40.27% C, 4.55% H, 13.27% N, 7.35% P. FAB MS, m/z (%): 401 (12) [M +
H]. 1H NMR (D2O): 8.37 d, 2 H an d 7.75 d, 2 H (H-arom .); 8.04 s, 1 H (H-6); 4.26 dd, 1 H,
J(1′a,2′) = 3.0, J(gem ) = 14.4 (H-1′a); 3.93 dd, 1 H, J(1′b,2′) = 8.2 (H-1′b); 3.88 dd, 1 H,
J(3′a,2′) = 3.9, J(gem ) = 12.2 (H-3′a); 3.84 m , 1 H (H-2′); 3.81 dd, 1 H, J(P,CHa) = J(P,CHb) =
9.4, J(gem ) = 13.2 (PCHa); 3.66 dd, 1 H, J(3′b,2′) = 3.4 (H-3′b); 3.61 dd, 1 H (PCHb). 13C NMR
(D2O): 158.49 (C-4); 149.99 (C-6); 148.99 (C-2); 148.13, 136.24, 130.97, 2 C an d 124.52, 2 C
(C-arom .); 106.51 (C-5); 79.06 d, J(P,C) = 11.2 (C-2′); 65.87 d, J(P,C) = 157.7 (P-C); 59.82
(C-3′); 50.60 (C-1′).
5-(4-Chlorophenyl)-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (8c). Eluted from
Dowex 1 with 0.5 M acetic acid. Yield 525 m g (82%), wh ite crystals. M.p. 293 °C. [α]D –37.2
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Pyrimidine Acyclic Nucleotide Analogues
941
(c 0.329, 0.1 M HCl). For C14H17ClN3O6P·0.5H2O (398.7) calculated: 42.17% C, 4.54% H,
8.90% Cl, 10.53% N, 7.77% P; foun d: 42.31% C, 4.31% H, 9.10% Cl, 10.27% N, 7.44% P.
FAB MS, m/z (%): 390 (100) [M + H]. 1H NMR (D2O): 7.63 s, 1 H (H-6); 7.51 d, 2 H an d 7.39 d,
2 H (H-arom .); 3.95 d, 2 H, J(1′,2′) = 5.9 (H-1′); 3.78 m , 1 H (H-2′); 3.78 dd, 1 H, J(3′a,2′) =
2.8, J(gem ) = 13.2 (H-3′a); 3.62 dd, 1 H, J(P,CHa) = 8.4, J(gem ) = 12.4 (PCHa); 3.55 dd, 1 H,
J(3′b,2′) = 6.1 (H-3′b); 3.48 dd, 1 H, J(P,CHb) = 9.5 (PCHb). 13C NMR (D2O): 164.60 (C-4);
157.95 (C-2); 146.12 (C-6); 133.93, 130.83, 2 C, 130.79 an d 129.24, 2 C (C-arom .); 108.92
(C-5); 80.14 d, J(P,C) = 9.8 (C-2′); 68.46 d, J(P,C) = 150.4 (P-C); 60.77 (C-3′); 50.01 (C-1′).
5-Benzyl-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (25a). Eluted from Dowex 1
with 0.5 M acetic acid. Yield 421 m g (68%), wh ite solid. [α]D –32.5 (c 0.412, H2O). For
C
15H20N3O6P·H2O (387.3) calculated: 46.52% C, 5.72% H, 10.85% N, 8.00% P; foun d:
46.53% C, 5.72% H, 10.80% N, 7.75% P. ESI MS, m/z (%): 370.1 (50) [M + H], 738.9 (100)
[2 M + H]. 1H NMR (D2O): 7.65 s, 1 H (H-6); 7.43 m , 2 H an d 7.33 m , 3 H (H-arom .); 4.11 dd,
1 H, J(1′a,2′) = 3.4, J(gem ) = 14.2 (H-1′a); 3.86 dd, 1 H, J(1′b,2′) = 7.8 (H-1′b); 3.84 s, 2 H (CH2);
3.82 dd, 1 H, J(3′a,2′) = 3.9, J(gem ) = 11.7 (H-3′a); 3.79 m , 1 H (H-2′); 3.74 dd, 1 H an d 3.59 dd,
1 H, J(P,CH) = 9.2, J(gem ) = 13.2 (PCH2); 3.59 dd, 1 H, J(3′b,2′) = 3.2 (H-3′b). 13C NMR (D2O):
158.84 (C-4); 149.36 (C-6); 148.98 (C-2); 135.96, 129.03, 2 C, 128.51, 2 C an d 127.28 (Ph );
105.53 (C-5); 79.17 d, J(P,C) = 10.7 (C-2′); 65.86 d, J(P,C) = 157.7 (P-C); 59.94 (C-3′); 49.94
(C-1′); 31.12 (CH2-Ph ).
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-(1-naphthylmethyl)cytosine (25b ). Eluted
from Dowex 1 with 1 M form ic acid. Yield 389 m g (58%), wh ite solid. [α]D –40.4 (c 0.136,
CH3OH + 0.1
C
M HCl, 10:1). FAB MS, m/z (%): 420 (100) [M + H]. HR MS (FAB): For
19H23N3O6P [M + H] calculated: 420.1324; foun d: 420.1329. 1H NMR (D2O): 7.95 d, 1 H,
7.86 m , 2 H, 7.56 m , 2 H, 7.47 t, 1 H, 7.30 d an d 1 H (H-arom .); 6.79 s, 1 H (H-6); 3.99 m , 2 H
(CH2); 3.60 dd, 1 H, J(1′a,2′) = 5.3, J(gem ) = 12.9 (H-1′a); 3.43–3.55 m , 4 H (H-1′b, H-2′,
H-3′a, OCH2Pa); 3.36 dd, 1 H, J(P,CHb) = 9.2, J(gem ) = 12.7 (PCHb); 3.23 dd, 1 H, J(3′b,2′) =
5.0, J(gem ) = 12.4 (H-3′b). 13C NMR (D2O): 165.95 (C-4); 158.51 (C-2); 146.47 (C-6); 134.22,
133.33, 131.94, 129.47, 128.42, 127.42, 127.34, 126.89, 126.57 an d 124.22 (1-n aph th yl);
106.93 (C-5); 80.24 d, J(C,P) = 10.0 (C-2′); 68.22 d, J(C,P) = 153.0 (P-C); 61.12 (C-3′); 50.10
(C-1′); 30.24 (CH2-n aph th yl).
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-(2-naphthylmethyl)cytosine (25c). Eluted from
Dowex 1 with
1 M form ic acid. Yield 389 m g (58%), wh ite solid. [α]D n ot determ in ed
(opalescen t solution s in H2O, CH3OH or 0.1 M NaOH). FAB MS, m/z (%): 420 (100) [M + H].
HR MS (FAB): For C19H23N3O6P [M + H] calculated: 420.1324; foun d: 420.1345. 1H NMR (D2O):
7.90–7.79 m , 4 H (H-arom .); 7.77 s, 1 H (H-6); 7.52–7.41 m , 3 H (H-arom .); 4.05 m , 1 H
(H-1′a); 3.88 s, 2 H (CH2); 3.75–3.51 m , 4 H (H-1′b, H-2′, PCH2); 3.45 m , 2 H (H-3′). 13C NMR
(D2O): 161.01 (C-4); 150.64 (C-2); 149.04 (C-6); 135.72, 133.24, 132.04, 128.08, 127.71,
127.68, 127.49, 126.68, 126.31 an d 125.81 (2-n aph th yl); 103.89 (C-5); 79.72 d, J(C,P) = 8.5
(C-2′); 65.88 d, J(C,P) = 159.0 (P-C); 60.58 (C-3′); 49.70 (C-1′); 31.39 (CH2-n aph th yl).
