Preparation of C-5 substituted cidofovir derivatives

Article abstract of DOI:10.1135/cccc20060579

1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir) was modified by substitution on the base moiety in positions C-S and N⁴. Key intermediates of these syntheses, diisopropyl esters of (S)-1-[2-(phosphonomethoxy)-3-(tripheny

Full text of DOI:10.1135/cccc20060579

C-5 Substituted Cidofovir Derivatives
579
PREPARATION OF C-5 SUBSTITUTED CIDOFOVIR DERIVATIVES
1,
2
3
Marcela KREČMEROVÁ *, Milena MASOJÍDKOVÁ and Antonín HOLÝ
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
2
166 10 Prague 6, Czech Republic; e-mail: marcela@uochb.cas.cz, masojid@uochb.cas.cz,
holy@uochb.cas.cz
3
Received Jan uary 9, 2006
Accepted March 10, 2006
1-[(S)-3-Hydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (HPMPC, cidofovir) was m odified
by substitution on th e base m oiety in position s C-5 an d N4. Key in term ediates of th ese syn -
th eses, diisopropyl esters of (S)-1-[2-(ph osph on om eth oxy)-3-(triph en ylm eth oxy)propyl]-
5-alkylcytosin es (6 an d 7) prepared from 5-alkyl-4-m eth oxypyrim idin -2(1H)-on es were tran s-
form ed to th e correspon din g 5-substituted cytosin e or N4-alkylcytosin e derivatives by th e ac-
tion of am m on ia or prim ary am in es, respectively. Th ese fully protected ph osph on ate esters
gave by treatm en t with brom otrim eth ylsilan e followed by h ydrolysis free ph osph on ic acids:
1-[(S)-3-h ydroxy-2-(ph osph on om eth oxy)propyl]-5-m eth ylcytosin e (5-m eth yl-HPMPC, 10),
5-eth yl-1-[(S)-3-h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (5-eth yl-HPMPC, 11) an d
a series of 5-eth yl-HPMPC an alogues 17–21 bearin g various substituen ts in N4 position
(cyclopropyl, cyclopen tyl, 2-h ydroxyeth yl, allyl, 2-(dim eth ylam in o)eth yl). 5-Eth yn yl-1-[(S)-
3-h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (5-eth yn yl-HPMPC, 26) was prepared
from 5-iodocytosin e derivative 23 usin g Son ogash ira couplin g with (trim eth ylsilyl)acetylen e,
CuI an d [PdCl2(Ph 3P)2]. Non e of th e prepared com poun ds exh ibited an tiviral activity in vitro.
Keyw ord s: Acyclic n ucleotide an alogues; Ph osph on ates; Pyrim idin es; Nucleosides; 5-Alkyl-
cytosin es; An tivirals; HPMPC; Son ogash ira cross-couplin g.
Cidofovir (HPMPC, CDV, (S)-1-[3-h ydroxy-2-(ph osph on om eth oxy)propyl]-
cytosin e)1 is a powerful an tiviral agen t active again st all types of DNA
viruses. Several reviews refer to its activity2–5, m ech an ism of action 6, ph ar-
m acology7,8 an d th erapeutic application s9,10. As th e system ic drug Vistide
it is approved for in traven ous treatm en t of cytom egalovirus retin itis11 in
AIDS patien ts, but at th e presen t tim e, it is clin ically im portan t particularly
in severe cases of (m align izin g) papillom atoses (an ogen ital, laryn geal)12,
progressive m ultifocal leukoen ceph alopath y13, aden ovirus in fection s14 an d
som e rath er obscure severe in fection s caused by poxviruses (vaccin ia15, orf,
m olluscum con tagiosum )16. Th e attractivity of cidofovir is dram atically en -
h an ced by its suprem e activity again st sm allpox virus17an d related m on key-
pox virus18.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 4, pp. 579–594
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20060579

580
Krečmerová, Masojídková, Holý:
NH2
N
N
O
OH
O
P(O)(OH)2
(S)-HPMPC (cidofovir)
Cidofovir is n ot yet available for clin ical use in an oral prodrug form , it is
curren tly available on ly for in traven ous use as a solution ; th e testin g of
cidofovir in th e form of aerosols or eye drops is still un der way in an im al
m odels. Orally bioavailable cidofovir prodrugs are un der preclin ical in vesti-
gation . In th is area th e m ain atten tion was paid to in vestigation of n eutral
ester prodrugs19 an d m odification of th e ph osph on ate-bearin g side ch ain 20
.
Th e side effect of cidofovir, its n eph rotoxicity, is also th e lim itin g factor in
its widespread use. Th ese facts substan tiate our effort in developin g addi-
tion al cidofovir derivatives with poten tially im proved ph arm acological
properties. In con trast to m odification s of th e ph osph on ate-bearin g side
ch ain , th e m eth odology of cytosin e m oiety m odification in cidofovir is n ot
worked out in detail. Recen tly we publish ed two papers dealin g with prepa-
ration of its N4-substituted derivatives21,22 an d at presen t tim e we deal sys-
tem atically with in vestigation of th e in fluen ce of 4-am in o group substitu-
tion of cytosin e in acyclic n ucleotide an alogues on th eir an tiviral activity.
Im portan t m odification of pyrim idin e n ucleosides an d/or n ucleobases
gen erally, is th eir substitution in position C-5. Man y com poun ds of th is
group are efficien t virostatics, especially 2′-deoxyuridin es with th e follow-
in g substituen ts: eth yl (aedurid), brom ovin yl (BVDU), trifluorom eth yl
(trifluridin ), vin yl, eth yn yl an d also th eir 2′-deoxycytidin e coun terparts23.
Som e 5-substituted n ucleobases are used as an tican cer drugs, e.g. 5-fluoro-
uracil (5-FU). 5-Eth yn yluracil24 (en iluracil) is utilized as accom pan yin g sub-
stan ce with 5-fluorouracil. 5-Fluorouracil itself is easily decom posed by
en zym e dih ydropyrim idin e deh ydrogen ase (DPD). En iluracil is a poten t,
suicide substrate for DPD, becom es covalen tly lin ked to th e en zym e25 an d
so en ables also oral adm in istration of 5-FU. In th e field of acyclic n ucleo-
tide an alogues (ANPs), an tiviral activity was foun d at som e 5-substituted
2,4-diam in o-6-[2-(ph osph on om eth oxy)eth oxy]pyrim idin es26. Oth er types
of 5-alkylated pyrim idin e ANPs were n ot system atically studied.
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C-5 Substituted Cidofovir Derivatives
581
Th ese facts substan tiate our effort to prepare a series of cidofovir an a-
logues bearin g various substituen ts in C-5 position an d also “double substi-
tuted” an alogues, berin g substituen ts in C-5 an d N4 position s sim ultan eous-
ly an d to study th e in fluen ce of such substitution to biological activity with
com parison with th e m oth er com poun d, HPMPC (cidofovir).
RESULTS AND DISCUSSION
Th e first approach to substituted cytosin e derivatives in cludin g C-5 substi-
tuted HPMPC exploits 5-alkyl-4-m eth oxypyrim idin -2(1H)-on es (1 an d 2)
wh ich are accessible on large scale by th ree-step syn th esis from th e corre-
spon din g 5-alkyluracils (th ym in e, 5-eth yluracil).
Th is procedure, described origin ally for uracil an d th ym in e, in cludes th e
treatm en t with POCl3 givin g 2,4-dich loropyrim idin e, reaction with sodium
m eth oxide leadin g to 2,4-dim eth oxypyrim idin e an d its fin al tran sform a-
tion to 4-m eth oxypyrim idin -2(1H)-on e by th e action of acetyl ch loride fol-
lowed by sodium m eth oxide27. We used th is m eth od for tran sform ation of
5-eth yluracil to 5-eth yl-4-m eth oxypyrim idin -2(1H)-on e (2). Th e startin g
5-eth yluracil was syn th esized from eth yl butyrate accordin g to a gen eral
procedure for preparation of 5-alkyluracils (form ylation of carboxylic esters,
followed by cyclization reaction with th iourea an d subsequen t h ydrolysis
with trich loroacetic acid)28.
Reaction of 4-m eth oxypyrim idin -2(1H)-on es 1 an d 2 with (2S)-2-[(trityl-
oxy)m eth yl]oxiran e (3) perform ed in DMF an d catalyzed with cesium
carbon ate afforded (S)-5-alkyl-1-[2-h ydroxy-3-(triph en ylm eth oxy)propyl]-
4-m eth oxypyrim idin -2(1H)-on es 4 an d 5 wh ich were subsequen tly treated
with diisopropyl [(tosyloxy)m eth yl]ph osph on ate in th e presen ce of sodium
h ydride to give fully protected ph osph on ate esters 6 an d 7 as key in term e-
diates for th e followin g syn th eses (Sch em e 1). Th e preparation of 5-m eth yl
derivatives 4 an d 6 h as been publish ed recen tly in th e paper dealin g with
preparation of HPMPT an d its side-ch ain fluorin ated an alogue29
.
