CHEMMEDCHEM FULL PAPERS DOI: 10.1002/cmdc.201402179

Ribonuclease A Inhibition by CarboxymethylsulfonylModified Xylo- and Arabinopyrimidines Dhrubajyoti Datta, Swagata Dasgupta,* and Tanmaya Pathak*[a] A group of acidic nucleosides were synthesized to develop a new class of ribonuclease A (RNase A) inhibitors. Our recent study on carboxymethylsulfonyl-modified nucleosides revealed some interesting results in RNase A inhibition. This positive outcome triggered an investigation of the role played by secondary sugar hydroxy groups in inhibiting RNase A activity. Uri-

dines and cytidines modified with SO2CH2COOH groups at the 2’- and 3’-positions show good inhibitory properties with low inhibition constant (Ki) values in the range of 109–17 mm. The present work resulted in a set of inhibitors that undergo more effective interactions with the RNase A active site, as visualized by docking studies.

Introduction Interest in ribonuclease inhibition has increased over the years, with the aim to diminish the harmful biological activities of a few members of the ribonuclease superfamily such as angiogenin,[1] eosinophil-derived neurotoxin (EDN),[2] and bovine seminal RNase.[3] All these enzymes bear active site homology with ribonuclease A (RNase A).[4–6] The biological activities of these proteins, which are detrimental to cells, are entirely dependent on their ribonuclease activities.[7a–e] Bovine pancreatic RNase A[8, 9] is often used as a model system to understand structure–function relationships of other members of this superfamily. The ribonuclease catalytic center of RNase A is composed of multiple subsites that bind the phosphate, base, and sugar components of RNA.[8, 10] Among them, the P1 subsite, consisting of His12, Lys41, and His119, is involved in phosphodiester bond cleavage of the substrate (Figure 1). The B1 and B2 sites recognize the nucleobases; the B1 site imparts pyrimidine specificity and B2 site has affinity for purines.[11] All members of the ribonuclease superfamily are active against singlestranded RNA substrates and catalyze RNA breakdown with different degrees of ligand recognition.[12, 13] Despite having obtained notable results for allosteric inhibitors of RNase A,[14] the search for reversible competitive RNase A inhibitors has always attracted chemists and enzymologists because it is easier to carry out structure–activity relationship studies of such compounds. Most of the reported potent and mechanism-based inhibitors of RNase A are phosphate- or pyrophosphate-containing nucleotides.[15–19] However, the utility of nucleotides as inhibitors is limited due to various reasons. First, there is difficulty in the transport of these [a] D. Datta, Prof. S. Dasgupta, Prof. T. Pathak Department of Chemistry Indian Institute of Technology Kharagpur, Kharagpur (India) Fax: (+ 91) 3222-255303 E-mail: [email protected] [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201402179.

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compounds through biological membranes given their polyanionic nature.[20] The negatively charged phosphate group makes them difficult to use in vivo.[19b, 20–23] Moreover, the phosphate group(s) are subject to hydrolysis by various enzymes present in the body, namely Ap3A hydrolase, Ap4A hydrolase, and diphosphoinositol polyphosphate phosphohydrolases (DIPP proteins). Apart from these drawbacks, there is the potential for toxicity through the accumulation of degradation products of those inhibitors, which can disrupt essential biological pathways.[21] Earlier reports revealed that the pKa values of histidine residues present at the RNase A active site P1 (His12 and His119)

Figure 1. Schematic representation of the RNase A active site, where Bn, Rn, and Pn are nucleobase-, ribose-, and phosphate-binding subsites, respectively, and are labeled with their constituent residues.

change from ~ 5.22/6.78 for the free enzyme to ~ 6.30/8.10 for the enzyme in complex with substrate.[24] Inhibitors that can disrupt this equilibrium would be able to decrease enzymatic activity. It is important to note that an overwhelming majority of small-molecule RNase A inhibitors are equipped with acidic ChemMedChem 2014, 9, 2138 – 2149

