Synthesis of phospholipid-Ribavirin Conjugates

Article abstract of DOI:10.1002/hlca.201200203

New conjugates of antiviral nucleoside Ribavirin (=1-(β-D- ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide; 1) with 1,2- and 1,3-diacyl glycerophosphates have been synthesized by the phosphoramidite method. A combination of 2',3'-phenylboronate protecting group for the sugar moiety of the ribonucleoside 1 and 2-cyanoethyl protection for the phosphate fragment ensured the preparation of the desired compounds with reasonable yields via a small number of synthetic steps. Copyright

Full text of DOI:10.1002/hlca.201200203

Helvetica Chimica Acta – Vol. 96 (2013)
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Synthesis of PhospholipidꢀRibavirin Conjugates
by Ilona A. Oleynikova, Tamara I. Kulak*, Dmitry A. Bolibrukh, and Elena N. Kalinichenko
Institute of Bioorganic Chemisrty, National Academy of Sciences of Belarus, 220141 Minsk, Belarus
(e-mail: kulak@iboch.bas-net.by)
New conjugates of antiviral nucleoside Ribavirin (¼1-(b-d-ribofuranosyl)-1H-1,2,4-triazole-3-
carboxamide; 1) with 1,2- and 1,3-diacyl glycerophosphates have been synthesized by the phosphor-
amidite method. A combination of 2’,3’-phenylboronate protecting group for the sugar moiety of the
ribonucleoside 1 and 2-cyanoethyl protection for the phosphate fragment ensured the preparation of the
desired compounds with reasonable yields via a small number of synthetic steps.
1. Introduction. – Nucleoside and nucleotide analogs are successfully used in the
therapy of various malignances as well as for the treatment of viral infections [1][2].
However, the usage of such compounds is often restricted by their low bioavailability
and pure pharmacokinetic properties, which necessitate the application of these drugs
at high, often toxic, doses. One of the ways to overcome this disadvantage is a chemical
modification of biologically active nucleosides and nucleotides aimed at the prepara-
tion of their prodrugs, including the lipid derivatives [3]. The type of an active-
compound modification is dictated both by the expected point of its action (intestine,
brain, lymphatic system, etc.) and the knowledge on possible metabolic pathways of
lipoconjugates [4].
This approach was successfully employed for preparing the prodrugs of various
biologically active nucleosides and nucleotides such as 3’-azido-3’-deoxythymidine
(AZT) [5 – 8], 2’,3’-dideoxyinosine (ddI) [6], 2’,3’-didehydro-3’-deoxythymidine (d4T)
[9], 1-(b-d-arabinofuranosyl)cytosine (Cytarabine) [10 – 12], 2-fluoro-9-(b-d-arabino-
furanosyl)adenine (Fludarabine) [13], 2’,2’-difluoro-2’-deoxycytidine (dFdC, Gemci-
tabine) [12][14].
One of the important nucleosides used in medical practice is Ribavirin (¼1-(b-d-
ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide; 1), a drug exhibiting pronounced
activity against various RNA and DNA viruses [15]. Oral Ribavirin is approved (in a
combination with recombinant human interferon 2a) for the therapy of chronic
hepatitis C in adults [16], and its nebulized form – for the treatment of diseases caused
by respiratory sincitial virus (RSV) in children [17]. Intravenous Ribavirin was
effectively used against RSV viral infections in adults after lung transplantation [18],
and for treatment of viral hemorrhagic fevers [19][20]. In recent studies on rats, it was
demonstrated that intraperitoneal (i.p.) Ribavirin induced amelioration of experimen-
tal autoimmune encephalomyelitis, a prototypical animal model of human multiple
sclerosis [21][22]. The use of Ribavirin is often limited by the toxicity of the drug
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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connected mainly with its property to accumulate in erythrocytes causing severe
hemolytic anemia. At present, the studies are aimed at the development of reasonable
schemes for the application of Ribavirin prodrug, Viramidine (¼1-b-d-ribofuranosyl-
1H-1,2,4-triazole-3-carboxamidine), which, being transformed to Ribavirin in the liver,
does not accumulate in the erythrocytes and, consequently, does not cause severe
hemolysis, thus reducing the risks of side-effects connected with anemia [23]. In animal
model of viral hepatitis, targeted delivery of Ribavirin to liver was accomplished using
Ribavirin conjugates with macromolecular carriers such as lactosaminated poly-l-
lysine [24] and hemoglobin [25]. This approach was associated with reduced systemic
toxicity, increased chemotherapeutic index of the drug, and amelioration of liver injury.
The efficacy of liposome-encapsulated Ribavirin in liver targeting has also been
demonstrated [26][27].
