An alternative synthesis of HPMPC and HPMPA diphosphoryl derivatives

Article abstract of DOI:10.1135/cccc20010500

In (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC) and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) with 3-hydroxy functions protected with 4,4′-dimethoxytrityl (DMTr) groups, phosphonate groups were transformed to the morpholides and treated with bis(tributylammonium) diphosphate. Selective cleavage of the DMTr group in the presence of the labile diphosphate residue was achieved in water at pH 2.5. Purification by charcoal adsorption followed by anion exchange chromatography afforded phosphonate-diphosphate compounds (HPMPC_pp, HPMPA_pp).

Full text of DOI:10.1135/cccc20010500

500
Otmar, Masojídková, Votruba, Holý:
AN ALTERNATIVE SYNTHESIS OF HPMPC AND HPMPA
DIPHOSPHORYL DERIVATIVES
1,
2
3
Miroslav OTMAR *, Milena MASOJÍDKOVÁ, Ivan VOTRUBA and Antonín HOLÝ
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: otmar@uochb.cas.cz,
2
3
votruba@uochb.cas.cz, holy@uochb.cas.cz
Received February 20, 2001
Accepted March 14, 2001
In (S)-1-[3-h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (HPMPC) an d (S)-9-[3-h ydroxy-
2-(ph osph on om eth oxy)propyl]aden in e (HPMPA) with 3-h ydroxy fun ction s protected with
4,4′-dim eth oxytrityl (DMTr) groups, ph osph on ate groups were tran sform ed to th e
m orph olides an d treated with bis(tributylam m on ium ) diph osph ate. Selective cleavage of th e
DMTr group in th e presen ce of th e labile diph osph ate residue was ach ieved in water at pH
2.5. Purification by ch arcoal adsorption followed by an ion exch an ge ch rom atograph y af-
forded ph osph on ate-diph osph ate com poun ds (HPMPCpp, HPMPApp).
Keyw ord s: Acyclic n ucleotide an alogs; Nucleotides; HPMPCpp; HPMPApp; Triph osph ates;
Diph osph ates; Ph osph on ates; Ph osph orylation .
HPMPC (Cidofovir, Vistide®; 1a), an acyclic n ucleoside ph osph on ate active
again st a broad spectrum of DNA viruses, is n ow m arketed for treatm en t of
CMV retin itis1,2. Man y studies were devoted to its m ech an ism of action 2–8
.
In cells, n ucleotide ph osph on ates are usually ph osph orylated to form a par-
allel to n atural n ucleoside triph osph ates. In th at form (bearin g th e
diph osph ate residue lin ked to th e ph osph on ate fun ction ), th ey in teract
with en zym es wh ose in h ibition is con sidered respon sible for th eir biologi-
cal action (e.g., DNA polym erases)9,10. Syn th etic diph osph orylated n ucleo-
side ph osph on ates are required for th e kin etic studies on th ese en zym es.
Th e stan dard m eth odology for th e syn th esis of n ucleoside triph osph ates
from n ucleotides con sists usually of a tran sform ation of th e ph osph ate in to
its activated in term ediate (m orph olide11, im idazolide12 or a m ixed an h y-
dride with diph en yl h ydrogen ph osph ate13) followed by treatm en t with in -
organ ic diph osph ate14. Th is activation prin ciple was easily applicable to
ph osph on ate an alogues of n ucleotides; for exam ple, 2-(ph osph on o-
m eth oxy)eth yl derivatives of n ucleobases were routin ely tran sform ed to th e
di- an d triph osph ate an alogues by m orph olide procedure15. However, th is
Collect. Czech. Chem. Commun. (Vol. 66) (2001)
doi: 10.1135/cccc20010500

HPMPC and HPMPA Diphosphoryl Derivatives
501
approach repeatedly failed in on e special case. It was in 3-h ydroxy-2-
(ph osph on om eth oxy)propyl derivatives of cytosin e an d aden in e
1
(HPMP-type; Sch em e 1), wh erein th e ph osph on om eth oxy group n eigh -
bours on a prim ary h ydroxy group. In th is case, a sterically favoured
six-m em bered cyclic ester (e.g., com poun d 2) was form ed durin g th e prepa-
ration of an activated in term ediate (e.g., m orph olide 3). Even use of an ex-
cess of th e activatin g agen t did n ot avoid form ation of th e cyclic ester 2 as
th e m ain product15. It is apparen t th at, for overcom in g th is obstacle, th e
prim ary h ydroxy group m ust be protected. Th e correspon din g 3-ben zyloxy
derivatives, wh ose usin g for th is purpose was recen tly described5,6, are n ot
accessible from 3-h ydroxy-2-(ph osph on om eth oxy)propyl derivatives 1.
