Featured Article pubs.acs.org/joc
Synthesis and Biological Properties of C-2 Triazolylinosine Derivatives Mahesh K. Lakshman,*,† Amit Kumar,† Raghavan Balachandran,‡ Billy W. Day,‡,§ Graciela Andrei,∥ Robert Snoeck,∥ and Jan Balzarini∥ †
Department of Chemistry, The City College and The City University of New York, 160 Convent Avenue, New York, New York 10031, United States ‡ Department of Pharmaceutical Sciences, and §Department of Chemistry, University of Pittsburgh, Pennsylvania 15213, United States ∥ Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Minderbroedersstraat 10, B 3000 Leuven, Belgium S Supporting Information *
ABSTRACT: O6-(Benzotriazol-1H-yl)guanosine and its 2′-deoxy analogue are readily converted to the O6-allyl derivatives that upon diazotization with t-BuONO and TMS-N3 yield the C-2 azido derivatives. We have previously analyzed the solventdependent azide·tetrazole equilibrium of C-6 azidopurine nucleosides, and in contrast to these, the O6-allyl C-2 azido nucleosides appear to exist predominantly in the azido form, relatively independent of solvent polarity. In the presently described cases, the tetrazole appears to be very minor. Consistent with the presence of the azido functionality, each neat C-2 azide displayed a prominent IR band at 2126−2130 cm−1. A screen of conditions for the ligation of the azido nucleosides with alkynes showed that CuCl in t-BuOH/H2O is optimal, yielding C-2 1,2,3-triazolyl nucleosides in 70−82% yields. Removal of the silyl groups with Et3N·3HF followed by deallylation with PhSO2Na/Pd(PPh3)4 gave the C-2 triazolylinosine nucleosides. In a continued demonstration of the versatility of O6-(benzotriazol-1H-yl)purine nucleosides, one C-2 triazolylinosine derivative was converted to two adenosine analogues via these intermediates, under mild conditions. Products were desilylated for biological assays. The two C-2 triazolyl adenosine analogues demonstrated pronounced antiproliferative activity in human ovarian and colorectal carcinoma cell cultures. When evaluated for antiviral activity against a broad spectrum of DNA and RNA viruses, some of the C-2 triazolylinosine derivatives showed modest inhibitory activity against cytomegalovirus.
■
INTRODUCTION The Cu-catalyzed version1,2 of the classic Huisgen azide− alkyne cycloaddition3−9 is a highly atom-economical reaction, often requiring mild conditions. Both factors render Cucatalyzed azide−alkyne cycloaddition (CuAAC) highly attractive for the modification of complex and sensitive molecules such as nucleosides. We have recently reported a facile and general synthesis of C-6 azidopurine nucleosides and their use in CuAAC reactions, where some of these new compounds showed low cytostatic activity against ovarian carcinoma cell lines.10 In general, azide−alkyne cycloaddition reactions have been a highly important transformation in the chemistry of nucleosides and DNA.11−13 To our knowledge, our previous report on the CuAAC reactions of 6-azidopurine nucleosides was the first to describe such reactions at the C-6 position of purine nucleosides.10 On © XXXX American Chemical Society
the other hand, CuAAC chemistry involving C-2 azidopurine nucleosides has been reported in the context of adenosine A3 receptor research14 as well as in the development of antituberculosis agents,15 and both describe reactions of 2azidoadenosine analogues. In general, there are three methods for the synthesis of 2-azidoadenosine derivatives. The first involves treatment of 6-amino-2-hydrazino ribofuranosylpurine with nitrous acid,16 the second is a Cu-catalyzed azidation of triO-silyl 6-amino-2-iodo ribofuranosylpurine,17 and the third involves diazotization/azidation of tri-O-acetyl 2-amino-6chlororibofuranosylpurine with isoamylONO/azidotrimethylsilane (TMS-N3), followed by replacement of the chloride with an amino group.18 Received: March 26, 2012
A
dx.doi.org/10.1021/jo300628y | J. Org. Chem. XXXX, XXX, XXX−XXX
The Journal of Organic Chemistry
Featured Article
Scheme 1. Synthesis of Protected O6-Allyl-2-azidoinosine and O6-Allyl-2-azido-2′-deoxyinosine
Table 1. Optimization of Azide−alkyne Ligation Conditions Using Trisilyl O6-Allyl-2-azidoinosine 2a and Phenylacetylenea
entry 1 2 3 4
catalytic system 20 20 20 20
mol mol mol mol
% % % %
CuSO4/40 mol % Na ascorbate CuSO4/40 mol % Na ascorbate Cu(I) thiophene-2-carboxylate CuCl
solvent (1:1)
time (h)
% yield of 3b
CH2Cl2/H2O t-BuOH/H2O t-BuOH/H2O t-BuOH/H2O
24 36 48 36
30 (60% of 2a recovered) 54 68 82
a Conditions: 0.1 M 2a in the solvents indicated, room temperature (reactions were monitored for completion by TLC analysis). bYield of isolated and purified product.