5-(4-Ch loroph en yl)-1-[(S)-3-h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (8d )
A solution of 4d (447 m g, 0.7 m m ol) an d 5 (350 m g, 1 m m ol) in DMF (25 m l) was cooled to
–20 °C an d 60% oil dispersion of NaH (84 m g, 2.1 m m ol) was added. Th e m ixture was
stirred at –20 °C for 30 m in , th en left to warm to room tem perature an d th e stirrin g con tin -
ued for addition al 16 h . Th e solution was n eutralized with acetic acid to pH 7, evaporated
an d th e residue coevaporated with toluen e. 30% m eth an olic am m on ia (20 m l) was added,
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Krečmerová, Holý, Masojídková:
th e resultin g solution kept at 4 °C for 24 h an d evaporated. Th e crude product 7d was
deprotected with brom otrim eth ylsilan e by th e sam e procedure as described for com poun ds
8a–8c. Th e product 8d was eluted from Dowex 1 with 0.3 M acetic acid. Yield 190 m g (70%),
wh ite am orph ous solid. [α]D –28.6 (c 0.219, H2O). FAB MS, m/z (%): 390 (34) [M + H]. HR MS
(FAB): For C14H18ClN3O6P [M + H] calculated: 390.0622; foun d: 390.0615. 1H NMR (D2O +
NaOD): 7.64 s, 1 H (H-6); 7.60 m , 1 H an d 7.47 m , 3 H (H-arom .); 3.98 br dd, 1 H, J(1′a,2′) =
3.0, J(gem ) = 13.9 (H-1′a); 3.91 dd, 1 H, J(1′b,2′) = 5.7 (H-1′b); 3.80 m , 1 H (H-2′); 3.76 dd,
1 H, J(3′a,2′) = 3.0, J(gem ) = 12.6 (H-3′a); 3.65 br dd, 1 H an d 3.59 br dd, 1 H, J(P,CH) = 9.0,
J(gem ) = 13.2 (PCH2); 3.56 dd, 1 H, J(3′b,2′) = 5.9 (H-3′b). 13C NMR (D2O + NaOD): 164.61
(C-4); 158.07 (C-2); 146.70 (C-6); 134.26, 132.59, 130.89, 130.46, 129.87 an d 127.80
(C-arom .); 107.45 (C-5); 80.60 d, J(P,C) = 10.7 (C-2′); 68.59 d, J(P,C) = 150.4 (P-C); 60.95
(C-3′); 49.85 (C-1′).
1-[(S)-3-Hydroxy-2-(ph osph on om eth oxy)propyl]-5-ph en yluracils (9a–9d , 15).
Gen eral Procedure
A solution of appropriate cytosin e ph osph on ate 8a–8d or 14 (0.28 m m ol) in 80% acetic acid
(3.5 m l) was kept with 3-m eth ylbutyl n itrite (0.5 m l, 3.7 m m ol) at room tem perature for
3 days. Th e solution was evaporated, th e residue coevaporated with water (2 × 5 m l) an d
applied on to a colum n of Dowex 50 (H+, 25 m l). Th e colum n was eluted with water, th e
UV-absorbin g eluate adjusted to pH 3 an d deion ized on activated ch arcoal. Th e fin al purifi-
cation was perform ed on preparative reverse ph ase HPLC colum n , isocratically in aqueous
m eth an ol (con cen tration given bellow), flow rate 12 m l/m in . Th e pure products were
lyoph ilized from water, som e of th em tran sform ed to dilith ium salts before lyoph ilization .
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-phenyluracil (9a). Yield 50 m g (49%) of
dilith ium salt, wh ite am orph ous solid. M.p. 180–185 °C. Elution from HPLC colum n : 10%
CH3OH. [α]D –18.2 (c 0.233, H2O). For C14H15Li2N2O7P (368.1) calculated: 45.68% C, 4.11% H,
7.61% N, 8.41% P; foun d: 45.34% C, 4.63% H, 7.71% N, 8.33% P. FAB MS, m/z (%): 369
(0.5) [M + H]. 1H NMR (D2O): 7.73 s, 1 H (H-6); 7.46 m , 5 H (H-arom .); 4.03 dd, 1 H,
J(1′a,2′) = 4.5, J(gem ) = 14.4 (H-1′a); 3.89 dd, 1 H, J(1′b,2′) = 7.2 (H-1′b); 3.84 dd, 1 H,
J(3′a,2′) = 3.7, J(gem ) = 12.4 (H-3′a); 3.79 m , 1 H (H-2′); 3.72 dd, 1 H, J(P,CHa) = 9.6, J(gem ) =
12.9 (PCHa); 3.67 dd, 1 H, J(P,CHb) = 9.2 (PCHb); 3.62 dd, 1 H, J(3′b,2′) = 4.4 (H-3′b). 13C NMR
(D2O): 165.09 (C-4); 151.97 (C-2); 145.13 (C-6); 131.81, 128.63, 2 C, 128.56, 2 C an d 128.19
(C-arom .); 114.79 (C-5); 79.75 d, J(P,C) = 11.7 (C-2′); 66.51 d, J(C,P) = 156.2 (P-C); 60.27
(C-3′); 49.07 (C-1′).
1-[(R)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-phenyluracil (15). Yield 61 m g (59%) of
dilith ium salt. [α]D +20.0 (c 0.459, H2O). M.p. 181–187 °C. MS an d NMR data are iden tical
with th ose of com poun d 9a.
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-(4-nitrophenyl)uracil (9b). Yield 49 m g (42%)
of dilith ium salt, yellow am orph ous solid. [α]D +11.64 (c 0.272, H2O). Elution from HPLC
colum n : 10% CH3OH. HR MS (FAB): For C14H15Li2N3O9P [M + H] calculated: 414.0866;
foun d: 414.0878. FAB MS, m/z (%): 414 (2) [M + H]. 1H NMR (D2O): 8.29 d, 2 H (H-arom .);
8.02 s, 1 H (H-6); 7.77 d, 2 H (H-arom .); 4.08 dd, 1 H, J(1′a,2′) = 4.4, J(gem ) = 14.4 (H-1′a);
3.99 dd, 1 H, J(1′b,2′) = 7.0 (H-1′b); 3.86 dd, 1 H, J(3′a,2′) = 3.0, J(gem ) = 12.2 (H-3′a); 3.80 m ,
1 H (H-2′); 3.65 dd, 1 H, J(P,CHa) = 9.4, J(gem ) = 12.4 (PCHa); 3.61 dd, 1 H, J(3′b,2′) = 4.4
(H-3′b); 3.60 dd, 1 H, J(P,CHb) = 9.4 (PCHb). 13C NMR (D2O): 164.55 (C-4); 151.98 (C-2);
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Pyrimidine Acyclic Nucleotide Analogues
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146.82 (C-arom .); 146.54 (C-6); 139.19, 129.30, 2 C an d 123.72, 2 C (C-arom .); 79.62 d,
J(P,C) = 10.2 (C-2′); 65.50 d, J(P,C) = 156.0 (P-C); 60.41 (C-3′); 49.23 (C-1′).
5-(4-Chlorophenyl)-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]uracil (9c). Yield 44 m g
(40%) of free acid, wh ite am orph ous solid. [α]D –3.8 (c 0.325, H2O). Elution from HPLC
colum n : 15% CH3OH. FAB MS, m/z (%): 391 (15) [M + H]. HR MS (FAB): For C14H17ClN2O7P
[M + H] calculated: 391.0462; foun d: 391.0451. 1H NMR (D2O): 7.75 s, 1 H (H-6); 7.47 m , 4 H
(H-arom .); 4.07 dd, 1 H, J(1′a,2′) = 3.8, J(gem ) = 14.6 (H-1′a); 3.89 dd, 1 H, J(1′b,2′) = 7.7
(H-1′b); 3.85 dd, 1 H, J(3′a,2′) = 3.7, J(gem ) = 12.7 (H-3′a); 3.79 m , 1 H (H-2′); 3.75 dd, 1 H,
J(P,CHa) = 4.7, J(gem ) = 13.0 (PCHa); 3.66 dd, 1 H, J(P,CHb) = 9.2 (PCHb); 3.63 dd, 1 H,
J(3′b,2′) = 4.1 (H-3′b). 13C NMR (D2O): 164.88 (C-4); 151.90 (C-2); 145.29 (C-6); 133.35,
130.43, 130.01, 2 C an d 128.51, 2 C (C-arom .); 113.66 (C-5); 79.76 d, J(P,C) = 12.2 (C-2′);
66.22 d, J(P,C) = 158.2 (P-C); 60.18 (C-3′); 49.20 (C-1′).