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CHEMMEDCHEM FULL PAPERS groups such as phosphate, carboxylate, or sulfonic acid moieties.[25, 26] At physiological pH these acidic groups are expected to be present in the deprotonated form and would therefore interact electrostatically with the protonated basic residues of the P1 site.[27] A large number of modified nucleoside inhibitors containing carboxylic acid groups were reported by our research group in which the COOH function is attached either with an amide backbone[28, 29] or with cyclic alkyl amino groups[30–32] of pyrimidine nucleosides. Modified dinucleosides, in which the phosphate group is replaced with sulfonamides[33] or amide groups,[34] were reported as inhibitors of RNase A. Triazole-bridged polyphenols[35] and triazole pyrimidine nucleosides have also emerged as RNase A inhibitors.[36] We recently documented that an increase in inhibitory efficiency is directly proportional to the increasing acidity of the carboxylic groups of -XCH2COOH (X = O, NH, S, SO2) attached to the nucleosides.[37, 38] Earlier, we screened a series of 3’-amino-substituted arabinonucleosides 1 a–e to monitor the effects of pKa and non-phosphate groups on RNase A inhibition (Figure 2).[30a] We observed that the isonipecotyl-modified compound 1 e was the most effective inhibitor (Ki = 103 mm), and the corresponding ester 1 d, with a comparatively higher Ki value, was a weaker inhibitor. On the other hand, other aminonucleosides 1 a–c were found to be weak inhibitors of RNase A. We therefore concluded that an acidic functionality at the 3’-position of arabinouridines contributed significantly in RNase A inhibition. The P1 and B1 sites of RNase A were recognized by the 3’-isonipecotic acid group of two units of 1 e which was evident from the enzyme–inhibitor co-crystal structure (PDB ID: 2G8R, 1.7  resolution).[30b] We therefore turned our attention to a group of arabinonucleosides modified with acyclic linkers carrying a carboxylic

www.chemmedchem.org on the inhibitory properties requires a thorough study. Notably, the 2’-hydroxy group of the sugar ring plays an important role in the hydrolysis of RNA by RNase A in a two-step process involving transphosphorylation and hydrolysis.[8] Again, the substrates of RNase A in its bound state with the enzyme interact favorably with the B1 subsite through the cytosine nucleobase of RNA. Therefore, we planned to synthesize 2’- and 3’carboxymethylthio-/sulfonyl-modified uridines as well as carboxymethylsulfonylcytidines. We expected the cytidine analogues to show better inhibitory properties than the uridinemodified nucleosides on the basis of nucleobase recognition.

Results and Discussion Chemistry To synthesize carboxymethylthio- and carboxymethylsulfonylmodified uridines, tritylated b-epoxide 2[39] of uridine was selected as the starting material to afford xylo and arabino analogues. Nucleophilic attack and hence epoxide ring opening by methylthioglycolate afforded xylo analogue 3 and arabino analogue 4 as minor (30 %) and major (52 %) products, respectively, in good overall yield (Scheme 1). The minor xylo isomer 3 was converted into the corresponding thioester 5 by acidic hydrolysis with trifluroacetic acid (TFA) in dichloromethane. Consecutive hydrolysis with lithium

Scheme 1. Synthesis of xylo- and arabinouridine compounds 3 and 4. Reagents and conditions: a) HSCH2CO2CH3, NaH, DMF, RT, 4 h.