In the literature, there are few data on the synthesis and biological properties of
lipophilic ribavirin derivatives. For example, in the in vivo experiments it was shown
that 2’,3’,5’-tri-O-acetate derivative of Ribavirin exhibited essentially higher activity
against encephalitis caused by Dengue virus type 2 in mice [28] and viral hemorrhagic
fewer in monkeys [29]. On the other hand, in several studies on animals, it was
demonstrated that 1,2-diacylphosphatidyl derivatives of antiviral dideoxynucleosides
(dideoxycytidine, dideoxyguanosine) were characterized by enhanced hepatic uptake
after i.p. administration of their liposomal preparations [30][31], and were substan-
tially more effective in the treatment of experimental hepatitis viral infection as
compared with neat nucleoside [31].
The present paper is devoted to the preparation of previously unknown lipid
derivatives of Ribavirin, namely, the conjugates of a given nucleoside with 1,2- and 1,3-
diacyl glycerophosphates.
2. Synthesis. – To conjugate the lipids with the nucleosides, one can use the
approaches similar to those employed in oligonucleotide chemistry, including
phosphodiester [9][13], H-phosphonate [6][7][9][32], phosphotriester [8], phosphor-
amidite [5][12][14][33], and enzymatic [34] methods. For the synthesis of conjugates,
we have chosen the method based on the preparation of phosphoramidite derivatives of
the lipids, followed by their condensation with the selectively protected nucleoside with
a free OH group at C(5’).
1,2-Diacyl-sn-glyceroles 2 and 3 containing palmitoyl or myristoyl groups were
prepared by the known methods starting from d-mannitol [35][36]. 1,3-Diacyl
derivatives 4 and 5 were obtained by the thermal isomerization of compounds 2 and 3,
respectively, as described in [37]. The treatment of lipids 2 – 5 with commercial
chloro(diisopropylamino)(2-cyanoethoxy)phosphane and EtNiPr2 [38] in CH2Cl2 led
to phosphoramidite derivatives 6 – 9, respectively (Scheme). After purification by
chromatography on alumina, phosphoramidites 6 – 9 were isolated in 81 – 87% yields.
The structures of phosphoramidites 6 – 9 have been confirmed by 1H-NMR
spectroscopy. 31P-NMR Spectra of compounds 6 – 9 exhibit signals at d(P) 149 –
150 ppm, characteristic for both nucleoside and lipid phosphoramidite derivatives
[5][38].
For the preparation of Ribavirin building block with free OH group at C(5’),
suitable for the condensation with lipid phosphoramidites 6 – 9, an easily removable

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465
Scheme
2’,3’-O-phenylboronylidene protecting group was chosen. It is well-known that a wide
range of molecules with cis, or coaxial 1,2- or 1,3-diol functionalities, including sugars
and nucleosides, interact with phenylboronic acid (PhB(OH)2) to give the correspond-
ing phenylboronates, which usually can be hydrolyzed under mild conditions, e.g., in the
case of nucleosides, by a simple aqueous workup or treatment with i-PrOH in
anhydrous media [39].
2’,3’-O-phenylboronate 10 has been synthesized by refluxing Ribavirin (1) with
equimolar PhB(OH)2 in anhydrous pyridine, as described for the preparation of
phenylboronate derivatives of other ribonucleosides [39]. After crystallization, 10 was
separated from the reaction mixture in 84% yield. The presence of phenylboronate
protecting group in nucleoside 10 is confirmed by characteristic signals of five aromatic
1
H-atoms at d(H) 7.88 – 7.32 ppm in its H-NMR spectrum.
The condensation of nucleoside 10 with lipid phosphoramidites 6 – 9 in MeCN in the
presence of 1H-tetrazole, followed by I2/H2O oxidation, afforded the phosphotriesters
11 – 14. The oxidation step was accompanied by the hydrolysis of 2’,3’-phenylboronate

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protecting group that was evident from the absence of phenyl H-atoms signals in
1H-NMR spectra of the compounds 11 – 14 (data not shown). The 3-phosphoglyceride
derivatives 11 and 12 were obtained in 79 and 71% yield, respectively, while the yields
of isomeric 2-phosphoglyceride-nucleoside conjugates 13 and 14 did not exceed 56 –
57%, probably due to the steric hindrances in the course of nucleoside coupling with
phosphoramidite functionality adjacent to two long-chain acyl groups.
The removal of 2-cyanoethyl protecting group from the phosphate moiety of 11 –
14 has been performed by their treatment with Py/Et3N 1:1. The triethylammonium
salts, 15 – 18, of nucleosideꢀlipid conjugates were isolated by column chromatography
on silica gel in 56 – 76% yields and then converted to the Na salts 19 – 22, respectively,
by the treatment of their MeOH solutions with NaI/acetone.
3. Physical Data. – All newly synthesized lipid derivatives were characterized by
TLC, 1H- and 31P-NMR, and elemental analysis, and conjugates 19 – 22 also by UVand
13C-NMR.