5-(2-Chlorophenyl)-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]uracil (9d ). Yield 90 m g
(76%) of m on oam m on iu m salt, wh ite solid . [α ]D –24.7 (c 0.120, H2O). Elu tion from
HPLC colum n : 15% CH3OH. FAB MS, m/z (%): 413 (8) [M + Na] of free acid. For
C
14H19ClN3O7P·H2O (425.8) calculated: 39.49% C, 4.97% H, 8.33% Cl, 9.87% N, 7.27% P;
foun d: 39.19% C, 4.68% H, 8.18% Cl, 9.73% N, 6.90% P. 1H NMR (D2O): 7.78 s, 1 H (H-6);
7.56 m , 1 H an d 7.43 m , 3 H (H-arom .); 4.09 dd, 1 H, J(1′a,2′) = 4.0, J(gem ) = 14.4 (H-1′a);
3.92 dd, 1 H, J(1′b,2′) = 7.2 (H-1′b); 3.85 dd, 1 H, J(3′a,2′) = 3.8, J(gem ) = 12.1 (H-3′a); 3.82 m ,
1 H (H-2′); 3.77 dd, 1 H an d 3.71 dd, 1 H, J(P,CH) = 9.6, J(gem ) = 13.2 (PCH2); 3.64 dd, 1 H,
J(3′b,2′) = 3.9 (H-3′b). 13C NMR (D2O): 164.915 (C-4); 152.19 (C-2); 146.79 (C-6); 134.13,
132.28, 130.45, 130.39, 129.42 an d 127.29 (C-arom .); 113.34 (C-5); 79.79 d, J(P,C) = 11.7
(C-2′); 66.33 d, J(P,C) = 157.2 (P-C); 60.25 (C-3′); 49.12 (C-1′).
5-Ph en yl-1-[(R)-2-(ph osph on om eth oxy)propyl]cytosin e (18)
Th e syn th on 16 (530 m g, 1.34 m m ol) was added in several portion s durin g 5 h to a m ixture
of 2a (437 m g, 1.5 m m ol) an d cesium carbon ate (85 m g, 0.25 m m ol) in DMF (5 m l) stirred
at 120 °C. Addition al portion of 16 (530 m g, 1.34 m m ol) was added an d th e h eatin g con -
tin ued for 20 h. The m ixture was evaporated, the residue coevaporated with toluene (2 × 10 m l),
th en set aside for 48 h with 30% m eth an olic am m on ia (20 m l) at 4 °C an d evaporated. Th e
residue was stirred in aceton itrile (20 m l) with brom otrim eth ylsilan e (3 m l, 22.2 m m ol) at
room tem perature for 24 h , evaporated an d coevaporated with aceton itrile (5 m l). Th e m ix-
ture of eth an ol, water an d trieth ylam in e (10, 10 an d 2 m l) was added an d th e solution after
5 m in evaporated. Th e residue was dissolved in water (5 m l) an d desalted on Dowex 50, fol-
lowed by purification on Dowex 1 (th e sam e procedure as described for com poun ds 8a–8d ).
Elution from Dowex 1 was perform ed with 0.3 M acetic acid. Yield 234 m g (46%) of wh ite
solid. M.p. 289–293 °C. HR MS (FAB): For C14H19N3O5P [M + H] calculated: 340.1062; foun d:
340.1067. FAB MS, m/z (%): 340 (100) [M + H]. 1H NMR (D2O + NaOD): 7.63 s, 1 H (H-6);
7.52 m , 2 H an d 7.43 m , 3 H (H-arom .); 4.00 dd, 1 H, J(1′a,2′) = 6.0, J(gem ) = 13.6 (H-1′a);
3.92 m , 1 H (H-2′); 3.84 dd, 1 H, J(1′b,2′) = 5.2 (H-1′b); 3.84 dd, 1 H, J(PCHa) = 8.9, J(gem ) =
12.6 (PCHa); 3.52 dd, 1 H, J(P,CHb) = 9.3 (PCHb); 1.19 d, 3 H, J(3′,2′) = 6.2 (CH3). 13C NMR
(D2O + NaOD): 164.73 (C-4); 158.16 (C-2); 146.12 (C-6); 132.23, 129.37, 2 C, 129.32, 2 C
an d 128.49 (C-arom .); 75.54 d, J(P,C) = 10.3 (C-2′); 67.28 d, J(P,C) = 152.3 (P-C); 53.54
(C-1′); 16.67 (C-3′).
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944
Krečmerová, Holý, Masojídková:
5-Ph en yl-1-[(R)-2-(ph osph on om eth oxy)propyl]uracil (19)
Th e com poun d was prepared from cytosin e derivative 18 (150 m g, 0.44 m m ol) by th e sam e
procedure as described for 9a–9d . Yield 50 m g (33%) of wh ite am orph ous solid. [α]D +16.1
(c 0.222, H2O). Elution from HPLC colum n : 10% CH3OH. FAB MS, m/z (%): 341 (44) [M + H].
HR MS (FAB): For C14H18N2O6P [M + H] calculated: 341.0903; foun d: 341.0914. 1H NMR
(D2O): 7.75 s, 1 H (H-6); 7.51 m , 4 H an d 7.45 m , 1 H (H-arom .); 3.96 dd, 1 H, J(1′a,2′) =
3.4, J(gem ) = 13.9 (H-1′a); 3.90 m , 1 H (H-2′); 3.82 dd, 1 H, J(1′b,2′) = 7.2 (H-1′b); 3.71 dd,
1 H an d 3.56 dd, 1 H, J(P,CH) = 9.3, J(gem ) = 13.2 (PCH2); 1.23 d, 3 H, J(3′,2′) = 6.2 (H-3′).
13C NMR (D2O): 165.18 (C-4); 152.10 (C-2); 145.30 (C-6); 114.69 (C-5); 75.89 d, J(P,C) =
11.2 (C-2′); 65.00 d, J(P,C) = 158.7 (P-C); 52.59 (C-1′); 16.1 (C-3′).
Tran sform ation of 5-Substituted Uracils to 4-Meth oxy Derivatives 21a–21c
A suspen sion of a uracil derivative 20a–20c (10.45 m m ol) in POCl3 (6 m l, 64.5 m m ol) was
h eated with N,N-dim eth ylan ilin e (2 m l, 15.7 m m ol) at 110 °C for 3 h (till dissolution ).
POCl3 was distilled off, th e liquid residue poured on to ice an d th e product (dich loro
derivative) extracted with eth er (200 m l). Th e organ ic layer was extracted with cold water
(100 m l), followed by saturated solution of sodium h ydrogen carbon ate (2 × 100 m l), dried
with an h ydrou s sodiu m su lfate an d evaporated. Th e residu e was dissolved in m eth an ol
(5 m l), 1 M sodium m eth oxide (20 m l) was added, th e m ixture refluxed for 8 h an d th en set
aside at room tem perature for 12 h . Th e precipitated NaCl was filtered off, wash ed with
m eth an ol an d com bin ed filtrates were evaporated. Th e residue was partition ed between wa-
ter (40 m l) an d eth er (3 × 40 m l), th e com bin ed eth er extracts dried with an h ydrous m agn e-
sium sulfate an d evaporated. Th e liquid residue was stirred with acetyl ch loride (6 m l) at
room tem perature for 24 h . Acetyl ch loride was distilled off un der reduced pressure, th e resi-
due codistilled with toluen e (2 × 20 m l) an d th en dissolved in m eth an ol (5 m l). 1 M sodium
m eth oxide was added un til th e solution was alkalin e, th e m ixture was h eated at 40 °C for
1 h , th en n eutralized with acetic acid to pH 7 an d evaporated. A m ixture of aceton e–eth er
(1:1) was added, th e wh ite crystallin e m aterial filtered by suction , wash ed successively with
aceton e, water, aceton e an d fin ally with a m ixture aceton e–eth er (1:1) an d dried in vacuo.
