ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 6, pp. 651–671. © Pleiades Publishing, Ltd., 2011. Original Russian Text © L.A. Alexandrova, 2011, published in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 6, pp. 723–744.
4'CNucleoside Derivatives: Synthesis and Antiviral Properties L. A. Alexandrova1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova32, 119991 Moscow, Russia Received November 22, 2010; in final form, December 22, 2010
Abstract—Synthesis of 4'Cmodified nucleosides and their derivatives, as well as the structure–activity rela tionship toward hepatitis C virus were discussed. Keywords: hepatitis C virus, HCV NTPase/helicase, HCV RNAdependent RNA polymerase, 4'Cmodified nucleosides, nucleosides, nucleoside 5'Otriphosphates, pyrimidine nucleosides DOI: 10.1134/S1068162011060021
1
INTRODUCTION New antiviral agents are required for treatment of virusinduced infections. Nucleosidederived anti metabolites play an important role in antiviral and anticancer chemotherapy. A number of nucleosides substituted at the carbohydrate residues were shown to be antiviral and anticancer agents. Thus far some effective nucleosidebased drugs have been devel oped for treatment of diseases caused by DNA viruses (hepatitis B virus and herpes viruses) and ret roviruses (HIV) [1–3]. Viral DNA and RNA poly merases are the main targets for this type of com pounds. The mechanism of action of nucleoside ana logues involves successive phosphorylation to nucleoside 5'triphosphates catalyzed by the corre sponding kinases, and incorporation of the triphos phates into 3' termini of viral DNA or RNA resulted in the inhibition of virus replication [1, 3]. For the herpes virus family, the selectivity of nucleoside effects can be explained by the presence in infected cells of virusencoded thymidine kinase, which cata lyzes nucleoside phosphorylation. It is noteworthy that in host noninfected cells monophosphates of modified nucleosides are not formed [1–3]. Other enzymes involved in nucleic acid biosynthesis, for Abbreviations: araC, arabinocytidine; mCPBA, mchloroper benzoic acid; DMDO, dimethoxydioxirane; HIV, human immunodeficiency virus; HCV, hepatitis C virus; CC50, dose causing death of 50% noninfected cells; IC50, minimal dose inhibiting the virusinduced pathogenic effect by 50%. 1 Corresponding author: phone: +7 (499) 1356065; fax: +7 (499) 1351405; email:
[email protected].
example, synthases, kinases, etc., can also serve as targets for nucleoside analogues [1–3]. Since the beginning of the 1990s, a large set of branched nucleosides has been synthesized and evalu ated as potential antiviral agents. In 1992 it was reported that 4'substituted* thymidine derivatives, namely, 4'cyanothymidine (1a) [4] and 3'O,4'meth ylenethymidine (2) [5], inhibited HIV replication. Various 4'modified 2'deoxynucleosides with a high antiviral activity have been created thus far, particu larly, (1a–d), (2), and (3) [4–7] including those con taining sulfur in the 2'deoxyribose cycle, for example, (4a–c) [8]. The information on the capacity of 4'modified ribonucleosides to inhibit replication of RNA viruses is much poorer. Recently, a series of 4'azidoribonu cleoside derivatives effectively inhibiting HCV repli cation in cell cultures has been synthesized, first of all, 4'azidocytidine (5) and its derivatives [9, 10]. Its pro drug (Balapiravir, 4'azidocytidine 2',3',5'tris(2 methylpropanoate)) is currently undergoing clinical trials [11]. In this review we discuss methods of synthesis and the antiviral activities of 4'modified nucleosides. The parent compound of this class is a natural antibiotic nucleocidine (6) [12], whose antiviral properties are unknown. *4'modified nucleosides are nucleosides with a substituent at 4'C of the carbohydrate fragment.
651
652
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O O
HO
NH
N
O
HO
O
R HO
NH
N
O
O
O O
HO
NH
N
O
O O (2)
O
R HO
O
(1 а–d) R=CN (а), C2H5 (b), CH=CH2 (c), C≡CH (d)
NH
N
S
HO
(3)
(4 а–c) R=N3 (а), CN (b), C≡CH (c)
Formulas 1. Some of the 4'modified 2'deoxynucleosides displayed a high antiviral activity: (1a) 4'cyanothymidine; (1b) 4'ethylth ymidine; (1c) 4'vinylthymidine; (1d) 4'ethynylthymidine; (2) 3'O,4'methylenethymidine; (3) 3'O,4'ethylenethymidine; (4a) 4'Cazido4'thiothymidine; (4b) 4'Ccyano4'thiothymidine; (4c) 4'Cethynyl4'thiothymidine.
NH2
O
N3 HO
O
N
N
O
HO
O S
H2N
O
O
N
N N
F
OH
NH2
N
HO
OH
(5)
(6)
Formulas 2. 4'Azidocytidine (5) and nucleocidine (6).
R
O
HO N3 HO
N
R
O NH
HO O
OH
(12 а–d) R=H (а), CH3 (b), OH (c), F (d)
N3 HO
O
N
NH2 N O
OH
O
N
HO N3 HO
(13 а–c) R=СH3 (а), OH (b), F (c)
O
NH
N N OH
NH2
N
NH2
HO N3 HO
(14)
N
N
O
N OH (15 а–c)
Formulas 3. 4'Azido derivatives of the ribo series: (12a) 4'azidouridine; (12b) 4'azidothymidine; (12c) 4'azido5hydroxyuridine; (12d) 4'azido5fluorouridine; (13a) 4'azido5methylcytidine; (13b) 4'azido5hydroxycytidine; (13c) 4'azido5fluorocytidine; (14) 4'azidoguanosine; (15) 4'azidoadenosine.
4'AZIDONUCLEOSIDE DERIVATIVES 4'Azidonucleosides were first synthesized by J. Moffatt et al. in 1975 [13]. In 1992 these researchers obtained 4'derivatives of 2'deoxy series [14] using the procedure described in general in [13] (as an example, the synthesis of 4'azidouridine (12a) is shown in Scheme 1). Unprotected nucleosides (7) were found to be the most suitable compounds for iodination of posi tion 5'. They were treated with triphenylphosphine, iodine, and pyridine and/or imidazole in dioxane [15] to give 29–59% of iodinated products. The treatment of 5'iodo derivative (8) with DBN (1,5 diazabicyclo[4.3.0]non5ene [16]) or sodium
methylate [17] in methanol resulted in the rapid formation of 4'olefin (9). Better yields were achieved with sodium methylate. The addition of an iodine atom and azido groups at the double bond of 4'olefine (9) was carried out with iodoazide obtained in situ from sodium azide and iodine monochloride. The resulting product (10) was ben zoylated to give compound (11). Oxidative nucleo philic substitution of the 5'iodide residue of (11) with mCPBA in dichloromethane saturated with water and successive debenzoylation with 1 M CH3ONa led to 4'azidouridine (12a). 5Modifed 4'azidouridines (12b–d) were similarly obtained (Scheme 1).
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O
O NH
N
O
HO
i
HO
OH (7)
iii
I N3 HO
N
NH
iv
O (10)
(11)
OH (9)
O NH
v
OBz
O NH
N
O
HO N3
O
BzO
OH
HO
N
O
I N3
NH
N
O
O
OH (8)
O O
ii
O
O HO
O
NH
N
O
I
653
O
HO
OH (12а)
Scheme 1. Synthesis of 4'azidouridine (12а). Reagents and conditions: i: Ph3P, I2, Py, dioxane; ii: 1 M CH3ONa, methanol; iii: ICl, NaN3, DMF; iv: BzCl; v: 1) mCPBA, CH2Cl2, H2O; 2) CH3ONa, methanol.
In 2003 and 2005 two research groups synthesized a set of 4'azido derivatives of the ribo series (12a–c), (14), and (15a–c) [18, 19]. The method shown in Scheme 1 served as the basis for the synthesis [13]. For the preparation of 4'azidocytidines (5) and (13a–c)
from the corresponding uridines, triOacetylated uri dine (16) was coupled with phosphoro(tristriazolide) followed by ammonolysis of derivative (17) by the K.J. Divakar and C.B. Riese method [20] or its modi fied version [21] (Scheme 2). N
O (12а)
i
O
AcO N3 AcO
NH
N
ii
O (16)
NH2
N N
OAc
NH
N
O
AcO N3
iii
O
AcO
(17)
O
AcO N3
NH O
AcO
OAc
N
(5)
OAc
Scheme 2. Synthesis of 4'azidocytidine (5). Reagents and conditions: i: Ac2O, Py; ii: triazole, POCl3, Et3N, CH3CN; iii: NH3/MeOH or aq. NH3, dioxane, 20°C, 17 h [20] or ii: 4ClPhOPOCl2 [21], Py, 20°C, 2 – 96 h; iii: NH3/MeOH or aq. NH3, dioxane, 20°C, 17 h [21].
been approved for treatment of hepatitis C virus infec tion [23] despite its rather high toxicity [24]. There fore, 4'azidonucleosides have been comprehensively studied as potential antiHCV agents. In further stud ies more effective HCV inhibitors were obtained, namely, 4'azidoarabinocytidine (18) [25, 26], 4'azido2'fluoro2'deoxyarabinocytidine (19), and 4'azido2',2'difluoro2'deoxycytidine (20) [25, 27].