Figure 2. 3’-Amino-modified arabinonucleosides 1 a–e[30a] as moderate RNase A inhibitors.

group. Depending on the nature of the linker as mentioned above,[37, 38] these nucleosides would be expected bind to the active site of RNase A. Based on our experience, we hypothesized that a combination of the acidity of the carboxylic function and the hydrogen bonding capacity of the sulfonyl oxygen atoms of SO2CH2COOH should make it the appropriate choice for attachment to nucleosides. However, the combined effects of the positions of such groups on the sugar ring (2’ or 3’) and the configurations of the hydroxy group (3’ or 2’)  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

hydroxide in tetrahydrofuran (THF) followed by TFA treatment yielded the thio acid 6 (xylo-U2’SCH2COOH) from compound 3 in moderate yield (Scheme 2). The 5’-O-tritylsulfone ester 7 was obtained in good yield from compound 3 after its oxidation with magnesium bis(monoperoxyphthalate)hexahydrate (MMPP) in methanol. Compound 7 was converted into the deprotected sulfone ester 8 by acidic hydrolysis and also to the corresponding acid 9 (xylo-U2’SO2CH2COOH) by concomitant mild base hydrolysis[40] followed by acidic hydrolysis in moderate yields (Scheme 2). The major isomer 4 was then hydrolyzed under acidic conditions to obtain the thioester 10. Again, consecutive hydrolysis of 4 by lithium hydroxide followed by TFA treatment led to the thio acid 11 (ara-U3’SCH2COOH) in moderate yield. Compound 4 was then oxidized by MMPP to the corresponding 5’O-tritylated sulfone ester 12 in good yield. After cleavage of the acid-labile trityl group, compound 12 afforded sulfone ester 13. The sulfone acid 14 (ara-U3’SO2CH2COOH) was obtained after base hydrolysis followed by acid hydrolysis of 12 in moderate yield (Scheme 3). ChemMedChem 2014, 9, 2138 – 2149

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Scheme 2. Synthesis of xylo-carboxymethylthio (xylo-U2’SCH2COOH) and xylo-carboxymethylsulfonyl (xylo-U2’SO2CH2COOH) uridines 6 and 9, respectively. Reagents and conditions: a) TFA (20 % in CH2Cl2), RT, 0.5 h, 72 %; b) MMPP, CH3OH, RT, 6 h, 80 %; c) TFA (20 % in CH2Cl2), RT, 0.5 h, 72 %; d) 1. LiOH·H2O, THF, RT, 0.5 h, 2. TFA (20 % in CH2Cl2), RT, 0.25 h, 73 % (two steps); e) 1. (CH3)3SnOH, DCE, 70 8C, 4 h, 2. TFA (20 % in CH2Cl2), RT, 0.25 h, 75 % (two steps).

After synthesizing the uridine-modified inhibitors, a synthetic route was sought for the corresponding carboxymethylsulfonyl-modified cytidine analogues. Initially, an attempt was made to convert the sulfone analogues of uridine into the corresponding cytidine analogues. For this purpose, the free hydroxy group of arabino compound 12 was acylated by acetic anhydride to afford fully protected compound 15. This step is essential for the attachment of the 1H-1,2,4-triazole ring to the

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Scheme 4. Attempted synthesis of compound 17. Reagents and conditions: a) Ac2O, py, 0 8C!RT, 8 h, 78 %; b) 1,2,4-triazole, POCl3, Et3N, CH2Cl2, 0 8C!RT, 3 h, 79 %; c) NH3 in THF, RT.

C4 position of uridine. Compound 15 was then converted into the C4 triazolyl derivative with 1H-1,2,4-triazole in presence of POCl3[41] to obtain compound 16. However, the ammonolysis[41] of 16 did not afford the required nucleoside 17 (Scheme 4). As an alternative pathway, epoxide ring opening by methylthioglycolate, similar to that shown in Scheme 1, was followed. Due to the presence of a free amine at C4 of tritylated cytidine b-epoxide 19,[39c] it is difficult to carry out subsequent synthetic steps and to purify the compounds. Hence, di-tert-butoxycarbonyl-protected cytidine epoxide 20 was synthesized from compound 2 in three steps, which first involved triazolylation of 2 to afford 18. Upon ammonolysis, compound 18 produced the free NH2 group to yield the cytidine analogue of 2; protection of this polar NH2 group by tert-butoxycarbonyl anhydride (Boc2O) yielded compound 20. Compound 20 was treated with methylthioglycolate to obtain two regioisomers, 21 and 22, in good yields (Scheme 5) with the loss of one Boc group. After separation of the mixture of xylo and arabino isomers, xylo compound 21 was oxidized to 23 by moist MMPP in dichloromethane.[42] Protected sulfone ester 23, after consecutive basic and acidic hydrolysis, afforded acid 24 in moderate yield. Similarly, compound 22 was also oxidized to the corresponding sulfone 25, which was then hydrolyzed under conditions identical to those applied for the synthesis of 24 to afford arabino analogue 26 in moderate yield (Scheme 5).