5-Benzyl-4-methoxypyrimidin-2(1H)-one (21a ). Yield 1.7 g (76%), wh ite crystals. M.p.
207–209 °C. FAB MS, m/z (%): 217 (100) [M + H]. For C12H12N2O2 (216.2) calculated:
66.65% C, 5.59% H, 12.95% N; foun d: 66.15% C, 5.59% H, 12.59% N. 1H NMR (DMSO-d6):
11.20 brs, 1 H (NH); 7.51 s, 1 H (H-6); 7.30 m , 2 H an d 7.20 m , 3 H (H-arom .); 3.78 s, 3 H
(OCH3); 3.60 s, 2 H (CH2). 13C NMR (DMSO-d6): 170.46 (C-4); 156.34 (C-2); 143.42 (C-6);
139.68, 128.53, 4 C an d 126.31 (C-arom .); 106.02 (C-5); 53.90 (OCH3); 31.62 (CH2).
4-Methoxy-5-(1-naphthylmethyl)pyrimidin-2(1H)-one (21b). Yield 1.6 g (56%), wh ite crystals.
M.p. 231 °C. FAB MS, m/z (%): 267 (100) [M + H]. For C16H14N2O2·0.25H2O (270.8) calcu-
lated: 70.97% C, 5.40% H, 10.34% N; foun d: 71.01% C, 5.53% H, 10.01% N. 1H NMR
(DMSO-d6): 11.15 brs, 1 H (NH); 8.04 d, 1 H, 7.93 d, 1 H, 7.82 d, 1 H, 7.54 m , 2 H, 7.44 t, 1 H
an d 7.27 d, 1 H (H-arom .); 7.18 s, 1 H (H-6); 4.07 s, 2 H (CH2); 3.82 s, 3 H (OCH3). 13C NMR
(DMSO-d6): 170.52 (C-4); 156.29 (C-2); 143.43 (C-6); 134.90, 133.59, 131.48, 128.82, 127.22,
126.49, 126.47, 125.97, 125.86 an d 123.85 (C-arom .); 105.21 (C-5); 54.02 (OCH3); 28.57
(CH2).
4-Methoxy-5-(2-naphthylmethyl)pyrimidin-2(1H)-one (21c). Yield 2.2 g (78%), wh ite crystals.
M.p. 220–223 °C. FAB MS, m/z (%): 267 (20) [M + H]. For C16H14N2O2·0.25H2O (270.8) cal-
culated: 70.97% C, 5.40% H, 10.34% N; foun d: 71.03% C, 5.29% H, 10.29% N. 1H NMR
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Pyrimidine Acyclic Nucleotide Analogues
945
(DMSO-d6): 11.25 brs, 1 H (NH); 7.87–7.82 m , 3 H (H-arom .); 7.66 m , 1 H (H-arom .); 7.59 s,
1 H (H-6); 7.49–7.43 m , 2 H an d 7.38 dd, 1 H (H-arom .); 3.78 s, 5 H (OCH3, CH2). 13C NMR
(DMSO-d6): 170.50 (C-4); 156.36 (C-2); 143.65 (C-4); 137.33, 133.25, 131.86, 128.00, 127.63,
127.58, 127.34, 126.44, 126.24 an d 126.61 (C-arom .); 105.76 (C-5); 53.91 (OCH3); 31.78
(CH2).
Reaction of (2S)-2-[(Trityloxy)m eth yl]oxiran e (3) with Com poun ds 21a–21c.
Gen eral Procedure. Meth od A
Com poun d 3 (2.1 g, 6.7 m m ol) an d cesium carbon ate (220 m g, 0.68 m m ol) were added to a
stirred suspen sion of 4-m eth oxy derivative 21 (6.3 m m ol) in DMF (60 m l). Th e m ixture was
h eated at 80 °C un til th e con version was com plete (10–12 h , TLC ch eck). Th e m ixture was
cooled to room tem perature, evaporated an d th e residue coevaporated with toluen e (2 × 50 m l).
Th e crude product was purified by ch rom atograph y on silica gel (750 m l) in system toluen e–
eth yl acetate (1:1) con tain in g 1% trieth ylam in e.
5-Benzyl-1-[(S)-2-hydroxy-3-(trityloxy)propyl]-4-methoxypyrimidin-2(1H)-one (22a). Yield 2 g
(60%), wh ite foam . [α]D –16.5 (c 0.427, CHCl3). ESI MS, m/z (%): 555 (100) [M + Na], 1087
(86) [2 M – H + Na], 1088 (50) [2 M + Na]. For C34H32N2O4 (532.7) calculated: 76.67% C,
6.06% H, 5.26% N; foun d: 76.63% C, 6.11% H, 5.08% N. 1H NMR (DMSO-d6): 7.70 s, 1 H
(H-6); 7.41 d, 6 H, 7.33 t, 6 H, 7.26 m , 5 H an d 7.18 t, 3 H (H-arom .); 5.31d, 1 H, J(OH,2′) =
5.7 (OH); 4.09 dd, 1 H, J(1′a,2′) = 3.7, J(gem ) = 13.2 (H-1′a); 3.99 m , 1 H (H-2′); 3.78 s, 3 H
(OCH3); 3.60 d, 1 H an d 3.56 d, 1 H, J(gem ) = 15.4 (CH2); 3.53 dd, 1 H, J(1′b,2′) = 8.8
(H-1′b); 2.99 dd, 1 H, J(3′a,2′) = 5.3, J(gem ) = 9.4 (H-3′a); 2.90 dd, 1 H, J(3′b,2′) = 5.3 (H-3′b).
13C NMR (DMSO-d6): 169.715 (C-4); 155.54 (C-2); 148.54 (C-6); 143.91, 3 C (Tr); 139.61 an d
128.48, 2 C (Ph ); 128.45, 6 C (Tr); 128.38, 2 C (Ph ); 128.06, 6 C an d 127.19, 3 C (Tr); 126.95
(Ph ); 105.41 (C-5); 86.06 (Tr); 67.07 (C-2′); 66.30 (C-3′); 54.06 (OCH3); 53.28 (C-1′); 31.73
(CH2).
1-[(S)-2-Hydroxy-3-(trityloxy)propyl]-4-methoxy-5-(1-naphthylmethyl)pyrimidin-2(1H)-one (22b).
Yield 2.5 g (67%), wh ite crystals. M.p. 150 °C (toluen e). [α]D –27.6 (c 0.243, CHCl3). ESI MS,
m/z (%): 605 (100) [M + Na], 606 (36) [M + H + Na]. For C38H34N2O4 (582.7) calculated:
78.33% C, 5.88% H, 4.81% N; foun d: 77.94% C, 5.76% H, 4.59% N. 1H NMR (DMSO-d6):
8.08 d, 1 H, 7.93 d, 1 H an d 7.80 d, 1 H (H-arom ); 7.64 s, 1 H (H-6); 7.54 m , 2 H, 7.41 t, 1 H,
7.39 d, 6 H, 7.31 t, 6 H, 7.25 t, 3 H an d 7.18 d, 1 H (H-arom .); 5.23 d, 1 H, J(OH,2′) = 5.9
(OH); 4.07 dd, 1 H, J(1′a,2′) = 3.4, J(gem ) = 13.0 (H-1′a); 4.06 s, 2 H (CH2); 3.97 m , 1 H
(H-2′); 3.79 s, 3 H (OCH3); 3.47 dd, 1 H, J(1′b,2′) = 8.8 (H-1′b); 2.97 dd, 1 H an d 2.86 dd,
1 H, J(3′,2′) = 5.4, J(gem ) = 9.2 (H-3′). 13C NMR (DMSO-d6): 169.84 (C-4); 155.56 (C-2); 148.86
(C-6); 143.92, 3 C (Tr); 135.02, 133.52, 131.51 an d 128.74 (n aph th yl); 128.44, 6 C, 128.05,
6 C an d 127.19, 3 C (Tr); 127.09, 126.40, 125.98, 125.92, 125.75 an d 123.76 (1-n aph th yl);
104.50 (C-5); 86.06 (Tr); 67.08 (C-2′); 66.23 (C-3′); 54.12 (OCH3); 53.29 (C-1′); 28.57 (CH2).