In 2006 it was reported that 4'azidocytidine (5) inhibited hepatitis C virus replication in cell cultures [9, 10]. Hepatitis C is a widespread and dangerous dis ease, whose etiological agent, a singlestranded RNA virus, was identified as late as 1989 [22]. Several nucle oside analogues inhibiting HCV replication in cell cul tures have been identified thus far (cf. review [23]). However, ribavirin (Virazole®) in combination with interferon α is the only nucleosidebased drug that has NH2 HO
O
N
N
HO O
N3 HO
OH (18)
NH2 N
N
O
HO O
N3 HO
NH2 O
N3 HO
F (19)
N F F
N O
(20)
Formulas 4. (18) 4'Azidoarabinocytidine; (19) 4'azido2'fluoro2'deoxycytidine; (20) 4'azido2',2'difluoro2'deoxycytidine.
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Among 4'azidonucleosides studied before 2006, only 4'azidocytidine (5) effectively inhibited HCV replication in vitro with IC50 1.28 µM in the HCV rep licon system [9, 10, 25, 26, 28]. It was shown that HCV RNAdependent RNA polymerase (NS5B protein [29]) was effectively inhibited by 4'azidouridine and 4'azidoadenosine 5'triphosphates. The inhibitory properties of these triphosphates were nearly the same (IC50 0.29 and 0.23 µM, respectively) [9, 10], which supported a conjecture that 4'azidouridine was not phosphorylated by cellular kinases [30].
O ArO P ONuc NH O R RI O (21)
iii
O HO P ONuc NH HO R O (24)
i
iv
The first phosphorylation resulting in the forma tion of 5'monophosphates is often the limiting stage of the intracellular synthesis of nucleoside triphos phates. At the same time, charged 5'phosphates can not pass through cell membranes and are unstable in physiological media [30, 31]. The recently developed ProTide approach [32, 33] made it possible to deliver into cells 5'monophosphorylated nucleosides in the form of membranepermeable ProTide Oacylated amino acid derivatives, particularly nucleoside 5'arylphosphoroamidates (21) (Scheme 3).
O ArO P ONuc NH HO R O (22)
O HO P ONuc OH NMP
ii
O
O O P ONuc NH R (23)
v
O O O HO P O P O P ONuc OH OH OH NTP
Scheme 3. Potential mechanism of the formation of nucleoside 5'monophosphates from Oacylated amino acid derivatives, nucleoside 5'(aryl)phosphoramidates (21). Conditions: i: esterase or carboxypeptidase; ii: spontaneously; iii: phosphoroamidase; iv and v: nucleotide kinase, –OC(O)CHRNH – amino acid residue, R1 – alkyl or aryl.
Potentially, the mechanism of nucleoside 5'phos phate formation (Scheme 3) involves enzymatic deg radation of amino acid esters (21) followed by a nucleophilic attack by the carboxylic group of com pound (22) on the phosphorus atom and substitution of the aryloxy residue [32]. As was demonstrated both in vivo and in vitro, the intermediate cyclic mixed anhydride (23) was spontaneously hydrolyzed to give the corresponding phosphoramide (24) [32]. Then, the successive formation of nucleoside mono, di, and triphosphates takes place in the presence of phos phoramidases and nucleotide kinases. The triphos phates are actual inhibitors of DNA and RNA poly merases and, hence, of virus replication. In 2007, McGuigan et al. reported the synthesis and study of antiHCV data of two 5'monophosphorylated mem branepermeable ProTide derivatives of 4'azidouri dine (31) [30] and 4'azidoadenosine (32) [34] (Scheme 4). Earlier, the use of this approach resulted in the aryloxyphosphoramidate derivatives of 2',3'dide hydro2',3'dideoxyadenosine (d4A) [33, 35], 2',3' dideoxyadenosine (ddA) [33], 2',3'didehydro2',3'
dideoxythymidine (d4T) [35, 36], and a carbocyclic analogue of 2',3'dideoxy2',3'didehydroadenosine [37] with enhanced activity. For the synthesis of aryloxyphosphoramidate derivatives of 4'azidouridines (31), the Uchiyama procedure was used [30, 34] (Scheme 4), which is based on nucleoside treatment with a strong organo metallic base, for example, tertbutylmagnesium chlo ride resulted in the formation of a metal alkoxide [38]. In the case of 4'azidouridine (12a), the reaction proceeded very rapidly with 3–20% product yields even if the 2',3'hydroxy groups were not protected. The low reactivity of these groups supported a regiose lective reaction at the 5' position. With this approach, 4'azidouridine 5'O[phenyl(butoxyLalanyl)]phos phoramidate (31) and some other derivatives were obtained. To increase the reaction selectivity and sol ubility of reaction components in THF, 2',3'diO cyclopentylidiene protection was used in further experiments (compounds (27) and (28)) [40].
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R R1
O
R
+
NH3 Cl
i
–
R1
O (25)
O
O H N
1
RO
O B HO N3 R2O OR2 (12a), (27), (28)
655
OAr P
R
ii
O N P Cl O (26)H OAr
O
B
O
O N3 O
O
(29), (30)
OR1 O
R iii
ArO
O
NH P
O
OR1
ArO O
O
N
O
O
O
NH2 N
N
HO (32) OH
–OC(O)CHRNH– R1 – – – – – – Me, Et, Pri, Amino acid resi dues: Gly, L, D Bui, But or Bzl Ala or βAla, LPhe, LLeu, LVal, Pro, LGln or LMet LAla residue
N
N3
HO (31) OH
B Ura Ura Ade – – Ura Ura Ura Ade
N
NH P
NH O
N3
Compound (12a) (27) (28) (25) (26) (29) (30) (31) (32)
O
R
R2, R2 H, H Cyclopentyl idene – – Cyclopentyl idene H, H
Ar – – – Phenil or naphtyl
Et, But or Bzl
Scheme 4. Synthesis of 4'azidouridine (31) and 4'azidoadenosine (32). Reagents and conditions: i: ArOPOCl2, Et3N, CH2Cl2; ii: 1) 1 M tBuMgCl in THF (2 eq), THF, 15 min; 2) sat. NH4Cl; iii: 80% formic acid, 4 h.
The developed scheme (Scheme 4) involved the inter action of phenyl dichlorophosphate with various salts of amino acid esters (25). The resulting phosphorochlori dates (26) were coupled with 4'azido2',3'diOcyclo pentylidieneuridine (27) in the presence of tBuMgCl and then deblocked with formic acid. Based on the NMR data, two diastereoisomers were always formed (due to a chiral phosphorus atom). As a result, 22 phenylphosphoramidates (31) were pre pared. Glycine, L, D and βalanines, phenylalanine, leucine, valine, proline, glutamine, and methionine were used as amino acid components. The amino acids were esterified to give methyl, ethyl, isopropyl, iso and tert butyl, and benzyl esters. Similarly, 4'azidouridine 5'О [1naphtyl(benzoyloxyLalanyl)] phosphate was obtained with 1naphtyl phosphorodichloridate (Scheme 4). Using HPLC, S and Rdiastereoisomers were isolated. The synthesized compounds were nontoxic (СС50 > 100 µM) and effectively inhibited in vitro the RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
HCV replication in the replicon system [9]. Their inhibitory activity was more than 450 times higher than that of parent 4'azidouridine and varied within one order of magnitude depending on the amino acid and ether. Among phenyl phosphoroamidates (31) the best properties were demonstrated by D and Lala nine derivatives with IC50 0.6–1.3 µM (for 4'azidocy tidine, IC50 was 1.28 µM without any differences between R and Sisomers). Benzyl and isopropyl ala nyl esters were among the most effective compounds in this series. However, the highest activity was found for 4'azidouridine 5'O[1naphtyl(benzyloxyLala nyl] phosphate (31). It is noteworthy that in this case the activity of the diastereoisomer mixture was higher (IC50 0.22 µM) than that of Risomer (IC50 0.39 µM) or Sisomer (IC50 0.43 µM) [30]. The next stage of the search for effective inhibitors of HCV replication and evaluation of the structure–function relationship was the synthesis of 4'azidoadenosine Pro Tide derivatives (32) and the study of their properties [34]. Vol. 37
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ALEXANDROVA
and 3'hydroxy groups were protected with tertbutyldim ethylsilyl residues. Then, compound (34) was subjected to HI elimination using the known procedure with potassium tertbutylate in pyridine [14]. The key stages were the prep aration of epoxide (37) and its opening.