The 1H- and 13C-NMR spectra of the synthesized Ribavirinꢀlipid conjugates
contained the signals of the H- and C-atoms, respectively, of every structural fragment
including sugar, heterocyclic base, glycerol residue, and acyl groups. The 31P-NMR
spectra of 19 – 22 exhibited the peaks near ꢀ 0.7 ppm, i.e., in the field characteristic for
nucleoside phosphodiesters [40]. The two-bond C, P couplings observed in 13C-NMR
spectra of isomeric conjugates 19 and 21 correspond to C-atoms in both nucleoside and
glycerol fragments attached to the phosphate group (2J(C(5’),P) ¼ 5 and 2J(C(1),P) ¼
5 Hz for 2,3-diacyl glycerophosphate derivative 19, and 2J(C(5’),P) ¼ 5 and
2J(C(2),P) ¼ 4.5 Hz for their 1,3-diacyl isomer 21). The 13C-NMR spectra of synthe-
sized conjugates also showed the expected three-bond C, P coupling involving C(4’)-
3
atom of carbohydrate moiety (3J(C(4’),P) ¼ 7 and J(C(4’),P) ¼ 6.5 Hz for 19 and 21,
resp.). Besides, in the 13C-NMR spectra of 2,3-diacyl glycerophosphate derivative 19, a
coupling between P- and C(2)-atom of glycerol fragment was observed (3J(C(2),P) ¼
5 Hz), whereas a distinctive feature of 1,3-diacyl isomer 21 was the three-bond C,P
couplings involving C(1)- and C(3)-atoms of glycerol skeleton (3J(C(1),P) ꢁ
3J(C(3),P) ꢁ 5 Hz; the doublets were not completely resolved).
4. Enzymatic Hydrolysis by Pancreatic Phospholipase A2. – The well-known
investigations on the antiretroviral activity of phosphatidyl derivatives of AZT clearly
demonstrated that phosphatidyl-AZT exerted its antiviral action by metabolic
conversion to AZT-5’-triphosphate [41]. It has been shown that, in the cells, these
conjugates are metabolized by sequential deacylation catalyzed by phospholipases A
and lysophospholipases, followed by the hydrolysis of the produced glycerophospho-
AZT by cellular phosphodiesterases to release the nucleoside/5’-nucleotide moiety
which are further phosphorylated by kinases to an active metabolite [41].
To verify the possibility for entering the first stage of the above described metabolic
pathway by the regioisomeric Ribavirinꢀlipid conjugates 19, 20 and 21, 22, we have
conducted preliminary experiments on the hydrolysis of these compounds by porcine
pancreatic phospholipase A2, the most common enzyme of phospholipase A2 (PLA2)
superfamily. The enzymatic reactions were carried out by incubation of 19 – 22 with
PLA2 (4.8 mg per 1 mmol of substrate) at 378 in 0.05m Tris · HCl buffer (pH 8.0)

Helvetica Chimica Acta – Vol. 96 (2013)
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containing sodium deoxycholate and Ca2þ ions. In all cases, the formation of
lysophospholipid derivatives with lower mobility than the corresponding parent
compound was observed by TLC. In the control mixtures (without PLA2), only initial
19 – 22 were detected in the same periods of time; hence, the formation of the
compounds with low Rf was attributable to the enzymatic hydrolysis. The initial rates of
hydrolytic cleavage (V0) and half-time of hydrolysis (t1/2) were determined at the 0.6-
mm concentration of 19 – 22. The probes of the reaction mixtures were collected in
fixed periods of time (t), and hydrolysis was stopped by addition of ethylenediami-
netetraacetic acid (EDTA). The components of the mixtures were resolved by TLC,
the phosphate derivatives were visualized by the formation of phosphomolybdenum
blue. The content of non-hydrolyzed 19 – 22 was determined by the measurement of the
absorbance (D) at 820 – 830 nm as described in [42]. The values of V0 and t1/2 for each
of 19 – 22 were calculated from the corresponding Dꢀt plot.
Under the above mentioned experimental conditions, the initial rates of hydrolysis
of phosphatidyl derivatives 19 and 20 were 29 and 16 mmolminꢀ1 mgꢀ1, respectively. The
hydrolytic cleavage proceeded much more slowly in the case of regioisomeric 1,3-
diacylglycerophosphateꢀribavirin conjugates 21 and 22 for which the values of V0 were
1.7 and 1.5 mmolminꢀ1 mgꢀ1, respectively. This dependency was also reflected in t1/2
values (5 and 15 min for compounds 19 and 20, resp.; 80 and 150 min for conjugates 21
and 22, resp.).
The ability of PLA2 to hydrolyze not only 2-acyl group of naturally occuring 1,2-
diacyl-sn-glycerophospholipids but also 1-acyl residue of their artificial 1,3-diacyl
regioisomers was established with the use of diacylglycerophosphocholines as
substrates [43]. It has been shown that the affinity of 1,3-diacylglycerophosphocholines
to phospholipases A2 are similar to that of natural 1,2-diacyl derivatives, whereas their
hydrolysis is essentially decelerated [44]. The enzyme-kinetic study revealed that, in
the case of porcine pancreatic PLA2, the maximum rates (Vmax,app) were lower by the
factor 13 – 20 for 1,3-diacylglycerophosphocholines with different chain lengths
compared to the corresponding 1,2-diacyl-sn-glycerophosphocholines [44].