Th e reaction of 4-m eth oxypyrim idin -2(1H)-on e ph osph on ates 6 an d 7
with am m on ia at 120 °C in an autoclave afforded th e correspon din g cyto-
sin e derivatives 8 an d 9; deprotection of ester groups with brom otrim eth yl-
silan e in aceton itrile followed by h ydrolysis gave free ph osph on ic acids:
1-[(S)-3-h ydroxy-2-(ph osph on om eth oxy)propyl]-5-m eth ylcytosin e (10) an d
5-eth yl-1-[(S)-3-h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (11). Th e
in term ediate 7, diisopropyl ester of 5-eth yl-4-m eth oxy-1-[(S)-2-(ph osph on o-
m eth oxy)-3-(trityloxy)propyl]pyrim idin -1(2H)-on e was also utilized for th e
reaction with various prim ary am in es th e selection of wh ich was m ade on
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582
Krečmerová, Masojídková, Holý:
th e basis of activity of oth er ANP an alogues substituted in th e am in o
group30. Th is reaction , perform ed in refluxin g dioxan e, afforded N4-alkyl-
5-eth ylcytosin e ph osph on ates 12–16 wh ich , after sim ultan eous deprotect-
ion of ester an d trityl groups with brom otrim eth ylsilan e, afforded a series
of free HPMP-5-eth ylcytosin es bearin g N4-substituen ts 17–21 as fin al prod-
ucts for biological screen in g (Sch em e 1, Table I).
OCH3
OCH3
OCH3
N
OTr
R
R
N
R
N
O
(ii)
3
N
O
N
O
N
H
O
(i)
OTr
OTr
O
P(O)(OiPr)2
OH
4, R = CH3
5, R = C2H5
6, R = CH3
7, R = C2H5
1, R = CH3
2, R = C2H5
(iii)
(v)
NH2
NHR´
N
NH2
NHR´
C2H5
N
R
C2H5
R
N
N
(iv)
(iv)
N
O
N
O
N
O
N
O
OTr
P(O)(OiPr)2
OTr
OH
OH
P(O)(OH)2
O
O
O
O
P(O)(OiPr)2
P(O)(OH)2
10, R = CH3
11, R = C2H5
8, R = CH3
9, R = C2H5
12-16
17-21
(i) Cs2CO3, DMF, 80 °C, 10 h; (ii) TsOCH2P(O)(OiPr)2, NaH, THF, r.t. ;
(iii) NH3/CH3OH, 120 °C; (iv) (CH3)3SiBr, CH3CN, r.t.; (v) R´ NH2, dioxane, reflux
SCHEME 1
Quite a differen t approach to C-5 substituted cidofovir derivatives is
based on PdII catalyzed couplin g reaction s23b. We h ave used Son ogash ira
couplin g for in troduction of eth yn yl (or oth er alkyn yl groups) to th e C-5
position (Sch em e 2). Th e startin g com poun d, fully protected cidofovir ester
22 was first iodin ated with N-iodosuccin im ide. Th e th us obtain ed 5-iodo
derivative 23 was th en treated with (trim eth ylsilyl)acetylen e un der
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C-5 Substituted Cidofovir Derivatives
583
TABLE I
Preparation of som e N4-substituted 5-eth yl-HPMPC derivatives from com poun d 7
Yield of
ph osph on ic
acid, %
Ph osph on ate
ester
Yield of
ester, %
Ph osph on ic
acid
R′
Cyclopropyl
12
13
14
15
16
46
17
18
19
20
21
74
48
62
69
41
Cyclopen tyl
52
HOCH2CH2–
CH2=CH–CH2–
(CH3)2NCH2CH2–
78
52
n ot isolated
NHBz
NHBz
N
NHBz
I
C
C
Si(CH3)3
N
N
(i)
(ii)
O
N
O
N
O
N
OTr
P(O)(OiPr)2
OTr
P(O)(OiPr)2
OTr
O
O
O
P(O)(OiPr)2
22
23
24
(iii)
NH2
O
NH2
N
NH2
C
CH
C
CH
N
N
N
(iv)
+
O
N
O
O
N
OH
P(O)(OH)2
OTr
OH
O
26
O
P(O)(OiPr)2
O
P(O)(OH)2
25
27
(i) N-Iodosuccinimide, AIBN, toluene, 80 °C; (ii) (trimethylsilyl)acetylene, [PdCl2(Ph3P)2],
CuI, (C2H5)3N, dioxane, r.t.; (iii) NH3/CH3OH, 4 °C; (iv) (CH3)3SiBr, CH3CN, 0 °C
SCHEME 2
[PdCl2(Ph 3P)2] an d CuI catalysis to give 5-[2-(trim eth ylsilyl)eth yn yl] deriva-
tive 24, wh ich was subsequen tly deprotected to com poun d 25 with
m eth an olic am m on ia.
Th e Son ogash ira couplin g ran ks am on g th e m eth ods utilized in n ucleo-
side ch em istry very frequen tly31. In m ost cases, it is perform ed with n ucleo-
sides or n ucleobases an d, due to h igh reactivity of eth yn yl group, it is
always used as a last syn th etic step. If th ese con dition s are fulfilled, usually
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 4, pp. 579–594

584
Krečmerová, Masojídková, Holý:
n o problem s occur an d such reaction s can be perform ed even on a large
scale (e.g. in dustrial syn th esis of 5-eth yn yluracil32). In our case, com poun d
25 was n ot a fin al product; its deprotection with brom otrim eth ylsilan e
turn ed out to be a critical step of th e syn th esis. Th is reaction wh en per-
form ed un der usual con dition s (room tem perature, h igh excess of brom o-
trim eth ylsilan e added in on e portion an d tim e-con sum in g purification on
ion exch an ge resin colum n s) afforded n o desired HPMP-5-eth yn ylcytosin e
(26) but 5-acetyl derivative 27 on ly as a product of a n ucleoph ilic addition
of water to triple bon d. Th is product was also con tam in ated with a sm all
am oun t of th e 5-brom o derivative. In order to get over th is problem we h ad
to elaborate m ild reaction an d purification con dition s con sistin g in a
dropwise addition of brom otrim eth ylsilan e at low tem perature in dry aceto-
n itrile an d a very sh ort con tact with Dowex 50 at low tem perature in th e
desaltin g process perform ed un der stirrin g. Subsequen t purification on
Dowex 1 in acetate form is n ot possible. Th erefore, fin al purification of
HPMP-5-eth yn ylcytosin e (26) was perform ed by preparative HPLC in n eu-
tral con dition s to ach ieve th e separation of a sm all am oun t of 5-acetyl de-
rivative 27 presen t as im purity.
In con clusion , we syn th esized a series of various base-m odified cidofovir
an alogues as com poun ds for an tiviral screen in g. Special atten tion was paid
to preparation of 5-eth yn yl-HPMPC wh ich was obtain ed by th e
Son ogash ira reaction ; for th is purpose, we optim ized reaction con dition s
to preven t tran sform ation of acid-sen sitive eth yn yl group durin g rem oval
of ester groups an d subsequen t purification . All target com poun ds were
tested for an tiviral activity in vitro in cell cultures. Non e of th e com poun ds
was active again st CMV, VZV, h erpes viruses, vaccin ia virus, VSV an d retro-
viruses (HIV).
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, Germ an y), ch rom atograph ic system s
are described in th e 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 easured on
a Varian Un ity 500 in strum en t (at 500 MHz) in DMSO-d6 solution s (referen ced to th e sol-
ven 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 ic acid (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 an d decouplin g experim en ts. 13C NMR
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 4, pp. 579–594

C-5 Substituted Cidofovir Derivatives
585
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 (referen 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 alyti-
cal) spectrom eter usin g FAB (ion ization with xen on , acceleratin g voltage 8 kV, glycerol
m atrix). Optical rotation s were m easured on an 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 an d solution con cen -
tration s in g/100 m l.
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, tetrah ydrofuran 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 literature proce-
dure1. (2S)-2-[(Trityloxy)m eth yl]oxiran e (3) was purch ased from DAISO, Co. Ltd (Japan ).
5-Eth yl-4-m eth oxypyrim idin -2(1H)-on e (2)
A m ixture of 5-eth yl-2,4-dim eth oxypyrim idin e33 (20 g, 117 m m ol) an d acetyl ch loride
(110 m l, 1.55 m ol) was stirred at room tem perature for 24 h , th en evaporated, th e solid resi-
due coevaporated with toluen e (2 × 200 m l) an d m ixed with m eth an ol (25 m l). 1 M Sodium
m eth oxide was added un til alkalin e reaction persisted an d th e solution was h eated for 40 °C
for 1 h . Th e m ixture was n eutralized with acetic acid to pH 7 an d taken down . Th e crude
product was crystallized from th e m ixture m eth an ol–eth an ol (1:3), crystals were collected by
suction , wash ed with a sm all am oun t of aceton e, th en with water an d fin ally again with
aceton e an d dried in vacuo to give 7 g (39%) of 2. Moth er liquors were evaporated an d
ch rom atograph ed on a colum n of silica gel (1500 m l) in system ch loroform –m eth an ol (20:1)
to give addition al portion of 2. Overall yield 14.9 g (83%), wh ite crystals, m .p. 166 °C. For
C7H10N2O2 (154.2) calculated: 54.54% C, 6.54% H, 18.17% N; foun d: 54.09% C, 6.49% H,
17.91% N. FAB MS, m/z (%): 155 (100) [M + H]. 1H NMR (DMSO-d6): 11.15 br, 1 H (NH);
7.45 s, 1 H (H-6); 3.82 s, 3 H (OCH3); 2.26 q, 2 H, J(CH2,CH3) = 7.4 (CH2); 1.02 t, 3 H (CH3).