O
B
B
B
(i)
O
N
O
OR
OR
P
O
P
O
O
P
O
O
O
O
O
O
1, R = OH
3, R = H
2
(ii)
5, R = DMTr
4, R = DMTr
(iii)
B
a
b
1 - 7
O O
O
OR
O
P
P
O
NH2
NH2
O
O P
O
O
O
N
N
B
N
N
O
N
N
6, R = DMTr
7, R = H
(iv)
(i) 1,3-Dicyclohexylcarbodiimide, morpholine, 2-methylpropan-2-ol, reflux;
(ii) 4,4'-dimethoxytrityl chloride, tributylamine, DMSO; (iii) (Bu3NH)2H2P2O7, DMSO; (iv) pH 2.5
SCHEME 1
Hen ce, th is approach requires a total syn th esis of 3-ben zyloxy derivatives
(i) by alkylation of (protected) h eterocyclic bases with an appropriate
ph osph on ate-bearin g syn th on or (ii) by an in troduction of ph osph on o-
m eth oxy groupin g on to th e 3-ben zyloxy-2-h ydroxypropyl fun ction of th e
acyclic n ucleotide in term ediate. Because of th e stron g liability of cytosin e
rin g to reduction un der form ation of th e 5,6-dih ydro derivative, th e fin al
rem oval of th e ben zyl group can n ot be don e by h ydrogen olysis, but it was
accom plish ed by a h ydrogen -tran sfer reaction with am m on ium form ate
an d Pd/C (refs5,6). Neverth eless, th ere is still a risk of con tam in ation of th e
fin al product with 5,6-dih ydro derivative of HPMPCpp, wh ich is difficult to
Collect. Czech. Chem. Commun. (Vol. 66) (2001)

502
Otmar, Masojídková, Votruba, Holý:
detect an d wh ich could substan tially in terfere in en zym e assays. Th erefore,
we sough t an oth er protectin g group wh ich could be directly in troduced at
th e prim ary h ydroxy group of th e HPMP derivatives 1 an d wh ich could be
easily rem oved leavin g in tact both th e cytosin e rin g an d th e labile
diph osph ate fun ction . Th e use of acyl protectin g groups is con train dicated
because of th e alkalin e con dition s required for th e deprotection . We se-
lected th e 4,4′-dim eth oxytrityl group (DMTr) as th e m ost con ven ien t can di-
date for th e purpose.
RESULTS AND DISCUSSION
HPMPC (1a) was tran sform ed to its tributylam m on ium salt in m eth an ol.
Selective tritylation of th e prim ary h ydroxy group was ach ieved in DMSO
with a th ree-fold excess of DMTr-Cl an d 20 equivalen ts of tributylam in e.
