Acyclic nucleoside phosphonates with 5-azacytosine base moiety substituted in C-6 position

Article abstract of DOI:10.1016/j.bmc.2009.10.044

Two methods for preparation of 6-substituted derivatives of anti DNA-viral agent 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC) were developed: (1) ammonia mediated ring-opening reaction of diisopropyl esters of HPMP-5-azaC (4) t

Full text of DOI:10.1016/j.bmc.2009.10.044

Bioorganic & Medicinal Chemistry 18 (2010) 387–395
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acyclic nucleoside phosphonates with 5-azacytosine base moiety substituted
in C-6 position
*
ˇ
´
Marcela Krecmerová , Milena Masojídková, Antonín Holy
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Gilead Sciences & IOCB Research Centre, Flemingovo nám.
2, CZ-166 10, Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Two methods for preparation of 6-substituted derivatives of anti DNA-viral agent 1-(S)-[3-hydroxy-2-
(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC) were developed: (1) ammonia mediated
ring-opening reaction of diisopropyl esters of HPMP-5-azaC (4) to carbamoylguanidine derivatives fol-
lowed by ring-closure reaction with orthoesters and (2) condensation reaction of 6-substituted 5-azacyto-
sines with diisopropyl (1S)-[2-hydroxy-1-tosyloxymethyl)ethoxy]methylphosphonate (15). Deprotection
of diisopropyl esters to free phosphonic acids was performed with bromotrimethylsilane in acetonitrile fol-
lowedbyhydrolysis. In contrastto parent compound HPMP-5-azaC, asubstantial decrease of antiviral activ-
ity in case of 6-substituted analogues occurred. Surprisingly, N-3 isomer of 6-methyl-HPMP-5-azaC in the
form of isopropyl ester revealed activity against RNA viruses (Sindbis virus).
Received 20 August 2009
Revised 22 October 2009
Accepted 24 October 2009
Available online 29 October 2009
Keywords:
5-Azacytosine
Acyclic nucleoside phosphonates
Antivirals
Orthoesters
Ó 2009 Elsevier Ltd. All rights reserved.
Condensation
Carbamoylguanidine
1. Introduction
acyclic nucleoside phosphonates containing as a base moiety
5-azacytosine; among them 1-(S)-[3-hydroxy-2-(phosphonometh-
Triazine compounds, especially derivatives of 5-azacytosine
play an important role in medicinal chemistry. In particular, 5-aza-
cytosine nucleosides are known for their antileukemic activity.
oxy)propyl]-5-azacytosine (HPMP-5-azaC, 1, Fig. 1) its cyclic form
2 and ester prodrugs 3a–d exerted remarkable activity against all
types of DNA viruses.12,13 From the structural point of view,
HPMP-5-azaC is a 5-azacytosine analog of cidofovir, 1-(S)-[3-hy-
droxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, Fig. 1).
This compound with exceptional activity against DNA viruses
was approved in clinical practice for the treatment of cytomegalo-
virus retinitis in AIDS patients14 but it is also used off label, either
systemically or topically, for the treatment of papillomatosis (ano-
genital or laryngeal)15 infections, progressive multifocal leukoen-
cephalopathy (caused by JC polyomavirus),16 adenovirus17
infections and some severe infections caused by poxviruses18
(e.g., vaccinia, orf and molluscum contagiosum). Comparison of
cidofovir and its 5-aza analog 1 showed that antiviral activity data
of 1 (based on 50% effective concentration (EC50 values)) are simi-
lar or in some cases higher, and the antiviral selectivity index (ratio
of 50% cytotoxic concentration (CC50) to EC50) was 2–16-fold high-
er than cidofovir.12 To improve bioavailability of 1, several types of
lipophilic ester prodrugs were synthesised. Esterification process
was carried out on the level of the cyclic phosphonate 2. The most
active compound was found hexadecyloxyethyl ester with EC50
Two of them, 5-azacytidine1 (azacitidine, Vidaza ) and 20-deoxy-
TM
2
TM
5-azacytidine (decitabine, Dacogen ), compounds developed orig-
inally in our Institute in early 1960s were approved recently in
clinical practice for the therapy of myelodysplastic syndrome.3
Clinical investigation of both compounds for the treatment of solid
tumors, especially metastatic lung cancer4 and hormone-indepen-
dent prostate cancer5 is currently under way. In principle, the effi-
cacy of 5-azacytosine nucleosides is related to their inhibitory
activity towards DNA methylations.6–10 Systematic investigation
of new biologically active compounds resulted among others also
in introduction of a sym-triazine component to the chemistry of
acyclic nucleoside phosphonates (ANPs). These compounds have
been developed in our Institute for many years and exerted a broad
spectrum of diverse biological activities (mostly antiviral, but also
cytostatic, antiparasital and immunomodulatory).11 At present,
three of them are utilized in clinical practice (adefovir, tenofovir,
cidofovir) while several others undergo intensive preclinical or
clinical investigation. Recently, we have described a new class of
values in the range of 0.003–0.008
60.0014 g/ml for VZV, 60.00014 to 60.00038
0.008–0.037 g/ml for HHV-6 and 0.037 g/ml for vaccinia virus.
The activity of ester prodrugs was decreasing in the order: 2-
l
g/ml for HSV, 60.0008 to
l
l
g/ml (HCMV),
* Corresponding author. Tel.: +420 220183475; fax: +420 220183560.
l
l
ˇ
E-mail addresses: marcela@uochb.cas.cz (M. Krecmerová), masojid@uochb.cas.cz
(M. Masojídková), holy@uochb.cas.cz (A. Holy´).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.044

ˇ
M. Krecmerová et al. / Bioorg. Med. Chem. 18 (2010) 387–395
388
NH2
N
NH2
N
NH2
NH2
N
N
N
N
N
O
N
O
N
O
N
O
O
N
O
P
O
P
OH
P(O)(OH)2
OH
O
OH
O
OR
O
O
O
P(O)(OH)2
3a, R = C18H37
(CH2)12-CH=CH -(CH2)7CH3
1
HPMPC
(cidofovir)
2
(HPMP-5-azaC)
3b, R = cis
O
CH2
3c, R =
O
CH2CH2 -O -(CH2)15CH3
3d, R =
Figure 1. Structures of cidofovir and its 5-azacytosine analogues: HPMP-5-azaC (1), cyclic HPMP-5-azaC (2) and ester prodrugs of cyclic HPMP-5-azaC (3a–3d).
(hexadecyloxy)ethyl > pivaloyloxymethyl ꢀ octadecyl > erucyl
(compounds 3a–d, Fig. 1).13 Progression in investigation of
HPMP-5-azaC and its derivatives is currently still under way.
In contrast to cidofovir, HPMP-5-azaC has more complicated
metabolic profile due to its chemical (and also enzymatic) instabil-
ity. Concerning to chemical instability, in aqueous solutions ring
opening between C-6 and N-1 of the triazine moiety occurs and
HPMP-5-azaC is successively degraded to 2-{[(2S)-3-hydroxy-2-
ylformamide. Unfortunately, in such solvents free phosphonic
acids are completely insoluble. Therefore, we avoided the use of
HPMP-5-azaC and its open-ring form as free phosphonic acids,
selecting instead the protected diisopropyl ester of HPMP-5-azaC
(compound 4, Scheme 1) as the starting compound for our synthe-
sis. This compound is an intermediate in preparation of HPMP-5-
azaC (1) starting from (2S)-2-[(trityloxy]methyl]oxirane.12
Ring-opening reaction of 4 was performed using highly concen-
trated aqueous-alcoholic solution of ammonia. Regarding the
chemical instability of 5-azacytosine nucleosides22 or free ANPs
(Ref. 19) in alkaline or even neutral solutions, we expected very ra-
pid decomposition. In fact, the degradation of triazine ring in 4 was
surprisingly slow: its complete degradation with 7 M-NH4OH at
25 °C required 6 days. The formed carbamoylguanidine intermedi-
ate 5 was subjected to ring-closure reaction with a series of diverse
orthoesters to give 6-substituted azacytosine phosphonate esters 6
(Table 1). The final deprotection of trityl group and isopropyl ester
groups was performed in one step by reaction with bromotrimeth-
ylsilane followed by hydrolysis giving free 6-alkyl/aryl-HPMP-5-
azaC (7) (Scheme 1).