Biophysical assays Agarose gel assays

Scheme 3. Synthesis of arabino-carboxymethylthio (ara-U3’SCH2COOH) and arabino-carboxymethylsulfonyl (ara-U3’SO2CH2COOH) uridines 11 and 14, respectively. Reagents and conditions: a) TFA (20 % in CH2Cl2), RT, 3 h, 68 %; b) MMPP, CH3OH, RT, 6 h, 84 %; c) TFA (20 % in CH2Cl2), RT, 3 h, 65 %; d) 1. LiOH·H2O, THF, RT, 2 h, 2. TFA (20 % in CH2Cl2), RT, 0.25 h, 65 % (two steps); e) 1. LiOH·H2O, THF, RT, 0.5 h, 2. TFA (20 % in CH2Cl2), RT, 0.25 h, 78 % (two steps).

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To get a qualitative assessment of the inhibitory properties of synthesized compounds 6, 9, 11, 14, 24, and 26 against RNase A, agarose gel assays were performed. In these experiments, the band in lanes 1 showed maximum intensity due to the presence of tRNA only. The band in lanes 2 was least intense due to degradation of tRNA by RNase A. Lanes 3, 4, 5, and 6 of each gel contained tRNA and RNase A with increasing ChemMedChem 2014, 9, 2138 – 2149

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www.chemmedchem.org pared. The activity of RNase A was decreased by the modified nucleosides by up to 37 %. Among these synthetic nucleosides, carboxymethylthio-modified nucleoside 6 (xylo-U2’SCH2COOH) showed the weakest inhibitory potency (16 % decreased activity), whereas compound 26 (ara-C3’SO2CH2COOH) exhibited the best result with 37 % inhibition of ribonuclease activity. Cytidine derivatives turned out to be better than the corresponding uridines. Again, 2’modified nucleosides exhibited lower efficacy than corresponding 3’-modified analogues (Table 1). Thus, compound 14 (ara-U3’SO2CH2COOH), which decreased ribonuclease activity by 33 % in these assays, is a better inhibitor than the xylo compound 9 (xylo-U2’SO2CH2COOH). Similarly, modified nucleosides 11 (ara-U3’SCH2COOH) and 26 (araC3’SO2CH2COOH) show better inhibitory properties than 6 (xylo-U2’SCH2COOH) and 24 (xyloC2’SO2CH2COOH), respectively (Figure 5). Kinetic studies

Scheme 5. Synthesis of xylo-C2’SO2CH2COOH 24 and ara-C3’SO2CH2COOH 26, respectively. Reagents and conditions: a) 1,2,4-triazole, POCl3, Et3N, CH2Cl2, 0 8C!RT, 3 h, 74 %; b) NH3, THF, RT, 4 h; c) (Boc)2O, DMAP, THF, RT, 2 h; d) HSCH2CO2CH3, NaH, DMF, RT, 4 h; e) moist MMPP, CH2Cl2, RT, 3 h; f) 1. LiOH·H2O, THF, RT, 2 h, 2. TFA (20 % in CH2Cl2), RT, 0.25 h, two steps.