Th e desired O-dim eth oxytrityl derivative 4a was obtain ed in alm ost quan ti-
tative yield. Usin g a larger excess of DMTr-Cl leads to a form ation of com -
poun d with M = 883, wh ich we con sider to be th e N4,O-bis(dim eth oxy-
trityl) derivative of HPMPC. Durin g attem pts of its isolation by crystalliza-
tion from eth yl acetate it was very liable to partial deprotection , wh ich
gives predom in an tly a com poun d with a sligh tly lower RF th an 4a, wh ich
we con sider to be N4-dim eth oxytrityl derivative. Crystallization from eth yl
acetate was foun d to be a con ven ien t m eth od for isolation an d purification
of th e product 4a. Surprisin gly, it was obtain ed as a free acid, even th ough
th e reaction m ixture h ad con tain ed th e excess of tributylam in e. Th e crys-
tallin e free acid 4a can be stored in freezer for several m on th s with out sig-
n ifican t decom position ; in protic solven ts it is deprotected by its own
acidity after several h ours. Th e ph osph on ate 4a was tran sform ed in to th e
m orph olidate 5a wh ich , on treatm en t with bis(tributylam m on ium )
diph osph ate, gave th e protected ph osph on ate-diph osph ate com poun d 6a.
Selective rem oval of DMTr group in th e presen ce of th e labile diph osph ate
fun ction was ach ieved in water at pH 2.5. Rem oval of th e residual
diph osph ate an ion was accom plish ed by adsorption of th e crude product
7a on activated ch arcoal at pH 2.5, wash of un adsorbed in organ ic salts with
water, an d liberation of th e partially purified product with 10% am m on ia.
Fin al isolation was accom plish ed via ch rom atograph y on a DEAE Seph adex
an ion -exch an ger colum n by applyin g a lin ear gradien t of 0–0.5
M
trieth ylam m on ium h ydrogen carbon ate (TEAB). Iden tity an d purity of th e
diph osph oryl derivative 7a was proved by NMR, MS an d by com parison
with th e auth en tic product, obtain ed by en zym atic ph osph orylation 3, on
HPLC with diode-array detector. Th e aden in e derivative, HPMPApp (7b),
Collect. Czech. Chem. Commun. (Vol. 66) (2001)

HPMPC and HPMPA Diphosphoryl Derivatives
503
was obtain ed in th e sam e m an n er. Th e presen ted protocol for ch em ical
ph osph orylation of HPMP derivatives avoids th e disadvan tages con n ected
with th e use of th e described5,6 ben zyl protection for th e prim ary h ydroxy
group. Moreover, our m eth od is sign ifican tly m ore sim ple an d less labori-
ous.
EXPERIMENTAL
HPLC was perform ed on
a W aters HPLC system (996 PDA detector, PDA software
Millen ium 32, version 3.05, 616 pum p with 600 S con troller an d Waters fraction collector II)
equipped with 15 cm × 4 m m Supelcosil™ LC 18T 3 µm reverse-ph ase colum n . Th e lin ear
gradien t (curve No. 6) of coun ter-ion solven t system at a flow rate 0.75 m l/m in was used:
0 (100% A)–100% B, 30 m in (solven t A: 50 m M potassium dih ydrogen ph osph ate, 3 m M
tetrabutylam m on ium h ydrogen sulfate, pH 3.19; solven t B: 50 m M potassium dih ydro-
gen ph osph ate, 3 m M tetrabutylam m on ium h ydrogen sulfate, 30% aceton itrile, pH 3.19).