(phosphonomethoxy)propyl]carbamoylguanidine.12,19
Recently,
we carried out a detailed study of this process under different
conditions using NMR methodology for identification and quantifi-
cation of all reaction products and intermediates.19 The ring-open-
ing reaction is a general property of all 5-azacytosine compounds;
the first study of this type was carried out by Pithova on 5-azacyti-
dine.20 It was proved that triazine ring in 5-azacytosine opens be-
tween C-6 and N-1 to form N-(formylamidino)-N0-b-
ribofuranosylurea in a reversible reaction followed by irreversible
loss a formyl group to form 1-b-
-ribofuranosyl-3-guanylurea.20
D-
D
The reaction is initiated by nucleophilic attack of hydroxyl ion in
position 6 of 5-azacytosine whose electron density is lower com-
pared to cytosine.
In our previous paper we studied a course of chemical decom-
position of several ANPs with 5-azacytosine base in diverse buf-
fers.19 The most labile phosphonate was found HPMP-5-azaC (1)
with a half-life time 10.3 days at pH 10.0 (phosphate buffer). In
contrast to other studied compounds of the series, HPMP-5-azaC
was the only one having a free hydroxyl group in 30 position of
an aliphatic chain. The reaction rate of HPMP-5-azaC was approx-
imately four times higher than the decomposition rates of the
other compounds and the formyl derivative 9 was formed as an
intermediate. This formyl derivative 9 was then hydrolyzed to
form carbamoylguanidine derivative 10 and formic acid. Formation
of the formiate 9 was not observed in the decomposition reaction
of other 5-azacytosine ANPs. The explanation for this different
reaction pathway is the following: the first intermediary formed
formylamido derivative 8 (this structural type was previously
shown to be an intermediate in the 5-azacytosine nucleoside
decomposition16) can react in a transformylation reaction with a
spatially close hydroxy group to form the formiate 9 (see Scheme
2). As a result, we can say that triazine can serve as a formylating
The aim of this work was finding appropriate modification of
5-azacytosine moiety in HPMP-5-azaC (and related compounds)
in order to increase its stability, especially in aqueous solutions.
Regarding above described facts, we focused to preparation of ana-
logs modified in C-6 position with various alkyl or aryl substitu-
ents. Electron donating effect of these substituents causing
decrease of electrophilicity of C-6 position as well as steric hin-
drance of C-6 position are predicted to have a positive influence
to overall stability of 5-azacytosine ANPs.
2. Results and discussion
It is known that sym-triazine ring (5-azacytosine and its 6-
substituted derivatives) can be formed by condensation of
guanylurea with orthoesters. This methodology was described by
Piskala in one of his pioneer works on 5-azacytosine compounds.21
3-Guanylureas substituted with a sugar residue or with an ali-
phatic chain are formed as final products of alkali catalyzed ring-
opening degradation of 5-azacytosine compounds. In a series of
5-azacytosine acyclic nucleoside phosphonates, we proposed that
such degradation products could be appropriate starting material
for synthesis of their stabilized 6-substituted derivatives. In this ef-
fort we focused on antiviral agent HPMP-5-azaC and utilization of
its triazine ring degradation product for reaction with diverse
types of orthoesters. Such reactions require both elevated temper-
ature and performance in dipolar aprotic solvents, usually dimeth-
agent when
arrangement.
a free hydroxyl group is in a suitable spatial
We believe that the surprisingly slow decomposition rate of 4 is
caused by protection of hydroxyl group in the 30 position, making
the transformylation reaction impossible. Another factor is esteri-
fication of phosphonomethyl group in 5-azacytosine ANPs. Numer-
ous observations made during a long time revealed that cyclic
phosphonates, esters of cyclic phosphonates and phosphonate

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M. Krecmerová et al. / Bioorg. Med. Chem. 18 (2010) 387–395
389
NH2
NH2
N
aq. NH4OH
MeOH
orthoester
(Table 1)
N
NH2
NH
N
O
O
N
DMF, 60-150 °C
OTr
OTr
P(O)(Oi-Pr)2
O
P(O)(Oi-Pr)2
O
4
5
NH2
N
NH2
N
N
N
Me3SiBr
MeCN
O
N
R
O
N
R
6a, 7a: R = Me
6b, 7b: R = Et
6c, 7c: R = Pr
6d, 7d: R = Bu
6e, 7e: R = Ph
OTr
OH
O
P(O)(Oi-Pr)2
O
P(O)(OH)2
6a-e
7a-e
Scheme 1. Transformation of tritylated ester of 1-(S)-[3-hydroxy-2(phosphonomethoxy)propyl]-5-azacytosine (4) to C-6 substituted HPMP-5-azaC (7a–e).
Table 1
nate, the trityl group removed and the intermediary formed 13 tosy-
lated by standard procedure (Scheme 3). In contrast to other
nucleobases, 5-azacytosine is sensitive towards catalytic hydrogena-
tion and 5,6-dihydro derivatives would be formed during hydrogeno-
lytic removal of benzylgroup. Therefore, removalofbenzyl group was
performed first, still in a stage of the synthon and thus formed diiso-
propyl (1S)-[2-hydroxy-1-tosyloxymethyl)ethoxy]methylphospho-
nate (15) was used for condensation with a sodium salt of
5-azacytosine and/or its 6-substituted analogues.
Reaction of the ‘open-ring’ intermediate 5 with orthoesters
Orthoester
Product
Conditions
Yield (%) of 6a
CH3C(OEt)3
6a
6b
6c
6d
6e
150 °C, 3 h
60 °C, 2 h
120 °C, 2 h
110 °C, 1.5 h
150 °C, 2 h
67
47
62
73
64
CH3CH2C(OEt)3
CH3(CH2)2C(OMe)3
CH3(CH2)3C(OMe)3
C6H5C(OMe)3
a
Overall yield from 4.
Interestingly, there is a substantial difference in regioselectivity
of condensation of 15 with 6-substituted and usubstituted 5-aza-
cytosines. Condensation with a sodium salt of 5-azacytosine
proceeds exclusively to form N-1 isomer, diisopropyl ester of
HPMP-5-azaC (16), accompanied by N-3 and O-isomers (17 and
18) in small amount only. This reaction thus revealed as advanta-
geous approach to antivirally active HPMP-5-azaC, especially for its
larger-scale syntheses. Its synthesis from 16 utilizes a standard
procedure with bromotrimethylsilane followed by hydrolysis.
The same procedure performed with 17 afforded antivirally inac-
tive N-3 isomer of HPMP-5-azaC while in case of O-isomer 18, its
treatment with bromotrimethylsilane lead to a complete degrada-
tion of the compound. Contrary to unsubstituted 5-azacytosine,
reaction of a sodium salt of 6-methyl-5-azacytosine with 15 af-
fords N-3 isomer 19 as a main product accompanied by N-1 isomer
20 as by-product in small amount. It can be explained by electron
donating effect of C-6 methyl group: in neighborhood of N-1 posi-
tion it probably partially compensates its nucleophilic character
diesters in 5-azacytosine ANPs are generally more stable compared
to free phosphonic acids. Besides the stabilizing effect, esterifica-
tion of a phosphonate function in HPMP-5-azaC (and ANPs gener-
ally), has also a beneficial effect to increase of their antiviral
activity. Regarding these facts, it is preferable their further phar-
macological investigation in a form of ester prodrugs.13
The second approach to 6-substituted 5-azacytosine ANPs is alkyl-
ation of 6-alkyl 5-azacytosines with appropriate synthon, that is, an
aliphatic part activated with a suitable leaving group, usually tosyl
or halogen. 6-Alkyl-5-azacytosines are accessible by condensation
reaction of guanylurea with orthoesters.21 Preparation of ‘(S)-HPMP
synthon’, diisopropyl (1S)-[2-benzyloxy-1-(tosyloxymethyl) ethoxy]
methylphosphonate (14) followed the protocol originally developed
for its racemic form.23 The starting (2S)-1-benzyloxy-3-
trityloxypropan-2-ol (12) accessible from (2S)-2-[(trityloxy]methyl]
oxirane (11) was treated with diisopropyl bromomethanephospho-
NH2
NH2
NH2
NH2
OH
NH2
NH2
N
N
N
NH
CHO
OH
N
N
CHO
O
NH
O
O
NH
O
NH
N
OH
O
O
P(O)(OH)2
O
P(O)(OH)2
O
P(O)(OH)2
O
P(O)(OH)2
1
8
9
10
Scheme 2. The course of hydrolytic triazine-ring opening in HPMP-5-azaC (1).