The synthesis of the source 4'azidoadenosine (15) as described in [14] resulted in a low product yield. There fore, after the authors optimized each stage, they suggested a new method for its preparation (Scheme 5). First, for an increase in the selectivity of 5'iodination, adenosine 2'
NH2
N NH2
N
N
N
O
HO
i
HO
OH
O
O
O
O
O
Si
O
iv
(35)
O
O
O
N
O
Si
N
HO
vii
N OH
N
N
N
(39)
(38)
N
N
O
HO N3
Si
NH2
O vi
O
Si N
HN
N
N
O
HO N3
v
(37)
(36)
HN
N
N
N
Si
O
O
HN
N
Si
HO N3
Si
Si
N
N Si
ii
O
N
N
N
(34)
O
HN
N iii
O
Si
(33)
N
N
O
N O
N
N
N
O
I
NH2
N
O
O (28)
Scheme 5. Synthesis of 2',3'diOcyclopentylidene4'azidoadenosine (28). Reagents and conditions: i: 1) I2, Ph3P, Py; 2) TBDMSCl, imidazole, Py; ii: ButOK, Py; iii: pivaloyl chloride, DIEA, CH2Cl2; iv: 1 M dimethoxydoxypyrane in acetone; v: TMSN3, SnCl4; vi: TBFA, THF; vii: 1) dimethoxycyclopentane, H+, 2) NH3/MeOH.
Earlier, epoxides were not used for the introduction of an azido group into the 4'position of ribose residues of purine nucleosides. The N1 adenosine amino group was acylated in order to avoid its oxidation in the pres ence of DMDO. The N6pivaloyl derivative (36) was oxidized with DMDO, which was earlier found to pro vide stereoselectivity in reactions of this type [40]. The epoxide ring of compound (37) was opened in the presence of a Lewis acid (tin tetrachloride) and trime thylsilylazide. The product (38) was deblocked with tetrabutylammonium fluoride to give N6pivaloyl4' azidoadenosine (39)with a total yield of about 13%. As in the case of the synthesis of 4'azidouridine 5'aryloxyphosphoramidate derivatives (31) [30], the authors used the Uchiyama procedure [38]. To increase the reaction selectivity and solubility in the reaction medium (THF), 2',3'diOcyclopentylidene derivatives were obtained. Treatment with ammonia in methanol resulted in compound (28), which was used for the synthesis of 4'azidoadenosine 5'aryloxyphos
phoramidate derivatives (32) as described in [30] with out any significant modifications (Scheme 4). Since in early experiments the highest antiviral activity was observed for 4'azidouridine Lalanyl phosphoramidate derivatives bearing aryl and ester substituents (31) [30, 37], the authors synthesized modified 4'azidoadenosines (32) containing various Lalanyl esters (tertbutyl, ethyl, and benzyl) and two aryl groups, 1naphtyl and phenyl. The synthesized compounds were nontoxic (CC50) > 100 µM) and some of them effectively inhib ited in vitro the HCV replication in the HCV replicon system [9]. It was the first example of antiHCV activ ity for purine 4'azidonucleosides. The starting 4'azi doadenosine (15) hardly inhibited HCV (IC50 > 100 µM). Among the three esters, tertbutyl esters were nearly inactive, unlike the benzyl and ethyl ones. The lack of antiviral activity of phosphoramidate tert butyl esters can be associated with relatively high sta bility towards enzymatic hydrolysis. Ethyl esters were
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3 to 4 times more potent inhibitors than benzyl esters, whereas 1naphtyl derivatives were 5 to 20 times more active than phenyl phosphoramidates. 1Naphtyl derivatives (32) bearing benzyl or ethylLalanyl res idues were the most active (IC50 0.22 and 0.59 μM, respectively). The synthesized 5'monophosphorylated ProTide derivatives of 4'azidouridine (31) [30] and 4'azi doadenosine (32) [34] demonstrated high antiHCV activities in the HCV replicon system. Since uridine and 4'azido adenosine 5'triphosphates were effec tive inhibitors of HCV RNAdependent RNA poly merase in the replicon system, one can assume that active phosphoramidates (a) could be transformed into 4'azidonucleoside 5'triphosphates; (b) 4'azido nucleosides were not recognized by kinases; and (c) 4'azidonucleoside 5'monophosphates could be phosphorylated to give the corresponding 5'triphos O
i
Further studies of effective HCV inhibitors within 4'azidonucleosides involved modifications of the car bohydrate residue [25–27]. 4'Azidoarabinocytidine (18) was synthesized as shown in Scheme 6. 2,2'Anhydro4'azido1(βara binofuranosyl)uracil (40) was obtained from 4'azidouri dine (12a) by modification of the Moffatt method [41]. The treatment of derivative (40) with 1 M NaOH in etha nol resulted in 4'azidoarabinouridine (41). The stan dard transformation of the uridine derivative (41) to the cytidine one using the triazolide approach [20] led to 4' azidoarabinocytidine (18) with a total yield of 25% [26]. O
N
O
HO
O
N3
phates, which inhibited HCV RNAdependent RNA polymerase and, hence, HCV replication. Thus, the use of the phosphoramidate approach supported the formation of effective inhibitors of HCV replication from inactive analogues.
O
NH
N
O
HO
HO (40)
HO (12a) OH
O
N
N
O
HO
iii
N3
O
O
NH
N
O
HO
ii
N3
657
O
N3
HO (41) OH
HO (18) OH
Scheme 6. Synthesis of 4'azidoarabinocytidine (18). Reagents and conditions: i: 1) (PhO)2CO, NaHCO3, DMF, Δ; ii: NaOH, EtOH; iii: 1)Ac2O, Py; 2) triazole, POCl3, Et3N, MeCN; 3) aq. NH3.
ribo (45) and αLlyxoisomers (the latter is not shown in the scheme). Oxidative nucleophilic substitution of the 5'iodine atom in the presence of mCPBA followed by deprotection and acetylation of azide (46) resulted in an isomeric mixture of 2',5'diacetates. The isomers were separated and 2',5'diOacetyl4'azidouridine (47) was isolated. Compound (47) was transformed into 3'deoxy cytidine (48) by treatment with 1Htetrazole and 4chlo rophenyl dichlorophosphate followed by ammonolysis.
Since 3'deoxycytidine 5'triphosphate was earlier shown to be an effective terminating substrate of HCV RNAdependent RNA polymerase [23], 4'azido3' deoxycytidine (48) was prepared from 3'deoxyuridine (42). The researchers used the Verhayden and Moffatt approach [13], which was modified to provide substan tially higher total yields (to 6%, Scheme 7). Olefin (44) was treated with iodine and benzyltriethylammonium azide followed by benzoylation to give compound (45) as an isomeric mixture (1 : 1) at the C4'atom, namely, βD O HO
N
O
NH
O i
O
I
N
O
iv
NH
ii
NH
N
O
O
(42) OH
O
N
O
I
v
O
BzO
OH
O (45) OH
NH2 vi
O
HO O
N3 (47)
NH
N
NH
N
N3
(44) OH O
O
N3 (46)
NH
iii
O
(43) OH O
HO
O
O
N
O
N3
OBz
(48)
N
OH
Scheme 7. Synthesis of 4'azido3'deoxycytidine (48). Reagents and conditions: i: I2, PPh3, imidazole, THF or CH3CN; ii: NaOMe, MeOH; iii: [Bn(Et)3N]Cl, NaN3:I2, CH3CN, Nmethylmorpholine, THF; iv: BzCl, DMAP, Nmethylmorpholine, THF; v: (Bu4N)HSO4, K2HPO4, mCPBA, mCBA, CH2Cl2; vi: 1) NH3, MeOH; 2) Ac2O, DMAP, Py; 3) 1Htetrazole Cl2P(O)OC6H4Cl, Py; 4)NH3, dioxane; 5) NH3, MeOH. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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compound (49). The reaction of trimethylaluminum with 2'ketone (50) obtained in a yield of 83% using the Dess–Martin periodinane followed by deprotec tion led to a 1 : 1 mixture of 2'methyl isomers (51) and (52) separated by HPLC [26].
The synthesis of 4'azido2'βmethylcytidine (51) and 4'azido2'αmethylcytidine (52) from 4'azi docytidine (5) is shown in Scheme 8. Protection of azide (5) by the successive introduction of tetraisopro pyldisiloxane and benzoyl residues resulted in 25%
NHBz
NH2 N
O
HO
N
Si N3 O Si O
O
N3 HO
(5)
N
N
O
O
i
O
R' R''
(49) R'=H, R''=OH
OH ii
(50) R', R''=O NH2
NH2 iii
N
O
HO
N +
O
N3 HO
(51)
O
HO N3
OH
N
N OH
O
HO (52)
Scheme 8. Synthesis of 4'azido2'βmethylcytidine (51) and 4'azido2'αmethylcytidine (52). Reagents and conditions: i: 1) TIPDSCl, Py; 2) BzCl, Py; ii: 1,1,1tris(acetoxy)1,1dihydro1,2benziodoxol3(Н)one (Dess–Martin periodinane), CH2Cl2; iii: 1) AlMe3, THF; 2) HCl, MeOH; 3) NH3, MeOH.