1-[(S)-2-Hydroxy-3-(trityloxy)propyl]-4-methoxy-5-(2-naphthylmethyl)pyrimidin-2(1H)-one (22c).
Yield 2.6 g (72%), wh ite foam . [α ]D –25.0 (c 0.220, CHCl3). FAB MS, m/z (%): 605 (17)
[M + Na], 583 (2) [M + H], 243 (100) [trityl]. For C38H34N2O4 (582.7) calculated: 78.33% C,
5.88% H, 4.81% N; foun d: 78.25% C, 5.77% H, 4.67% N. 1H NMR (DMSO-d6): 7.88–7.80 m ,
4 H (H-6, H-arom .); 7.65 m , 1 H, 7.49–7.43 m , 2 H, 7.41 m , 6 H, 7.36 dd, 1 H, 7.33 m , 6 H
an d 7.25 m , 3 H (H-arom .); 5.32 d, 1 H, J(OH,2′) = 5.8 (OH); 4.12 dd, 1 H, J(1′a,2′) = 3.5,
J(gem ) = 13.1 (H-1′a); 4.02 m , 1 H (H-2′); 3.78 s, 3 H (OCH3); 3.76 m , 2 H (CH2); 3.55 dd,
1 H, J(1′b,2′) = 8.8, J(gem ) = 13.1 (H-1′b); 3.01 dd, 1 H, J(3′a,2′) = 5.2, J(gem ) = 9.4 (H-3′a);
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946
Krečmerová, Holý, Masojídková:
2.91 dd, 1 H, J(3′b,2′) = 5.4. 13C NMR (DMSO-d6): 169.79 (C-4); 155.59 (C-2); 148.78 (C-6);
143.92 (Tr); 137.24, 133.26 an d 131.91 (2-n aph th yl); 128.46 an d 128.07 (Tr); 127.98,
127.67, 127.62 an d 127.30 (2-n aph th yl); 127.20 (Tr); 126.44, 126.24 an d 125.63 (n aph th yl);
105.23 (C-5); 86.08 (Tr); 67.08 (C-2′); 66.33 (C-3′); 54.11 (OCH3); 53.36 (C-1′); 31.88 (CH2).
Reaction of (2S)-2-[(Trityloxy)m eth yl]oxiran e (3) with 21a. Meth od B
Th e sam e procedure as described for m eth od A but th e reaction was perform ed at 110 °C for
4 h . Th e ch rom atograph y afforded four fraction s in th e followin g elution order: 28, 27, 22a
(1.5 g, 45%) an d 5-ben zyluracil (153 m g, 12%).
5-Benzyl-1,3-bis[(2S)-2-hydroxy-3-(trityloxy)propyl]uracil (28). Yield 421 m g (8%), wh ite
foam . [α]D –12.4 (c 0.349, CHCl3). ESI MS, m/z (%): 857 (100) [M – H + Na], 858 (55) [M + Na].
For C55H50N2O6 (835.0) calculated: 79.11% C, 6.04% H, 3.35% N; foun d: 78.95% C, 5.99% H,
3.28% N. 1H NMR (DMSO-d6): 7.49 s, 1 H (H-6); 7.40–7.15 m , 35 H (H-arom .); 5.26 d, 1 H,
J = 5.6 an d 5.00 d, 1 H, J = 5.8 (OH); 3.87 dd, 1 H, J = 5.0 an d 13.2, 3.76 dd, 1 H, J = 6.1 an d
13.2, 3.37 dd, 1 H, J = 8.8 an d 13.2 an d 3.34 dd, 1 H, J = 8.3 an d 13.2 (H-1′); 3.98 m , 1 H
an d 3.88 m , 1 H (H-2′); 3.88 d, 2 H an d 3.85 d, 2 H, J(gem ) = 15.6 (CH2); 3.00 dd, 1 H, J =
5.5 an d 9.4, 2.99 dd, 1 H, J = 4.8 an d 9.4, 2.86 dd, 1 H, J = 5.6 an d 9.4 an d 2.80 dd, 1 H, J =
5.4 an d 9.4 (H-3′). 13C NMR (DMSO-d6): 162.97 (C-4); 151.17 (C-2); 144.00 an d 143.87 (Tr);
142.70 (C-6); 139.92 an d 128.56, 2 C (Ph ); 128.42, 12 C (Tr); 128.33, 2 C (Ph ); 128.06, 6 C,
127.93, 6 C, 127.20, 3 C an d 127.08, 3 C (Tr); 126.15 (Ph ); 110.57 (C-5); 86.12 an d 86.11
(Tr); 67.37 an d 66.78 (C-2′); 67.27 an d 66.14 (C-3′); 52.74 an d 44.53 (C-1′); 32.65 (CH2).
5-Benzyl-1-[(S)-2-hydroxy-3-(trityloxy)propyl]uracil (27). Yield 849 m g (26%), wh ite foam .
[α]D –27.4 (c 0.460, CHCl3). For C33H30N2O4 (518.6) calculated: 76.43% C, 5.83% H, 5.40% N;
foun d: 76.89% C, 6.05% H, 5.01% N. 1H NMR (DMSO-d6): 7.57 s, 1 H (H-6); 7.40 d, 6 H,
7.32 t, 6 H an d 7.28–7.14 m , 8 H (H-arom .); 5.31 d, 1 H, J(OH,2′) = 5.4 (OH); 4.03 dd, 1 H,
J(1′a,2′) = 3.9, J(gem ) = 13.4 (H-1′a); 3.95 m , 1 H (H-2′); 3.51 dd, 1 H, J(1′b,2′) = 8.8 (H-1′b);
3.13 s, 2 H (CH2); 3.00 dd, 1 H, J(3′a,2′) = 5.1, J(gem ) = 9.5 (H-3′a); 2.87 dd, 1 H, J(3′b,2′) =
5.6 (H-3′b). 13C NMR (DMSO-d6): 162.97 (C-4); 151.19 (C-2); 142.545 (C-6); 143.955, 3 C
(Tr); 139.96 an d 128.62, 2 C (Ph ); 128.43, 6 C (Tr); 128.34, 2 C (Ph ); 128.08, 6 C an d 127.22,
3 C (Tr); 126.18 (Ph ); 110.48 (C-5); 86.13 (Tr); 67.34 (C-2′); 66.175 (C-3′); 52.72 (C-1′).
Ph osph on ate Esters 23a–23c. Gen eral Procedure
A solution of appropriate h ydroxy derivative 22 (2.5 m m ol) an d 5 (3 m m ol) in tetra-
h ydrofuran (30 m l) was cooled to –20 °C an d 60% oil dispersion of NaH (150 m g, 3.7 m m ol)
was added. Th e m ixture was stirred at –20 °C for 15 m in , th en at room tem perature for 7 h
an d filtered th rough Celite. Th e filtrate was evaporated, th e residue coevaporated with tolu-
en e an d ch rom atograph ed on a colum n of silica gel (200 m l) in th e system described below.