■
For the current study, we were interested in the CuAAC reactions of 2-azidoinosine and its 2′-deoxy analogue. Syntheses of 2-azidoinosine derivatives are less well developed in comparison to the adenosine analogues. Reaction of 2chloroinosinic acid with NaN3 has been shown to yield 2azidoinosine-5′-monophosphate, which was subsequently converted to 2-azidoinosine by reaction with acid phosphatase.19 Similarly, 2-fluoro-2′-deoxyinosine, which requires relatively nontrivial synthesis,20 has been converted to the 2-azido derivative within the context of a DNA oligomer.21 For our purposes, we needed a simple, general access to 2-azidoinosine and 2-azido-2′-deoxyinosine. In this paper we report the synthesis of suitable O6-protected 2-azidoinosine derivatives and their applicability toward CuAAC reactions. We have evaluated the anticancer and antiviral properties of C-2 (1,2,3triazol-1H-yl)inosine and 2′-deoxyinosine analogues after appropriate deprotection protocols. We also report methodology for conversion of C-2 triazolylinosine derivatives to adenosine analogues via their O6-(benzotriazol-1H-yl) derivatives, as well as the development of doubly reactive 2-azido-O6(benzotriazol-1H-yl)purine ribonucleoside.
RESULTS AND DISCUSSION
Synthesis of C-2 Triazolylinosine and 2′-Deoxyinosine Derivatives. We have recently reported that O6-(benzotriazol1H-yl)inosine and -guanosine derivatives, as well as the corresponding 2′-deoxy analogues, are exceptionally effective reagents for the introduction of substituents at the C-6 position of these purine nucleosides.22−26 On the basis of these results, we reasoned that O6-allylguanosine and 2′-deoxyguanosine would be excellent precursors for the present work.25 We have previously developed the synthesis of 2-chloro-3′,5′-di-O-(tertbutyldimethylsilyl)-2′-deoxyinosine from an O6-allyl-2′-deoxyguanosine precursor,27 and as reported for 2-chloroinosinic acid,19 this can potentially be used to generate the 2azidoinosine derivative. Despite this, as shown in Scheme 1, it is more expeditious to directly install the azido group at the C-2 position by diazotization of the amino group in the presence of TMS-N3.18 Silyl-protected O6-allyl-2-azidoinosine 2a(A) and the 2′deoxyinosine analogue 2b(A) could be synthesized via this procedure in ca. 60% yield. C-2 azido derivatives of purines28,29 and purine nucleosides16,30−33 can exist in equilibrium with two possible tetrazolyl isomers. Similarly, 2a,b can exist as two B
dx.doi.org/10.1021/jo300628y | J. Org. Chem. XXXX, XXX, XXX−XXX
The Journal of Organic Chemistry
Featured Article
Table 2. Azide−alkyne Ligation Reactions of Nucleosides 2a and 2ba
a
Conditions: 0.5 M 2a or 2b in 1:1 t-BuOH/H2O, 20 mol % of CuCl, room temperature (reactions were monitored for completion by TLC analysis). bYields are of isolated and purified products.