4'Azidoxylocytidine (54) was obtained by benzoy lation of 4'azidocytidine (5) followed by partial deblock ing (Scheme 9). The resulting benzoylated products were chromatographed on silica gel to give N 4,2',5'tribenzoyl derivative (53) in a yield of 28%. The interaction of com pound (53) with trifluoromethanesulfonic acid anhy dride in pyridine followed by water treatment also led to a mixture of benzoylated 4'azidonucleosides, deprotec tion of which using ammonia in methanol resulted in 63% 4'azidoxylocytidine (54) as the only isomer [26]. All the synthesized compounds were nontoxic (СС50 > 100 μM) or cytotoxic [25, 26] unlike the ref erence araC (arabinocytidine, cytarabine), a known cytostatic agent [25]. Introduction of the 4'azido group into araC resulted in 4'azidoarabinocytidine (18), an effective in vitro inhibitor of the HCV replica
tion in the HCV replicon system (IC50 171 nM), which did not affect cell proliferation. The selectivity of 4'azidoarabinocytidine (18) was more than 3000 times higher than that of araC. Such a high antiHCV activ ity was hardly expected because in the molecule there was no 2'hydroxy group, assumed essential for recog nition by RNA polymerase [25, 26]. It is likely that similar to 4'azidocytidine (5), the 4'azido group made matching of the 3'hydroxyl with the enzyme active site incorrect [25]. At the same time, 4'azido 3'deoxycytidine (48) was inactive in the HCV repli con system [25] unlike its prototype, 3'deoxycytidine (42). Probably it may be explained by the fact that both cytidinederived compound (48) and 4'azidouridine (12a) could not be phosphorylated by cell kinases to the corresponding 5'triphosphates.
O
HO
N
O
N3 HO
N
(5)
OH
NH2
NHBz
NH2 i
O
BzO N3 HO
N
N
O (53)
OBz
ii
O
HO
N
O
N3 HO
N
(54)
OH
Scheme 9. Synthesis of 4'azidoxylocytidine (54). Reagents and conditions: i: 1) BzCl, Py; 2) MeONa, THF; ii: 1) Tf2O, Py, CH2Cl2; 2) H2O; 3) NH3/MeOH.
In contrast to 4'azido3'deoxycytidine (48), 4'azidoxylocytidine (54) demonstrated a noticeable antiviral activity in the HCV replicon system (IC50 27.8 μM) [25, 26]. However, its inhibitory
capacity was 20 times lower than that of 4'azidocyti dine (5), which implies an important role of the 3'hydroxy group in the formation of an active ana logue of nucleoside 5'triphosphate. 4'Azido2'β
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methylcytidine (51), a hybrid of the most wellknown in vitro inhibitors of HCV replication, 2'βmethylcy tidine [42] and 4'azidocytidine (5) [9, 10] was com pletely inactive in the HCV replicon system, most probably due to its inability to be phosphorylated in the presence of cell kinases. As was shown earlier, prototypes of compound (51) could be phosphorylated with various cell enzymes, particularly, 2'hydroxy2'βmethylcytidine, with deoxycytidine kinase, and 4'azidocytidine, with uri dine cytidine kinase 1 [25]. A combination of struc tural peculiarities in the molecule of nucleoside (51) may cause the loss of binding to the active sites of both enzymes. For the confirmation of this assumption, 50 mem branepermeable ProTide phosphate derivatives of
659
4'azido2'βmethylcytidine (31) were synthesized. As a result, inactive nucleoside (51) gave rise to effective inhibitors of HCV replication. The activity of the most active compounds was observed in the HCV replicon system at the submicromolar level (IC50 0.5 μM) [43]. Thus, it was shown that 4'azidoarabinocytidine (18) was the most effective inhibitor of HCV replica tion in the HCV replicon system despite the lack of the 2'αhydroxy group, whereas 3'modified 4'azidocy tidines lost the inhibitory properties [25,26]. Further studies directed at modifying 4'azidocyti dine involved the introduction of fluorine atoms [27]. Fluoronucleosides have been described well in the lit erature. They are smoothly phosphorylated by cell kinases and can be good substrates for DNA and RNA polymerases [44–46].
O N
O
HO
O
HO
NH O
N3
O
N
O
HO
ii
MthpO
O
iii
iv
O
N3 MthpO
(55)
O
OH
MthpO
N
N
O
HO
v
N3
(56)
NH2
NH
N
O
MthpO
O
N
O
O
NH
N
O
MthpO
N
N3
(40)
HO (12a) OH
O
MthpO
N
N3
i
O
N3
F
HO
(57)
(58)
F
Scheme 10. Synthesis of 4'azido2'fluoro2'deoxycytidine (58). Reagents and conditions: i: (PhO)2CO, NaHCO3, DMF, Δ; ii: MTHP, H+ (camphorsulfonic acid); iii: NaOH, EtOH; iv: DAST, Py, CH2Cl2; v: 1) tetrazole, Cl2P(O)C6H4Cl, Et3N, MeCN; 2) NH3, dioxane; 3) AcOH/MeOH/H2O (4 : 2: 1), 50°C, 5 h.
O O
HO
O
NH
N
i
O
I
O HO
(59)
NH
N
O
ii
HO
O
HO
iii
(60)
O
F
HO
O iv
NH
N
O
F
N
N3 HO (63) F
NH O
v
O
BzO
O
O
N
NH
HO
(62)
F NH2
vi
O
N3
O
N3
F (61) O
NH
N
O
I
O
HO
N
N3
BzO (64) F
N O
HO (19) F
Scheme 11. Synthesis of 4'azido2'fluoro2'deoxyarabinocytidine (19). Reagents and conditions: i: I2, PPh3, imidazole, THF; ii: NaOMe, MeOH; iii: [BnEt)3N]N3:I2, CH3CN, Nmethylmorpholine, THF; iv: BzCl, DMAP, Nmethylmorpholine, THF; v: (Bu4N)HSO4, K2HPO4, mCPBA, mCBA, CH2Cl2; vi: 1) NH3, MeOH; 2) BzCl, DMAP, Py; 3) 1Htetrazole, Cl2P(O)OC6H4Cl, Py; 4) NH3, dioxane; 5) NH3, MeOH.
The synthesis of 4'azido2'fluoro2'deoxycytidine (58) is shown in Scheme 10. Anhydro derivative (40) obtained by the reaction of diphenyl carbonate and 4' RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
azidouridine (12a) using the Reese approach [20] was protected by a 4methoxytetrahydropyranyl group, and the resulting compound (55) was treated with an NaOH Vol. 37
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ALEXANDROVA
alcohol solution to give arabino derivative (56). The use of DAST led to the only isomer (57). 4'Azido2'fluoro 2'deoxycytidine (58) was obtained by a standard proce dure of uridine–cytidine transformation [20] followed by deprotection (the total yield after seven stages was 7%). 4'Azido2'fluoro2'deoxyarabinocytidine (19) was synthesized from 2'fluoro2'deoxyarabinouri dine (59) by the Moffatt method [13] with the modifica tion used for the preparation of 4'azido2'deoxyara binocytidine (18) (Scheme 11) [26]. The yield of nucle oside (19) was 29%. O
O
NH
N
O
HO
i
HO
ii
OH (12a)
HO
NH
N
O
TrO
O
N3
NH2
O
NH
N
O
TrO
O
N3
The preparation of 4'azido3'fluoro3'deoxy xylocytidine (67) is shown in Scheme 12. The key stage, fluorination of the ditrityl derivative of 4'azido uridine (65) carried out by heating with DAST in a pyridine–dichloromethane mixture, gave 4'azido3' fluoro3'deoxy2',5'diOtritylxylouridine (66) in a yield of 31% relative to compound (65). Further transformations of uridine to cytidine using the triaz olide method followed by deprotection resulted in the target (67) in a yield of 47% (Scheme 12).
iii
O
N3
OTr
F
(65)
O
N3 F
OTr
N
N
O
HO
OH (67)
(66)
Scheme 12. Synthesis of 4'azido3'fluoro3'deoxyxylocytidine (67). Reagents and conditions: i: TrCl, Py; ii: DAST, Py, CH2Cl2; iii: 1) triazole, POCl3, Et3N, MeCN; 2) NH3, dioxane; 3) Amberlite 15, MeOH.
For the synthesis of 4'azido3'fluoro3'deoxy cytidine (74) from 3'fluoro3'deoxycytidine (68), the modified Moffatt method was also used (Scheme 13) [13, 26]. The interaction of olefin (70) with iodine and benzyltriethylammonium azide resulted in a 1 : 1 mixture of diastereomers, which O O
HO
O
NH
N
i
OH
F
O
N
iii
F
O
N3 F
OH (70)
iv
OTr (71)
O
NH O
N3
F
NH
N
O
I
O
OH (69) O v
O
NH
N
O
ii
O
(68)
mClBzO
O
NH
N
O
I
O F
was separated to give 31% 4'azide (71). A similar loss of stereoselectivity was earlier reported for the 3'deoxycytidine derivative (45) [26]. Further treat ment using the above described methods led to compound (74) in a yield of about 50% after four stages.