concentrations of synthesized compounds (Figures 3 and 4). A significant difference in intensity between lanes 2 and 3 was observed for all compounds, and these differences gradually increased from lanes 3 to 6. From this qualitative study, compound carboxymethylthionucleosides 6 (xylo-U2’SCH2COOH) and 11 (ara-U3’SCH2COOH) were identified as weaker inhibitors than the carboxymethylsulfonyl-modified nucleosides 9, 14, 24, and 26 (Figure 3). Among the carboxymethylsulfonylnucleosides, arabino analogues 14 (ara-U3’SO2CH2COOH) and 26 (ara-C3’SO2CH2COOH) were found to be superior to the xylo-configured derivatives 9 (xylo-U2’SO2CH2COOH) and 24 (xylo-C2’SO2CH2COOH) (Figure 4). Precipitation assays were performed with compounds 6, 9, 11, 14, 24, and 26 against RNase A to obtain quantitative data. The decrease in ribonuclease activity by these compounds for RNase A (1.30 mm) at a fixed concentration (0.34 mm) was com-

Steady-state kinetic experiments were performed to determine the mode of inhibition and inhibition constants (Ki) of 6, 9, 11, 14, 24 and 26. Ki values were determined from the resulting Lineweaver–Burk plots (Supporting Information). From these, it was concluded that the carboxymethylsulfonyl-modified nucleosides were more potent than the thio analogues. Nucleoside 26 (ara-C3’SO2CH2COOH) exhibited the

Figure 4. Agarose gel for the inhibition of RNase A (2.0 mm): tRNA (lane 1), tRNA + RNase A (lane 2), and tRNA + RNase A + compounds 9, 14, 24, or 26 at 0.05, 0.10, 0.15, and 0.20 mm (lanes 3–6, respectively).

Table 1. Decreased ribonuclease activity by RNase A inhibitors. Inhibitor

Figure 3. Agarose gel for the inhibition of RNase A (2.0 mm): tRNA (lane 1), tRNA + RNase A (lane 2), and tRNA + RNase A + compounds 6 or 11 at 0.05, 0.10, 0.15, and 0.20 mm (lanes 3–6, respectively).

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Decreased ribonuclease activity [%] 16  1 21  3 20  2 33  4 25  3 37  2

6 (xylo-U2’SCH2COOH) 9 (xylo-U2’SO2CH2COOH) 11 (ara-U3’SCH2COOH) 14 (ara-U3’SO2CH2COOH) 24 (xylo-C2’SO2CH2COOH) 26 (ara-C3’SO2CH2COOH) Data are the mean  SD (n = 4).

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www.chemmedchem.org compound 3’-CMP, partition coefficients of the two most effective inhibitors 14 and 26 were measured in an octanol–water system. The data were compared (Table 3) with those of 3’-cytidine monophospate (3’-CMP), one of the more potent ribonuclease inhibitors.[43] The experimental data also compared well with the computationally generated partition coefficient values. The results indicated better lipophilicity of these modified nucleosides over that of 3’-CMP.

Docking studies Figure 5. Reduction of ribonuclease activity of RNase A (1.30 mm) in the presence of inhibitors 6, 9, 11, 14, 24, and 26 (all at 0.34 mm) by precipitation assay.

lowest Ki value (Table 2). A representative Lineweaver–Burk plot depicts the kinetics of RNase A inhibition by compound 26 (Figure 6). Among these inhibitors, carboxymethylsulfonylfunctionalized arabino nucleosides 14 (ara-U3’SO2CH2COOH) and 26 (ara-C3’SO2CH2COOH) were the most effective against RNase A, as was indicated in preliminary studies.

Table 2. Inhibition constants of RNase A inhibitors. Inhibitor

Ki [mm]

6 (xylo-U2’SCH2COOH) 9 (xylo-U2’SO2CH2COOH) 11 (ara-U3’SCH2COOH) 14 (ara-U3’SO2CH2COOH) 24 (xylo-C2’SO2CH2COOH) 26 (ara-C3’SO2CH2COOH)

109  6 43  3 50  3 19  2 33  4 17  2

Data are the mean  SD (n = 4).