13C NMR (DMSO-d6): 170.65 (C-4); 156.43 (C-2); 141.93 (C-6); 107.98 (C-5); 53.81 (OCH3);
19.20 (CH2); 13.54 (CH3).
3´´
1´´
2´´
HN
4
R
3
N
5
6
2
N
O
1
3´
2´
1´
OH
P(O)(OR´) 2
O
FIG. 1
Gen eral n um berin g sch em e for assign m en t of NMR sign als
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586
Krečmerová, Masojídková, Holý:
(S)-5-Eth yl-1-[2-h ydroxy-3-(triph en ylm eth oxy)propyl]-
4-m eth oxypyrim idin -2(1H)-on e (5)
(2S)-2-[(Trityloxy)m eth yl]oxiran e (3; 9.0 g, 28.5 m m ol) an d cesium carbon ate (945 m g,
2.9 m m ol) were added to a solution of 5-eth yl-4-m eth oxypyrim idin -2(1H)-on e (2; 4.39 g,
28.5 m m ol) in DMF (200 m l) an d th e m ixture stirred at 75 °C for 10 h . After coolin g to
room tem perature, th e m ixture was taken down , th e residue codistilled with toluen e (150 m l)
an d ch rom atograph ed on a colum n of alum in um oxide (500 m l). Elution was perform ed
with toluen e un til residues of 3 an d TrOH were com pletely rem oved, th en with eth yl acetate
(1 l) an d fin ally, th e pure 5 was eluted with a m ixture eth yl acetate–eth an ol (10:1). Yield
12.78 g (93%) of a wh ite foam , [α]D –36.2 (c 0.443, CHCl3). For C29H30N2O4·0.5H2O (479.6)
calculated: 72.63% C, 6.51% H, 5.84% N; foun d: 72.98% C, 6.54% H, 5.89% N. FAB MS, m/z
(%): 471 (3) [M + H], 243 (100) [trityl]. 1H NMR (DMSO-d6): 7.60 s, 1 H (H-6); 7.42 d, 6 H,
7.33 t, 3 H an d 7.26 t, 3 H (H-arom .); 5.27 d, 1 H, J(OH,2′) = 5.9 (OH); 4.05 dd, 1 H, J(1′a,2′) =
4.0, J(gem ) = 12.9 (H-1′a); 3.99 m , 1 H (H-2′); 3.83 s, 3 H (OCH3); 3.58 dd, 1 H, J(1′b,2′) =
8.3 (H-1′b); 2.97 dd, 1 H, J(3′a,2′) = 5.0, J(gem ) = 9.4 (H-3′a); 2.90 dd, 1 H, J(3′b,2′) = 5.4
(H-3′b); 2.23 q, 2 H, J(CH2,CH3) = 7.4 (CH2); 1.00 t, 3 H (CH3). 13C NMR (DMSO-d6): 169.79
(C-4); 155.56 (C-2); 146.82 (C-6); 143.69, 3 C, 128.42, 6 C, 128.02, 6 C an d 127.16, 3 C
(trityl); 107.60 (C-5); 86.03 (trityl); 67.08 (C-2′); 66.25 (C-3′); 53.95 (OCH3); 53.16 (C-1′);
19.20 (CH2); 13.28 (CH3).
(S)-1-{2-[(Diisopropoxyph osph oryl)m eth oxy]-3-(triph en ylm eth oxy)propyl}-
5-eth yl-4-m eth oxypyrim idin -2(1H)-on e (7)
Sodium h ydride (60% dispersion in oil, 1.52 g, 38 m m ol) was added to a solution of 5 (12 g,
25.5 m m ol) an d diisopropyl [(tosyloxy)m eth yl]ph osph on ate (10.7 g, 30.6 m m ol) in tetra-
h ydrofuran (350 m l) at –20 °C an d stirred for 20 m in . Th e stirrin g was th en con tin ued at
25 °C for 24 h an d fin ally at 40 °C for 2 h . Th e m ixture was filtered th rough Celite, th e fil-
trate evaporated an d th e rem ain in g oily residue (18 g) ch rom atograph ed on a colum n of
silica gel (1500 m l), startin g with ch loroform –trieth ylam in e (100:1) un til th e residue of
diisopropyl [(tosyloxy)m eth yl]ph osph on ate was rem oved an d th en con tin uin g with ch loro-
form –m eth an ol–trieth ylam in e (100:7:1). Yield 13 g (79%) of 7 as colourless oil, [α]D –29.0 (c
0.186, CHCl3). For C36H45N2O7P (648.7) calculated: 66.65% C, 6.99% H, 4.32% N, 4.77% P;
foun d: 66.85% C, 6.98% H, 4.36% N, 4.74% P. FAB MS, m/z (%): 672 (0.6) [M + Na], 649
(0.3) [M + H], 407 (1.4) [M – trityl + 2 H], 243 (100) [trityl]. 1H NMR (DMSO-d6): 7.60 s, 1 H
(H-6); 7.41 d, 6 H, 7.33 t, 6 H an d 7.26 t, 3 H (H-arom .); 4.56 m , 2 H (P-OCH); 3.95 m , 3 H
(H-1′, H-2′); 3.82 s, 3 H (OCH3); 3.78 dd, 1 H, J(P,CHa) = 8.8, J(gem ) = 13.7 (PCHa); 3.68 dd,
1 H, J(P,CHb) = 9.7 (PCHb); 3.24 dd, 1 H, J(3′a,2′) = 5.0, J(gem ) = 9.5 (H-3′a); 2.97 dd, 1 H,
J(3′b,2′) = 5.0 (H-3′b); 2.23 q, 2 H, J(CH2,CH3) = 7.4 (CH2); 1.21 d, 3 H, 1.20 d, 3 H, 1.185 d,
3 H an d 1.16 d, 3 H, J(CH3,CH) = 6.1 (CH3); 1.02 t, 3 H, J(CH2,CH3) = 7.4 (CH3). 13C NMR
(DMSO-d6): 169.86 (C-4); 155.39 (C-2); 146.40 (C-6); 143.64, 3 C, 128.39, 6 C, 128.04, 6 C
an d 127.23, 3 C (trityl); 107.96 (C-5); 86.34 (trityl); 78.18 d, J(P,C) = 12.7 (C-2′); 70.27 d, 2 C,
J(P,C) = 6.4 (P-OCH); 64.08 d, J(P,C) = 165.5 (P-C); 62.85 (C-3′); 53.96 (OCH3); 50.50 (C-1′);
23.88 d, 2 C, J(P,C) = 3.4, 23.83 d, J(P,C) = 4.9 an d 23.72 d, J(P,C) = 4.4 (CH3); 19.29 (CH2);
13.04 (CH3).
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Diisopropyl Esters of C-5 Substituted (S)-1-[2-(Ph osph on om eth oxy)-
3-(triph en ylm eth oxy)propyl]cytosin es. Gen eral Procedure
A solution of an appropriate 4-m eth oxy derivative 6 or 7 (4.7 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 16 h . After coolin g to room
tem perature, th e m ixture was taken down an d th e residue ch rom atograph ed on a colum n of
silica gel (200 m l) in system eth yl acetate–aceton e–eth an ol–water (18:3:2:2).
(S)-1-{2-[(Diisopropoxyphosphoryl)methoxy]-3-(triphenylmethoxy)propyl}-5-methylcytosine (8).