O
mClBzO
N
OH
F
(72)
NH vi
O
N3
NH2 N
O
HO
O
N3
OBz (73)
N
F
OH (74)
Scheme 13. Synthesis of 4'azido3'fluoro3'deoxycytidine (74). Reagents and conditions: i: I2, PPh3, Py, CH3CN; ii: NaOMe, MeOH; iii: I2, [Bn(Et)3N]N3, CH3CN; iv: (Bu4N)HSO4, K2HPO4, mCPBA, mCBA, CH2Cl2; v: Bz2O, Py; vi: 1) 1Htetrazole, Cl2P(O)OC6H4Cl, Py; 2) NH3, dioxane; 3) NH3, MeOH.
Based on the Moffatt method [13], 5'iodo deriva tive (76) was synthesized by the interaction of Nben zoylgemcytarabine (75) [47] with iodine and triphe nylphosphine in a yield of 90% (Scheme 14). After elimination, protection of the hydroxy group with a 4'methoxytrityl residue, and the reaction with iodine
and benzyltriethylammonium azide, 4'azide (78) was obtained (a yield of 60% for three stages). Nucleo philic substitution of a 5'iodine atom in (78) by a ben zoyl group followed by deblocking of compound (79) resulted in 4'azido2',2'difluoro2'deoxycytidine (20) in a yield of 35%.
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N
O O
HO HO
O iii
O
I N3 PMBzO
N
NHBz
i
F F (75) NHBz N N F F (78)
iv
F F (76)
HO
O BzO N3
O
PMBzO
N
N
O
ii
F F (77)
HO NHBz
O
N
v
F F (79)
NHBz
N
O
N
O
I
NHBz
N
O
661
HO N3 HO
O
N
NH2
N F F (20)
Scheme 14. Synthesis of 4'azido2',2'difluoro2'deoxycytidine (20). Reagents and conditions: i: I2, PPh3, MeCN, Py; ii: ButOK, DMF; iii: 1) I2, [Bn(Et)3N]N3, THF, MeCN; 2) PMBzCl, 4methylmorpholine, DMAP, CH3CN, THF; iv: NaOBz, 15crown5, DMF, 60°C; v: 2 M NH3/MeOH.
The synthesized fluoro derivatives were studied as potential HCV inhibitors in the HCV replicon system in the 220923 cell line as described in [9, 10]. All the compounds were nontoxic (CC50 > 100 μM) or cyto toxic unlike the control 2'fluoro2'deoxyarabino cytidine (dFaC), a known cytostatic agent [25]. The presence of a 4'azido group in 4'azido2'flu oro2'deoxyarabinocytidine (19) did not appreciably affect the inhibition of cell proliferation but made com pound (19) much more selective than dFaC (by more than 4000 times). As a result, the most effective inhibitor of HCV replication in the HCV replicon system was obtained (IC50 24 nM). Its activity was 50 times as high as that of cytidine (5) and 7 times as high as that of 4'azido arabinocytidine (18) [25, 26]. As with 4'azidoarabino cytidine (18), such a high activity of compound (19) was not expected because of the lack of a 2'hydroxy group assumed essential for recognition by RNA polymerase [25, 26]. It is noteworthy that the inversion of a 2'fluoro residue resulted in 4'azido2'fluoro2'deoxycytidine (58) causing a 150fold decrease in the HCV activity as compared with arabinocytidine (19). The introduction of a 4'azido group into gemcit abine (75), a highly toxic compound with CC50 at the nanomolar level in some human cells [25], used for treatment of tumors, led to compound (20), which displayed very specific properties. On the one hand, it was a highly effective inhibitor of HCV replication in the in vitro replicon system (IC50 66 nM), and on the other hand, it was only negligibly cytotoxic. On the contrary, 3'fluoro derivatives of 4'azidocytidine (67) and (74) were completely inactive and nontoxic in the HCV replicon system. We can assume that the lack of a 3'hydroxy group prevented the interaction of these compounds with kinaseactive sites required for their phosphorylation to the corresponding triphosphates. To summarize, the synthesized 4'azidoarabino cytidine (18), 4'azido2'fluoro2'deoxyarabino cytidine (19), and 4'azido2',2'difluoro2'deoxycyti dine (20), (IC50 171, 24, and 66 nM, respectively) were the most effective in vitro inhibitors of HCV replication RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
in the replicon system, although they did not contain 2'hydroxy groups, whereas 3'modified derivatives of 4'azidocytidine lost the inhibitory properties. On the other hand, the introduction of a 4'azido group into the molecules of highly toxic araC and its fluoro deriv atives led to a nearly complete loss of toxicity of the resulting compounds (18), (19), and (20). 4'FLUORONUCLEOSIDE DERIVATIVES In the mid1950s, it was found that Streptomyces cal vus isolated from Indian soil could produce nucleoci dine, an antibiotic with a wide range of antibacterial activity, an relative of adenosine [48]. However, its use was strictly limited by its toxicity: following intravenous or intraperitoneal injections, its CC50 was 0.2 mg/kg [49]. Using NMR and mass spectral data, it was identified in 1969 as 4'fluoro5'Osulfamoyladenosine (6) [50]. The structure of nucleocidine is unique, because it is the only known natural product bearing both fluoro and sulfamoyl groups. This was the first example of nucleosides modified at the carbohydrate C4 atom. The main problem in synthesizing compound (6) was the stereospecificity of introduction of the fluorine atom into the ribose 4'position. In early publications, the preparation of both purine [51, 52] and pyrimidine [53, 54] 4',5'unsturated nucleosides was reported. It seemed attractive to introduce a 4'fluorine atom by addition of a fluorinecontaining pseudohalogen, for example, iodine fluoride IF, to the protected 4',5'unsturated adenosine derivative. Iodine fluoride can be obtained either by iodine and fluorine in situ interaction [55] or from AgF and I2 in the course of the reaction with the nucleoside. The nucleocidine synthesis is shown in Schemes 15 and 16. The interaction of 2,3'diOisopropylideneole fin (80) with IF (obtained in situ from AgF and I2) was carried out under various conditions [12, 56]. In nearly all cases, a mixture of two isomeric 4'fluoro5'deoxy5' iodonucleosides (81) and (82) was formed in yields of 60 to 90%. As is seen in the NMR spectrum of compounds Vol. 37
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ALEXANDROVA
Under these conditions, βDriboisomer (81) yielded product (84) (UV monitoring), whereas in the case of (82), this product was not observed. Configurations of the isomers were confirmed by comparison of C3' proton resonances of compounds (81) and (82) [56–60]. The coupling constant J3', F of βDriboisomer (81) was 11.5 Hz, and that of αDlyxoisomer (82), 5.5 Hz [56].
(81) and (82), the presence of a 4'fluoro atom resulted in an extra proton–fluorine decoupling of the proton 3' and 5'resonances of the ribose residues. The NMR spec trum of the 5'deoxy4'fluoronucleoside (83) obtained by hydrogenolysis of compound (81) contained a doublet of the 5'methyl group with J5’,F 17 Hz. With the goal of ascribing the right configuration of the isomers, βD riboisomers (81) and (82) were heated at 140°С for 16 h. NBz2
N
N
N
O
NBz2
N
N
N
N
O
I
i
F +
N
O
O
O
(80)
NBz2
N N H3C
NBz2
N
H2C
F
(82)
iii
N
N
N
O
O
iii
N
O
N
(81)
ii
N
N
O
I
F O
NBz2
N
I–
N
O +
F O
O
O
O (83)
(84)
Scheme 15. Determination of configurations of isomeric 4'fluoro5'deoxy5'iodonucleosides (81) and (82). Reagents and conditions: i: IF (из I2 and AgF), ii: H2, 10% Pd/C, MeOH, Et3N, 4 h, 20°C; iii: 140°C, 16 h.
NH2
N
N
N
O
I
N
O
O
O
O
O
O (85)
NH2
N
N
N
O
iii
N
N
O
O
O (88)
(87)
NH2
N
O S
H2N
N
N
O
Bu3SnO F
O
O
(86) NH2
N
F
iv
N F
(81)
HO
N
N
O
O
ii
F
F
NH2
N
N
N
O
N3
i
N
NH2
N
N
N
O
O
O
N F O
S v
H2N
O
O
(89)
N
N N
F HO
O
NH2
N
O
(6)
OH
Scheme 16. Synthesis of nucleocidine (6). Reagents and conditions: i: LiN3, DMF, 95°C, 20 h; ii: 1) photolysis, THF, benzene; 2) 0.5 N HCl, dioxane; 3) aq NaHCO3; 4) NaBH4, H2O; iii: benzene, hexabutyldistannoxane; iv: sulfamoyl chloride, dioxane; v: 90% CF3COOH.