Measurement of partition coefficient To support the argument that this new class of modified nucleosides would have better permeability than the reference

Figure 6. Lineweaver–Burk plots for the inhibition of RNase A (10.5 mm) by 26 (ara-C3’SO2CH2COOH) at 9.00 (~), 4.50 (&), and 0.00 (^) mm and substrate 2’,3’-cCMP concentrations in the range 0.63–0.43 mm.

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Docking experiments were performed to identify the probable binding patterns of the inhibitors with RNase A. In this study thio- and sulfone-acid-modified compounds 6, 9, 11, 14, 24, and 26 were docked with RNase A (PDB ID: 1FS3). In general, the number of hydrogen bonding interactions for carboxymethylthio derivatives was lower than for carboxymethylsulfonylmodified inhibitors owing to the absence of two oxygen

Table 3. Comparison of partition coefficient (log P) values obtained experimentally and by computational methods. Inhibitor

SFlog Poctanol/water[a]

QPlog Poctanol/water[b]

Alog Poctanol/water[b]

3’-CMP 14 26

2.11  0.12 1.39  0.10 1.67  0.14

2.04  0.08 1.59  0.10 1.79  0.07

2.00  0.06 1.45  0.05 1.69  0.06

[a] Experimentally obtained values. [b] Computationally generated values (see Experimental Section). Data are the mean  SD (n = 4).

atoms in the former group of nucleosides. The carboxylic group of 6 (xylo-U2’SCH2COOH) interacts with His119 of the P1 site, but the nucleobase is inclined toward Lys41 instead of B1, which is a pyrimidine-base recognition site (Figure 7 A). Although two hydrogen bonding interactions are observed for 11 (ara-U3’SCH2COOH) in B1 site recognition, the total number of interactions remained only six (Figure 7 C and Table I in the Supporting Information). In the docked pose, the carboxylic groups of the carboxymethylsulfonyl-modified arabinonucleosides 14 (ara-U3’SO2CH2COOH) and 26 (ara-C3’SO2CH2COOH) form hydrogen bonds with the P1 subsite residues (Figure 7 D and 8 B) consisting of His12, Lys41, and His119 (highlighted in green); simultaneously, nucleobases were found to occupy the B1 subsite consisting of Val43, Asn44, and Thr45 (in cyan). For the analogous xylo compounds 9 (xylo-U2’SO2CH2COOH) and 24 (xylo-C2’SO2CH2COOH) nucleobases of inhibitors shifted away from the B1 subsite (Figures 7 B and 8 A), and the acidic groups also interacted poorly with the active site P1 and B1. The overall number of hydrogen bonding interactions was fewer (7 and 8, respectively, for compound 9 and 24) than for 14 and 26. From the hydrogen bonding distances obtained (Tables I and II, Supporting Information), it was observed that the better binding in sulfone-acid-modified nucleosides is attributed to the oxygen atoms of the sulfone group. Inhibitor 26 (araC3’SO2CH2COOH) exhibits the highest number of hydrogen bonding interactions (14 overall) with active site P1 and other ChemMedChem 2014, 9, 2138 – 2149

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www.chemmedchem.org Energetic analysis by PEARLS After docking studies, experimentally obtained inhibition constant (Ki) values were subjected to a correlational study with docking parameters by the help of PEARLS.[44] In this analysis, protein–ligand merged PDB-format files were selected for all nucleosides, viz. 6, 9, 11, 14, 24, and 26, to calculate the total energy of ligand–receptor interactions. The calculated protein–ligand interaction values were obtained from the automated program PEARLS. The total interaction energy data thus obtained were tabulated (Table 4), and a correlation graph was plotted against inhibition constants (Figure 9). A linear relationship was achieved from these theoretically and experimentally obtained data sets.