The data collected in our work show a similar tendency. The possibility of the
hydrolysis of both 1,2-diacyl-sn- and 1,3-diacylglycerophosphateꢀRibavirin conjugates
by PLA2 indicates in principle that these compounds can enter the first stage of
metabolic deacylation pathways under the action of cellular phospholipases.
5. Conclusions. – The described approach to the preparation of phospholipid
Ribavirin derivatives by the phosphoramidite technique, implying a combination of
easily removable 2’,3’-O-phenylboranylidene protecting group for the carbohydrate
moiety of the ribonucleoside and cyanoethyl group for the phosphate fragment of
conjugate molecule, allows the preparation of the desired compounds with reasonable
yields via a small number of synthetic steps. Besides, it ensures a stability of the acyl
groups at glycerol moiety in the course of all synthetic stages, avoiding impure
structural isomers in the intermediate compounds and final nucleosideꢀphospholipid
conjugates.
The glycerophosphate derivatives 19 – 22 can be considered as Ribavirin prodrugs
potentially useful for medicinal purposes. The study of the biochemical and antiviral
properties of the synthesized conjugates is in progress.

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Experimental Part
General. Porcine pancreatic PLA2 for enzymatic hydrolysis from Sigma-Aldrich, (cat. No P6534).
TLC: Precoated Al2O3 thin-layer sheets (Fluka) and SiO2 thin-layer sheets (60 F 254; Merck); lipid
derivatives visualized by spraying with 30% H2SO4 and charring. Prep. column chromatography (CC):
Al2O3 (Brockmann activity II, neutral; Reanal), SiO2 (60, 63 – 200 mm; Merck). M.p.: Barnstead
1
Electrothermal IA9300; uncorrected. UV/VIS Spectra: Cary 100 (Varian); lmax (log e) in nm. H- and
13C-NMR spectra: Avance 500 (Bruker); d in ppm rel. to Me4Si as internal standard, J in Hz, assignments
carried out with the use of 1H,1H and 1H,13C correlation spectroscopy. 31P-NMR: Avance 500 (Bruker); d
in ppm rel. to external H3PO4. Elemental analysis: CHNS-O Analyser EA-3000 (EuroVector).
Lipid Phosphoramidites 6 – 9. General Procedure. To a soln. of a lipid, 2 – 5 (1 mmol) in CH2Cl2
(5 ml), EtNiPr2 (0.52 g, 0.68 ml, 4 mmol) and chloro(diisopropylamino)(2-cyanoethoxy)phosphane
(0.47 g, 0.45 ml, 2 mmol) were added under Ar. After stirring for 1 h at r.t., the mixture was diluted with
CH2Cl2 (30 ml) and hexane (60 ml), and extracted with a mixture of sat. NaCl and NaHCO3 solns. 1:1 (v/
v; 5 ꢂ 15 ml). The org. layer was dried (Na2SO4) and evaporated. The residue was purified by CC (Al2O3
(2.5 ꢂ 4 cm); hexane, hexane/Et2O 9 :1, and then hexane/Et2O 7 :3). After evaporation of the
appropriate fractions, the phosphoramidites 6 – 9 were isolated as white powders.
3-({[Bis(1-methylethyl)amino](2-cyanoethoxy)phosphanyl}oxy)propane-1,2-diyl Dihexadecanoate
(6; mixture of diastereoisomers). From 2 (0.6 g, 1.05 mmol): 0.69 g (85%) of 6. Rf (Al2O3; hexane/
1
Et2O 1:2) 0.72. H-NMR (CDCl3): 5.21 – 5.17 (m, HꢀC(2)); 4.36 (dd, J(1,2) ¼ 4, J(1,1) ¼ 12, CH2(1));
4.32 (dd, J(1,2) ¼ 4, J(1,1) ¼ 12, CH2(1)); 4.18 (dd, J(1,2) ¼ 6, J(1,1) ¼ 12, CH2(1)); 4.15 (dd, J(1,2) ¼ 6.5,
J(1,1) ¼ 12, CH2(1)); 3.88 – 3.75 (m, CH2(3), CH2CH2CN); 3.73 – 3.66 (m, CH2(3)); 3.63 – 3.56 (m,
2 Me2CH); 2.65 – 2.62 (m, CH2CH2CN); 2.33 – 2.29 (m, 2 CH2CO); 1.64 – 1.58 (m, 2 CH2CH2CO); 1.30 –
1.25 (m, 2 Me(CH2)12); 1.19 – 1.17 (m, 2 Me2CH); 0.89 – 0.87 (m, 2 Me(CH2)14). 31P-NMR (CDCl3): 149.7;