An ion -exch an ger colum n ch rom atograph y was don e on DEAE Seph adex purch ased from
Sigm a. TLC was carried out on silica gel pre-coated alum in ium plates with fluorescen t in di-
cator (Merck 5554, 60 F254) in th e system propan -1-ol–con cen trated aqueous am m on ia–
water (11 : 7 : 2) an d propan -2-ol–con cen trared aqueous am m on ia–water (7 : 1 : 2), an d
on PEI-cellulose pre-coated plastic sh eets Polygram ® Cel 300 PEI/UV254 (Mach erey–Nagel,
Germ an y) in th e system 4 M LiCl – 1 M AcOH (1 : 4). Paper electroph oresis (20 V/cm ) was
perform ed on a Wh atm an No. 3MM paper in 1 M acetic acid. Acid-wash ed activated ch arcoal
was purch ased from Sigm a. FAB m ass spectra were recorded on a ZAB-EQ (VG An alytical)
spectrom eter, usin g ion ization by Xe, acceleratin g voltage 8 kV, an d th ioglycerol–glycerol
3 : 1 m ixture as a m atrix. 1H NMR spectra were recorded at 500 MHz on a Varian UNITY
500 in strum en t in DMSO-d6 or in deuterium oxide with th e solven t sign al (δ 2.50) or so-
dium 3-(trim eth ylsilyl)propan e-1-sulfon ate (DSS) as an in tern al stan dard. 13C NMR APT
spectra were recorded at 127.5 MHz on a Varian UNITY 500 in strum en t in DMSO-d6 with
th e solven t sign al as an in tern al referen ce (δ 39.7) or in deuterium oxide with dioxan e as an
extern al stan dard (δ 66.86). 31P NMR spectra were recorded at 80.98 MHz on a Varian
UNITY 200 in strum en t with th e use of ph osph oric acid as an extern al stan dard. Ch em ical
sh ifts are given in ppm (δ-scale), couplin g con stan ts (J) in Hz. HPMPC was obtain ed from
Gilead Scien ces (U.S.A.), HPMPA was syn th esized accordin g to ref.16. 4,4′-Dim eth oxytrityl
ch loride was purch ased from Sigm a.
(S)-1-{3-[4,4′-Dim eth oxytrityl)oxy]-2-(ph osph on om eth oxy)propyl}cytosin e (Free Acid) (4a)
To a suspen sion of (S)-1-[3-h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e dih ydrate (free
acid, 1a, 158 m g, 0.5 m m ol) in m eth an ol (15 m l), tributylam in e (1.6 g, 10 m m ol) was
added; th e suspen sion of th e free acid easily dissolved to a clear solution of tributyl-
am m on ium salt. Meth an ol was evaporated; th e residue, co-evaporated with aceton itrile, was
dissolved in DMSO, 4,4′-dim eth oxytrityl ch loride (510 m g, 1.5 m m ol) was added an d th e re-
action m ixture was stirred for 3 h . Proccedin g of th e reaction was m on itored by TLC in
propan –1-ol-am m on ia–water (11 : 7 : 2). If th e reaction was n ot com pleted, an addition al
portion of 4,4′-dim eth oxytrityl ch loride was added (200 m g, 0.6 m m ol) an d th e reaction
tim e prolon ged overn igh t. DMSO an d tributylam in e were extracted down with eth er; prod-
Collect. Czech. Chem. Commun. (Vol. 66) (2001)

504
Otmar, Masojídková, Votruba, Holý:
uct 4a was collected by filtration , recrystallized from eth yl acetate (tributylam m on ium
ch loride is soluble in eth yl acetate), an d dried in vacuo. Yield 275 m g (95%), wh ite crystals,
m .p. 140–142 °C. MS (FAB–): m/z 580 (100%, M – H). HRMS (FAB–): for C29H31N3O8P (M –
H) calculated 580.1849, foun d 580.1793. 1H NMR (500 MHz, DMSO-d6): 8.70 br, 1 H (P-OH);
8.45 br, 1 H (P-OH); 7.80 d, 2 H (DMTr); 7.64 d, 1 H, J = 7.1 (6-H); 7.60 br, 1 H (Ha-NH);
7.55 m , 5 H (DMTr); 7.19 t, 2 H (DMTr); 6.88 d, 4 H (DMTr); 6.70 br, 1 H (Hb-NH); 5.77 d,
1 H, J = 7.1 (5-H); 4.00 dd, 1 H, J = 12.0, 3.0 (1′a-H); 3.83 m , 1 H (2′-H); 3.71 s, 6 H (DMTr);
3.64 dd, 1 H, J = 12.4, 10.0 (Ha-CP); 3.63 dd, 1 H, J = 12.0, 7.5 (1′b-H); 3.40 dd, 1 H, J =
12.4, 9.3 (Hb-CP); 3.10 dd, 1 H, J = 10.5, 3.0 (3′a-H); 2.95 dd, 1 H, J = 10.5, 4.0 (3′b-H).