Yield 1.63 g (54%) of a colourless foam , [α]D –19.0 (c 0.297, CHCl3). For C34H42N3O6P·0.5H2O
(637.3) calculated: 64.08% C, 6.96% H, 6.59% N, 4.86% P; foun d: 64.10% C, 6.96% H,
6.72% N, 4.79% P. FAB MS, m/z (%): 642 (0.5) [M + Na], 378 (2) [M – trityl + 2 H], 243 (100)
[trityl], 126 (8) [5-m eth ylcytosin e + H]. 1H NMR (DMSO-d6): 7.40 d, 6 H, 7.34 t, 6 H an d
7.26 t, 3 H (H-arom .); 7.21 br s, 1 H (H-6); 7.15 br, 1 H an d 6.65 br, 1 H (NH2); 4.59 m , 2 H
(P-OCH); 3.84 m , 2 H (H-1′a, H-2′); 3.74 dd, 1 H, J(P,CH) = 8.9, J(gem ) = 13.7 (PCHa);
3.72 dd, 1 H, J(1′b,2′) = 8.4, J(gem ) = 14.6 (H-1′b); 3.69 dd, 1 H, J(P,CHb) = 9.6 (PCHb);
3.19 dd, 1 H, J(3′a,2′) = 3.2, J(gem ) = 10.6 (H-3′a); 2.89 dd, 1 H, J(3′b,2′) = 3.3 (H-3′b);
1.73 d, 3 H, J(CH3,H-6) = 1.0 (CH3); 1.24 d, 3 H, 1.22 d, 3 H, 1.21 d, 3 H an d 1.19 d, 3 H,
J(CH3,CH) = 6.1 (CH3). 13C NMR (DMSO-d6): 165.82 (C-4); 155.91 (C-2); 144.37 (C-6);
143.69, 3 C, 128.40, 6 C, 128.07, 6 C an d 127.22, 3 C (trityl); 100.11 (C-5); 86.23 (trityl);
78.63 d, J(P,C) = 12.7 (C-2′); 70.33 d, J(P,C) = 6.3 (P-OCH); 70.31 d, J(P,C) = 5.9 (P-OCH);
64.17 d, J(P,C) = 165.5 (P-C); 62.81 (C-3′); 49.88 (C-1′); 23.94 d, 2 C, J(P,C) = 3.9, 23.87 d,
J(P,C) = 4.4 an d 23.78 d, J(P,C) = 4.4 (CH3).
(S)-1-{2-[(Diisopropoxyphosphoryl)methoxy]-3-(triphenylmethoxy)propyl}-5-ethylcytosine (9).
Yield 1.5 g (50%) of a colourless foam , [α]D –9.0 (c 0.321, CHCl3). For C35H44N3O6P (633.7)
calculated: 66.34% C, 7.00% H, 6.63% N, 4.89% P; foun d: 65.73% C, 7.03% H, 6.46% N,
4.90% P. FAB MS, m/z (%): 656 (0.5) [M + Na], 243 (100) [trityl]. 1H NMR (DMSO-d6): 7.38 d,
6 H, 7.32 t, 6 H an d 7.26 t, 3 H (H-arom .); 7.18 s, 1 H (H-6); 7.10 br, 1 H an d 6.75 br, 1 H
(NH2); 4.55 m , 2 H (P-OCH); 3.85 m , 2 H (H-1′a, H-2′); 3.75 dd, 1 H, J(1′b,2′) = 8.7, J(gem ) =
14.0 (H-1′b); 3.66 dd, 1 H, J(P,CHa) = 9.5, J(gem ) = 13.4 (PCHa); 3.60 dd, 1 H, J(P,CHb) = 9.8
(PCHb); 3.18 dd, 1 H, J(3′a, 2′) = 2.8, J(gem ) = 10.6 (H-3′a); 2.90 dd, 1 H, J(3′b,2′) = 3.2
(H-3′b); 2.14 br q, 2 H (CH2); 1.205 d, 3 H, 1.19 d, 3 H, 1.18 d, 3 H an d 1.16 d, 3 H,
J(CH3,CH) = 6.1 (CH3); 0.96 t, 3 H, J(CH3,CH2) = 7.4 (CH3). 13C NMR (DMSO-d6): 165.40
(C-4); 156.06 (C-2); 143.85, 3 C (trityl); 143.70 (C-6); 128.56, 6 C, 128.26, 6 C an d 127.44,
3 C (trityl); 106.38 (C-5); 86.43 (trityl); 78.84 d, J(P,C) = 13.2 (C-2′); 70.67 d, 2 C, J(P,C) =
6.3 (P-OCH); 64.29 d, J(P,C) = 165.5 (P-C); 63.07 (C-3′); 50.29 (C-1′); 24.09 d, 2 C, J(P,C) =
3.4, 24.02 d, J(P,C) = 4.4 an d 23.93 d, J(P,C) = 4.4 (CH3); 19.985 (CH2); 12.76 (CH3).
Reaction of Com poun d 7 with Prim ary Am in es. Gen eral Procedure
An appropriate prim ary am in e (40 m m ol) was added to a solution of com poun d 7 (1 m m ol)
in dry dioxan e (20 m l) an d th e m ixture stirred un der reflux for 5–15 h un til a com plete con -
version was ach ieved (TLC ch eck). Th e solution was evaporated, th e residue coevaporated
with xylen e (3 × 20 m l) an d ch rom atograph ed on a colum n of silica gel (200 m l) in th e sys-
tem described below. Th e followin g com poun ds were prepared by th is procedure:
N4 -Cyclopropyl-1-{(S)-2-[(diisopropoxyphosphoryl)methoxy]-3-(triphenylmethoxy)propyl}-
5-ethylcytosine (12). Ch rom atograph ed in th e system ch loroform –m eth an ol–trieth ylam in e
(100:7:1). Yield 307 m g (46%) of a colourless syrup, [α]D –13.4 (c 0.209, CHCl3). For
C
38H48N3O6P (673.8) calculated: 67.74% C, 7.18% H, 6.24% N, 4.60% P; foun d: 67.63% C,
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588
Krečmerová, Masojídková, Holý:
7.13% H, 6.49% N, 4.51% P. FAB MS, m/z (%): 674 (0.5) [M + H], 432 (2) [M – trityl + 2 H],
243 (100) [trityl]. 1H NMR (DMSO-d6): 7.40 d, 6 H, 7.33 t, 6 H an d 7.26 t, 3 H (H-arom .);
7.14 s, 1 H (H-6); 6.95 d, 1 H, J(NH,CH) = 4.0 (NH); 4.55 m , 2 H (P-OCH); 3.87 m , 2 H an d
3.75 m , 1 H (H-1′, H-2′); 3.76 dd, 1 H, J(P,CHa) = 8.8, J(gem ) = 13.6 (PCHa); 3.67 dd, 1 H,
J(P,CHb) = 9.8 (PCHb); 3.20 dd, 1 H, J(3′a,2′) = 2.9, J(gem ) = 10.7 (H-3′a); 2.90 dd, 1 H,
J(3′b,2′) = 4.4 (H-3′b); 2.83 m , 1 H (cyclopropyl); 2.13 m , 2 H (CH2); 1.21 d, 3 H, 1.20 d, 3 H,
1.18 d, 3 H an d 1.16 d, 3 H, J(CH3,CH) = 6.2 (CH3); 0.94 t, 3 H, J(CH2,CH3) = 7.3 (CH3);
0.67 m , 2 H an d 0.54 m , 2 H (cyclopropyl). 13C NMR (DMSO-d6): 163.97 (C-4); 155.64 (C-2);
143.70, 3 C (trityl); 142.45 (C-6); 128.41, 6 C, 128.055, 6 C an d 127.22, 3 C (trityl); 106.40
(C-5); 86.24 (trityl); 78.74 d, J(P,C) = 12.7 (C-2′); 70.30 d, 2 C, J(P,C) = 6.3 (P-OCH); 64.13 d,
J(P,C) = 165.0 (P-C); 62.91 (C-3′); 50.07 (C-1′); 24.235 (NCH); 23.94 d, 2 C, J(P,C) = 3.4,
23.86 d, J(P,C) = 4.9 an d 23.76 d, J(P,C) = 3.9 (CH3); 19.39 (CH2); 12.65 (CH3); 6.34 an d
6.29 (cyclopropyl CH2).
N4 -Cyclopentyl-1-{(S)-2-[(diisopropoxyphosphoryl)methoxy]-3-(triphenylmethoxy)propyl}-
5-ethylcytosine (13). Ch rom atograph ed in th e system eth yl acetate–eth an ol (12:1). Yield
440 m g (52%) of a colourless syrup, [α]D –11.8 (c 0.196, CHCl3). FAB MS, m/z (%): 702 (1)
[M + H], 243 (48) [trityl]. 1H NMR (DMSO-d6): 7.39 d, 6 H, 7.31 t, 6 H an d 7.27 t, 3 H
(H-arom .); 7.12 s, 1 H (H-6); 6.66 d, 1 H, J(NH,CH) = 7.5 (NH); 4.56 m , 2 H (P-OCH);
3.88 m , 1 H (H-2′); 3.84 dd, 1 H, J(1′a,2′) = 3.4, J(gem ) = 12.9 (H-1′a); 3.76 dd, 1 H,
J(P,CHa) = 9.2, J(gem ) = 13.6 (PCHa); 3.74 dd, 1 H, J(1′b,2′) = 6.7 (H-1′b); 3.67 dd, 1 H,
J(P,CHb) = 9.8 (PCHb); 3.19 dd, 1 H, J(3′a,2′) = 2.6, J(gem ) = 10.7 (H-3′a); 3.04 m , 1 H (NCH);
2.89 dd, 1 H, J(3′b,2′) = 4.2 (H-3′b); 2.19 m , 2 H (CH2); 1.66 m , 2 H, 1.58 m , 2 H, 1.50 m ,
2 H an d 1.44 m , 2 H (cyclopen tyl CH2); 1.21 d, 3 H, 1.20 d, 3 H, 1.18 d, 3 H an d 1.16 d,
3 H, J(CH3,CH) = 6.2 (CH3); 0.97 t, 3 H, J(CH3,CH2) = 7.3 (CH3). 13C NMR (DMSO-d6):
162.28 (C-4); 155.66 (C-2); 143.74, 3 C (trityl); 142.44 (C-6); 128.41, 6 C, 128.055, 6 C an d
127.22, 3 C (trityl); 106.40 (C-5); 86.24 (trityl); 78.71 d, J(P,C) = 12.7 (C-2′); 70.30 d, 2 C,
J(P,C) = 6.3 (P-OCH); 64.12 d, J(P,C) = 165.5 (P-C); 62.89 (C-3′); 51.68 (NCH); 50.02 (C-1′);
31.95 an d 31.88 (cyclopen tyl CH2); 24.01 d, J(P,C) = 3.9, 23.94 d, J(P,C) = 4.9, 23.90 d,
J(P,C) = 4.9 an d 23.84 d, J(P,C) = 3.9 (CH3); 23.73 an d 23.63 (cyclopen tyl CH2); 19.44 (CH2);
12.66 (CH3).