149.6. Anal. calc. for C44H85N2O6P (769.1): C 68.71, H 11.14, N 3.64; found: C 69.11, H 10.94, N 3.59.
3-({[Bis(1-methylethyl)amino](2-cyanoethoxy)phosphanyl}oxy)propane-1,2-diyl Ditetradecanoate
(7; mixture of diastereoisomers). From 3 (0.6 g, 1.17 mmol): 0.77 g (92%) of 7. Rf (Al2O3; hexane/
Et2O 1:2) 0.72. 1H-NMR (CDCl3): 5.22 – 5.17 (m, CH(2)); 4.36 (dd, J(1,2) ¼ 4, J(1,1) ¼ 12, CH2(1)); 4.32
(dd, J(1,2) ¼ 4, J(1,1) ¼ 12, CH2(1)); 4.18 (dd, J(1,2) ¼ 6.5, J(1,1) ¼ 12, CH2(1)); 4.15 (dd, J(1,2) ¼ 6.5,
J(1,1) ¼ 12, CH2(1)); 3.88 – 3.75 (m, CH2(3), CH2CH2CN); 3.72 – 3.65 (m, CH2(3)); 3.63 – 3.55 (m,
2 Me2CH); 2.65 – 2.62 (m, CH2CH2CN); 2.33 – 2.30 (m, 2 CH2CO); 1.64 – 1.58 (m, 2 CH2CH2CO); 1.30 –
1.25 (m, 2 Me(CH2)10); 1.19 – 1.16 (m, 2 Me2CH); 0.89 – 0.87 (m, 2 Me(CH2)12). 31P-NMR (CDCl3): 149.7;
149.6. Anal. calc. for C40H77N2O6P (713.0): C 67.38, H 10.88, N 3.93; found: C 67.67, H 11.12, N 3.75.
2-({[Bis(1-methylethyl)amino](2-cyanoethoxy)phosphanyl}oxy)propane-1,3-diyl Dihexadecanoate
(8). From 4 (0.6 g, 1.05 mmol): 0.71 g (88%) of 8. Rf (SiO2; hexane/AcOEt 9 :1) 0.52. 1H-NMR
(CDCl3): 4.26 – 4.12 (m, CH2(1)CH(2)CH2(3)); 3.89 – 3.76 (m, CH2CH2CN); 3.65 – 3.55 (m, 2 Me2CH);
2.65 – 2.63 (m, CH2CH2CN); 2.33 – 2.29 (m, 2 CH2CO); 1.65 – 1.58 (m, 2 CH2CH2CO); 1.31 – 1.25 (m,
2 Me(CH2)12); 1.19 – 1.17 (m, 2 Me2CH); 0.89 – 0.87 (m, 2 Me(CH2)14). 31P-NMR: 150.2. Anal. calc. for
C44H85N2O6P (769.1): C 68.71, H 11.14, N 3.64; found: C 69.08, H 10.99, N 3.65.
2-({[Bis(1-methylethyl)amino](2-cyanoethoxy)phosphanyl}oxy)propane-1,3-diyl Ditetradecanoate
(9). From 5 (0.6 g, 1.17 mmol): 0.66 g (79%) of 9. Rf (SiO2, hexane/AcOEt 9 :1) 0.52. 1H-NMR
(CDCl3): 4.26 – 4.12 (m, CH2(1)CH(2)CH2(3)); 3.89 – 3.77 (m, CH2CH2CN); 3.65 – 3.54 (m, 2 Me2CH);
2.65 – 2.63 (m, CH2CH2CN); 2.34 – 2.29 (m, 2 CH2CO); 1.65 – 1.59 (m, 2 CH2CH2CO); 1.31 – 1.25 (m,
2 Me(CH2)10); 1.19 – 1.17 (m, 2 Me2CH); 0.89 – 0.87 (m, 2 Me(CH2)12). 31P-NMR: 150.2. Anal. calc. for
C40H77N2O6P (713.0): C 67.38, H 10.88, N 3.93; found: C 67.59, H 10.69, N 3.71.
1-(2,3-Di-O-phenylboranylidene-b-d-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide (¼1-[Tetrahy-
dro-6-(hydroxymethyl)-2-phenylfuro[3,4-d][1,3,2]dioxaborol-4-yl]-1H-1,2,4-triazole-3-carboxamide;
10). To a soln. of Ribavirin (1; 0.2 g, 0.82 mmol) in pyridine (40 ml), a soln. of PhB(OH)2 (0.1 g,
0.82 mmol) in pyridine (20 ml) was added under stirring for 5 min at r.t. The mixture was refluxed for 2 h
under anh. conditions and then evaporated. The residue was dissolved in dioxane (5 ml), and Et2O
(25 ml) was added. After keeping the mixture for 16 h at þ 48, the precipitate was filtered and dried in
vacuum to yield crystalline 10 (0.23 g, 85%). M.p. 213 – 2158. 1H-NMR ((D5)pyridine): 9.12 (s, HꢀC(5));
8.85, 8.80 (2s, CONH2); 7.88 – 7.32 (m, 5 arom. H); 6.58 (d, J(1’,2’) ¼ 1.5, HꢀC(1’)); 5.37 (dd, J(2’,3’) ¼ 6,

Helvetica Chimica Acta – Vol. 96 (2013)
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HꢀC(2’)); 5.11 (dd, J(3’,4’) ¼ 2.5, HꢀC(3’)); 4.88 – 4.86 (m, HꢀC(4’)); 4.16 (dd, J((5’a,4’) ¼ 5, J(5’a,5’b) ¼
11.5, HaꢀC(5’)); 4.08 (dd, J(5’b,4’) ¼ 5.5, HbꢀC(5’)). Anal. calc. for C14H15BN4O5 (330.1): C 50.94, H 4.58,
N 16.97; found: C 51.12, H 4.75, N 16.73.