13C NMR (127.5 MHz, DMSO-d6): 163.30 (4-C); 158.25, 2 C (DMTr); 152.48 (2-C); 148.50
(6-C); 144.94 (DMTr); 135.69 (DMTr); 135.62 (DMTr); 129.87, 4 C (DMTr); 128.04, 2 C
(DMTr); 127.88, 2 C (DMTr); 126.81 (DMTr); 113.395, 4 C (DMTr); 93.34 (5-C); 85.68
(DMTr); 78.22 d, J = 7.8 (2′-C); 67.02 d, J = 161.1 (C-P); 62.80 (3′-C); 55.185, 2 C (DMTr);
50.15 (1′-C).
(S)-9-[3-(4,4′-Dim eth oxytrityl)oxy-2-(ph osph on om eth oxy)propyl]aden in e (Free Acid) (4b)
Th is com poun d was prepared from (S)-9-[3-h ydroxy-2-(ph osph on om eth oxy)propyl]aden in e
(1b, 150 m g, 0.5 m m ol) as described above for 4a. Yield 225 m g (74%), wh ite crystals, m .p.
129–131 °C. MS (FAB–): 604 (100%, M – H). HRMS (FAB–): for C30H31N5O7P (M – H) calcu-
lated 604.1961, foun d 604.1934. 1H NMR (500 MHz, DMSO-d6): 8.105 s, 1 H (2-H or 8-H);
8.06 s, 1 H (2-H or 8-H); 7.35 br, 2 H (NH2); 7.35 d, 2 H (DMT); 7.27 t, 2 H (DMTr); 7.19 m ,
5 H (DMTr); 6.83 d, 2 H (DMTr); 6.82 d, 2 H (DMTr); 4.38 dd, 1 H, J = 14.2, 4.5 (1′a-H);
4.32 dd, 1 H, J = 14.4, 5.7 (1′b-H); 3.95 br quin t, 1 H, ΣJ = 19.9 (2′-H); 3.72 s, 6 H (DMTr);
3.70 dd, 1 H, J = 12.9, 10.3 (Ha-CP); 3.57 dd, 1 H, J = 12.9, 10.0 (Hb-CP); 3.005 dd, 1 H, J =
10.0, 4.9 (3′a-H); 2.91 dd, 1 H, J = 10.0, 5.0 (3′b-H). 13C NMR (127.5 MHz, DMSO-d6): 158.20
(DMTr); 155.60 (6-C); 151.80 (2-C); 149.63 (4-C); 144.85 (DMTr); 141.62 (8-C); 135.52, 2 C
(DMTr); 129.81 (DMTr); 127.97 (DMTr); 127.83, 2 C (DMTr); 126.79 (DMTr); 118.40 (5-C);
113.30, 4 C (DMTr); 85.75 (DMTr); 78.94 d, J = 11.7 (2′-C); 66.23 d, J = 161.2 (C-P); 62.83
(3′-C); 55.17, 2 C (DMTr); 43.76 (1′-C).