1 -{(S)-2 -[(Diisopropoxyph osph oryl)m eth oxy]-3 -(triph en ylm eth oxy)propyl}-5 -eth yl-N4 -
(2-hydroxyethyl)cytosine (14). Ch rom atograph ed in th e system eth yl acetate–m eth an ol (4:1).
Yield 528 m g (78%) of a colourless syrup, [α]D –17.6 (c 0.203, CHCl3). FAB MS, m/z (%): 678
(1.3) [M + H], 243 (100) [trityl]. 1H NMR (DMSO-d6): 7.39 d, 6 H, 7.32 t, 6 H an d 7.26 t, 3 H
(H-arom .); 7.15 s, 1 H (H-6); 7.01 t, 1 H, J(NH,CH2) = 5.5 (NH); 4.85 t, 1 H, J(OH,CH2) = 5.5
(OH); 4.55 m , 2 H (P-OCH); 3.87 m , 1 H (H-2′); 3.86 dd, 1 H, J(1′a,2′) = 4.8, J(gem ) = 14.6
(H-1′a); 3.76 dd, 1 H, J(1′b,2′) = 8.4 (H-1′b); 3.76 dd, 1 H, J(P,CHa) = 8.9, J(gem ) = 13.6
(PCHa); 3.68 dd, 1 H, J(P,CHb) = 9.8 (PCHb); 3.49 br q, 2 H, J = 5.6 (NCH2); 3.19 dd, 1 H,
J(3′a,2′) = 2.2, J(gem ) = 10.5 (H-3′a); 3.09 br q, 2 H, J = 5.6 (OCH2); 2.90 dd, 1 H, J(3′b,2′) =
4.2 (H-3′b); 2.16 m , 2 H (CH2); 1.21 d, 3 H, 1.20 d, 3 H, 1.18 d, 3 H an d 1.17 d, 3 H,
J(CH3CH) = 6.2 (CH3); 0.98 t, 3 H, J(CH3,CH2) = 7.4 (CH3). 13C NMR (DMSO-d6): 162.85
(C-4); 155.62 (C-2); 143.71, 3 C (trityl); 142.61 (C-6); 128.42, 6 C, 128.07, 6 C an d 127.23,
3 C (trityl); 106.44 (C-5); 86.26 (trityl); 78.72 d, J(P,C) = 13.2 (C-2′); 70.34 d, 2 C, J(P,C) =
6.4 (P-OCH); 63.86 d, J(P,C) = 165.5 (P-C); 60.10 (C-3′); 59.75 (OCH2); 50.02 (NCH2); 43.15
(C-1′); 23.95 d, 2 C, J(P,C) = 3.4, 23.88 d, J(P,C) = 4.4 an d 23.78 d, J(P,C) = 3.9 (CH3); 19.49
(CH2); 12.60 (CH3).
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N4-Allyl-1-{(S)-2-[(diisopropoxyphosphoryl)methoxy]-3-(triphenylmethoxy)propyl}-5-ethylcytosine
(15). Ch rom atograph ed in th e system ch loroform –m eth an ol (9:1). Yield 349 m g (52%) of a
wh ite foam , [α]D –30.6 (c 0.239, CHCl3). For C38H48N3O6P (673.8) calculated: 67.74% C,
7.18% H, 6.24% N, 4.60% P; foun d: 67.41% C, 7.42% H, 5.98% N, 4.93% P. FAB MS, m/z
(%): 674 (1) [M + H], 243 (100) [trityl]. 1H NMR (DMSO-d6): 7.39 d, 6 H, 7.32 t, 6 H an d
7.26 t, 3 H (H-arom .); 7.23 t, 1 H, J(NH,1′′) = 5.7 (NH); 7.17 s, 1 H (H-6); 5.87 ddt, 1 H,
J(2′′,1′′) = 5.2, J(2′′,3′′cis) = 10.3, J(2′′,3′′tran s) = 17.2 (H-2′′); 5.08 dq, 1 H, J(3′′tran s,1′′) =
J(gem ) =1.7 (H-3′′tran s); 5.04 dq, 1 H, J(3′′cis,1′′) = J(gem ) = 1.7 (H-3′′cis); 4.56 m , 2 H
(P-OCH); 3.93 br tt, 2 H (H-1′′); 3.86 m , 2 H (H-1′a, H-2′); 3.76 dd, 1 H, J(P,CHa) = 9.0,
J(gem ) = 13.6 (PCHa); 3.75 dd, 1 H, J(1′b,2′) = 7.8, J(gem ) = 14.0 (H-1′b); 3.67 dd, 1 H,
J(P,CHb) = 9.6 (PCHb); 3.19 dd, 1 H, J(3′a,2′) = 2.7, J(gem ) = 10.6 (H-3′a); 2.89 dd, 1 H,
J(3′b,2′) = 4.4 (H-3′b); 2.18 m , 2 H (CH2); 1.21 d, 3 H, 1.20 d, 3 H, 1.18 d, 3 H an d 1.16 d, 3 H,
J(CH3,CH) = 6.1 (CH3); 0.99 t, 3 H, J(CH3,CH2) = 7.3 (CH3). 13C NMR (DMSO-d6): 162.47
(C-4); 155.64 (C-2); 143.71, 3 C (trityl); 142.77 (C-6); 135.58 (C-2′′); 128.42, 6 C, 128.07, 6 C
an d 127.23, 3 C (trityl); 114.98 (C-3′′); 106.38 (C-5); 86.24 (trityl); 78.68 d, J(P,C) = 13.2
(C-2′); 70.31 d, 2C, J(P,C) = 6.3 (P-OCH); 64.13 d, J(P,C) = 165.5 (P-C); 62.91 (C-3′); 50.04
(C-1′); 42.35 (C-1′′); 23.95 d, 2 C, J(P,C) = 4.4, 23.88 d, J(P,C) = 4.9 an d 23.78 d, J(P,C) = 4.4
(CH3); 19.53 (CH2); 12.78 (CH3).
1-{(S)-2-[(Diisopropoxyphosphoryl)methoxy]-3-(triphenylmethoxy)propyl}-N4-[2-(dimethylamino)-
ethyl]-5-ethylcytosine (16). Th e crude product (1.2 g, ca. 75% purity) was used for th e syn th e-
sis of 21 with out ch rom atograph y. FAB MS, m/z (%): 705 (1) [M + H], 243 (100) [trityl].