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Another key stage of nucleocidine synthesis was the replacement of a 5'iodo residue of compound (81) by a hydroxy group. The substitution in the presence of oxygenbearing nucleophiles (such as lithium acetate, lithium benzoate, or dimethylsulfoxide) was ineffec tive. The only suitable nucleophile proved to be an azide ion. Compound (81) reacted with lithium azide in DMF at 95°С producing only 5'azido4'fluoronu cleoside (85). It was shown earlier that photolysis of alkylazide led to the elimination of nitrogen and the formation of nitrene [61], which gave a carbonylcontaining com pound when hydrolyzed [62]. Based on this method, compound (85) was subjected to photolysis and the resultant 5'aldehyde was in situ reduced by sodium borohydride to 4'fluoro2',3'diOisopropylidene
663
adenosine (87) [63]. The product was purified by chromatography on silica gel in a yield of 20–30% rel ative to two stages. Then compound (87) was treated with hexabutyldistannoxane in benzene followed by the reaction of ester (88) with sulfamoyl chloride at 5°С to give 87% 4'fluoro2',3'diOisopropylidene5'Osul famoyladenosine (89). The isopropylidene protective group was removed by 90% trifluoroacetic acid. The total yield of the target nucleocidine (6) was 6%. In the course of these studies 4'fluorouridine derivatives (97) were obtained (Scheme 17) [64]. The synthesis was based on the coupling of 2',3'diO protected 1(5deoxyβDerythropent4enofuran syl)uracils with iodine in methanol, which resulted in a mixture of 5'iodo4'methoxynucleosides of βD and αLconfigurations [13]. O
H N
O
O
O
N
O
H N
O
N O
N
O i
+
(90)
O ii
O
O
O (94)
(93)
H N
O
N
O
F O
O
(95)
HO
(96) v vi
a) R=H b) R=SO2NH2 c) PO(OCH2CCl3)2
H N
O
N
F
O
O
O
RO v
F
O
O
N
O
RO iv
O
O
O iii
F
O
N H2C
N
O
I
(92)
O
O
N
O
N3
N
O
F
O
O (91)
H N
O
H2C
O
O
O
H N
viii
OH
(97) a) R=H b) R=SO2NH2 c) PO(OCH2CCl3)2 d) PO3H2
Scheme 17. Synthesis of 5'sulfamoyl4'fluorouridineа (97b) and 4'fluorouridine 5'phosphate (97d). Reagents and conditions: i: AgF, I2, CH2Cl2, 20°C, 30 min; ii: NaN3, DMF, 95°C, 17 h; iii: 1) NOBF4, CH3CN, 0°C, 20 min; 2) 20°C, 45 min; iv: CF3COOH, THF, H2O, 20°C, 15 min; v: sulfamoyl chloride, dioxane, 20°C, 18 h; vi: bis(2,2,2trichloroethyl)phosphate, 2,4,6TPSCl, Py, 20°C, 18 h; vii: 90% HCOOH, 20°C, 1.5 h; viii: 1) Zn powder, AgOAc, AcOH, DMF, 20°C, 1.5 h; 2) Dowex 50 (NH4+).
The nature of the 2',3'protective group consider ably affected the strereochemistry of the addition reaction. Particularly, a 2',3'diOisopropylidene group supported the formation of a 3 : 2 βD and αL 5'iodo4'methoxynucleoside mixture, whereas a 2',3'diOcarbonate group maintained mainly the βDisomer [13, 66]. As was shown above, a similar coupling of 4',5'unsat urated adenosine derivatives with iodine fluoride led to an epimer mixture [56, 66]. Therefore, even more unex RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
pected was the formation of the only product, 4'fluoro 5'iodo5'deoxy2',3'diOisopropylideneuridine (93) in the reaction of 1(5deoxy2,3diOisopropylidene βDerythropent4enofuransyl)uracil (90) [52, 67] with iodine fluoride (obtained from AgF and I2). The structure of (93) was confirmed by comparison of its NMR spectrum (J3', F = 11.5 and J1', F = 0 Hz) with those of βD and αL4'fluoro5'iodo5'deoxy2',3'diO isopropylideneadenosines (81) and (82), respectively Vol. 37
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ALEXANDROVA
[56]. Its coupling constants were nearly identical to those of isomer (81). The unexpected stereospecificity of this reaction was explained by the involvement of the uracil residue. Particularly, the interaction of iodine with olefin (90) led to a threemember cycle (91) followed by the con version to O2,4'cyclonucleoside (92). The opening of the latter in the presence of a fluorine ion could only result in the βDriboderivative (93). It is noteworthy that compound (93) was rather labile under both acidic and strongly alkaline conditions. As in the nucleocidine (6) synthesis, the substitu tion of a 5'iodide residue in compound (93) for a hydroxyl group was difficult. The reaction of (93) with sodium azide in DMF at 95°C also resulted in one product, 5'azido4'fluoronucleoside (94). For the replacement of the 5'azido group by a hydroxy group, nitrozonium tetrafluoroborate [68] was used, but the product differed from the expected 4'fluoro2',3'di Oisopropylideneuridine (96a). It was found to be O 2,4'anhydronucleoside (95), which was hydrolyzed by diluted acid to give compound (96a). In general Nglycoside bonds of 4'fluoronucleo sides are labile. However, modifications of 2',3' or 5'hydroxy groups of the carbohydrate residue consid erably increased stability of these nucleosides. The authors failed to isolate the exhaustivelydeprotected 4'fluorouridine (97a). For the synthesis of a nucleoci H N
O
O
N
O
H N
O F
I
dine uridine analogue, 5'sulfamoyl4'fluorouridine (97b), 4'fluoro2',3'diOisopropylideneuridine (96a) was treated with sulfamoyl chloride in dioxane fol lowed by deblocking of compound (96b) by 90% for mic acid. 4'Fluorouridine 5'phosphate (97d) was also obtained from 4'fluoro2',3'diOisopropylide neuridine (96a) by successive treatment with bis(2,2,2trichloroethyl) phosphate in pyridine, 90% formic acid, and acetic acid in the presence of zinc powder and silver acetate. As was supposed, 5'substituted derivatives of 4'fluorouridine (96b) and (97d) were much more sta ble than parent 4'fluorouridine (97a). Particularly, phosphate (97d) was hydrolyzed by 20–30% in the presence of 0.05 M NaOH at room temperature for 30 min to give uracil. Under acidic conditions it was more stable and was not cleaved in 1M HCl at room temperature for 15 min. However, when the tempera ture was increased to 60°C, the degree of hydrolysis achieved 58% in 30 min. Thus, the stability of 5'sub stituted derivatives of 4'fluorouridine proved to be high enough for biological studies. The studies by Moffatt et al. [11,12, 59, 66] on the synthesis of nucleocidine derivatives opened a new approach for the preparation of 4'fluronucleoside derivatives. Based on these studies, in 1988 S. Ajmera et al. described the preparation of 4',5difluoro5' deoxyuridine (107) (Schemes 18 and 19) [69].
O F
N
O
i
O
O
O F
O
O
(100) iv
iii
O
F i
O
O
O (99)
N
O
F
N
O F
(98)
H N
O
ii
F O
H N
O
O
O (101)
F
HN N H (102)
Scheme 18. Synthesis of 4'fluoro5'deoxy2',3'diOisopropylidene5fluorouridine (101). Reagents and conditions: i: IF; ii: H2, 5% Pd/C; iii: C5H5NH+(HF)xF–, 0°C; iv: H+.
The first attempts to obtain compound (107) included the interaction of 4',5'unsaturated 5fluo rouridine (98) with IF with the goal of stereospecifi cally introducing a fluorine atom into the sugar 4'position, a method similar to that described in [64, 66] (Scheme 18). The glycoside bond of the iso lated compound (99) was found to be acidlabile. Although Moffatt et al. reported that the isopropy lidene protective group could be removed with 90%
formic acid to give 25% of the unprotected nucleoside, deblocking of compounds (100) and (101) under these conditions resulted in fluorouracil (102). It was also shown that 4'fluorouridine was unstable under alka line conditions [64]. Therefore, a good alternative for protection of a 2',3'hydroxy group seemed to be a benzyl group that can be removed by hydrogenolysis under neutral conditions.
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H N
O O
TrO
O F
N
O iii
O F BnO
BnO H O N
O F
O iv
O F HO
OBn (106)
O F
N
O
ii
OBn (104)
F
N
H N
O
N
O
MsO
i
OH (103)
HO
H N
O
665
OBn (105)
BnO H O N F
N OH (107)
Scheme 19. Synthesis of 4'fluoro5'deoxy5fluorouridine (107). Reagents and conditions: i: 1) BnCl, KOH; 2) HCOOH; 3) MsCl; ii: ButOK; iii: C5H5NH+(HF)xF–, –50°C; iv: H2, Pd(OH)2.
counterpart. A drop in temperature to –50°C resulted in βDriboisomer (106) in a yield of 80%. The ben zyl groups were removed by hydrogenolysis with Pd/C [72] to give 4',5difluoro5'deoxyuridine (107) in a total yield of 11%.
2',3'Hydroxy groups of 5'Otrityl5fluorouridine (103) [70] were successively protected with benzyl groups using the A.M. Michelson and A.J.Todd method [71]. Then the 5'Otrityl group was substi tuted by a methansulfonyl group to give derivative (104) (Scheme 19). The interaction of the latter with sodium tertbutoxide yielded 67% 1(5deoxy2,3 bis(benzyloxy)βDerythropent4enofuranosyl) 5fluorouracil (105). The reaction of compound (105) with poly(fluorohydrogen) in pyridine proceeded at 0°C as described for compound (98). As a result, a 1 : 1 mixture of fluorinated products was obtained. Based on the available NMR data, the compounds were identified as βDriboisomer (106) and its αLlyxo
Antiviral properties of the abovedescribed 4'fluo ronucleosides have not been studied. Due to evalua tion of effective inhibitors of HCV replication, par ticularly 4'azidocytidine [9, 10] and its derivatives [25–27], there is renewed interestin synthesis and antiviral studies of 4'fluoronucleosides. The synthesis of some pyrimidine 4'fluoronucleosides was reported in 2010 [73].