5 -Ben zyl-1 -{(S)-2 -[(diisopropoxyph osph oryl)m eth oxy]-3 -(trityloxy)propyl}-4 -m eth oxy-
pyrimidin-2(1H)-one (23a). Ch rom atograph y in system ch loroform –m eth an ol–trieth ylam in e
(100:5:1). Yield 1.12 g (63%), wh ite foam . [α]D –20.0 (c 0.178, CHCl3). For C41H47N2O7P
(710.8) calculated: 69.28% C, 6.66% H, 3.94% N, 4.36% P; foun d: 69.29% C, 6.67% H,
3.82% N, 4.40% P. ESI MS, m/z (%): 733.3 (100) [M + Na], 243.3 (14) [trityl]. 1H NMR
(DMSO-d6): 7.78 s, 1 H (H-6); 7.39 d, 6 H, 7.33 t, 6 H, 7.26 m , 6 H an d 7.18 t, 2 H
(H-arom .); 3.98 dd, 1 H, J(1′a,2′) = 2.8, J(gem ) = 11.8 (H-1′a); 3.94 m , 1 H (H-2′); 3.90 dd, 1 H,
J(1′b,2′) = 7.8 (H-1′b); 3.76 s, 3 H (OCH3); 3.76 dd, 1 H an d 3.68 dd, 1 H, J(P,CH) = 9.5,
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Pyrimidine Acyclic Nucleotide Analogues
947
J(gem ) = 13.6 (PCH2); 3.55 s, 2 H (CH2); 3.22 dd, 1 H, J(3′a,2′) = 2.8, J(gem ) = 10.6 (H-3′a);
2.96 dd, 1 H, J(3′b,2′) = 4.0 (H-3′b). 13C NMR (DMSO-d6): 169.785 (C-4); 155.44 (C-2);
148.05 (C-6); 143.675, 3 C (Tr); 139.45, 128.48, 2 C an d 128.47, 2 C (Ph ); 128.43, 6 C,
128.11, 6 C an d 127.29, 3 C (Tr); 126.32 (Ph ); 105.89 (C-5); 86.37 (Tr); 78.16 d, J(P,C) = 12.7
(C-2′); 70.35 d, 2 C, J(P,C) = 6.3 (P-O-C); 64.06 d, J(P,C) = 165.0 (P-C); 62.95 (C-3′); 54.12
(OCH3); 50.52 (C-1′); 31.87 (CH2); 23.95 d an d 23.93 d, J(P,C) = 3.9 (CH3); 23.89 d an d
23.78 d, J(P,C) = 4.9 (CH3).
1-{(S)-2-[(Diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-4-methoxy-5 -(1 -n a ph t h yl-
m eth yl)pyrim idin -2 (1 H)-on e (2 3 b ). Ch rom atograph y in system ch loroform –m eth an ol–
trieth ylam in e (100:2:1). Yield 1.5 g (81%), wh ite foam . [α]D –17.1 (c 0.253, CHCl3). For
C
45H49N2O7P (760.9) calculated: 71.04% C, 6.49% H, 3.68% N, 4.07% P; foun d: 70.79% C,
6.59% H, 3.47% N, 4.14% P. FAB MS, m/z (%): 783.7 (20) [M + Na], 243.3 (100) [trityl].
1H NMR (DMSO-d6): 8.07 d, 1 H, 7.94 d, 1 H an d 7.80 d, 1 H (H-arom .); 7.66 s, 1 H (H-6);
7.54 m , 2 H, 7.38 t, 1 H, 7.36 d, 6 H, 7.31 t, 6 H, 7.25 t, 3 H an d 7.13 d, 1 H (H-arom .); 4.52 d
sept, 2 H, J(CH,CH3) = 6.2, J(P,OCH) = 7.7 (P-OCH); 4.04 d, 1 H an d 4.00 d, 1 H, J(gem ) =
16.4 (CH2); 3.95 dd, 1 H, J(1′a,2′) = 4.0, J(gem ) = 12.2 (H-1′a); 3.92 m , 1 H (H-2′); 3.86 dd,
1 H, J(1′b,2′) = 6.5 (H-1′b); 3.72 dd, 1 H, J(P,CH) = 8.7, J(gem ) = 13.7 (PCHa); 3.65 dd, 1 H,
J(P,CHb) = 9.2 (PCHb); 3.17 dd, 1 H, J(3′a,2′) = 2.6, J(gem ) = 10.8 (H-3′a); 2.96 dd, 1 H,
J(3′b,2′) = 4.4 (H-3′b); 1.18 d, 6 H, 1.14 d, 3 H and 1.13 d, 3 H, J(CH3,CH) = 6.2 (CH3). 13C NMR
(DMSO-d6): 169.88 (C-4); 155.44 (C-2); 148.36 (C-6); 143.65, 3 C (Tr); 134.90, 133.52,
131.435 an d 128.79 (1-n aph th yl); 128.41, 6 C, 128.09, 6 C an d 127.27, 3 C (Tr); 127.07,
126.43, 125.91, 2 C, 125.76 an d 123.58 (1-n aph th yl); 104.93 (C-5); 86.38 (Tr); 78.22 d,
J(P,C) = 11.2 (C-2′); 70.40 d, 2 C, J(P,C) = 6.4 (P-O-C); 64.12 d, J(P,C) = 161.1 (P-C); 63.05
(C-3′); 50.45 (C-1′); 23.94 d, 23.90 d, 23.84 d an d 23.78 d, J(P,C) = 3.9 (CH3).
1-{(S)-2-[(Diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-4-methoxy-5-(2-naphthyl-
methyl)pyrimidin-2(1H)-one (23c). Ch rom atograph y in system ch loroform –m eth an ol–
trieth ylam in e (100:2:1). Yield 1.1 g (58%), wh ite foam . [α]D –18.4 (c 0.217, CHCl3). For
C
45H49N2O7P (760.9) calculated: 71.04% C, 6.49% H, 3.68% N, 4.07% P; foun d: 70.51% C,
6.72% H, 3.52% N, 4.27% P. ESI MS, m/z (%): 1542.9 (43) [2 M + Na], 783.3 (100) [M + Na],
243.2 (21) [trityl]. 1H NMR (DMSO-d6): 7.87–7.79 m , 3 H (H-arom .); 7.83 s, 1 H (H-6); 7.65 m ,
1 H, 7.49–7.43 m , 2 H, 7.39 m , 6 H, 7.36 dd, 1 H, 7.32 m , 6 H an d 7.25 m , 3 H (H-arom .);
4.53 dh , 2 H, J(CH,CH3) = 6.2, J(CH,P) = 7.7 (P-OCH); 4.01 dd, 1 H, J(1′a,2′) = 3.3, J(gem ) =
12.1 (H-1′a); 3.97–3.89 m , 2 H (H-1′b, H-2′); 3.76 s, 3 H (OCH3); 3.80–3.67 m , 4 H (PCH2,
CH2-2-n aph th yl); 3.22 dd, 1 H, J(3′a,2′) = 3.3, J(gem ) = 10.6 (H-3′a); 2.99 dd, 1 H, J(3′b,2′) =
4.3 (H-3′b); 1.18 d, 3 H, 1.17 d, 3 H, 1.15 d, 3 H an d 1.13 d, 3 H (CH3). 13C NMR (DMSO-d6):
169.84 (C-4); 155.47 (C-2); 148.20 (C-6); 143.66, 3 C (Tr); 137.05, 133.26 and 131.91 (naphthyl);
128.42, 6 C an d 128.10, 6 C (Tr); 127.97, 127.65, 2 C an d 127.31 (2-n aph th yl); 127.28, 3 C
(Tr); 126.48, 126.18 an d 125.61 (2-n aph th yl); 105.72 (C-5); 86.38 (Tr); 78.20 d, J(C,P) = 12.4
(C-2′); 70.35 d, 2 C, J(C,P) = 6.2 (P-O-C); 64.09 d, J(C-P) = 165.3 (P-C); 63.00 (C-3′); 54.14
(OCH3); 50.64 (C-1′); 32.09 (CH2); 23.95–23.75 m , 4 C (CH3).