Preparation of 1-[(S)-3-Hydroxy-2-(ph osph on om eth oxy)propyl] Derivatives of
Substituted Cytosin es 10, 11 an d 17–21. Gen eral Procedure
Brom otrim eth ylsilan e (1.5 m l, 11 m m ol) was added to a solution of an appropriate ph os-
ph on ate ester 8, 9, 12–16 (1 m m ol) in aceton itrile (15 m l) an d kept in th e dark at room
tem perature for 48 h . Th e m ixture was evaporated an d th e residue coevaporated with aceto-
n itrile (20 m l). 90% Aqueous m eth an ol (25 m l) was added, th e solution n eutralized with 1 M
trieth ylam m on ium h ydrogen carbon ate (TEAB) to pH 7–8 an d evaporated. Th e residue was
codistilled with water (2 × 25 m l) to decom pose rem ain in g TEAB, th en dissolved in water
(150 m l) an d extracted with eth er (2 × 50 m l). Th e aqueous layer was con cen trated to th e
5 m l volum e an d applied on to a colum n of Dowex 50 (H+ form , 40 m l). Elution was per-
form ed with water (600 m l), followed by 2.5% aqueous am m on ia. Th e UV-absorbin g am -
m on ia fraction was evaporated to dryn ess, th en dissolved in water (5 m l) an d applied on to a
colum n of Dowex 1 (acetate form , 40 m l). After elution with water (200 m l), th e product
was eluted with 0.5 M acetic acid. Th e product-con tain in g fraction was evaporated, th e resi-
due coevaporated with water (4 × 30 m l) an d th e pure ph osph on ic acid crystallized from
water or lyoph ilized. Th e followin g free ph osph on ic acids were prepared by th is procedure:
1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-methylcytosine (10). Yield 232 g (76%) of
wh ite crystals, m .p. 162–165 °C, [α]D –64.5 (c 0.372, H2O). For C9H16N3O6P·2/3H2O (305.2)
calculated: 35.42% C, 5.72% H, 13.77% N, 10.14% P; foun d: 35.60% C, 5.88% H, 13.45% N,
9.73% P. FAB MS, m/z (%): 294 (100) [M + H]. 1H NMR (D2O): 7.73 br q, 1 H, J(6,CH3) = 1.0
(H-6); 4.13 dd, 1 H, J(1′a,2′) = 3.3, J(gem ) = 14.4 (H-1′a); 3.86 dd, 1 H, J(1′b,2′) = 8.1 (H-1′b);
3.82 dd, 1 H, J(3′a,2′) = 3.9, J(gem ) = 12.4 (H-3′a); 3.77 m , 1 H (H-2′); 3.75 dd, 1 H, J(P,CHa) =
9.1, J(gem ) = 12.9 (PCHa); 3.60 dd, 1 H, J(3′b,2′) = 4.2 (H-3′b); 3.57 dd, 1 H, J(P,CHb) = 9.6
(PCHb); 2.02 d, 3 H, J(CH3,6) = 1.0 (CH3). 13C NMR (D2O): 160.37 (C-4); 150.42 (C-2);
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590
Krečmerová, Masojídková, Holý:
147.64 (C-6); 102.99 (C-5); 79.49 d, J(P,C) = 11.7 (C-2′); 66.08 d, J(P,C) = 157.7 (P-C); 60.07
(C-3′); 49.81 (C-1′); 11.47 (CH3).
5-Ethyl-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (11). Yield 225 m g (73%) of
wh ite crystals, m .p. 126 °C, [α]D –106.6 (c 0.357, H2O). For C10H18N3O6P (307.3) calculated:
39.09% C, 5.90% H, 13.68% N, 10.08% P; foun d: 39.12% C, 6.10% H, 13.38% N, 9.79% P.
FAB MS, m/z (%): 308 (100) [M + H], 140 (17) [5-eth yluracil + H]. 1H NMR (D2O): 7.72 s, 1 H
(H-6); 4.14 dd, 1 H, J(1′a,2′) = 2.5, J(gem ) = 14.2 (H-1′a); 3.89 dd, 1 H, J(1′b,2′) = 8.0 (H-1′b);
3.83 dd, 1 H, J(3′a,2′) = 3.9, J(gem ) = 12.4 (H-3′a); 3.78 m , 1 H (H-2′); 3.76 dd, 1 H, J(P,CHa) =
9.4, J(gem ) = 13.1 (PCHa); 3.62 dd, 1 H, J(3′b,2′) = 3.9 (H-3′b); 3.58 dd, 1 H, J(P,CHb) = 9.5
(PCHb); 2.42 q, 2 H (CH2); 1.18 t, 3 H, J(CH3,CH2) = 7.4 (CH3). 13C NMR (D2O): 159.08
(C-4); 149.27 (C-2); 147.18 (C-6); 108.32 (C-5); 79.36 d, J(P,C) = 11.7 (C-2′); 66.03 d, J(P,C) =
157.2 (P-C); 59.98 (C-3′); 49.94 (C-1′); 18.95 (CH2); 11.25 (CH3).
N4-Cyclopropyl-5-ethyl-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (17). Yield 257 m g
(74%) of a wh ite am orph ous solid, [α]D –69.5 (c 0.679, H2O). HR MS (FAB): for C13H23N3O6P
[M + H] calculated 348.1325, foun d 348.1345. FAB MS, m/z (%): 348 (100) [M + H]. 1H NMR
(D2O): 7.62 s, 1 H (H-6); 4.14 dd, 1 H, J(1′a,2′) = 3.4, J(gem ) = 14.3 (H-1′a); 3.90 dd, 1 H,
J(1′b,2′) = 7.9 (H-1′b); 3.83 dd, 1 H, J(3′a,2′) = 3.9, J(gem ) = 12.2 (H-3′a); 3.79 m , 1 H (H-2′);
3.75 dd, 1 H, J(P,CHa) = 9.4, J(gem ) = 13.2 (PCHa); 3.69 dd, 1 H, J(3′b,2′) = 4.0 (H-3′b);
3.57 dd, 1 H, J(P,CHb) = 9.4 (PCHb); 2.77 m , 1 H (NCH); 2.36 br q, 2 H, J(CH2,CH3) = 7.5
(CH2); 1.13 t, 3 H (CH3); 1.06 m , 2 H an d 0.84 m , 2 H (CH2). 13C NMR (D2O): 159.08 (C-4);
149.52 (C-2); 145.28 (C-6); 108.64 (C-5); 79.40 d, J(P,C) = 12.2 (C-2′); 66.09 d, J(P,C) = 156.7
(P-C); 60.03 (C-3′); 49.93 (C-1′); 23.46 (NCH); 18.88 (CH2); 11.23 (CH3); 6.87, 2 C (CH2).
N4-Cyclopentyl-5-ethyl-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (18). Yield 179 m g
(48%) of a wh ite am orph ous solid, [α]D –70.0 (c 0.165, H2O). HR MS (FAB): for C15H27N3O6P
[M + H] calculated 376.1637, foun d 376.1622. FAB MS, m/z (%): 376 (1.5) [M + H]. 1H NMR
(D2O): 7.51 s, 1 H (H-6); 4.22 m , 1 H (NCH); 4.12 dd, 1 H, J(1′a,2′) = 3.4, J(gem ) = 14.4
(H-1′a); 3.88 dd, 1 H, J(1′b,2′) = 7.9 (H-1′b); 3.83 dd, 1 H, J(3′a,2′) = 3.8, J(gem ) = 12.3
(H-3′a); 3.79 m , 1 H (H-2′); 3.74 dd, 1 H, J(P,CHa) = 9.5, J(gem ) = 13.1 (PCHa); 3.62 dd, 1 H,
J(3′b,2′) = 4.0 (H-3′b); 3.58 dd, 1 H, J(P,CHb) = 9.5 (PCHb); 2.41 q, 2 H, J(CH2,CH3) = 7.4
(CH2); 2.10 m , 2 H an d 1.78 m , 2 H (CH2); 1.69 m , 4 H (CH2); 1.16 t, 3 H (CH3). 13C NMR
(D2O): 156.53 (C-4); 149.85 (C-2); 144.64 (C-6); 108.84 (C-5); 79.48 d, J(P,C) = 11.7 (C-2′);
66.14 d, J(P,C) = 157.2 (P-C); 60.09 (C-3′); 54.685 (NCH); 49.81 (C-1′); 31.50, 31.48 an d
23.40, 2 C (CH2); 18.96 (CH2); 11.25 (CH3).
5-Ethyl-N4-(2-hydroxyethyl)-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (19). Yield
217 m g (62%) of a wh ite solid (after lyoph ilization ), [α]D –57.8 (c 0.250, H2O). For
C
12H22N3O7P·0.5H2O (360.3) calculated: 40.00% C, 6.43% H, 11.66% N, 8.60% P; foun d:
39.98% C, 6.42% H, 11.38% N, 8.06% P. FAB MS, m/z (%): 352 (13) [M + H]. 1H NMR (D2O):
7.60 s, 1 H (H-6); 4.12 dd, 1 H, J(1′a,2′) = 3.5, J(gem ) = 14.3 (H-1′a); 3.87 dd, 1 H, J(1′b,2′) =
7.8 (H-1′b); 3.83 t, 2 H (NCH2); 3.82 dd, 1 H, J(3′a,2′) = 3.8, J(gem ) = 12.4 (H-3′a); 3.78 m ,
1 H (H-2′); 3.74 dd, 1 H, J(P,CHa) = 9.4, J(gem ) = 13.1 (PCHa); 3.68 t, 2 H, J(CH2,CH2) = 5.2
(OCH2); 3.61 dd, 1 H, J(3′b,2′) = 4.2 (H-3′b); 3.58 dd, 1 H, J(P,CHb) = 9.4 (PCHb); 2.42 q,
2 H, J(CH2,CH3) = 7.6 (CH2); 1.18 t, 3 H (CH3). 13C NMR (D2O): 158.64 (C-4); 150.73 (C-2);
144.92 (C-6); 109.01 (C-5); 79.54 d, J(P,C) = 11.7 (C-2′); 66.17 d, J(P,C) = 157.7 (P-C); 60.012
(C-3′); 59.40 (OCH2); 49.91 (C-1′); 44.39 (NCH2); 19.06 (CH2); 11.27 (CH3).