ˇ
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M. Krecmerová et al. / Bioorg. Med. Chem. 18 (2010) 387–395
1. BrCH2P(O)(OiPr)2 ,
NaH
OTr
BnONa
BnO
OH
BnO
OTr
O
O
P(O)(OiPr)2
OH
2. AcOH
11
12
13
TsCl
pyridine
H2/Pd
BnO
OTs
P(O)(OiPr)2
HO
OTs
P(O)(OiPr)2
O
O
14
15
Scheme 3. Preparation of (S)-HPMP synthons 14 and 15 from (2S)-2-[(trityloxy]methyl]oxirane.
and an alternative formation of anion at more remote N-3 position
in creation of salt can be thus preferable. Deprotection of esters to
free phosphonic acids was performed by a standard procedure
with bromotrimethylsilane. In case of N-3 isomer, the final product
21 was accompanied by a large amount of monoester 22 due to its
increased tendency to crystallization and lower solubility in reac-
tion mixture. Both compounds can be separated by a reverse phase
HPLC technique (see Scheme 4).
ester of N-3 isomer 22 gives an impetus for preparation of other
(especially lipophilic) esters of this type of phosphonates and their
subjection to detailed biological investigation.
5. Experimental
5.1. General
Unless stated otherwise, solvents were evaporated at 40 °C/
2 kPa and compounds were dried at 13 Pa. Melting points were
determined on a Kofler block and are uncorrected. Analytical TLC
was performed on Silica Gel 60 F254 plates (Merck KGaA, Darms-
tadt, Germany); chromatographic systems are described in text.
3. Biological activity
Free phosphonic acids (7a–e, 21, 23) and the isopropyl ester 22
were tested for activity against DNA viruses [in human embryonic
lung (HEL) cells], retroviruses (HIV-1, HIV-2) in human T-lympho-
cyte (CEM) cells and against RNA viruses (in Vero cells). Concerning
DNA viruses, none of the tested compounds was active against her-
pes viruses HSV-1 and HSV-2. Activity of 6-substituted derivatives
7a–e was decreased against poxviruses (vaccinia virus, cowpox
virus, orf virus) and varicella zoster virus (50–100-fold compared
to parent HPMP-5-azaC) and 1000–1500-fold against cytomegalo-
virus. All prepared compounds were inactive against HIV-1 and
HIV-2. Surprisingly, in case of N-3 isomers we found anti RNA-viral
Column chromatography was performed on silica gel 60 lm (Flu-
ka). Preparative reverse phase HPLC separations were performed
on a Waters Delta 600 instrument with a Waters 2487 Dual k
Absorbance Detector using a column Luna PhenomenexÒ C-18
(10
l
m, 21 ꢁ 250 mm), flow 12 ml/min. 1H and 13C NMR spectra
were measured on a Bruker Avance 500 spectrometer (1H at
500 MHz, 13C at 125.7 MHz) in D2O or DMSO-d6 solutions (refer-
enced to sodium 3-(trimethylsilyl)propane-1-sulfonic acid (DSS)
or residual solvent signal). The numbering system for assignment
of NMR signals is outlined in Figure 2. Mass spectra were measured
on a ZAB-EQ (VG Analytical) spectrometer using FAB (ionization
with xenon, accelerating voltage 8 kV, glycerol matrix) or by ESI
technique. Optical rotations were measured on Autopol IV polar-
activity at isopropyl ester 22 (20 lg/ml for Sindbis virus). Since the
corresponding free phosphonic acid 21 was antivirally inactive, we
suppose that esterification of these structures (N-3 isomers) is nec-
essary to allow passage through the cell membrane to achieve anti-
viral activity.
The activity of 22 against RNA virus is rather atypical; acyclic
nucleoside phosphonates of HPMP structural type are typically ac-
tive against DNA viruses. One of the rare exceptions is recently de-
scribed activity of alkoxyalkyl esters of HPMPA against HCV (but
HPMPA as free phosphonic acid has no anti-HCV activity).24
imeter (Rudolph Research Analytical, USA) at 20 °C, [
a
]
values
D
are given in 10ꢂ1 deg cm2 gꢂ1. Most of chemicals and ion-exchange
resins (Dowex 1 ꢁ 2-400) were purchased from Sigma–Aldrich
(Czech Republic). Compound 4 was prepared according to previ-
ously described procedure.12
All prepared compounds are non-toxic (CC50 >100
none of the compounds revealed cytostatic activity.
lg/ml) and
5.2. C-6. Substituted 5-azacytosine phosphonate esters: general
procedure
25% Aqueous ammonia (30 mL) was added to a solution of 4
(950 mg, 1.57 mmol) in methanol (30 mL), the mixture was stirred
for 6 days at rt and then evaporated under reduced pressure. The
residue (intermediate 5) was coevaporated with absolute ethanol
(2 ꢁ 50 ml), and after drying in vacuo at 50 °C, dissolved in DMF
(7 mL). An appropriate orthoester (10 mmol) was added and the
mixture was heated under conditions given in Table 1. Then, the
solution was evaporated, the residue coevaporated with xylene
(20 mL) and subjected to column chromatography on silica gel
(150 mL) in system described below.
4. Conclusions
A new series of acyclic nucleoside phosphonates with 6-substi-
tuted 5-azacytosine base moiety was prepared using two different
synthetic procedures. Synthesis of enantiomerically pure modified
‘(S)-HPMP synthon’, that is, diisopropyl (1S)-[2-hydroxy-1-tosyl-
oxymethyl)ethoxy]methylphosphonate (15) has a general impor-
tance for preparations of HPMP derivatives whose base moiety is
sensitive to catalytic hydrogenation. Especially in case of antiviral-
ly active HPMP-5-azaC, synthesis based on utilization of this syn-
thon is easier and gives higher yields compared to the originally
described procedure.12 The described methodology is applicable
especially for large-scale syntheses of HPMP-5-azaC. Unfortunately,
compared to HPMP-5-azaC, introduction of an alkyl (aryl) group to
C-6 position of a base moiety leads to a substantial decrease of anti-
viral activity. On the other hand, anti RNA-viral activity of isopropyl
5.2.1. 1-(2S)-2-[(Diisopropoxyphosphoryl)methoxy-3-(triphen-
ylmethoxy)propyl-6-methyl-5-azacytosine (6a)
Chromatographed in system EtOAc–acetone–EtOH–water
(18:3:1:1), Rf = 0.24. White foam. [
NMR (DMSO-d6, ppm) d: 1.13, 1.18, 1.19 and 1.20 (4 ꢁ d, 4 ꢁ 3H,
a
]
ꢂ38.7 (c 0.626, CHCl3). 1H
D

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M. Krecmerová et al. / Bioorg. Med. Chem. 18 (2010) 387–395
391
NH2
NH2
N
NH2
1. CH3ONa
N
N
O
N
N
N
O
(isolation of salt)
N
N
O
N
H2N
N
O
O
N
O
N
H
2. 15, 120°C
OH
P(O)(Oi-Pr)2
DMF + DMSO
OH
P(O)(OR)2
OH
P(O)(Oi-Pr)2
O
. 16
. 17, R = iPr
14 % (N-3)
. 18
8 % (O2)
62 % (N-1)
(CH3)3SiBr
CH3CN
. 23, R = H
1. CH3ONa
CH3
NH2
N
(isolation of a sodium
NH2
N
salt of 6-methyl-5-
azacytosine)
N
N
N
N
H2N
N
O
O
N
CH3
2. 15, 120°C, DMF
O
N
H
CH3
OH
P(O)(OiPr)2
OH
O
O
P(O)(Oi-Pr)2
19
42 % (N-3)
20
6% (N-1)
(CH3)3SiBr
CH3CN
(CH3)3SiBr
CH3CN
CH3
CH3
NH2
N
N
N
N
N
N
H2N
N
O
H2N
N
O
O
N
CH3
OH
P(O)(OH)2
OH
P(O)(Oi-Pr)(OH)
OH
P(O)(OH)2
O
O
O
21
7a
. 22
Scheme 4. Alkylation of (S)-HPMP synthon 15 with sodium salts of 5-azacytosine and 6-methyl-5-azacytosine.
168.19 (C-6). FAB-MS, m/z: 621 [M+H]+, 379 [M+2HꢂTr]+, 243
[Tr], 165. Anal. Calcd for C33H41N4O6P: C, 63.86; H, 6.66; N, 9.03;
P, 4.99. Found: C, 63.53; H, 6.51; N, 8.74; P, 4.76.
NH2
4
5
6
3
N
2
N
R
3´
O
N
1
5.2.2. 1-(2S)-2-[(Diisopropoxyphosphoryl)methoxy-3-
(triphenylmethoxy)propyl-6-ethyl-5-azacytosine (6b)
Chromatographed in system chloroform–MeOH (9:1), Rf = 0.21.