Cytosin e Derivatives 26a–26c. Gen eral Procedure
A solution of appropriate 4-m eth oxy derivative 23a–23c (0.74 m m ol) in 30% m eth an olic
am m on ia (40 m l) was h eated in an autoclave at 110 °C for 14 h . Th e solution was cooled to
room tem perature, evaporated an d th e residue ch rom atograph ed on silica gel colum n (100 m l)
in system ch loroform –m eth an ol 9:1.
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948
Krečmerová, Holý, Masojídková:
5-Benzyl-1-{(S)-2-[(diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}cytosine (26a). Yield
275 m g (53%) of a wh ite foam . [α]D –26.7 (c 0.238, CHCl3). For C40H46N3O6P (695.8)
calculated: 69.05% C, 6.66% H, 6.04% N, 4.45% P; foun d: 68.78% C, 6.55% H, 5.86% N,
4.48% P. ESI MS, m/z (%): 696.8 (41) [M + H], 243.3 (49) [trityl]. 1H NMR (CDCl3): 7.43 m ,
6 H an d 7.32–7.15 m , 15 H (H-6, H-arom .); 4.69 m , 2 H (P-OCH); 4.20 dd, 1 H, J(1′a,2′) =
4.0, J(gem ) = 13.6 (H-1′a); 3.92 m , 1 H (H-2′); 3.87 dd, 1 H, J(P,CHa) = 8.9, J(gem ) = 13.4
(PCHa); 3.75 dd, 1 H, J(1′b,2′) = 7.9 (H-1′b); 3.63 dd, 1 H, J(P,CHb) = 10.0 (PCHb); 3.62 d, 1 H
and 3.54 d, 1 H, J(gem ) = 16.4 (CH2); 3.38 dd, 1 H, J(3′a,2′) = 3.4, J(gem ) = 10.6 (H-3′a); 3.10 dd,
1 H, J(3′b,2′) = 4.1 (H-3′ b); 1.29 d, 6 H, 1.26 d, 3 H an d 1.245 d, 3 H, J(CH3,CH) = 6.2
(CH3). 13C NMR (CDCl3): 165.29 (C-4); 156.44 (C-2); 146.24 (C-6); 143.53, 3 C (Tr); 136.88
an d 129.05, 2 C (Ph ); 128.58, 6 C (Tr); 128.12, 2 C (Ph ); 127.90, 6 C (Tr); 127.19 (Ph );
127.13, 3 C (Tr); 103.42 (C-5); 86.75 (Tr); 79.25 d, J(P,C) = 13.7 (C-2′); 71.00 d, J(P,C) = 6.3
an d 70.81 d, J(P,C) = 6.3 (P-O-C); 65.02 d, J(P,C) = 169.4 (PCH2); 62.59 (C-3′); 51.24 (C-1′);
33.94 (CH2); 24.09 d an d 24.06 d, J(P,C) = 3.9 (CH3); 24.03 d an d 23.97 d, J(P,C) = 4.9
(CH3).
1-{(S)-2-[(Diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-5-(1-naphthylmethyl)cytosine
(26b). Yield 359 m g (65%) of a wh ite foam . [α]D –16.4 (c 0.520, CHCl3). For C44H48N3O6P
(745.9) calculated: 70.86% C, 6.49% H, 5.63% N, 4.15% P; foun d: 70.51% C, 6.56% H,
5.36% N, 4.25% P. ESI MS, m/z (%): 1513.1 (100) [2 M + Na], 768.4 (56) [M + Na], 243.2 (16)
[trityl]. 1H NMR (DMSO-d6): 7.95 m , 4 H, 7.81 d, 2 H, 7.55 m , 2 H, 7.38 t, 1 H, 7.36 d, 6 H,
7.30 t, 6 H, 7.25 t, 3 H an d 7.08 d, 2 H (H-arom .); 7.06 s, 1 H (H-6); 4.52 m , 2 H (P-OCH);
4.00 d, 1 H an d 3.95 d, 1 H, J(gem ) = 17.1 (CH2); 3.85 m , 1 H (H-2′); 3.77 dd, 1 H, J(1′a,2′) =
5.1, J(gem ) = 13.6 (H-1′a); 3.69 dd, 1 H, J(P,CH) = 9.0, J(gem ) = 13.8 (PCHa); 3.65 dd, 1 H,
J(1′b,2′) = 5.5 (H-1′b); 3.63 dd, 1 H (PCHb); 3.11 dd, 1 H, J(3′a,2′) = 3.4, J(gem ) = 10.4
(H-3′a); 2.89 dd, 1 H, J(3′b,2′) = 4.8 (H-3′b); 1.18 d, 3 H, 1.175 d, 3 H, 1.142 d, 3 H an d
1.14 d, 3 H, J(CH3,CH) = 6.2. 13C NMR (DMSO-d6): 165.39 (C-4); 155.72 (C-2); 145.76 (C-6);
143.67, 3 C (Tr); 134.61, 133.53, 131.64 an d 128.68 (1-n aph th yl); 128.37, 6 C, 128.03, 6 C
an d 127.19, 3 C (Tr); 127.03, 126.25, 125.87, 125.73, 125.53 an d 123.79 (1-n aph th yl);
102.78 (C-5); 86.28 (Tr); 78.65 d, J(P,C) = 11.7 (C-2′); 70.30 d, 2 C, J(P,C) = 6.3 (P-O-C);
64.23 d, J(P,C) = 165.0 (P-C); 63.16 (C-3′); 50.00 (C-1′); 29.60 (CH2); 23.93 d, J(P,C) = 3.9,
23.83 d, J(P,C) = 4.4 an d 23.78 d, 2 C, J(P,C) = 4.9 (CH3).
1-{(S)-2-[(Diisopropoxyphosphoryl)methoxy]-3-(trityloxy)propyl}-5-(2-naphthylmethyl)cytosine
(26c). Yield 353 m g (64%) of a wh ite foam . [α]D –20.5 (c 0.235, CHCl3). For C44H48N3O6P
(745.9) calculated: 70.86% C, 6.49% H, 5.63% N, 4.15% P; foun d: 69.93% C, 6.54% H,
5.77% N, 3.98% P. ESI MS, m/z (%): 1491.2 (71) [2 M + H], 768.3 (100) [M + Na], 243.2 (65)
[trityl]. 1H NMR (DMSO-d6): 7.89–7.78 m , 3 H, 7.69 bs, 1 H, 7.50–7.44 m , 2 H an d 7.39 m , 7 H
(H-arom .); 7.36 s, 1 H (H-6); 7.31 m , 6 H an d 7.25 m , 3 H (H-arom .); 4.54 m , 2 H (P-OCH);
3.93–3.83 m , 2 H (H-1′a, H-2′); 3.79–3.65 m , 5 H (H-1′b, PCH2, CH2-2-n aph th yl); 3.17 dd, 1 H,
J(3′a,2′) = 2.7, J(gem ) = 10.5 (H-3′a); 2.93 dd, 1 H, J(3′b,2′) = 4.5 (H-3′b); 1.19 d, 3 H, 1.18 d,
3 H, 1.16 d, 3 H an d 1.15 d, 3 H, J(CH3,CH) = 6.4 (CH3). 13C NMR (DMSO-d6): 165.19 (C-4);
155.74 (C-2); 145.80 (C-6); 143.70 (Tr); 136.67, 133.20 an d 131.94 (2-n aph th yl); 128.41 an d
128.07 (Tr); 127.94, 127.65, 127.59 an d 127.39 (2-n aph th yl); 127.23 (Tr); 126.52, 126.21
an d 125.63 (2-n aph th yl); 103.71 (C-5); 86.30 (Tr); 78.71 d, J(C,P) = 12.5 (C-2′); 70.36 d, 2 C,
J(C,P) = 6.3 (P-O-C); 64.24 d, J(C,P) = 165.5 (P-C); 63.17 (C-3′); 50.18 (C-1′); 32.53 (CH2);
23.98–23.79 m , 4 C (CH3).