N4-Allyl-5-ethyl-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (20). Yield 240 m g
(69%) of a wh ite foam , [α]D –54.7 (c 0.167, H2O). HR MS (FAB): for C13H23N3O6P [M + H]
calculated 348.1324, foun d 348.1330. FAB MS, m/z (%): 348 (100) [M + H]. 1H NMR (D2O):
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7.65 s, 1 H (H-6); 5.92 ddt, 1 H, J(2′′,1′′) = 4.6, J(2′′,3′′cis) = 10.6, J(2′′,3′′tran s) = 17.2 (H-2′′);
5.29 dq, 1 H, J(3′′cis,1′′) = J(gem ) =1.8 (H-3′′cis); 5.25 dq, 1 H, J(3′′tran s,1′′) = 1.8 (H-3′′tran s);
4.17 dt, 2 H (H-1′′); 4.13 dd, 1 H, J(1′a,2′) = 3.4, J(gem ) = 14.3 (H-1′a); 3.88 dd, 1 H, J(1′b,2′) =
8.0 (H-1′b); 3.83 dd, 1 H, J(3′a,2′) = 3.9, J(gem ) = 12.3 (H-3′a); 3.80 m , 1 H (H-2′); 3.78 dd,
1 H, J(P,CHa) = 9.3, J(gem ) = 13.2 (PCHa); 3.62 dd, 1 H, J(3′b,2′) = 4.9 (H-3′b); 3.60 dd, 1 H,
J(P,CHb) = 9.5 (PCHb); 2.45 q, 2 H (CH2); 1.19 t, 3 H, J(CH2,CH3) = 7.5 (CH3). 13C NMR
(D2O): 156.98 (C-4); 149.22 (C-2); 145.44 (C-6); 129.87 (C-2′′); 117.07 (C-3′′); 108.64 (C-5);
79.40 d, J(P,C) = 11.7 (C-2′); 65.85 d, J(P,C) = 157.7 (P-C); 60.03 (C-3′); 49.94 (C-1′); 44.15
(C-1′′); 19.09 (CH2); 11.30 (CH3).
N4-[2-(Dimethylamino)ethyl]-5-ethyl-1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine
(21). Yield 166 m g (41%) of a wh ite am orph ous solid, [α]D +15.9 (c 0.178, H2O). For
C
14H27N4O6P·1.5H2O (405.4) calculated: 41.48% C, 7.46% H, 13.82% N, 7.64% P; foun d:
41.80% C, 7.61% H, 13.81% N, 7.72% P. FAB MS, m/z (%): 379 (85) [M + H]. 1H NMR (D2O):
7.44 s, 1 H (H-6); 4.07 dd, 1 H, J(1′a,2′) = 3.5, J(gem ) = 13.8 (H-1′a); 3.85 dd, 1 H, J(1′b,2′) =
7.6 (H-1′b); 3.82 t, 2 H, J(CH2,CH2) = 5.6 (NCH2); 3.80 dd, 1 H, J(3′a,2′) = 4.0, J(gem ) = 12.2
(H-3′a); 3.78 m , 1 H (H-2′); 3.68 dd, 1 H, J(P,CHa) = 9.4, J(gem ) = 12.8 (PCHa); 3.60 dd, 1 H,
J(3′b,2′) = 4.2 (H-3′b); 3.56 dd, 1 H, J(P,CHb) = 9.4 (PCHb); 3.42 t, 2 H (NCH2); 2.98 s, 6 H
(NCH3); 2.34 q, 2 H, J(CH2,CH3) = 7.4 (CH2); 1.16 t, 3 H (CH3). 13C NMR (D2O): 164.09
(C-4); 158.14 (C-2); 143.57 (C-6); 110.19 (C-5); 80.13 d, J(P,C) = 12.2 (C-2′); 66.63 d, J(P,C) =
157.2 (P-C); 60.56 (C-3′); 57.57 (NCH2); 50.23 (C-1′); 43.14, 2 C (NCH3); 36.24 (NCH2);
19.21 (CH2); 11.38 (CH3).
N4-Ben zoyl-1-{(S)-2-[(diisopropoxyph osph oryl)m eth oxy]-3-(triph en ylm eth oxy)propyl}-
5-iodocytosin e (23)
1
A solution of protected HPMPC (22; 6.0 g, 8.5 m m ol) in dry toluen e (40 m l) was h eated to
80 °C. N-Iodosuccin im ide (2.9 g, 13 m m ol) was added, followed by AIBN (180 m g, 1.1 m m ol),
th e m ixture was h eated to 80 °C for 4 h an d th en set aside at room tem perature for 12 h .
Th e resultin g dark red solution was con cen trated to 1/3 of its origin al volum e, applied on to
a colum n of silica gel (800 m l) equilibrated in th e system toluen e–trieth ylam in e (100:1) an d
th e colum n eluted with th is system un til N-iodosuccin im ide an d all iodin ated (red-coloured)
by-products were rem oved. Th e desired product 23 was eluted with th e system toluen e–eth yl
acetate–trieth ylam in e (200:100:1). Yield 5 g (70%) of a yellowish foam , [α]D –44.3 (c 0.439,
CHCl3). For C40H43IN3O7P (835.7) calculated: 57.49% C, 5.19% H, 15.19% I, 5.03% N,
3.71% P; foun d: 57.71% C, 5.31% H, 14.89% I, 4.80% N, 3.97% P. FAB MS, m/z (%): 836.2
(0.3) [M + H], 243 (100) [trityl], 105 (30) (ben zoyl]. 1H NMR (DMSO-d6): 13.05 br s, 1 H
(NH); 8.32 s, 1 H (H-6); 8.25 d, 2 H, 7.61 t, 1 H, 7.53 t, 2 H, 7.41 d, 6 H, 7.34 t, 6 H an d
7.25 t, 3 H (H-arom .); 4.56 m , 2 H (P-OCH); 4.02 dd, 1 H, J(1′a,2′) = 7.9, J(gem ) = 13.9
(H-1′a); 3.94 m , 2 H (H-1′b, H-2′); 3.79 dd, 1 H, J(P,CHa) = 8.5, J(gem ) = 13.9 (PCHa);
3.73 dd, 1 H, J(P,CHb) = 8.7 (PCHb); 3.24 dd, 1 H, J(3′a,2′) = 3.8, J(gem ) = 10.6 (H-3′a);
2.99 dd, 1 H, J(3′b,2′) = 3.8 (H-3′b); 1.22 d, 3 H, 1.21 d, 3 H, 1.205 d, 3 H an d 1.19 d, 3 H,
J(CH3,CH) = 6.2 (CH3). 13C NMR (DMSO-d6): 178.39 (C=O); 157.87 (C-4); 153.35 (C-6);
148.01 (C-2); 143.63, 3 C (trityl); 136.71, 132.985, 129.68, 2 C an d 128.59, 2 C (ben zoyl);
128.41, 6 C, 128.15, 6 C, 127.305, 3 C an d 86.48 (trityl); 77.76 d, J(P,C) = 11.2 (C-2′); 70.45
d, J(P,C) = 6.3 (P-OCH); 70.40 d, J(P,C) = 5.9 (P-OCH); 68.29 (C-5); 64.00 d, J(P,C) = 164.5
(P-C); 62.71 (C-3′); 50.26 (C-1′); 23.97 d, 2 C, J(P,C) = 3.9 (CH3); 23.85 d, 2 C, J(P,C) = 4.4
(CH3).
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592
Krečmerová, Masojídková, Holý:
N4-Ben zoyl-1-{(S)-2-[(diisopropoxyph osph oryl)m eth oxy]-3-(triph en ylm eth oxy)propyl}-
5-[(trim eth ylsilyl)eth yn yl]cytosin e (24)
Dry dioxan e (20 m l) was added un der argon to a m ixture of iodo derivative 23 (3.70 g,
4.43 m m ol), CuI (44 m g, 0.22 m m ol) an d [PdCl2(Ph 3P)2] (62 m g, 0.089 m m ol), followed
by trim eth ylsilylacetylen e (1.06 m l, 7.53 m m ol) an d trieth ylam in e (0.66 m l, 4.8 m m ol).
Th e m ixture was stirred at room tem perature for 2 h , th en taken down an d th e residue par-
tition ed between eth yl acetate (250 m l) an d a saturated solution of am m on ium ch loride an d
EDTA (1:1, 200 m l). Th e organ ic layer was dried with an h ydrous MgSO4, evaporated in
vacuo an d th e residue ch rom atograph ed on silica gel colum n (750 m l) in th e system ch loro-
form –m eth an ol (95:5). Yield 2.5 g (70%) of a yellowish foam , [α]D –54.1 (c 0.376, CHCl3).