2´
1´
OH
White foam. [
a]
D ꢂ50.6 (c 0.419, CHCl3). 1H NMR (DMSO-d6, ppm)
O
P(O)(OH)2
d: 1.13 (t, 3H, J = 7.3, CH3CH2), 1.13, 1.18, 1.19 and 1.205 (4 ꢁ d,
Figure 2. General numbering scheme for assignment of NMR signals.
4 ꢁ 3H, JCH3,CH = 6.2, (CH3)2CH), 2.81 (m, 2H, CH2), 2.99 (dd, 1H,
J3 a,2 = 3.7, Jgem = 10.6, H-30a), 3.29 (dd, 1H, J3 b,2 = 3.4, H-30b),
3.54 (dd, 1H, JP,CHa = 10.2, Jgem = 13.8, PCHa), 3.71 (dd, 1H,
0
0
0
0
JP,CHb = 8.4, PCHb), 3.86 (m, 1H, H-20), 3.93 (dd, 1H, J1 a,2 = 4.2,
0
0
0
0
JCH3,CH = 6.2, CH3), 2.43 (s, 3H, CH3), 3.00 (dd, 1H, J3 a,2 = 3.8,
Jgem = 10.4, H-30a), 3.27 (dd, 1H, J3 b,2 = 3.4, H-30b), 3.55 (dd, 1H,
JP,CHa = 10.1, Jgem = 13.7, PCHa), 3.72 (dd, 1H, JP,CHb = 8.4, PCHb),
3.86 (m, 1H, H-20), 3.94 (m, 2H, H-10), 4.51 (m, 2H, P–OCH), 7.23
and 7.24 (2 ꢁ br s, 2 ꢁ 1H, NH2), 7.28 (t, 3H, H-arom.), 7.36 (t,
6H, H-arom.), 7.41 (d, 6H, H-arom.). 13C NMR (DMSO-d6, ppm) d:
22.41 (CH3), 23.63 and 23.82 (2 ꢁ d, JP,C = 4.4, 2 ꢁ CH3), 23.85 and
23.88 (2 ꢁ d, JP,C = 3.4, 2 ꢁ CH3), 46.44 (C-10), 62.52 (C-30), 63.95
(d, JP,C = 165.0, P–C), 70.15 and 70.31 (2 ꢁ d, JP,C = 6.3, P–OCH),
78.85 (d, JP,C = 12.7, C-20), 86.36 (Tr), 127.34 (3C, Tr), 128.17 (6C,
Tr), 128.41 (6C, Tr), 143.63 (3C, Tr), 155.19 (C-2), 165.36 (C-4),
Jgem = 14.3, H-10a), 3.97 (dd, 1H, J1 b,2 = 8.3, H-10b), 4.51 (m, 2H,
P–OCH), 7.21 and 7.23 (2 ꢁ br s, 2 ꢁ 1H, NH2), 7.27 (t, 3H, H-arom.),
7.30 (t, 6H, H-arom.), 7.37 (d, 6H, H-arom.). 13C NMR (DMSO-d6,
ppm) d: 10.14 (CH3CH2), 23.59 (d, JP,C = 4.4, CH3), 23.83 (d, 2C,
JP,C = 3.9, CH3), 23.87 (d, JP,C = 3.9, CH3), 26.66 (CH2), 45.35 (C-10),
62.36 (C-30), 63.98 (d, JP,C = 165.2, P–C), 70.14 and 70.29 (2 ꢁ d,
JP,C = 3.9, P–OCH), 78.66 (d, JP,C = 13.0, C-20), 86.31 (Tr), 127.33
(3C, Tr), 128.15 (6C, Tr), 128.39 (6C, Tr), 143.61 (3C, Tr), (155.32
(C-2), 165.40 (C-4), 170.99 (C-6). +ESI-MS, m/z: 1291 [2 M+Na]+,
1269 [2 M+H]+, 657 [M+Na]+, 243 [Tr]. Anal. Calcd for
0
0
0
0

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M. Krecmerová et al. / Bioorg. Med. Chem. 18 (2010) 387–395
392
C34H43N4O6P: C, 64.34; H, 6.83; N, 8.83, P, 4.88. Found: C, 63.96; H,
7.01; N, 8.64; P, 5.11.
[Tr]. Anal. Calcd for C38H43N4O6P: C, 66.85; H, 6.35; N, 8.21; P,
4.54. Found: C, 66.80; H, 6.64; N, 7.95; P, 4.83.
5.2.3. 1-(2S)-2-[(Diisopropoxyphosphoryl)methoxy-3-
(triphenylmethoxy)propyl-6-propyl-5-azacytosine (6c)
Chromatographed in system chloroform–MeOH (9:1), Rf = 0.22.
5.3. Cleavage of the phosphonate esters: general procedure
Bromotrimethylsilane (1 mL, 7.5 mmol) was added to a solution
of appropriate ester 6a–e (0.7 mmol) in acetonitrile (8 mL), the
mixture was set aside in the dark for 48 h at rt and evaporated.
The residue was coevaporated with toluene (2 ꢁ 20 ml). 90% Aque-
ous methanol (50 mL) was added, the solution was neutralized
drop wise with 0.2 M triethylammonium hydrogencarbonate to
pH 7 and evaporated. Water (10 mL) was added and the mixture
applied onto a column of Dowex 1 (AcOꢂ form, 25 mL). The column
was eluted with methanol till disappearance of UV absorption (re-
moval of TrOH), followed by water (300 mL), a linear gradient of
acetic acid (0.5–1.0 M, 500 mL) and 1 M formic acid. UV absorbing
formic acid eluate was evaporated, the residue coevaporated with
water (4 ꢁ 30 mL) and lyophilized from water or crystallized.
White foam. [
a]
D ꢂ46.6 (c 0.222, CHCl3). 1H NMR (DMSO-d6, ppm)
d: 0.97 (t, 3H, JCH3,CH2 = 7.4, CH3), 1.05 (m, 2H, CH2), 1.13, 1.19, 1.20
0
0
and 1.21 (4 ꢁ d, 4 ꢁ 3H, JCH3,CH = 6.2, CH3), 2.70 (dd, 1H, J3 a,2 = 6.3,
Jgem = 10.6, H-30a), 2.97 (dd, 1H, J3 b,2 = 3.5, H-30b), 2.79 and 3.31
(2 ꢁ m, 2 ꢁ 1H, CH2), 3.52 (dd, 1H, JP,CHa = 10.2, Jgem = 13.6, PCHa),
3.69 (dd, 1H, JP,CHb = 8.6, PCHb), 3.85 (m, 1H, H-20), 3.92 (dd, 1H,
0
0
J1 a,2 = 3.5, Jgem = 14.2, H-10a), 3.99 (dd, 1H, J1 b,2 = 9.0, H-10b),
4.52 (m, 2H, P–OCH), 7.21 and 7.22 (2 ꢁ br s, 2 ꢁ 1H, NH2), 7.28
(t, 3H, H-arom.), 7.36 (t, 6H, H-arom.), 7.41 (d, 6H, H-arom.). 13C
NMR (DMSO-d6, ppm) d: 13.85 (CH3), 19.14 (CH2), 23.60 (d,
JP,C = 4.2, CH3), 23.83 (d, JP,C = 3.9, CH3), 23.86 (d, JP,C = 4.6, CH3),
23.90 (d, JP,C = 3.9, CH3), 35.49 (CH2), 45.43 (C-10), 61.99 (C-30),
63.87 (d, JP,C = 165.5, P–C), 70.14 and 70.29 (2 ꢁ d, JP,C = 6.2, P–
OCH), 78.47 (d, JP,C = 13.4, C-20); 80.26 (Tr), 127.35 (3C, Tr),
128.14 (6C, Tr), 128.38 (6C, Tr), 143.61 (3C, Tr), 155.28 (C-2),
165.38 (C-4), 170.18 (C-6). FAB-MS, m/z: 671 [M+Na]+, 243 [Tr],
165. Anal. Calcd for C35H45N4O6P: C, 64.80; H, 6.99; N, 8.64; P,
4.77. Found: C, 64.44; H, 6.97; N, 8.46; P, 4.60.
0
0
0
0
5.3.1. 1-(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-6-
methyl-5-azacytosine (7a)
Yield: 165 mg, 80%. White powder. Mp: 137–140 °C (MeOH–
EtOH). [
a
]
ꢂ78.2 (c 0.253, H2O). 1H NMR (D2O, ppm) d: 2.68 (s,
D
3H, CH3), 3.67 (dd, 1H, JP,CHa = 8.7, Jgem = 14.4, PCHa), 3.96 (dd, 1H,
JP,CHb = 7.7, PCHb), 3.89 (m, 2H, H-30), 4.16 (m, 1H, H-20), 4.19 (m,
13
2H, H-10). C NMR (D2O, ppm) d: 20.00 (CH3); 44.57 (C-10), 57.22
5.2.4. 6-Butyl-1-(2S)-2-[(diisopropoxyphosphoryl)methoxy-3-
(triphenylmethoxy)propyl-5-azacytosine (6d)
(C-30), 65.08 (d, JP,C = 159.5, P–C), 76.58 (d, JP,C = 8.8, C-20), 145.57
(C-2), 154.67 (C-4), 171.40 (C-6). FAB-MS, m/z: 589 [2 M+H]+, 295
[M+H]+. FAB-HRMS calcd for C8H16N4O6P 295.0807, found
295.0809 [M+H+].