tautomers termed 2a,b(T1) and 2a,b(T3), depending upon the nitrogen atom of the purine that is involved. Among the two azide derivatives, synthesis of 2b from the C-2 triflate has been reported in 26% yield, and this compound was reported not to display any tetrazole tautomer.34 In our case, assessment by 1H NMR indicated that both 2a and 2b exist predominantly as the azide 2a(A) and 2b(A) (≥90% in CDCl3, acetone-d6, and DMSO-d6). Consistent with this, IR spectra of 2a and 2b showed absorptions at 2126 and 2130 cm−1, respectively. By comparison, 2-azidoadenosine derivatives demonstrated 17− 55% of the tetrazolyl isomer.14,15,30,31,33 Apart from factors such as temperature and solvent polarity,31,33 the electronic nature of substituents also influences the azide/tetrazole ratio of purinyl derivatives.33 Whereas electron-donating substituents favor the ring-closed tetrazolyl form, the azide form is preferred with electron-withdrawing groups.33 The significantly greater proportion of the azido rather than the tetrazolyl forms of 2-
azidoinosine derivatives 2a,b as compared to the 2azidoadenosines is possibly linked to this substituent effect. Between the two tetrazolyl forms, the T1 form is generally invoked for nucleosides.14,15,32,33 With the synthesis of the C-2 azido derivatives 2a,b completed, conditions for effectuating their ligation reactions with alkynes were evaluated (Table 1). In previous work with 6azidopurine nucleosides, we had observed that reduction of the azide to the amine was a competing process and that biphasic conditions were essential in order to obtain satisfactory azide− alkyne ligation.10 Interestingly, application of those conditions here resulted in an unsatisfactory outcome (entry 1). Switching the solvent to 1:1 t-BuOH/H2O gave an improved result (entry 2). Cu(I) thiophene-2-carboxylate as the catalyst gave a modest improvement (entry 3), whereas CuCl proved to be the best under the conditions tested, affording a product yield of 82% (entry 4). Therefore, the generality of azide−alkyne ligation C
dx.doi.org/10.1021/jo300628y | J. Org. Chem. XXXX, XXX, XXX−XXX
The Journal of Organic Chemistry
Featured Article
Scheme 2. Deprotection of O6-Allyl C-2 Triazolylinosine and 2′-Deoxyinosine Analogues
Scheme 3. Synthesis of C-2 Triazolyladenosine Analogues via O6-Benzotriazolyl Derivatives
atom, we considered methodology for facile synthesis of C-2 triazolyladenosine analogues (derivatives of 2,6-diaminopurine nucleosides). For this purpose, our previously described methodology for activation of the amide linkage, via the O6(benzotriazol-1H-yl) derivative, appeared to be a reasonable approach.22,25,26 As shown in Scheme 3, deallylation of 3 followed by exposure of the resulting silyl-protected C-2 triazolylinosine derivative to 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and i-Pr2NEt in THF at room temperature led to the formation of the corresponding O6-(benzotriazolyl) derivative 31 in 55% yield. Reactions of 31 with morpholine and benzyl amine were conducted in 1,2-dimethoxyethane (DME) to yield the adenosine derivatives 32 and 33 in 77% and 90% yields, respectively. The products were then desilylated to yield the C2 triazolyl adenosine analogues 34 and 35. Synthesis of a Doubly Reactive Purine Nucleoside Derivative. The foregoing experiments clearly showed that azide−alkyne ligation could be effectively utilized to synthesize C-2 triazolyl nucleoside analogues. This in combination with chemistry leading to O6-(benzotriazol-1H-yl)purine nucleosides produces a powerful new approach to difunctionalization at C-2 and C-6 of the purine scaffold. At this point, we considered whether an appropriately functionalized, doubly reactive purine nucleoside derivative could be obtained.
chemistry was next investigated using both the ribose derivative 2a as well as the 2′-deoxy analogue 2b. Results from these experiments are summarized in Table 2. With a series of O6-protected C-2 triazolylinosine and 2′deoxyinosine derivatives available, deprotection of the products became the focus. Two possible sequences could be envisioned; either deprotection of the O6-allyl group followed by desilylation or vice versa. Desilylation by Et3N·HF followed by deallylation was the preferred order. This is because we have noted that nucleoside desilylations with amine·HF complexes can leave a fluoride contaminant in some instances, which can be difficult to eliminate. Hence, our chosen deprotection sequence offers the possibility to chromatographically purify the products of desilylation