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Pyrimidine Acyclic Nucleotide Analogues
949
Ph osph on ic Acids 24a–24c. Gen eral Procedure
A solution of appropriate com poun d 23 (1.04 m m ol) in aceton itrile (10 m l) was set aside
with brom otrim eth ylsilan e (1.3 m l, 10 m m ol) in th e dark at room tem perature for 24 h . Th e
solution was evaporated, th e residue coevaporated with aceton itrile (10 m l), stirred with a
m ixture eth an ol–water–trieth ylam in e 15:15:1 (31 m l) for 5 m in an d evaporated. Th e residue
was partition ed between water (50 m l) an d eth er (50 m l), th e aqueous layer was con cen -
trated to ca. 1/3 volum e an d applied on to a colum n of DEAE Seph adex A-25 (Cl– form , acti-
vated with 0.02 M trieth ylam m on ium h ydrogen carbon ate; 10 m l). Elution was perform ed
with water (200 m l), followed by lin ear gradien t of trieth ylam m on ium h ydrogen carbon ate
(0.02–0.05 M, 500 m l). Th e UV-absorbin g fraction eluted with 0.3 M buffer con cen tration
was evaporated, codistilled with water (3 × 50 m l) an d deion ized on activated ch arcoal.
Fin al products were lyoph ilized as free ph osph on ic acids or tran sform ed to dilith ium salts
on Dowex 50 (Li+ form ) an d lyoph ilized. Th e followin g com poun ds were prepared:
5-Benzyl-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]uracil (24a). Isolated as dilith ium salt.
Yield 347 m g (80%), white am orphous solid. [α]D –3.6 (c 0.311, H2O). For C15H17Li2N2O7P·2H2O
(418.19) calculated: 43.08% C, 5.06% H, 6.70% N, 7.41% P; foun d: 43.26% C, 4.84% H,
6.63% N, 7.33% P. HR MS (TOF ES+): For C15H18Li2N2O7P [M + H] calculated: 383.1172;
foun d: 383.1166. ESI MS, m/z (%): 383.4 (100) [M + H]. 1H NMR (D2O): 7.56 s, 1 H (H-6);
7.37 m , 2 H an d 7.31 m , 3 H (H-arom .); 3.95 dd, 1 H, J(1′a,2′) = 4.9, J(gem ) = 14.3 (H-1′a);
3.87 dd, 1 H, J(1′b,2′) = 6.7 (H-1′b); 3.79 dd, 1 H, J(3′a,2′) = 3.4, J(gem ) = 12.2 (H-3′a); 3.74 m ,
1 H (H-2′); 3.67 s, 2 H (CH2); 3.66 dd, 1 H an d 3.63 dd, 1 H, J(P,CH) = 9.8, J(gem ) = 12.8
(PCH2); 3.57 dd, 1 H, J(3′b,2′) = 4.6 (H-3′b). 13C NMR (D2O): 166.065 (C-4); 152.305 (C-2);
144.95 (C-6); 138.97, 128.69, 2 C, 128.42, 2 C an d 126.55 (C-arom .); 113.75 (C-5); 79.76 d,
J(P,C) = 11.2 (C-2′); 66.93 d, J(P,C) = 154.8 (P-C); 60.39 (C-3′); 48.51 (C-1′); 31.81 (CH2).
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-(1-naphthylmethyl)uracil (24b ). Isolated as
free acid. Yield 188 m g (43%), wh ite am orph ous solid. [α]D was n ot determ in ed (opalescen t
solution , H2O). HR MS (FAB): For C19H22N2O7P [M + H] calculated: 421.1165; foun d:
421.1170. FAB MS, m/z (%): 443 (10) [M + Na], 421 (5) [M + H]. 1H NMR (D2O): 7.82 d, 1 H,
7.57 d, 1 H, 7.48 d, 1 H, 7.37 t, 1 H, 7.28 t, 2 H an d 7.24 d, 1 H (H-arom .); 6.76 s, 1 H (H-6);
3.84 d, 1 H an d 3.76 d, 1 H, J(gem ) = 16.6 (CH2); 3.75 dd, 1 H, J(1′a,2′) = 5.6, J(gem ) = 13.2
(H-1′a); 3.60 dd, 1 H an d 3.55 dd, 1 H, J(P,CH) = 9.0, J(gem ) = 13.6 (PCH2); 3.50 m , 1 H
(H-2′); 3.38 m , 1 H (H-1′b); 3.38 m , 1 H an d 3.31 m , 1 H (H-3′). 13C NMR (D2O): 165.31
(C-4); 151.65 (C-2); 144.21 (C-6); 134.16, 133.48, 131.26, 128.58, 127.22, 126.80, 126.28,
125.82, 125.70 an d 123.62 (C-arom .); 112.94 (C-5); 79.64, J(P,C) = 8.3 (C-2′); 65.02 d, J(P,C) =
161.1 (P-C); 60.14 (C-3′); 48.34 (C-1′); 37.71 (CH2).
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-[(2-naphthyl)methyl]uracil (24c). Isolated as
free acid. Yield 214 m g (49%), yellowish solid. [α]D –11.4 (c 0.129, H2O). HR MS (FAB): For
C
19H22N2O7P [M + H] calculated: 421.1165; foun d: 421.1176. FAB MS, m/z (%): 421 (20)
[M + H]. 1H NMR (D2O): 7.78–7.66 m , 3 H, 7.60 bs, 1 H an d 7.41–7.31 m , 3 H (H-arom .);
7.29 s, 1 H (H-6); 3.80–3.73 m , 2 H (H-1′a, PCHa); 3.70 dd, 1 H, J(3′a,2′) = 3.5, J(gem ) = 12.3
(H-3′a); 3.68–3.63 m , 4 H (CH2, PCHb, H-2′); 3.61 m , 1 H (H-1′b); 3.50 dd, 1 H, J(3′b,2′) =
4.2, J(gem ) = 12.3 (H-3′b). 13C NMR (D2O): 166.47 (C-4); 152.69 (C-2); 145.43 (C-6); 137.21,
133.79,132.47, 128.78, 128.16, 128.08, 127.87, 127.24, 126.96 an d 126.38 (C-arom .); 114.10
(C-5); 80.52 d, J(C,P) = 11.1 (C-2′); 66.03 d, J(C,P) = 159.3 (P-C); 60.84 (C-3′); 49.42 (C-1′);
32.60 (CH2).
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950
Krečmerová, Holý, Masojídková:
This study was performed as a research project of the Institute of Organic Chemistry and
Biochemistry, Academy of Sciences of the Czech Republic (Z4 055 0506) and the Centre for New
Antivirals and Antineoplastics (1M0508). We gratefully acknowledge the financial support of the
Program of targeted projects of Academy of Sciences of the Czech Republic (1QS400550501), the
European Commission (René Descartes Prize-2001, grant No. HPAW-2002-100096) and Gilead
Sciences, Inc. (Foster City, CA, U.S.A.). The authors’ thanks are due to Professor Erik De Clercq and
his group in the Rega Institute for Medical Research, Catholic University Leuven (Belgium) for
antiviral screening, Dr I. Votruba from the Institute of Organic Chemistry and Biochemistry for testing
inhibitory activity of compounds against thymidine phosphorylase and cytostatic activity, the
staff of the ma ss spectrometry (Dr J. Cvačka, Hea d), a na lytica l depa rtments of the Institute
(Dr L. Holasová, Head) and Dr M. Dračinský for measurement and interpretation of some NMR
spectra (compounds 24–26) . An excellent technical assistance of Mrs J. Křelinová is also
acknowledged.
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