Chromatographed in system EtOAc–acetone–EtOH–water
(18:3:1:1), Rf = 0.40. Yellowish syrup. [
a
]
ꢂ38.2 (c 0.169, CHCl3).
D
1H NMR (DMSO-d6, ppm) d: 0.92 (t, 3H, JCH3,CH2 = 7.3, CH3), 1.12,
1.18, 1.19 and 1.20 (4 ꢁ d, 4 ꢁ 3H, JCH3,CH = 6.1, CH3), 1.24, 1.38
0
0
and 1.62 (3 ꢁ m, 3 ꢁ 2H, CH2), 2.66 (dd, 1H, J3 a,2 = 5.4, Jgem = 10.5,
5.3.2. 6-Ethyl-1-(S)-3-hydroxy-2-(phosphonomethoxy)propyl]-
5-azacytosine (7b)
H-30a), 2.95 (dd, 1H, J3 b,2 = 2.9, H-30b), 3.51 (dd, 1H, JP,CHa = 10.3,
Jgem = 13.7, PCHa), 3.68 (dd, 1H, JP,CHb = 8.4, PCHb), 3.85 (m, 1H, H-
0
0
Yield: 149 mg, 69%, white amorphous solid. [
a]
D ꢂ89.1 (c 0.191,
20), 3.91 (dd, 1H, J1 a,2 = 2.7, Jgem = 14.1, H-10a), 3.99 (dd, 1H,
H2O). 1H NMR (D2O, ppm) d: 1.22 (s, 3H, CH3), 3.49 (dd, 1H,
0
0
J1 b,2 = 9.5, H-10b), 4.51 (m, 2H, P–OCH), 7.25 and 7.35 (2 ꢁ br s,
2 ꢁ 1H, NH2), 7.29 (t, 3H, H-arom.), 7.36 (t, 6H, H-arom.), 7.42 (d,
6H, H-arom.). 13C NMR (DMSO-d6, ppm) d: 13.99 (CH3), 22.13
(CH2), 23.75 (2 ꢁ d, 2C, JP,C = 4.9, CH3), 23.89 (2 ꢁ d, 2C, JP,C = 3.9,
CH3), 27.99 (CH2), 33.45 (CH2), 46.80 (C-10), 61.94 (C-30), 63.85
(d, JP,C = 165.5, P–C), 70.15 and 70.31 (2 ꢁ d, 2C, JP,C = 6.3, P–OCH),
78.54 (d, JP,C = 13.7, C-20), 86.25 (Tr), 127.36 (3C, Tr), 128.15 (6C,
Tr), 128.40 (6C, Tr), 143.60 (3C, Tr), 155.295 (C-2), 165.40 (C-4),
170.41 (C-6). FAB-MS, m/z: 685 [M+Na]+, 243 [Tr], 165. Anal. Calcd
for C36H47N4O6P: C, 65.24; H, 7.15; N, 8.45; P, 4.67. Found: C, 65.37;
H, 6.97; N, 8.24; P, 4.42.
0
0
0
0
JP,CHa = 9.1, Jgem = 13.3, PCHa), 3.65 (dd, 1H, J3 a,2 = 3.3, Jgem = 12.5,
H-30a), 3.77 (dd, 1H, JP,CHb = 8.8, PCHb), 3.82 (m, 1H, H-20), 3.89
(dd, 1H, J3 b,2 = 3.8, H-30b), 4.14 (d, 2H, J1 ,2 = 5.6, H-10). 13C NMR
(D2O, ppm) d: 8.90 (CH3); 45.78 (C-10), 59.53 (C-30), 65.72 (d,
JP,C = 157.1, P–C), 78.75 (d, JP,C = 10.6, C-20), 150.57 (C-2); 159.15
(C-4), 175.94 (C-6). FAB-MS, m/z: 617 [2M+H]+, 309 [M+H]+. FAB-
HRMS calcd for C9H18N4O6P 309.0964, found 309.0959 [M+H+].
0
0
0
0
5.3.3. 1-(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-6-
propyl-5-azacytosine (7c)
Yield: 111 mg, 49%, white amorphous solid. [
a]
D ꢂ90.0 (c 0.236,
H2O). 1H NMR (D2O, ppm) d: 0.91 (t, 3H, JCH3 CH2
,
= 7.3, CH3), 1.64
0
0
5.2.5. 1-(2S)-2-[(Diisopropoxyphosphoryl)methoxy-3-
(triphenylmethoxy)propyl-6-phenyl-5-azacytosine (6e)
Chromatographed in system EtOAc–acetone–EtOH–water
(m, 2H, CH2), 2.83 (m, 2H, CH2), 3.40 (dd, 1H, J3 a,2 = 9.0, Jgem = 12.6,
H-30a), 3.56 (dd, 1H, J3 b,2 = 3.3, H-30b), 3.67 (dd, 1H, J1 a,2 = 8.8,
0
0
0
0
Jgem = 12.4, H-10a), 3.73 (m, 1H, H-20), 3.79 (dd, 1H, J1 b,2 = 2.5, H-
10b), 4.05 (d, 2H, JP,CH = 8.6, PCH2). 13C NMR (D2O, ppm) d: 12.48
(CH3), 18.72 (CH2), 35.90 (CH2), 45.83 (C-10), 59.55 (C-30), 65.75
(d, JP,C = 159.0, P–C), 78.72 (d, JP,C = 10.2, C-20), 151.10 (C-2),
159.42 (C-4), 174.95 (C-6). FAB-MS, m/z: 323) [M+H]+. FAB-HRMS
calcd for C10H20N4O6P 323.1120, found 323.1113 [M+H+].