prior to deallylation. We also generally recommend checking products of desilylation by 19F NMR. Several conditions were tested for this purpose: Pd2(dba)3/ (±)-BINAP with morpholine in THF, Pd2(dba)3/(±)-BINAP with Et2NH2+HCO3−, in CH2Cl2, and Pd(PPh3)4 with sodium benzenesulfinate (PhSO2Na) in THF. The first two sets of conditions were not very successful, but use of Pd(PPh3)4/ PhSO2Na proved optimal. Scheme 2 shows the products prepared by deprotection. Synthesis of C-2 Triazolyladenosine Derivatives. Having synthesized a series of C-2 triazolylinosine and 2′deoxyinosine analogues, which can also be considered as modified guanine nucleosides by virtue of the C-2 nitrogen D
dx.doi.org/10.1021/jo300628y | J. Org. Chem. XXXX, XXX, XXX−XXX
The Journal of Organic Chemistry
Featured Article
Scheme 4. Synthesis of Difunctionalizable Purine Nucleoside Derivative
Therefore, the synthesis of a 2-azido-O6-(benzotriazol-1Hyl)purine nucleoside derivative was investigated. This would not only allow us to evaluate the stability of the benzotriazolyl moiety to the diazotization/azidation conditions but also provide a route to novel reactive nucleosides that can be difunctionalized at the C-2 and the C-6 positions of the purine. In this context, hydroxyl-protected O6-(benzotriazolyl)guanosine derivatives have been converted to the C-2 halo (F, Cl, and I) purine nucleosides.35 We chose to diazotize O6(benzotriazol-1H-yl)-2′,3,5′-tri-O-(tert-butyldimethylsilyl)guanosine (37)25 with t-BuONO/TMS-N3 (Scheme 4), along the lines of previously published methods.18,31 This reaction gave a 49% unoptimized yield of 38 indicating the general stability of the O6-(benzotriazol-1H-yl) group to the reaction conditions. The 1H and 13C NMR spectra of 38 in CDCl3 showed the presence of three isomers, presumably the azide and the two tetrazolyl forms (T1 and T3). For example, three singlets are observed in the 1H NMR spectrum (δ 8.63, 8.58, and 8.55 ppm) corresponding to the purinyl resonances. Although only two resonances are observed for H-1′ (δ 6.16 and 6.05 ppm), the HMQC spectrum clearly shows three C-1′ resonances (δ 89.61, 89.06, 88.99 ppm). The three H-2′ resonances at δ 4.58 (22%), 4.54 (42%), and 4.47 ppm (36%) ppm were used to determine the isomer ratio. At this time, no attempts have been made to assign structures to these isomers. The IR spectrum of 38 showed an absorption at 2128 cm−1. Doubly reactive compounds, such as 38, with two preinstalled reactive entities can potentially be functionalized at the C-2 and the C-6 by CuAAC and SNAr chemistry, respectively. Further work along these lines is forthcoming. Biological Activities of the New Compounds. The compounds were evaluated for their antiviral activity against a broad variety of DNA and RNA viruses. Several compounds (i.e., 18, 22, and 25, see Table 3) showed marginal activity against cytomegalovirus (CMV), whereas the anti-CMV activity of 23 was somewhat more pronounced. Indeed, the inosine derivative 23 showed activity against CMV in human embryonic lung (HEL) cells at an EC50 of 39−73 μM. None of the compounds showed antiviral activity against other viruses at subtoxic concentrations except the inosine derivative 17 that was endowed with moderate antivesicular stomatitis virus (VSV) activity (27 ± 2.4 μM) in human cervix carcinoma HeLa cell cultures. This activity could not be confirmed in human embryonic lung (HEL) fibroblast cell cultures against the same virus, making the moderate activity rather cell-type specific. Yet, in the HeLa and HEL cell cultures toxicity of 17 was observed at 100−240 μM. This may also mean that the anti-VSV activity noticed for 17 in the VSV/HeLa cell assay can be due to underlying toxicity to the host cells, rather than to a specific
Table 3. Anti-CMV Activity of the Test Compounds in HEL Cell Cultures anti-CMV activity EC50a (μM)
HEL cell effects (μM)
compd
AD-169 strain
Davis strain
cell morphology (MDC)b
cell growth (IC50)c
17 18 19 20 21 22 23 24 25 26 27 28 29 30 34 35
>50 118 ≥230 >270 ≥200 120 73 >230 ≥250 >240 >290 >210 ≥200 >270 >40 >40
>50 151 ≥230 >270 >200 123 39 >230 158 >240 >290 >210 ≥200 >270 >40 >40
240 >240 >230 >270 >200 ≥190 ≥260 >230 >250 >240 >290 >210 >200 >270 ≥40 200
ndd ≥240 >230 ndd >200 123 >250 ndd 188 ndd ndd ndd >200 ndd ndd ndd
a Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque-forming units (PFU). bMinimum cytotoxic concentration that caused a microscopically detectable alteration of cell morphology. cConcentration required to reduce cell growth by 50%. dNot determined.