HPMPCpp Sodium Salt (7a)
Com poun d 4a (290 m g, 0.5 m m ol) was dissolved in m eth an ol (15 m l) con tain in g m orph o-
lin e (130 m g, 1.5 m m ol). Meth an ol was evaporated, 2-m eth ylpropan -2-ol (10 m l) an d
1,3-dicycloh exycarbodiim ide (DCC, 60 m g, 0.3 m m ol) was added, an d th e reaction m ixture
was h eated to reflux. In 1 h in tervals, furth er four portion s of DCC (60 m g, 0.3 m m ol) in
2-m eth ylpropan -2-ol (1 m l) were added; th en reflux was prolon ged for addition al 2 h . Th e
solven t was evaporated an d DCC was extracted with eth er. Th e precipitate was collected
by filtration , dissolved in a solution of bis(tributylam m on ium ) diph osph ate (820 m g,
1.5 m m ol) in DMSO (5 m l), an d left at room tem perature overn igh t. Reaction m ixture was
diluted with water (40 m l) an d pH was adjusted to 2.5 with diluted h ydroch loric acid. Pro-
ceedin g of th e rem oval of DMTr group was m on itored by TLC in propan -1-ol–con cen trated
am m on ia–water (11 : 7 : 2). After 1 h , activated ch arcoal (10 g) was added an d th e reaction
m ixture was stirred for 30 m in . Ch arcoal, con tain in g adsorbed product 7a, was filtered over
celite an d wash ed with water (100 m l). Th e crude product was liberated by wash with 10%
aqueous am m on ia (100 m l). Th e am m on ia solution was evaporated to dryn ess; th e residue
was dissolved in water an d passed th rough a Dowex 50 (Na+) colum n (20 m l). Th e eluate
Collect. Czech. Chem. Commun. (Vol. 66) (2001)

HPMPC and HPMPA Diphosphoryl Derivatives
505
was con cen trated to 40 m l volum e, pH adjusted to 2.5, an d th e adsorption -desorption pro-
cess m en tion ed above was repeated. (N,N′-Dicycloh exyl(m orph olin e-4-carboxam idin ium )
salts of diph osph osph oric acid can be adsorbed on ch arcoal an d con tam in ate th e product.)
Th e evaporated crude product was purified on a DEAE Seph adex colum n (60 m l) applyin g a
lin ear gradien t of trieth ylam m on ium h ydrogen carbon ate (0–0.5 m ol/l). Appropriate frac-
tion s, iden tified by TLC or by UV detector, were collected, evaporated to dryn ess, an d
co-evaporated with water. Th e residue was dissolved in water an d passed th rough a Dowex
50 (Na+) colum n (20 m l). Th e eluen t was evaporated to dryn ess an d co-evaporated with eth -
an ol; th e product 7a was precipitated from a m eth an ol–eth er (1 : 4) m ixture. Yield 55 m g
(22%), coun ted for trisodium salt, a wh ite powder. MS (FAB+): 527 (M – 4 H + 4 Na), 505 (M
– 3 H + 3 Na), 483 (M – 2 H + 2 Na). HRMS (FAB+): for C8H14N3Na3O12P3 (M – 3 H + 3 Na)
calculated 505.9483, foun d 505.9431; for C8H15N3Na2O12P3 (M – 2 H + 2 Na) calculated
483.9664, foun d 483.9600. 1H NMR (500 MHz, D2O): 7.81 d, 1 H, J = 7.6 (6-H); 6.13 d, 1 H,
J = 7.6 (5-H); 4.13 dd, 1 H, J = 14.3, 3.5 (1′a-H); 3.91 dd, 1 H, J = 13.1, 9.6 (Ha-CP); 3.89 dd,
1 H, J = 14.3, 7.0 (1′b-H); 3.83 dd, 1 H, J = 12.4, 3.7 (3′a-H); 3.79 m , 1 H (2′-H); 3.78 dd, 1
H, J = 13.1, 9.8 (Hb-CP); 3.60 dd, 1 H, J = 12.4, 4.3 (3′b-H). 13C NMR (127.5 MHz, D2O):
161.08 (4-C); 152.04 (2-C); 147.18 (6-C); 92.995 (5-C); 77.82 d, J = 11.7 (2′-C); 63.70 d, J =
163.1 (C-P); 58.14 (3′-C); 47.71 (1′-C). 31P NMR (80.98 MHz, D2O): 9.21 d, J = 24.4 (α-P);
–9.67 d, J = 19.5 (γ-P); –22.04 dd, J = 26.4, 19.5 (β-P).