O N
O
HO
NH i
O
O
NH2 O
AcO F HO
N
N O
OH (112)
O
O iii, iv ii
ii
F HO
(108) NH2
N
O
RO
N
vii, ii
O
O v, vi
NH O
OH (109) NHOH O
AcO
O
F
N
O
AcO
O
F
(96a)
NH
N
O
AcO O
F
O
O
(110) R=H (111) R=Ac
F HO
N
N O
OH (113)
Scheme 20. Synthesis of 4'fluoro5'Oacetyluridine (109), 4'fluoro5'Oacetyltidine (112) and 4'fluoro5'Oacetyl N4hydroxycytidine (113). Reagents and conditions: i: Ac2O, Py, 20°C, 72 h; ii: 90% HCOOH, 20°C, 1.5 h; iii: triazole, 4CIPhOPOCI2, Py, 20°C, 96 h; iv: 34% aq NH3, dioxane, 20°C, 3 h; v: N,Ndimethylformamide diethylacetal, DMF, 20°C, 2.5 h; vi: 1) Ac2O, Py, 20°C, 18 h; 2) pBuOHH2OAcOH, 5 : 3 : 2, 20°C, 5 h; vii: aq NH2OH ⋅ HCl, NaOH to pH 5.2, 37°C, 24 h.
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As indicated above, the Nglycoside bond in 4'flu oronucleosides was rather labile. However, Moffatt et al. demonstrated that modifications of 2',3' and 5'hydroxy groups could significantly increase it [64, 65]. The biological activity of 5'acetyl nucleoside derivatives can be studied due to smooth cleavage of 5'acetyl groups by cell esterases (see, e.g., review [74]). Therefore, a set of 5'acetates of pyrimidine 4'fluoronucleosides was synthesized from 4'fluoro 2',3'diOisopropylideneuridine (96a) obtained as described in [64] (Scheme 20). Nucleoside (96a) was acetylated, and the isopropylidene group of (108) was removed by HCOOH to give 4'fluoro5'Oacetyluri dine (109). 4'Fluoro2',3'diOisopropylidenecyti dine (110) was prepared from nucleoside (108) by the triazolide method [21]. It was acetylated, and the iso propylidene residue was removed by formic acid to give 4'fluoro5'Oacetylcytidine (112). The interac tion of 4'fluoro2',3'diOisopropylidene5'Oace tylcytidine (111) with hydroxylamine, similar to [75], followed by deblocking of the 2',3'diOisopropy lidene group, led to 4'fluoro5'OacetylN4 hydroxycytidine (113). As expected, the synthesized
N
O
HO O
O
The cytotoxic effect in the human hepatoma Huh7 cell line measured as described in [9] was not observed for any of the synthesized 4'fluoronucleosides (109), (112), or (113) within the tested concentration range (up to 500 μM). The nucleosides were tested as poten tial inhibitors of HCV replication in the HCV replicon system [28]. Compounds (112) and (113) were inac tive in this system, whereas nucleoside (109) demon strated moderate activity (IC50 2 μM). The synthesis of 4'fluorouridine 5'Ophosphate (115) was performed according to the classical Scheme 21. Nucleoside (96a) was coupled with phosphoro(tristri azolide) [76] followed by the removal of the isopropy lidene group in the presence of 90% HCOOH (Scheme 21). The resulting monophosphate (114) was activated with carbonyldiimidazole and treated with tertbutylammonium pyrophosphate in DMF [77]. The target 4'fluorouridine 5'Otriphosphate (115) was obtained in a yield of 9.7% relative to nucleoside (96a).
O
O
O
NH
O NH O N HO P O O OH F OH HO (114)
O O O NH O N HO P O P O P O O OH OH OH F HO OH (115)
O
F
compounds were rather stable: no changes were observed following storage at 4°C for a year.
(96a)
i, ii
iii
Scheme 21. Synthesis of 4'fluorouridine 5'Otriphosphate (115). Reagents and conditions: i: phosphoro(tristriazolide), CH3CN, 20°C, 1 h; ii: 90% HCOOH, 20°C, 1.5 h; iii: 1) N,N'carbonyldiimidazole, Bu3N, DMF, 20°C 40 min; 2) (Bu3NH)2H2P2O7, DMF, 20°C, 24 h.
The stability of phosphates (114) and (115) was evaluated in PBS at 37°C [78]. In both cases uracil was formed. The halflife was about 18 h. When frozen (⎯20°C), both compounds were stable for several months.
same time, low inhibitory properties and the stability of 4'fluoronucleoside derivatives in comparison with those of 4'azidonucleosides offer little promise for further studies.
The study of the effect of triphosphate (115) on the key HCV enzymes, namely, RNAdependent RNA polymerase (the NS5B protein) [29] and NTP dependent NTP helicase (the NS3 protein) [79–81], showed that triphosphate (115) was an effective inhibitor of RNA synthesis catalyzed by viral RNA dependent RNA polymerase (IC50 2 μM) (IC50 for 4' azidouridine triphosphate was 0.2 μM [30]). Triph osphate (115) was a substrate of the NTPase reaction catalyzed by the NS3 protein and, also, activated the helicase reaction with an efficacylower than that of ATP by a only factor of 3. Thus, the antiviral activity of 4'fluoro5'Oacetyluridine (109) in cell cultures could be due to its conversion to triphosphate (115), which inhibited the virus replication catalyzed by viral HCV RNAdependent RNA polymerase. At the
4'ETHINYL, 4'PROPYNYL, 4'CYANO, AND 4'ALKOXYNUCLEOSIDES 4'Modified nucleosides were first synthesized by Moffatt et al. in the 1970s [12, 13, 16, 17, 51, 52, 56, 64–66, 83–85]. In later studies, these schemes served as the background for syntheses with modifications that resulted in an increase in product yields. In partic ular, Moffatt et al. developed a convenient method for the introduction of a hydroxymethyl group into the 4'position of protected ribo and 2'deoxyribonucle osides using an aldol condensation of the correspond ing 5'aldehydes and formaldehyde [83–85]. Mat suda et al. adapted this method to the preparation of 4'ethyl and chloroethynyl 2'deoxycytidine deriva tives as well as 4'ethynyl and 4'cyanouridine and
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cytidine derivatives [82]. Later, this approach was used for the preparation of 4'αvinyluridine and cytidine [10]. The synthesis of 4'substituted ribonu cleosides from 1(2,3,5triOTBDMS4'formylβ Dribopentafuranosyl)uracil (116) is shown in Scheme 22. The 5'OTBDMS group of uridine (116) was selectively removed to give bisTBDMS derivative (117), whose oxidation with DMSO–DCC [83, 84] or DMSO–EDC [82] followed by a shortterm treat
H N
O R1O
ment with aqueous formaldehyde and NaOH resulted in aldehyde (118). The latter was reduced with NaBH4 to give 44% diol (119). This aldol reaction had advan tages over the Kannizzaro reaction [82]. After manip ulations with protective groups and a slightly modified oxidation under the Swern conditions [86, 87], a key 4'formyl derivative (121) was obtained in a yield of 82%. Deblocking of compound (119) led to 4'hydroxymethyluridine (120).
H N
O O
N
O
ii
O iii
O v
O
HO HO HO
iv
TBDMSO O
H N
O
N
N HO O
N
OTBDMS
O O O
HO
OH (122)
vi
O
vii
O
HO
viii
iv/v
(119)
N
O
HO
OH (118) H N O O
H N
O
TBDMSO
O
N
O
HO HO
OR2 R2O TBDMSO OTBDMS 1 2 (116): R =R =TBDMS (118) (117): R1=H, R2=TBDMS
i
H N
O
N
O
O
667
H N
HO O
H N
O
N OH (123)
N
HO OH (124)
TBDMSO OTBDMS (121)
Scheme 22. Synthesis if 4'substituted ribonucleosides. Reagents and conditions: i: 4% CF3COOH in 80% AcOH, 1 h, 20°C; ii: EDC ⋅ HCl, DMSO, CF3COOH, Py, benzene, 30 min, 20°C; 2) 37% aq formaldehyde, 2 M NaOH, dioxane, 50 min, 20°C, iii: NaBH4, EtOAc, 30 min, 0°C; iv: 1) DMTrCl, Py, 1.5 h, 20°C; 2) TBDMSCl, imidazole, DMF, 13.5 h, 20°C; 3) 80% AcOH, 5 h, 20°C; 4) DMSO, (CF3CO)2O, CH2Cl2, 30 min, –78°C, then Et3N; v: TBAF, THF; vi: 1) NH2OH ⋅ HCl, Py, 40 min, 20°C; 2) NaOAc; Ac2O, 2.5 h, 130°С; 3) TBAF, THF; vii: Ph3P=CH2 (from methyl triphenylphosphonium bromide and BuLi in hexane) THF, –78°C, then 0°C, 30 min in argon), THF, 1.5 h, 20°C; 2) TBFA, THF; viii: 1) Ph3P=CHCl (from cloromethyl triphenylphosphonium chloride and BuLi in hexane) THF, –78°C, then 0°C, 30 min in argon, THF, 1.5 h, 20°C; 2) BuLi/hexane, THF, –78°C 2 h in argon; 3) TBAF, THF.