antiviral activity of the compound. From the antiviral assay systems performed, compound 34 had the highest impact on mammalian cell morphology, but this highly depended on the nature of the cell line used as the virus host [minimum detectable morphology-altering (cytotoxic) concentration (MCC): 8.3 μM against canine kidney MDCK, 42 μM against HeLa, 83 μM against feline kidney CRFK, 210 μM against green monkey kidney Vero, and ≥40 μM against HeLa cells]. The inosine derivatives 17−24 and 2′-deoxyinosine derivatives 25−30 were also evaluated for their cytostatic activity against murine leukemia L1210, human lymphocyte CEM, and HeLa cells. Modest cytostatic activity was noticed for several compounds. In particular, the HeLa cells were usually somewhat more sensitive to the inhibitory potential of these compounds than the other cell lines. Also, the ribose derivatives were consistently more cytostatic than their corresponding 2′deoxyribose derivatives. Among all compounds tested, 17 proved most cytostatic, irrespective the nature of the tumor cell line (IC50: 34−124 μM). Both adenosine derivatives 34 and 35 were poorly cytostatic (IC50 for 34 98−185 μM, for 35 90−120 μM). E
dx.doi.org/10.1021/jo300628y | J. Org. Chem. XXXX, XXX, XXX−XXX
The Journal of Organic Chemistry
Featured Article
to the O6-(benzotriazol-1H-yl) derivatives. SNAr substitution of the benzotriazolyloxy group with amines then furnished C-2 triazolyl adenosine analogues. In addition, we have converted silyl-protected O6-(benzotriazol-1H-yl)guanosine to a doubly reactive 2-azido-O6-(benzotriazol-1H-yl)purine nucleoside derivative. Interestingly, based upon the NMR results, this compound appears to exist as the azide and two tetrazolyl isomers. This nucleoside derivative, which can react at the C-2 via CuAAC and at the C-6 via SNAr, should find broad utility in greatly diversifying the purine nucleoside scaffold via generally simple operational procedures.
All of the newly synthesized C-2 triazolyl nucleoside analogues were also tested for their antiproliferative activity using human ovarian cancer 1A9 cells and their paclitaxelresistant clones, 1A9-PTX10 and 1A9-PTX22, and colorectal carcinoma HCT116 cells and their clones (Table 4). Several Table 4. GI50 (μM) of the Test Compounds against Ovarian (1A9), Two Paclitaxel-Resistant (PTX10 and PTX22) Ovarian, Colorectal (HCT116), and p53KO HCT116 Cancer Cell Lines compd
1A9
PTX10
PTX22
HCT116
p53KO
17 18 19 20 21 22 23 24 25 26 27 28 29 30 34 35 paclitaxel (nM)
22.8 38.0 29.5 20.4 6.32 29.7 37.0 34.0 31.0 37.2 49.4 49.6 41.6 46.1 0.18 5.0 1.51
>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 11.73 >50 79
4.76 13.4 8.56 3.53 14.1 12.9 28.1 31.0 14.8 15.7 32.9 32.2 18.0 26.9 0.95 24.5 68
>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 3.7 43.61 6.85
>50 >50 >50 42.6 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 10.4 >50 8.58
■
EXPERIMENTAL SECTION