0
0
(18:3:1:1), Rf = 0.42. White foam. [
a]
ꢂ24.3 (c 0.323, CHCl3). 1H
D
NMR (DMSO-d6, ppm) d: 1.20, 1.22 and 1.25 (3 ꢁ d, 12H,
JCH3,CH = 6.2, CH3), 3.19 (dd, 1H, J3 a,2 = 2.1, Jgem = 13.2, H-30a), 3.44
(dd, 1H, JP,CHa = 10.1, Jgem = 13.6, PCHa), 3.61 (dd, 1H, JP,CHb = 8.9,
0
0
PCHb), 3.71 (dd, 1H, J1 a,2 = 2.0, Jgem = 13.6, H-10a), 3.91 (dd, 1H,
0
0
J3 b,2 = 7.6, H-30b), 4.02 (dd, 1H, J1 b,2 = 9.5, H-10b), 4.54 (m, 2H, P–
OCH), 4.60 (m, 1H, H-20), 7.23 (t, 3H, H-arom.), 7.25 (br s, 1H,
NH2), 7.30 (t, 6H, H-arom.), 7.40 (br s, 1H, NH2), 7.42 (d, 6H, H-
arom.), 7.51 (t, 2H, H-arom.), 7.60 (t, 1H, H-arom.), 7.69 (d, 2H,
H-arom.). 13C NMR (DMSO-d6, ppm) d: d 23.89 (d, 2C, JP,C = 4.9,
CH3), 23.93 (d, 2C, JP,C = 3.9, CH3), 47.67 (C-10), 61.50 (C-30), 63.82
(d, JP,C = 166.2, P–C), 70.21 and 70.44 (2 ꢁ d, 2C, JP,C = 6.3, P–OCH),
77.45 (d, JP,C = 12.9, C-20), 85.92 (Tr), 127.24 (3C, Tr), 128.08 (6C,
Tr), 128.27 (8C, Tr, Ph), 128.55 (2C, Ph), 130.17 and 133.87 (Ph),
143.53 (3C, Tr). FAB-MS, m/z: 683 [M+H]+, 441 [M+2HꢂTr], 243
0
0
0
0
5.3.4. 6-Butyl-1-(S)-3-hydroxy-2-(phosphonomethoxy)propyl]-
5-azacytosine (7d)
Yield: 179 mg, 76%, white amorphous solid. [
a]
D ꢂ51.5 (c 0.253,
H2O). 1H NMR (D2O, ppm) d: 0.92 (t, 3H, JCH3,CH2 = 6.4, CH3), 1.43
(m, 2H, CH2), 1.69 (q, 2H, J = 7.6, CH2), 2.95 (m, 2H, CH2), 3.57
0
0
(dd, 1H, JP,CHa = 8.5, Jgem = 13.8, PCHa), 3.66 (dd, 1H, J3 a,2 = 2.7,
Jgem = 12.2, H-30a), 3.85 (dd, 1H, JP,CHb = 8.2, PCHb), 3.87 (m, 1H,
H-20), 3.89 (dd, 1H, J3 b,2 = 4.0, H-30b), 4.17 (d, 2H, J1 ,2 = 5.6, H-
10). 13C NMR (D2O, ppm) d: 10.44 (CH3), 19.02 (CH2), 24.81 (CH2),
0
0
0
0

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M. Krecmerová et al. / Bioorg. Med. Chem. 18 (2010) 387–395
393
30.0 (br, CH2), 43.72 (C-10), 57.20 (C-30), 62.85 (d, JP,C = 157.9, P–C),
5.4. Diisopropyl (1S)-[2-benzyloxy-1-(tosyloxymethyl)ethoxy]-
methylphosphonate (14)
76.38 (d, JP,C = 8.9, C-20), 146.23 (C-2), 155.11 (C-4), 173.77 (C-6).
FAB-MS, m/z: 337 [M+H]+. FAB-HRMS calcd for C11H22N4O6P
337.1277, found 337.1273 [M+H+].
The
compound
was
prepared
from
(2S)-2-[(trityl-
oxy]methyl]oxirane (11) according to a protocol described for the
racemic form.23
[a]D +13.7 (c 0.246, CHCl3). NMR data are in agree-
5.3.5. 1-(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-6-
phenyl-5-azacytosine (7e)
ment with the literature.23
Yield: 153 mg, 49%, white amorphous solid. [
a
]
D ꢂ36.7 (c 0.185,
H2O). 1H NMR (D2O, ppm) d: 3.41 (dd, 1H, JP,CHa = 9.2, Jgem = 13.5,
5.5. Diisopropyl (1S)-[2-hydroxy-1-tosyloxymethyl)ethoxy]me-
thylphosphonate (15)
PCHa), 3.42 (dd, 1H, J3 a,2 = 3.6, Jgem = 12.6, H-30a), 3.61 (dd, 1H,
0
0
JP,CHb = 8.7, PCHb), 3.65 (dd, 1H, J3 b,2 = 3.9, H-30b), 3.74 (m, 1H, H-
0
0
20), 4.15 (m, 2H, H-10), 7.61 (m, 2H, H-300-Ph), 7.68 (m, 1H, H-400-
10% Palladium on carbon (1.05 g) was added to a solution of 14
(11.5 g, 22.4 mmol) in methanol (150 mL) and the mixture was
hydrogenated at room temperature under atmospheric pressure
for 24 h. The solid was filtered off through Celite, washed with
methanol (2 ꢁ 100 mL) and combined filtrates were concentrated
in vacuo. The syrupy residue was chromatographed on a column
of silica gel (400 mL) in ethyl acetate. Product containing fractions
were evaporated to give 7.7 g (81%) of compound 8 as a colourless
13
Ph), 7.23 (m, 2H, H-200-Ph). C NMR (D2O, ppm) d: 48.19 (C-10),
60.30 (C-30), 66.18 (d, JP,C = 157.8, P–C), 79.02 (d, JP,C = 10.3, C-20),
128.66 (C-200-Ph), 129.59 (C-300-Ph), 131.85 (C-100-Ph), 132.74 (C-
400-Ph), 151.62 (C-2), 159.99 (C-4), 171.86 (C-6). FAB-MS, m/z:
357 [M+H]+. FAB-HRMS calcd for C13H18N4O6P 357.0964, found
357.0956 [M+H+].
syrup, [a]
D +21.4 (c 0.337, CHCl3). 1H NMR (DMSO-d6, ppm) d: 1.20
5.3.6. 3-(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-6-
methyl-5-azacytosine (21)
(d, 3H, JCH3,CH = 6.2, CH3), 1.205 (d, 3H, CH3), 1.225 (d, 6H, CH3),
2.42 (s, 3H, CH3), 3.37 (dt, 1H, JCH2,OH = JCH2,CH = 6.0, Jgem = 11.2,
OCH2), 3.45 (dt, 1H, JCH2,OH = JCH2,CH = 5.0, Jgem = 11.2), 3.62 (m,
1H, OCH), 3.71 (dd, 1H, JP,CH = 8.8, Jgem = 13.9, PCH2), 3.81 (dd, 1H,
PCH2), 3.98 (dd, 1H, JCH2,CH = 6.0, Jgem = 10.6, OCH2), 4.15 (dd, 1H,
JCH2,CH = 3.1, Jgem = 10.6, OCH2), 4.56 (m, 2H, P–OCH), 4.82 (t, 1H,
JOH,CH2 = 5.5, OH), 7.49 (d, 2H, H-arom.), 7.79 (d, 2H, H-arom.). 13C
NMR (DMSO-d6, ppm) d: 21.26 (CH3), 23.81 (d, JP,C = 4.4, CH3),
23.97 (d, JP,C = 4.4, CH3), 59.13 (OCH2), 69.73 (OCH2), 70.39 d and
70.41 (2 ꢁ d, JP,C = 6.3, P–OCH), 79.41 (d, JP,C = 11.2, OCH); 127.80
(2C, arom.), 130.34, (2C, arom.), 132.32 and 145.12 (arom.). FAB-
MS, m/z: 425 [M+H]+. Anal. Calcd for C17H29O8SP: C, 48.11; H,
6.89; S, 7.55; P, 7.30. Found: C, 47.71; H, 6.99; S, 7.80; P, 7.48.
The compound was eluted from Dowex 1 with 0.3 M acetic acid
(together with 22). Both compounds were separated by reverse
phase HPLC with a linear gradient of MeOH (0–50%): 21 was eluted
first at 1–5% MeOH followed by 22 (eluted with 10% MeOH). Yield
of 21: 72 mg (35%) of a white amorphous solid after lyophilization
from water. [
a
]
D ꢂ13.3 (c 0.318, H2O). 1H NMR (D2O, ppm) d: 2.43
(s, 3H, CH3), 3.60 (dd, 1H, JP,CHa = 8.8, Jgem = 13.2, PCHa), 3.68 (dd,
1H, J3 a,2 = 4.5, Jgem = 13.9, H-30 a), 3.82 (dd, 1H, JP,CHb = 9.3,
0
0
Jgem = 13.2, PCHb), 3.90 (dd, 1H, J3 b,2 = 3.9, Jgem = 13.9, H-30b),
0
0
3.91 (m, 1H, H-20), 4.12 (dd, 1H, J1 a,2 = 7.8, Jgem = 15.4, H-10a),
0
0
4.25 (dd, 1H, J 1 b,2 = 3.3, Jgem = 15.4, H-10b). Proton-coupled 13C
NMR (D2O, ppm) d: 21.61 (CH3), 45.83 (C-10), 59.70 (C-30), 66.02
(d, JP,C = 156.7, P–C), 79.12 (d, JP,C = 10.7, C-20), 148.75 (t, J = 3.9,
C-2), 159.91 (t, J = 3.9, C-4), 170.32 (q, J = 5.8, C-6). FAB-MS, m/z:
295 [M+H]+. FAB-HRMS calcd for C8H16N4O6P 295.0807, found
295.0801 [M+H+].
0
0
5.6. Condensation reactions of 15 with 5-azacytosine and 6-
methyl-5-azacytosine
1 M Sodium methoxide in methanol (10 mL) was added to a
suspension of appropriate base (10 mmol) in absolute methanol
(100 mL) and the mixture refluxed for 2 h. The mixture was stirred
for 12 h at 25 °C and evaporated to 1/4 of an original volume. Dry
ether (100 mL) was added, the precipitated sodium salt filtered by
suction, washed with ether and dried in vacuo.