Sodium 1-(5-O-{[2,3-Bis(hexadecanoyloxy)propoxy]phosphinato}-b-d-ribofuranosyl)-1H-1,2,4-tria-
zole-3-carboxamide (19). A soln. of 10 (72 mg, 0.22 mmol) in MeCN (15 ml) and 0.45m soln. of 1H-
tetrazole (1.47 mmol) in MeCN (3.3 ml) were added to 6 (261 mg, 0.34 mmol) under Ar. After stirring
for 16 h, a soln. of I2 (86 mg, 0.34 mmol) in pyridine/H2O/CH2Cl2 3 :1:1 (1.29 ml) [5] was added. The
mixture was diluted with CHCl3 (200 ml) and extracted with 2% soln. of Na2S2O3 in sat. NaCl (60 ml),
and the org. layer was dried (Na2SO4) and evaporated. The residue was purified by CC (SiO2 (1.8 ꢂ
20 cm); CHCl3 and then CHCl3/MeOH 15 :1) to give 144 mg (71%) of 11. Colorless syrup. Rf (SiO2;
CHCl3/MeOH 9 :1) 0.31.
A soln. of 11 (100 mg, 0.11 mmol) in pyridine/Et3N 1:1 (2.2 ml) was kept at r.t. for 24 h and
evaporated. The residue was evaporated with toluene (2 ml) and purified by CC (SiO2 (2 ꢂ 20 cm);
CHCl3/MeOH/Et3N 99 :1:0 ! 50 :50 :1) to give 15 (74 mg, 70%). Colorless syrup. The salt 15 was
dissolved in a small volume of CHCl3, and treated with 1m NaI/acetone (0.15 ml) and acetone (10 ml).
The precipitate was filtered and dried in vacuum dessicator to give 46 mg (67%) of 19. White powder. Rf
1
(SiO2, CHCl3/MeOH 2 :1) 0.26. UV (MeOH): 207 (4.06). H-NMR ((D6)DMSO): 8.90 (s, HꢀC(5));
7.98, 7.54 (2s, CONH2); 5.78 (d, J (1’,2’) ¼ 4, HꢀC(1’)); 5.62 – 5.51 (m, HOꢀC(2’), HOꢀC(3’)); 5.06 – 5.01
(m, CH2CHCH2OP); 4.40 – 4.37 (m, HꢀC(2’)); 4.31 – 4.24 (m, CH2CHCH2OP); 4.22 – 4.19 (m, HꢀC(3’));
4.10 – 4.03 (m, CH2CHCH2OP); 4.04 – 4.01 (m, 1 HꢀC(4’)); 3.83 – 3.79 (m, 1 HꢀC(5’)); 3.77 – 3.72 (m,
HꢀC(5’)); 3.73 – 3.66 (m, CH2CHCH2OP); 2.23 (t, J ¼ 7; 2 CH2CO); 1.53 – 1.43 (m, 2 CH2CH2CO);
1.28 – 1.21 (m, 2 Me(CH2)12); 0.84 (t, J ¼ 7, 2 Me(CH2)14). 13C-NMR ((D6)DMSO): 172.53; 172.28 (2
Me(CH2)14CO); 160.51 (CONH2); 157.04 (C(3)); 144.81 (C(5)); 92.08 (C(1’)); 84.15 (d, 3J(C(4’),P) ¼ 7,
C(4’)); 74.77 (C(2’)); 70.50 (C(3’)); 70.45 (d, 3J(C,P) ¼ 5, CH2CHCH2OP); 64.04 (d, 2J(C(5’),P) ¼ 5,
C(5’)); 62.44 (d, 2J(C,P) ¼ 5, CH2CHCH2OP); 62.36 (CH2CHCH2OP); 33.55; 33.38 (2 Me(CH2)13CH2);
31.25 (2 MeCH2CH2); 29.01; 28.97; 28.90; 28.88; 28.65; 28.40; 28.37 (2 Me(CH2)2(CH2)9); 28.73; 28.70
(2 Me(CH2)11CH2); 24.43, 24.37 (2 Me(CH2)12CH2); 22.04 (2 MeCH2); 13.87 (2 Me(CH2)14). 31P-NMR
((D6)DMSO): ꢀ 0.73. Anal. calc. for C43H78N4NaO12P · H2O (915.1): C 56.44, H 8.81, N 6.12; found: C
56.31, H 8.49, N 5.90.