Aldehyde (121) was coupled with hydroxylamine to give the oxime that was dehydrogenated by Ac2O in the presence of NaOAc and deprotected to give 4'α cyano derivative (122). The formyl group of compound (121) was trans formed to a 4'vinyl group using the Wittig reaction with Ph3P=CH2 and the protective groups were removed to give compound (123). The interaction of nucleoside (121) with Ph3P=CH2 under the Wittig reaction conditions and successive treatment with n butyl lithium and deblocking yielded 4'αethynyluri dine (124). It is noteworthy that a 20 : 1 mixture of Z RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
and E isomers in a yield of 92% was obtained in both Wittig reactions. 4'Methoxynucleosides have been first obtained by Moffatt et al. [13, 65]. The key stage was oxidation of the C4'=C5' double bond to epoxide (125) with mCPBA in methanol followed by the epoxide opening to give a mixture of 4'epimers (Scheme 23) [13]. As was mentioned above, the nature of the group protect ing 2',3'hydroxyls considerably affected the stere ochemistry of the addition reaction. Particularly, the 2',3'diOisopropylidene group supported the forma tion of a 3 : 2 mixture of βDribo and αLlyxo5' Vol. 37
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iodo4'methoxynucleosides, whereas the 2',3'diO carbonate protection yielded mainly the βDribo isomer [13, 66]. However, in further syntheses of 4' methoxyuridine [14] and 4'ethoxyuridine [10] unprotected olefin (9) was used as a starting nucleo
O
side. Configurations of the isomers were ascribed using 13 C NMR spectroscopy [14]. The resonance of 5'C of 4'methoxyuridine (126) was located downfield if compared with that of its αLlyxo counterpart (127) (60.7 and 55.8 ppm, respectively).
O O
O
NH
N
O
O
i/ii
N
O HO
NH O
OH
HO
OH
(9)
O
HO
(125)
RO HO
H N
H N
O O RO
N +
OH
HOH2C HO
(126) a) R=CH3 b) R=C2H5
O
N
O
OH
(127) a) R=CH3 b) R=C2H5
Scheme 23. Synthesis of 4'alkoxynucleosides. Reagents and conditions: i: mCPBA, MeOH (for the preparation of 126а), ii: mCPBA, EtOH (for the preparation of 126b).
4'Modified cytidines (128a–e) were prepared from the corresponding acetylated uridines by the Divakar and Reese method [10] or via 4triisopropyl sulfonyl derivatives obtained by treatment with 2,4,6 triisopropylsulfonyl chloride in the presence of Et3N and DMAP followed by treatment with ammonia [82]. The synthesized 4'modified uridine and cytidine derivatives were studied as potential inhibitors of HCV replication in the replicon system in the 220923 cell line [9, 10]. 4'Cyanocytidine (128b) effectively inhib ited HCV replication with the efficacy close to that of 4'azidocytidine (5) (99 and 97% inhibition at 20 μM, respectively). However, unlike nucleoside (5), its cyto toxicity was nearly as high as its activity [9, 10]. NH2 O
HO R HO
N
N O
OH (128 а–f)
R = а) CH2OH, b) CN, c) CH=CH2, d) C≡CH, e) CH3, f) C2H5 Formulas 5. 4'Modified cytidines (128 a–f): 4'αhydroxy methylcytidine (128a), 4'αcyanocytidine (128b), 4'α vinylcytidine (128c) 4'αethynylcytidine (128d) 4'meth oxycytidine (128e) and 4'ethoxycytidine (128f).
The lack of antiviral activity in the HCV replicon system of other tested compounds can be explained by either the inability to be phosphorylated or complete or partial inability of the corresponding triphosphates
to incorporate into the HCV RNA polymeraseactive site. However, the synthesized 4'azidouridine (5) and 4'ethynylcytidine (128d) 5'triphosphates were effec tive inhibitors of RNA synthesis catalyzed by viral RNAdependent RNA polymerase (IC50 0.29 and 2.7 μM, respectively) [9, 10]. This allowed a conclu sion that the lack of the antiviral effect of 4'modified nucleosides could be explained by unproductive phos phorylation. CONCLUSIONS A wide range of synthetic approaches provided a new class of compounds, 4'branched ribonucleo sides. As was shown in numerous publications, the nature of a 4'substituent is essential for the antiviral properties and toxicity of these compounds. For example, 4'azidocytidine and its derivatives demon strated in vitro a high inhibitory activity against HCV replication (at nanomolar concentrations) in the HCV replicon system. At the same time, the introduction of a 4'azido group in the highly toxic araC and its fluoro derivatives resulted in the nearly complete loss of tox icity. The inhibitory effect of 4'fluoronucleosides was considerably lower than that of 4'azidonucleosides. Other 4'modified nucleosides did not inhibit HCV replication. Since 4'branched nucleoside 5'triphosphates were effective inhibitors of RNA synthesis catalyzed by the HCV RNAdependent RNA polymerase, the lack of antiviral effects of 4'modified nucleosides could be due to the ineffective phosphorylation. The studies on the synthesis and antiHCV activity of phosphorylated membranepermeable ProTide derivatives of 4'azid
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ouridine and 4'azidoadenosine demonstrated that inactive analogues of 4'azidonucleosides can be transformed into effective inhibitors of HCV replica tion. To summarize, 4'branched ribonucleosides are promising compounds for the design of effective anti viral agents. ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research (project nos. 110400603a, 080400549, and 110412035ofim2011) and the Program for Fundamental Research of the Presidium of Russian Academy of Sciences “Molecular and Cell Biology.” REFERENCES 1. De Clercq, E., Adv. Virus Res., 2009, vol. 73, pp. 1–53. 2. De Clercq, E., Int. J. Antimicrob. Agents, 2009, vol. 33, pp. 307–20. 3. Watanabe, K.A. and Patterson, S.E., in Frontiers in Nucleosides and Nucleic Acids, Schinazi, R.F. and Liotta, D.C., Eds., IHL Press, 2004, pp. 3–55. 4. OYang, C., Wu, H.Y., FraserSmith, E.V., and Walker, K.A.M., Tetrahedron Lett., 1992, vol. 33, pp. 37–40. 5. OYang, C., Kurz, W., Eugui, E.M., McRoberts, M.J., Verheyden, J.P.H., Kurz, L.J., and Walker, K.A.M., Tetrahedron Lett., 1992, vol. 33, pp. 41–44. 6. Bousquie, I., Madiot, V., Florent, J.C., and Monneret, C., Bioorg. Med. Chem. Lett., 1996, vol. 6, pp. 1815–1818. 7. Hayakawa, H., Kohgo, S., Kitano, K., Ashida, N., Kodama, E., Mitsuya, H., and Ohru, H., Antivir. Chem. Chemother., 2004, vol. 15, pp. 169–187. 8. Haraguchi, K., Shimada, H., Tanaka, H., Hamasaki, T., Baba, M., Gullen, E.A., Dutschman, G.E., and Cheng, Y.Ch., J. Med. Chem., 2008, vol. 51, pp. 1885– 1893. 9. Klumpp, K., Leveque, V., Le Pogam, S., Ma, H., Jiang, W.R., Kang, H., Granycome, C., Singer, M., Laxton, C., Hang, J.Q., Sarma, K., Smith, D.B., Hei ndl, D., Hobbs, C.J., Merrett, J.H., Symons, J., Cam mack, N., Marttin, J.A., Devos, R., and Najera, I., J. Biol. Chem., 2006, vol. 281, pp. 3793–3799. 10. Smith, D.B., Martin, J.A., Klumpp, K., Baker, S.J., Blomgren, P., Devos, R., Granycome, C., Hang, J., Hobbs, C.J., Jiang, W.R., Laxton, C., Le Pogam, S., Leveque, V., Ma, H., Maile, G., Merrett, J.H., Pichota, A., Sarma, K., Smith, M., Swallow, S., Symons, J., Vesey, D., Najera, I., and Cammack, N., Bioorg. Med. Chem. Lett., 2007, vol. 17, pp. 2570–2576. 11. Li, F., Wu, X., Huang, S., Hong, L., Tran, T., Brandi, M., and Alfredson, T., Drag. Dev. Ind. Pharm., 2010, vol. 36, pp. 413–420. 12. Jenkins, I.D., Verheyden, J.P., and Moffatt, J.G., J. Am. Chem. Soc., 1971, vol. 93, pp. 4323–4324. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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