5.3.7. 3-[(2S)-2-(Isopropoxyphosphoryl)methoxy-3-hydroxypro-
pyl]-5-azacytosine (22)
Obtained as a by-product in preparation of 21. Yield: 47 mg (20
as a white amorphous solid after lyophilization from water. 1H
NMR (D2O, ppm) d: 0.17 (s, 6H, JCH3,CH = 6.2, CH3), 2.43 (s, 3H,
CH3), 3.58 (dd, 1H, JP,CHa = 9.0, Jgem = 13.2, PCHa), 3.63 (dd, 1H,
A mixture of thus obtained sodium salt of 5-azacytosine or
6-methyl-5-azacytosine and synthon 15 (4.2 g, 9.9 mmol) in
dimethylformamide (30 mL) was heated to 90 °C with exclusion
of moister for 5 h. Additional portion of 15 (1.2 g, 2.8 mmol) was
added together with a catalytic amount of cesium carbonate
(10 mg) and the heating was continued at 120 °C for 2 h. During
that time, a complete dissolution occurred. The reaction mixture
was taken down, the residue coevaporated with toluene
(2 ꢁ 50 mL) and xylene (50 mL) and applied onto a column of silica
gel (400 mL) in system chloroform–methanol (95:5). A column was
eluted first with this system, after elution of approx. 1.5 L volume,
the polarity of system was increased to the ratio chloroform–meth-
anol (85:15). Three types of products were obtained in the follow-
ing elution sequence: 2-O-isomer 18, N3-isomer (17 or 19) and
finally, the N-1 isomer (16 or 20). The product containing fractions
were taken down and coevaporated with absolute ethanol.
J3 a,2 = 3.3, Jgem = 11.4, H-30a), 3.74 (pent, 1H, JCH3,CH = 6.2, P–
0
0
0
b,20
OCH), 3.83 (dd, 1H, J3
= 4.5, Jgem = 11.4, H-30b), 3.84 (dd, 1H,
JP,CHb = 8.8, Jgem = 13.2, PCHb), 3.98 (m, 1H, H-20), 4.16 (dd, 1H,
J1 a,2 = 7.7, Jgem = 15.4, H-10a), 4.26 (dd, 1H, J1 b,2 = 3.5, Jgem = 15.4,
H-10b). 13C NMR (D2O, ppm) d: 20.75 (2C, CH3), 21.00 (CH3),
46.20 (C-10), 65.90 (C-30), 65.96 (d, JP,C = 156.2, P–C), 77.67 (d,
JP,C = 10.7, C-20), 73.36 (P–O–C), 148.73 (C-2), 159.91 (C-4),
170.28 (C-6).
0
0
0
0
5.3.8. 3-(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]-5-
azacytosine (23)
Yield 78 mg (40%), white foam, [
a]
+14.3 (c 0.224, H2O). 1H
D
NMR (D2O, ppm) d: 3.60 (dd, 1H, JP,CH = 8.8, Jgem = 13.2, PCHa),
3.69 (dd, 1H, J3 a,2 = 3.2, Jgem = 11.6, H-30a), 3.84 (dd, 1H, JP,CHb = 9.2,
0
0
Jgem = 13.2, PCHb), 3.91 (dd, 1H, J3 b,2 = 3.9, Jgem = 11.6, H-30b), 3.92
0
0
(m, 1H, H-20), 4.14 (dd, 1H, J1 a,2 = 7.8, Jgem = 14.9, H-10a), 4.27 (dd,
0
0
5.6.1. 1-[(2S)-2-(Diisopropoxyphosphoryl)methoxy-3-hydroxy-
propyl] -5-azacytosine (16)
1H, J1 b,2 = 3.2, Jgem = 14.9, H-10b), 8.32 (s, 1H, H-6). 13C NMR (D2O,
ppm) d: 46.05 (C-10), 59.70 (C-30), 65.94 d, J(P,C) = 156.7 (PCH2),
79.06 d, J(P,C) = 10.7 (C-20), 148.47 (C-2), 158.56 (C-6), 160.83 (C-
4). FAB-MS, m/z: 281.1 [M+H]+. FAB-HRMS calcd for C7H14N4O6P
281.0651, found: 281.0652 [M+H+].
0
0
Yield: 2.4 g (62%) as colourless oil crystallizing after several days
standing, [
a
]
ꢂ43.0 (c 0.49, ethanol). 1H NMR (DMSO-d6, ppm) d:
D
1.19, 1.21, 1.22 and 1.23 (4 ꢁ d, 12H, JCH3,CH = 6.2, CH3), 3.45 (ddd,
1H, J3 b,2 = 4.4, J3 b,OH = 5.8, Jgem = 12.0, H-30b), 3.51 (tt, 1H,
0
0
0

ˇ
M. Krecmerová et al. / Bioorg. Med. Chem. 18 (2010) 387–395
394
J3 a,OH = J3 a,2 = 4.8, Jgem = 12.0, H-30a), 3.59 (dd, 1H JP,CHa = 8.2,
Jgem = 14.0, PCHa), 3.63 (m, 1H, H-20), 3.70 (dd, 1H, JP,CHb = 9.4,
C-6). FAB-MS, m/z: 379.1 [M+H]+. Anal. Calcd for C14H27N4O6Pꢃ1/
2H2O: C, 43.41; H, 7.29; N, 14.46; P, 8.00. Found: C, 43.37; H,
7.36; N, 14.55; P, 7.72.
0
0
0
Jgem = 14.0, PCHb), 3.89 (dd, 1H, J1 a,2 = 8.1, Jgem = 14.1, H-10a), 3.96
0
0
(dd, 1H, J1 b, 2 = 2.4, Jgem = 14.1, H-10b), 4.54 (m, 2H, P–OCH), 4.83
0
0
0
(t, 1H, JOH,3 = 5.5, OH), 7.38 (br s, 2H, NH2), 8.06 (s, 1H, H-6). Pro-
5.6.5. 1-[(2S)-2-(Diisopropoxyphosphoryl)methoxy-3-
hydroxypropyl]-6-methyl-5-azacytosine (20)
ton-coupled 13C NMR (DMSO-d6, ppm) d: 23.78 and 23.87 (2 ꢁ d,
JP,C = 4.4, CH3), 23.91 and 23.95 (2 ꢁ d, JP,C = 4.9, CH3), 47.70 (C-10),
60.34 (C-30), 63.61 (d, JP,C = 165.0, P–C), 70.33 (d, 2C, JP,C = 6.4, P–O–
Yield: 240 mg (7%) of a white foam, [
a
]
D ꢂ68.5 (c 0.263, CHCl3).
1H NMR (DMSO-d6, ppm) d: 1.15, 1.19, 1.20 and 1.215 (4 ꢁ d, 12H,
C), 79.28 (d, JP,C = 10.7, C-20), 154.29 (dt, JC-2,H-6 = 4.9, JC-2,H-1 a
0
0
0
JCH3,CH = 6.2, CH3), 2.40 (s, 3H, CH3), 3.46 (ddd, 1H, J3 a,2 = 3.9,
0
= JC-2,H-1 b = 2.9, C-2), 166.63 d, JC-4,H-6 = 12.7, C-4), 159.785 (dt,
J3 a,OH = 5.9, Jgem = 12.0, H-30a), 3.59 (dd, 1H, JP,CHa = 10.3,
0
1J = 204.1, JC-6,H-1 a = JC-6,H-1 b =3.9, C-6). FAB-MS, m/z: 365 [M+H]+,
281 [free phosphonic acid + H]+, 113 [5-azacytosine + H]+. Anal.