Sodium 1-(5-O-{[2,3-Bis(tetradecanoyloxy)propoxy]phosphinato}-b-d-ribofuranosyl)-1H-1,2,4-tri-
azole-3-carboxamide (20). As described for 11, with 10 (30 mg, 0.09 mmol), 7 (100 mg, 0.14 mmol),
MeCN (7 ml), 0.45m soln. of 1H-tetrazole in MeCN (1.34 ml, 0.60 mmol), I2 (36 mg, 0.14 mmol), and
pyridine/H2O/CH2Cl2 3 :1:1 (0.54 ml); CC afforded 66 mg (79%) of 12. Colorless syrup. Rf (SiO2,
CHCl3/MeOH 9 :1) 0.31.
As described for 15, with 12 (53 mg, 0.06 mmol) and pyridine/Et3N 1:1 (1.2 ml); after CC, 16 (36 mg,
64%) was obtained. Treatment of 16 in CHCl3 with 1m NaI/acetone (0.08 ml) and acetone (6 ml) gave 20
1
(25 mg, 76%). White powder. Rf (SiO2; CHCl3/MeOH 2 :1) 0.26. UV (MeOH): 207 (4.06). H-NMR
((D6)DMSO): 8.90 (s, HꢀC(5)); 7.99, 7.59 (2s, CONH2); 5.80 (d, J(1’,2’) ¼ 4, HꢀC(1’)); 5.64 (d,
J(HOꢀC(2’),2’) ¼ 5, HOꢀC(2’)); 5.58 (d, J(HOꢀC(3’),3’) ¼ 4.5, HOꢀC(3’)); 5.06 – 5.02 (m,
CH2CHCH2OP); 4.41 – 4.38 (m, HꢀC(2’)); 4.31 – 4.24 (m, CH2CHCH2OP); 4.23 – 4.20 (m, HꢀC(3’));
4.10 – 4.03 (m, CH2CHCH2OP); 4.05 – 4.02 (m, HꢀC(4’)); 3.84 – 3.80 (m, 1 HꢀC(5’)); 3.78 – 3.73 (m,
1 HꢀC(5’)); 3.74 – 3.67 (m, CH2CHCH2OP); 2.24 (t, J ¼ 7, 2 CH2CO); 1.54 – 1.44 (m, 2 CH2CH2CO);
1.28 – 1.21 (m, 2 Me(CH2)10); 0.85 (t, J ¼ 7, 2 Me(CH2)12). 31P-NMR ((D6)DMSO): ꢀ 0.72. Anal. calc. for
C39H70N4NaO12P · 2 H2O (877.0): C 53.41, H 8.50, N 6.39; found: C 53.15, H 8.30, N 6.16.
Sodium 1-[5-O-({[1,3-Bis(hexadecanoyloxy)propan-2-yl]oxy}phosphinato)-b-d-ribofuranosyl]-1H-
1,2,4-triazole-3-carboxamide (21). As described for 11, with 10 (49 mg, 0.15 mmol), 8 (177 mg,
0.23 mmol), MeCN (13 ml), 0.45m soln. of 1H-tetrazole in MeCN (2.2 ml, 0.99 mmol), I2 (58 mg,
0.23 mmol), and pyridine/H2O/CH2Cl2 3 :1:1 (0.87 ml); CC gave 77 mg (56%) of 13. Colorless syrup. Rf
(SiO2; CHCl3/MeOH 9 :1) 0.31.
As described for 15, with 13 (68 mg, 0.073 mmol) and pyridine/Et3N 1:1 (1.46 ml), after CC, 17
(40 mg, 56%) was obtained. Treatment of 17 in CHCl3 with 1m NaI/acetone (0.082 ml) and acetone (7 ml)
gave 21 (26 mg, 70%). White powder. Rf (SiO2; CHCl3/MeOH 2 :1) 0.31. UV (MeOH): 207 (4.06).
1H-NMR ((D6)DMSO): 8.84 (s, HꢀC(5)); 7.72, 7.32 (2s, CONH2); 5.79 (d, J(1’,2’) ¼ 4, HꢀC(1’)); 5.36 –

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Helvetica Chimica Acta – Vol. 96 (2013)
5.31 (m, HOꢀC(2’), HOꢀC(3’)); 4.42 – 4.39 (m, HꢀC(2’)); 4.34 – 4.29 (m, (CH2)2CHOP); 4.24 – 4.21 (m,
HꢀC(3’)); 4.13 – 4.05 (m, (CH2)2CHOP, HꢀC(4’)); 3.90 – 3.84 (m, 1 HꢀC(5’)); 3.87 – 3.80 (m, 1 HꢀC(5’));
2.25 (t, J ¼ 7, 2 CH2CO); 1.52 – 1.46 (m, 2 CH2CH2CO); 1.29 – 1.23 (m, 2 Me(CH2)12); 0.84 (t, J ¼ 7, 2
Me(CH2)14). 13C-NMR ((D6)DMSO): 172.50 (2 Me(CH2)14CO); 160.49 (CONH2); 157.07 (C(3)); 144.71
(C(5)); 92.22 (C(1’)); 84.27 (d, 3J(C(4’),P) ¼ 6.5, C(4’)); 74.82 (C(2’)); 70.58 (C(3’)); 69.21 (d,2J(C,P) ¼
4.5, (CH2)2CHOP); 64.26 (d,2J(C(5’),P) ¼ 5, C(5’)); 63.09 (d, 3J(C,P) ¼ 5), 63.05 (d, 3J(C,P) ¼ 5)
((CH2)2CHOP); 33.39 (2 CH2CO); 31.16 (2 MeCH2CH2); 28.90; 28.86; 28.74; 28.58; 28.54; 28.36
(2 Me(CH2)2(CH2)9); 24.31 (2 Me(CH2)12CH2); 21.93 (2 MeCH2); 13.73 (2 Me(CH2)14). 31P-NMR
((D6)DMSO): ꢀ 0.73. Anal. calc. for C43H78N4NaO12P · 2 H2O (933.1): C 55.35, H 8.86, N 6.00; found:
C 55.22, H 8.45, N 6.02.