HPMPApp Sodium Salt (7b)
Com poun d 4b (300 m g, 0.5 m m ol) provided, usin g th e above protocol, product 7b (60 m g,
23%, coun ted for trisodium salt, a wh ite powder). MS (FAB+): 508 (M – H + 2 Na), 530 (M –
2 H + 3 Na), 552 (M – 3 H + 4 Na). 1H NMR (500 MHz, D2O): 8.305 s, 1 H (2-H or 8-H);
8.26 s, 1 H (2-H or 8-H); 4.51 dd, 1 H, J = 15.0, 4.0 (1′a-H); 4.43 dd, 1 H, J = 15.0, 6.3
(1′b-H); 3.94 m , 1 H (2′-H); 3.90 dd, 1 H, J = 12.8, 10.4 (Ha-CP); 3.795 dd, 1 H, J = 12.8, 9.3
(Hb-CP); 3.79 dd, 1 H, J = 12.6, 3.9 (3′a-H); 3.53 dd, 1 H, J = 12.6, 4.9 (3′b-H). 13C NMR
(127.5 MHz, D2O): 155.09 (6-C); 151.91 (2-C); 147.55 (4-C); 143.27 (8-C); 117.90 (5-C);
79.82 d, J = 11.7 (2′-C); 63.75 d, J = 166.0 (C-P); 59.95 (3′-C); 43.58 (1′-C). 31P NMR (80.98
MHz, D2O): 8.99 d, J = 26.4 (α-P); –9.32 d, J = 19.5 (γ-P); –22.03 dd, J = 26.4, 19.5 (β-P).
This study was made as a part of research project No. Z4055905 of the Institute. It was supported
by the Grant Agency of the Czech Republic (grant No. 203/96/K001) and by Gilead Sciences (Foster
City, U.S.A.). An excellent technical assistance of Ms Y. Černá is gratefully acknowledged.
REFERENCES
1. This work was presented in part: Otmar M., Votruba I., Holý A. in: Collection Symposium
Series (A. Holý and M. Hocek, Eds), Vol. 2, p. 252. Institute of Organic Chemistry and
Biochemistry, Academy of Sciences of the Czech Republic, Prague 1999.
2. De Clercq E.: Collect. Czech. Chem. Commun. 1998, 63, 480.
3. Cihlář T., Votruba I., Horská K., Liboska R., Rosenberg I., Holý A.: Collect. Czech. Chem.
Commun. 1992, 57, 661.
4. Johnson J. A., Gangemi J. D.: Antimicrob. Agents Chemother. 1999, 43, 1198.
Collect. Czech. Chem. Commun. (Vol. 66) (2001)

506
Otmar, Masojídková, Votruba, Holý:
5. Cherrington J. M., Allen S. J., McKee B. H., Chen M. S.: Biochem. Pharmacol. 1994, 48,
1986.
6. Ho H. T., Woods K. L., Bronson J. J., De Boeck H., Martin J. C., Hitchcock M. J.: Mol.
Pharmacol. 1992, 41, 197.
7. Birkuš G., Kramata P., Votruba I., Otová B., Otmar M., Holý A.: Collect. Czech. Chem.
Commun. 1998, 63, 723.
8. Kramata P., Černý J., Birkuš G., Votruba I., Otová B., Holý A.: Collect. Czech. Chem.
Commun. 1995, 60, 1555.
9. Holý A, Votruba I., Merta A., Černý J., Veselý J., Vlach J., Šedivá K., Rosenberg I.,
Otmar M., Hřebabecký H., Trávníček M., Vonka V., Snoeck R., De Clercq E.: Antiviral Res.
1990, 13, 295.
10. Kramata P., Birkuš G., Otmar M., Votruba I., Holý A.: Collect. Czech. Chem. Commun.
1996, 61 (Special Issue), S188.
11. Moffatt J. G., Khorana H. G.: J. Am. Chem. Soc. 1961, 83, 649.
12. Hoard D. E., Ott D. G.: J. Am. Chem. Soc. 1965, 87, 1785.
13. Michelson A. M.: Biochim. Biophys. Acta 1964, 91, 1.
14. Burgess K., Cook D.: Chem. Rev. (Washington, D. C.) 2000, 100, 2047.
15. Rosenberg I., Holý A.: Collect. Czech. Chem. Commun. 1987, 52, 2792.
16. Holý A., Rosenberg I.: Collect. Czech. Chem. Commun. 1987, 52, 2775.
Collect. Czech. Chem. Commun. (Vol. 66) (2001)