For C45H52N3O7PSi (806.0) calculated: 67.06% C, 6.50% H, 5.21% N, 3.84% P; foun d:
66.81% C, 6.41% H, 5.31% N, 3.92% P. FAB MS, m/z (%): 806.6 (0.3) [M + H], 243 (100)
[trityl], 105 (41) (ben zoyl]. 1H NMR (DMSO-d6): 12.70 br s, 1 H (NH); 8.00–7.20 m , 21 H
(H-6 + H-arom .); 4.57 m , 2 H (P-OCH); 4.00 m , 3 H an d 3.76 m , 2 H (H-1′, H-2′, PCH2);
3.25 dd, 1 H, J(3′a,2′) = 3.0, J(gem ) = 10.6 (H-3′a); 2.95 dd, 1 H, J(3′b,2′) = 3.5 (H-3′b);
1.22 d, 6 H an d 1.21 d, 6 H, J(CH3,CH) = 6.2 (CH3); 0.16 s, 9 H ( SiCH3). 13C NMR
(DMSO-d6): 177.15 (C=O); 156.70 (C-4); 153.87 (C-2); 151.69 (C-6); 143.63, 3 C (trityl);
136.58, 132.71, 129.54, 2 C an d 128.86, 2 C (ben zoyl); 128.40, 6 C, 128.12, 6 C an d 127.28,
3 C (trityl); 98.76 (C≡); 97.35 (C-5); 96.70 (C≡); 86.41 (trityl); 77.67 d, J(P,C) = 10.0 (C-2′);
70.40 d, 2 C, J(P,C) = 6.3 (P-OCH); 63.95 d, J(P,C) = 164.6 (P-C); 62.59 (C-3′); 50.26 (C-1′);
23.90 d, 2 C, J(P,C) = 4.9 (CH3); 23.80 d, 2 C, J(P,C) = 4.4 (CH3); –0.15 (SiCH3).
1-{(S)-2-[(Diisopropoxyph osph oryl)m eth oxy]-3-(triph en ylm eth oxy)propyl}-
5-eth yn ylcytosin e (25)
A solution of 24 (1.5 g, 1.86 m m ol) in 30% m eth an olic am m on ia was kept in refrigerator
(4 °C) for 24 h , th en evaporated an d th e residue ch rom atograph ed on silica gel (400 m l) in th e
system ch loroform –m eth an ol (95:5). Yield 1.13 g (96%) of a wh ite foam , [α]D –24.0 (c 0.240,
CHCl3). For C35H40N3O6P (629.7) calculated: 66.76% C, 6.40% H, 6.67% N, 4.92% P; foun d:
66.87% C, 6.32% H, 7.00% N, 5.18% P. FAB MS, m/z (%): 604.5 (0.3) [M – acetylen + H], 243
(100) [trityl]. 1H NMR (DMSO-d6): 7.82 s, 1 H (H-6); 7.60 brs, 1 H (NH2); 7.40 d, 6 H, 7.35 t,
6 H an d 7.27 t, 3 H (H-arom .); 6.72 br s, 1 H (NH2); 4.55 m , 2 H (P-OCH); 4.32 s, 1 H (≡CH);
3.94 dd, 1 H, J(1′a,2′) = 4.0, J(gem ) = 12.9 (H-1′a); 3.87 m , 1 H (H-2′); 3.81 dd, 1 H, J(1′b,2′) =
7.5 (H-1′b); 3.75 dd, 1 H, J(P,CHa) = 8.8, J(gem ) = 13.8 (PCHa); 3.64 dd, 1 H, J(P,CHb) = 9.7
(PCHb); 3.19 dd, 1 H, J(3′a,2′) = 3.4, J(gem ) = 10.6 (H-3′a); 2.89 dd, 1 H, J(3′b,2′) = 4.3
(H-3′b); 1.23 d, 3 H, 1.21 d, 3 H, 1.20 d, 3 H an d 1.18 d, 3 H, J(CH3,CH) = 6.2 (CH3).
13C NMR (DMSO-d6): 164.855 (C-4); 154.23 (C-2); 151.375 (C-6); 143.67, 3 C, 128.42, 6 C,
128.13, 6 C an d 127.28, 3 C (trityl); 88.205 (-C≡); 86.29 (trityl); 85.86 (≡CH); 78.19 d, J(P,C) =
12.7 (C-2′); 75.92 (C-5); 70.38 d an d 70.37 d, J(P,C) = 6.4 (P-OCH); 64.03 d, J(P,C) = 165.0
(P-C); 62.59 (C-3′); 50.53 (C-1′); 23.96 d an d 23.93 d, J(P,C) = 4.9 (CH3); 23.79 d, 2 C, J(P,C) =
4.4 (CH3).
5-Eth yn yl-1-[(S)-3-h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (26)
Brom otrim eth ylsilan e (0.9 m l, 6.6 m m ol) was added dropwise durin g 30 m in to a stirred
solution of ester 25 (1 g, 1.6 m m ol) in fresh ly distilled aceton itrile (20 m l) cooled to 0 °C.
After addition of brom otrim eth ylsilan e, th e m ixture was slowly warm ed up to room tem per-
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C-5 Substituted Cidofovir Derivatives
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ature an d set aside in th e dark for 24 h . 0.5 M Trieth ylam m on ium h ydrogen carbon ate (20 m l)
was added, th e solution evaporated at 30 °C an d coevaporated in water (2 × 20 m l). Th e resi-
due dissolved in water (5 m l) was applied on to a colum n of Dowex 50 (H+, 50 m l), elution
perform ed with cold (0 °C) 50% aqueous aceton e (200 m l) followed by water (0 °C, 1 l).
Dowex 50 was th en rem oved from th e colum n , stirred with cold water (0 °C) an d 10%
NH4OH was added un til pH 9. Th e ion exch an ger was filtered off by suction , wash ed with
water (50 m l) an d com bin ed filtrates were evaporated in vacuo. Th e crude product was puri-
fied by preparative HPLC usin g 1% aqueous m eth an ol (isocratic elution , flow rate 12 m l/m in ).
Two products were obtain ed: HPMP-5-eth yn ylcytosin e 26 (reten tion tim e 4.3 m in on an alyt-
ical colum n , 20 m in on preparative colum n ) an d HPMP-5-acetylcytosin e 27 (reten tion tim e
5.0 m in on an alytical colum n , 23 m in on preparative colum n ). Correspon din g fraction s
were evaporated to dryn ess an d lyoph ilized from water. Yield 250 m g (52%) of 26 as a wh ite
am orph ous solid, [α]D –22.1 (c 0.165, H2O). FAB MS, m/z (%): 304 (23) [M + H]. 1H NMR
(D2O): 8.01 s, 1 H (H-6); 4.10 dd, 1 H, J(1′a,2′) = 3.7, J(gem ) = 14.0 (H-1′a); 3.82 dd, 1 H,
J(1′b,2′) = 7.6 (H-1′b); 3.80 dd, 1 H, J(3′a,2′) = 3.3, J(gem ) = 12.2 (H-3′a); 3.79 s, 1 H (≡CH);
3.70 dd, 1 H, J(P,CHa) = 9.5, J(gem ) = 12.8 (PCHa); 3.58 dd, 1 H, J(3′b,2′) = 4.4 (H-3′b);
3.57 dd, 1 H, J(P,CHb) = 9.5 (PCHb). 13C NMR (D2O): 165.84 (C-4); 157.10 (C-2); 152.50
(C-6); 91.36 (C-5); 85.69 (-C≡); 80.39 d, J(C,P) = 12.1 (C-2′); 74.65 (≡CH); 66.96 d, J(C,P) =
157.3 (P-C); 60.92 (C-3′); 51.28 (C-1′).
5-Acetyl-1-[(S)-3-h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (27)
Th e com poun d was obtain ed as a by-product in preparation of 26. Yield 40 m g (8%) of a
wh ite am orph ous solid. HR MS (FAB): for C10H17N3O7P [M + H] calculated 322.0804, foun d
322.0792. FAB MS, m/z (%): 322 (10) [M + H]. 1H NMR (D2O): 8.65 s, 1 H (H-6); 4.25 dd, 1 H,
J(1′a,2′) = 3.3, J(gem ) = 14.0 (H-1′a); 3.88 dd, 1 H, J(1′b,2′) = 8.4 (H-1′b); 3.85 dd, 1 H,
J(3′a,2′) = 4.0, J(gem ) = 12.5 (H-3′a); 3.77 m , 1 H (H-2′); 3.74 dd, 1 H, J(P,CHa) = 9.5, J(gem ) =
12.8 (PCHa); 3.63 dd, 1 H, J(3′b,2′) = 4.3 (H-3′b); 3.52 dd, 1 H, J(P,CHb) = 9.5 (PCHb); 2.52 s,
3 H (CH3). 13C NMR (D2O): 198.99 (C=O);163.80 (C-4); 156.65 (C-6); 156.48 (C-2); 104.50
(C-5); 79.60 d, J(C,P) = 12.7 (C-2′); 66.50 d, J(C,P) = 156.7 (P-C); 60.22 (C-3′); 51.40 (C-1′);
26.30 (CH3).
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. We gratefully acknowledge
the financial support of the NIH grant 1UC1AI062540-01, the Program of targeted projects of
Academy of Sciences of the Czech Republic No. 1QS400550501 and the Centre for New Antivirals and
Antineoplastics 1M0508. The authors’ thanks are due to Prof. E. De Clercq and his group in the
Rega Institute, Catholic University Leuven (Belgium) for antiviral screening, the staff of the mass
spectrometry (Dr K. Ubik, Head) and analytical departments of the Institute (Dr L. Holasová, Head).
Excellent technical assistance of Ms J. Křelinová is also acknowledged.
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