Sodium 1-[5-O-({[1,3-Bis(tetradecanoyloxy)propan-2-yl]oxy}phosphinato)-b-d-ribofuranosyl]-1H-
1,2,4-triazole-3-carboxamide (22). As described for 11, with 10 (33 mg, 0.10 mmol), 9 (111 mg,
0.16 mmol), MeCN (8 ml), 0.45m soln. of 1H-tetrazole in MeCN (1.49 ml, 0.67 mmol), I2 (41 mg,
0.16 mmol), pyridine/H2O/CH2Cl2 3 :1:1 (0.62 ml); CC gave 50 mg (57%) of 14. Colorless syrup. Rf
(SiO2, CHCl3/MeOH 9 :1) 0.31.
As described for 15, with 14 (36 mg, 0.041 mmol) and pyridine/Et3N 1:1 (0.83 ml); after CC, 18
(29 mg, 76%) was obtained. Treatment of 18 in CHCl3 with 1m NaI/acetone (0.063 ml) and acetone (5 ml)
gave 22 (19 mg, 70%). White powder. Rf (SiO2, CHCl3/MeOH 2 :1) 0.31. UV (MeOH): 207 (4.06).
1H-NMR ((D6)DMSO): 8.85 (s, HꢀC(5)); 7.73, 7.34 (2s, CONH2); 5.80 (d, J (1’,2’) ¼ 4, HꢀC(1’)); 5.37 –
5.32 (m, HOꢀC(2’), HOꢀC(3’)); 4.43 – 4.40 (m, HꢀC(2’)); 4.35 – 4.30 (m, (CH2)2CHOP); 4.25 – 4.22 (m,
HꢀC(3’)); 4.13 – 4.06 (m, (CH2)2CHOP), HꢀC(4’)); 3.91 – 3.85 (m, 1 HꢀC(5’)); 3.88 – 3.81 (m,
1 HꢀC(5’)); 2.26 (t, J ¼ 7, 2 CH2CO); 1.53 – 1.47 (m, 2 CH2CH2CO); 1.28 – 1.23 (m, 2 Me(CH2)10); 0.85
(t, J ¼ 7, 2 Me(CH2)12). 31P-NMR ((D6)DMSO): ꢀ 0.73. Anal. calc. for C39H70N4NaO12P · 2 H2O (877.0):
C 53.41, H 8.50, N 6.39; found: C 53.23, H 8.33, N 6.22.
Enzymatic Hydrolysis of 19 – 22 by PLA2. To 1.2 mmol of Ribavirinꢀlipid conjugate, 19 – 22, 10 mm
sodium deoxycholate (0.36 ml) and 0.05m Tris · HCl buffer (pH 8.0, 1 mm Ca2þ; 1.64 ml) were added. The
mixture was sonicated in Elmasonic S 10 H untrasonic bath (4 ꢂ 10 min) until obtaining clear one-phase
dispersion. The reaction was started at 378 by addition of PLA2 soln. containing 5.8 mg of the protein. The
probes of the reaction mixture (0.25 ml) were collected in fixed periods of time (t) and the hydrolysis was
stopped by the addition of 10 mm EDTA (0.62 ml). Then, the probes were extracted by vortexing with
CHCl3/MeOH 2 :1 (2 ꢂ 0.5 ml), centrifuged at 1800 rpm for 15 min, the lower layers separated and
evaporated to dryness, the residues were dissolved in CHCl3/MeOH 2 :1 (60 ml) and applied on TCL
plate, which was further developed with CHCl3/MeOH 2 :1. After subsequent workup in accordance with
the method of Vaskovsky et. al. [42], the content of the phospholipid derivatives in each probe was
determined by the measurement of the absorbance (D) at 820 – 830 nm. The values of V0 and t1/2 for each
of 19 – 22 were calculated from the corresponding Dꢀt plot. The data presented are the average of at least
two experiments.
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Received April 19, 2012