General Experimental Considerations. Thin-layer chromatography was performed on 250 μm silica plates, and column chromatographic purifications were performed on 200−300 mesh silica gel. CH2Cl2 was distilled over CaCl2, and THF and 1,2-DME were distilled over LiAlH4 and then over Na prior to use. All other reagents were obtained from commercial sources and were used as received. 1H NMR spectra were recorded at 500 MHz, in the solvents indicated, and are referenced to residual protonated solvent resonance. 13 C NMR data were recorded at 125 MHz in the solvents indicated and are referenced to the solvent resonance. In all cases, for HRMS analyses ESI ionization and a TOF analyzer were used. Although 1a and 1b are reported in the literature,25 larger scale syntheses are described below. O 6 -Allyl-2′,3′,5′-tri-O-(tert-butyldimethylsilyl)guanosine (1a).25 In a clean, dry 100 mL round-bottomed flask equipped with a stirring bar were placed O6-(benzotriazol-1H-yl)-2′,3′,5′-tri-O-(tertbutyldimethylsilyl)guanosine25 (5.0 g, 6.7 mmol), allyl alcohol (50 mL), and Cs2CO3 (4.74 g, 14.1 mmol). The reaction mixture was flushed with nitrogen gas and stirred at room temperature for 2 h after which the mixture was evaporated to dryness. Chromatographic purification of the crude material on a silica gel column using 20% EtOAc in hexanes afforded 3.60 g (81% yield) of 1a as a white foam. Rf (SiO2/20% EtOAc in hexanes) = 0.52. 1H NMR (CDCl3): δ 7.96 (s, 1H, Ar-H), 6.16−6.08 (m, 1H, CH), 5.92 (d, 1H, H-1′, J = 5.3 Hz), 5.41 (dd, 1H, CHtrans, J = 1.4, 17.2 Hz), 5.25 (dd, 1H, CHcis, J = 1.4, 10.2 Hz), 5.05 (s, 1H, NH2), 4.98 (d, 2H, OCH2, J = 5.7 Hz), 4.48 (t, 1H, H-2′, J = 4.6 Hz), 4.27 (t, 1H, H-3′, J = 3.4 Hz), 4.09 (app q, 1H, H-4′, Japp ≈ 3.2 Hz), 3.96 (dd, 1H, H-5′, J = 3.6, 11.4 Hz), 3.77 (dd, 1H, H-5′, J = 2.5, 11.4 Hz), 0.96, 0.95, and 0.82 (3s, 27H, t-Bu), 0.15, 0.14, 0.13, 0.12, −0.02, and −0.16 (6s, 18H, SiCH3). 13C NMR (CDCl3): δ 160.7, 159.1, 153.8, 137.7, 132.7, 118.0, 115.6, 87.5, 85.2, 76.2, 72.0, 67.2, 62.6, 26.0, 25.8, 25.6, 18.5, 18.0, 17.9, −4.3, −4.7, −5.0, −5.4. HRMS: calcd for C31H60N5O5Si3 [M + H]+ 666.3897, found 666.3909. O6-Allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine (1b).25 As described for the synthesis of 1a, this compound was prepared by a reaction of O6-(benzotriazol-1H-yl)-3′,5′-di-O-(tertbutyldimethylsilyl)-2′-deoxyguanosine25 (5.0 g, 8.16 mmol), allyl alcohol (50 mL), and Cs2CO3 (5.64 g, 17.1 mmol). Chromatographic purification of the crude material on a silica gel column using 30% EtOAc in hexanes) afforded 3.71 g (87% yield) of 1b as a white foam. Rf (SiO2/40% EtOAc in hexanes) = 0.60. 1H NMR (CDCl3): δ 7.93 (s, 1H, Ar-H), 6.34 (t, 1H, H-1′, J = 6.5 Hz), 6.17−6.09 (m, 1H, CH), 5.43 (dd, 1H, CHtrans, J = 1.4, 17.2 Hz), 5.27 (dd, 1H, CHcis, J = 1.4, 10.4 Hz), 5.01 (d, 2H, OCH2, J = 5.7 Hz), 4.94 (s, 1H, NH2), 4.66−4.58 (m, 1H, H-3′), 3.99 (app q, 1H, H-4′, Japp ≈ 3.5 Hz), 3.83 (dd, 1H, H-5′, J = 4.4, 11.2 Hz), 3.77 (dd, 1H, H-5′, J = 3.4, 11.2 Hz), 2.57 (app quint, 1H, H-2′, Japp ≈ 6.5 Hz), 2.37 (ddd, 1H, H-2′, J = 4.0, 6.0, 13.0 Hz), 0.92 (s, 18H, t-Bu), 0.11 and 0.09 (2s, 12H, SiCH3). 13 C NMR (CDCl3): δ 160.0, 159.2, 153.5, 137.8, 132.8, 118.4, 116.0, 87.8, 88.8, 72.1, 67.5, 63.0, 41.1, 26.1, 25.9, 18.6, 18.1, −4.4, −4.6, −5.2, −5.3. HRMS: calcd for C25H46N5O4Si2 [M + H]+ 536.3083, found 536.3093. O 6 -Allyl-2-azido-2′,3′,5′-tri-O-(tert-butyldimethylsilyl)inosine (2a). To a solution of 1a (3.0 g, 4.5 mmol) in dry CH2Cl2 (40
compounds were modestly active against 1A9 and 1A9-PTX22, but all were inactive against 1A9-PTX10. Adenosine derivatives 34 and 35 showed the best activity against 1A9, and among the two, 34 bearing a morpholinyl group at C-6 was most active (0.18 μM). Inosine derivative 21 with a phthalimido triazolyl substituent also showed activity at