Calcd for C13H25N4O6Pꢃ1/2C2H5OH: C, 43.40; H, 7.29; N, 14.46; P,
8.00. Found: C, 43.58; H, 6.99; N, 14.45; P, 8.22.
0
0
00
0
0
Jgem = 13.9, PCHa), 3.61 (ddd, 1H, J3 b,2 = 3.8, J3 b,OH = 5.0, Jgem = 12.0,
H-30b), 3.66 (m, 1H, H-20), 3.76 (dd, 1H, J1 a,2 = 9.2, Jgem = 14.2, H-
10a), 3.92 (dd, 1H, JP,CHb = 7.7, Jgem = 13.9, PCHb), 3.98 (dd, 1H,
0
0
J1 b,2 = 2.8, Jgem 14.2, H-10b), 4.51 (m, 2H, P–OCH), 4.84 (t, 1H,
0
0
0
JOH,3 = 5.5, OH), 7.18 and 7.21 (2 ꢁ br s, 2H, NH2). Proton-coupled
5.6.2. 3-[(2S)-2-(Diisopropoxyphosphoryl)methoxy-3-
13C NMR (DMSO-d6, ppm) d: 22.47 (CH3), 23.67 (d, JP,C = 3.9, CH3),
23.83 and 23.85 (2 ꢁ d, JP,C = 4.4, CH3), 23.92 (d, JP,C = 3.9, CH3),
46.265 (C-10), 60.31 (C-30), 63.71 (d, JP,C = 164.5, P–C), 70.12 and
70.23 (2 ꢁ d, JP,C = 6.3, P–OCH), 80.23 (d, JP,C = 11.7, C-20), 155.32
(t, J = 2.9, C-2), 165.36 (C-4), 168.35 (qt, J = 2,9 and 5.9, C-6). FAB-
MS, m/z: 379.1 [M+H]+. Anal. Calcd for C14H27N4O6P: C, 44.44; H,
7.19; N, 14.81; P, 8.19. Found: C, 44.37; H, 7.26; N, 14.55; P, 7.85.
hydroxypropyl] -5-azacytosine (17)
Yield 0.5 g (14%), amorphous solid, [a D
NMR (DMSO-d6, ppm) d: 1.20 (d, 3H, JCH3,CH = 6.1, CH3), 1.22 (d, 6H,
]
ꢂ13.7 (c 0.352, CHCl3). 1H
0
0
0
CH3), 1.23 (d, 3H, CH3), 3.50 (dt, 1H, J3 a,2 ꢄ J3 a,OH = 5.2, Jgem = 11.8,
H-30a), 3.55 (dt, 1H, J3 b,2 ꢄ J3 b,OH = 4.6, Jgem = 11.8, H-30b), 3.75 (dd,
0
0
0
1H, JP,CHa = 8.5, Jgem = 14.2, PCHa), 3.78 (m, 1H, H-20), 3.90 (dd, 1H,
J1 a,2 = 4.7, Jgem = 14.5, H-10a), 3.91 (dd, 1H, JP,CHb = 8.0, Jgem = 14.2,
0
0
PCHb), 3.95 (dd, 1H, J1 b,2 = 7.6, Jgem = 14.5, H-10b), 4.55 (m, 2H, P–
0
0
Acknowledgements
0
OCH), 4.94 (t, 1H, JOH,3 = 5.2, OH), 7.50 (br s, 1H, NH2), 8.30 (br s, 1H,
NH2), 7.88 (s, 1H, H-6). Proton-coupled 13C NMR (DMSO-d6, ppm) d:
23.81 (d, JP,C = 4.4, CH3), 23.89 (d, JP,C = 4.9, CH3), 23.94 and 23.97
(2 ꢁ d, JP,C = 3.9, CH3), 43.76 (C-10), 60.95 (C-30), 64.20 (d, JP,C = 164.6,
P–C), 70.42 and 70.47 (2 ꢁ d, JP,C = 6.3, P–OCH), 79.32 (d, JP,C = 9.8, C-
This work is a part of the research project of the Institute
Z40550506. It was supported by the Centre of New Antivirals
and Antineoplastics 1M0508 by the Ministry of Education, Youth
and Sports of the Czech Republic, the Program of targeted projects
of Academy of Sciences of the Czech Republic 1QS400550501 and
by Gilead Sciences, Inc. (Foster City, CA, USA). The authors0 thanks
are due to Professor Jan Balzarini and his group in the Rega Insti-
tute for Medical Research, Catholic University Leuven (Belgium)
for antiviral and cytotoxicity assays of described compounds, to
Dr. P. Fiedler from this Institute for a measurement of IR spectra,
20), 155.59 (dt, JC-2,H-6 = 12.7, JC-2,H-1 a = JC-2,H-1 b = 3.9, C-2), 160.96
0
0
(dt, JC-4,H-6 = 12.7, JC-4,H-1 a = JC-4,H-1 b = 3.9, C-4), 166.46 (d, 1J = 195.3,
C-6). FAB-MS, m/z: 365 [M+H]+. Anal. Calcd for C13H25N4O6P: C,
42.86; H, 6.92; N, 15.38; P, 8.50. Found: C, 42.42; H, 7.05; N, 15.54;
P, 8.68.
0
0
5.6.3. Diisopropyl {[(1R)-2-[(4-amino-1,3,5-triazin-2-yl)oxy]-1-
ˇ
the staff of the mass spectrometry (Dr. J. Cvacka, Head) and
(hydroxymethyl)ethoxy]methyl}-phosphonate) (18)
Yield 0.3 g (8%), colourless oil, [a
]D + 2.3 (c 0.502, ethanol). 1H NMR
ˇ
analytical departments of the Institute (Dr. S. Matejková, Head).
ˇ
An excellent technical assistance of Mrs. Julie Krelinová is also
(DMSO-d6, ppm) d: 1.20, 1.21, 1.22 and 1.23 (4 ꢁ d, 12H, JCH3,CH = 6.1,
0
CH3), 3.53 (t, 2H, J3 ,2 = J3 ,OH = 5.5, H-30), 3.72 (m, 1H, H-20), 3.87 and
acknowledged.
0
0
0
0
3.91 (2 ꢁ dd, 2H JP,CH = 8.7, Jgem = 13.8, PCH2), 4.25 (dd, 1H, J1 a,2 = 5.9,
0
Jgem = 11.6,H-10a), 4.40(dd, 1H,J1 b,2 = 3.7, Jgem = 11.6,H-10b), 4.59(m,
0
References and notes
0
2H, P–OCH), 4.82 (t, 1H, JOH,3 = 5.5, OH), 7.45 and 7.51 (2 ꢁ br s, 2H,
NH2), 8.25 (s, 1H, H-6). Proton-coupled 13C NMR (DMSO-d6, ppm) d:
23.85 (d, 2C, JP,C = 4.4, CH3), 24.02 (d, 2C, JP,C = 3.9, CH3), 59.98 (C-30),
64.05 (d, JP,C = 165.0, P–C), 65.94 (C-10), 70.36 (d, JP,C = 6.4, P–OCH),
70.43 (d, JP,C = 6.4, P–OCH), 80.09 (d, JC,P = 11.7, C-20), 167.815 (d,
1J = 200.2, C-6), 168.05 (d, JC-4,H-6 = 10.7, C-4), 170.065 (dt,
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ˇ
Yield: 1.6 g (42%) as white crystals, Mp: 124 °C (EtOAc), [
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]
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ꢂ24.9 (c 0.700, CHCl3). 1H NMR (DMSO-d6, ppm) d: 1.19, 1.21,
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ˇ
´
3.48 (dt, 1H, J3 a,2 = J3 b,OH = 5.0, Jgem = 12.0, H-30a), 3.53 (dt, 1H,
0
0
0
Snoeck, R.; Naesens, L.; Neyts, J.; De Clercq, E. J. Med. Chem. 2007, 50, 1069.
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ˇ
´
13. Krecmerová, M.; Holy, A.; Pohl, R.; Masojídková, M.; Andrei, G.; Naesens, L.;
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0
0
0
0
0
1H, JP,CHa = 8.6, Jgem = 14.2, PCHa), 3.81 (dd, 1H, J1 a,2 = 5.1,
Jgem = 14.6, H-10a), 3.90 (dd, JP,CHb = 8.1, Jgem = 14.2, PCHb), 3.93
(dd, 1H, J1 b,2 = 7.6, Jgem = 14.6, H-10b), 4.55 (m, 2H, P–OCH), 4.93
0
0
0
(t, 1H, JOH,3 = 5.0, OH), 7.35 and 8.10 (2 ꢁ br s, 2H, NH2). Proton-
coupled 13C NMR (DMSO-d6, ppm) d: 23.78 and 23.80 (2 ꢁ d,
JP,C = 4.4, CH3), 23.91 and 23.23.96 (2 ꢁ d, JP,C) = 3.9, CH3), 25.575
(CH3), 43.36 (C-10), 60.94 (C-30), 64.195 (d, JP,C = 163.6, P–C),
70.42 and 70.47 (d, JP,C = 6.3, P–OCH), 79.44 (d, JP,C = 10.3, C-20),
155.70 (t, J = 2.9, C-2), 160.16 (t, J = 2.9, C-4), 175.775 (q, J = 5.8,

ˇ
M. Krecmerová et al. / Bioorg. Med. Chem. 18 (2010) 387–395
395
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