Figure 7. Docked poses of A) compound 6 (xylo-U2’SCH2COOH), B) compound 9 (xylo-U2’SO2CH2COOH), C) compound 11 (ara-U3’SCH2COOH), and D) compound 14 (ara-U3’SO2CH2COOH) with RNase A (PDB ID: 1FS3).

Conclusions

A set of xylo- and arabino-configured pyrimidine nucleosides were synthesized to evaluate the combined role of the secondary hydroxy groups and acidic functionalities at the 2’- and 3’-positions of the sugar ring. After qualitative and quantitative bioassays as well as docking studies, we concluded that several factors such as acidic group pKa values, orientation of nucleobases and hydroxy groups, and the additional (or lack of) hydrogen bonding capacities of the sulFigure 8. Docked poses of A) compound 24 (xylo-C2’SO2CH2COOH) and B) compound 26 (ara-C3’SO2CH2COOH) fone oxygen atoms contribute with RNase A (PDB ID: 1FS3). to the overall inhibitory properties of these synthetic nucleosubsites of RNase A (Table II in the Supporting Information). sides. In this series, the cytidine analogue 1-[3’-deoxy-3’-S-(carCorresponding uridine analogue 14 (ara-U3’SO2CH2COOH) also boxymethyl)sulfonyl-b-d-arabinofuranosyl]cytosine 26 exhibitshows similar interaction patterns (13 hydrogen bonding interTable 4. Theoretical total ligand–receptor interaction energies (Eint) and actions overall; Supporting Information Table I). 2’-Hydroxy experimentally obtained inhibition constants for correlation. groups of arabinonucleosides interacted with His119 through hydrogen bonding for effective recognition with the active site Eint [kcal mol1] Inhibitor/Ligand Ki [mm] P1 (Figures IV, V, and VII in the Supporting Information). Thus, 6 (xylo-U2’SCH2COOH) 109 4.70 the analysis of docked poses of inhibitor–enzyme interactions 43 9.01 9 (xylo-U2’SO2CH2COOH) substantiates the experimentally obtained qualitative and 50 8.05 11 (ara-U3’SCH2COOH) 19 12.11 14 (ara-U3’SO2CH2COOH) quantitative biophysical data. 24 (xylo-C2’SO2CH2COOH) 26 (ara-C3’SO2CH2COOH)

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33 17

10.50 13.12

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www.chemmedchem.org by H3PO4 and CH3OH). Applied gradient was 68 % buffer in CH3OH over 40 min. The observed purities of these two compounds were more than 95 % (Supporting Information).

Chemistry

Figure 9. Correlation of ligand–receptor interaction energy and inhibition constant values.

ed the lowest Ki value of 17  2 mm. Partition coefficient measurements also indicated the better lipophilicity of compounds 14 and 26 than a standard nucleotide. It should be noted that the efficiency of the arabino derivatives 14 and 26 as inhibitors is in line with the results obtained with ara-UMP.[45] From the docking studies it was evident that the 3’-modified nucleosides or arabinonucleosides interact favorably with enzyme active sites via the nucleobase, the carboxymethylsulfonyl group, as well as the 2’-hydroxy group, whereas the 2’-substituted analogues or the xylonucleosides furnished fewer such interactions. Such lack of interaction with the residues of RNase A for xylo-configured inhibitors is the most possible reason behind their lower inhibitory properties. Extensive docking studies, supported by the PEARLS software package, supported the experimentally obtained results.

Experimental Section Reagents were purchased from Sigma–Aldrich and SRL India. Column chromatographic separations were done using silica gel (60–120 and 230–400 mesh). Solvents were dried and distilled following standard procedures. TLC was carried out on pre-coated plates (Merck silica gel 60 F254), and spots were visualized with UV light or by charring the plates dipped in 5 % H2SO4 in CH3OH and 5 % vanillin in CH3OH solutions. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker NMR spectrometer unless stated otherwise. Chemical shifts (d) are reported in parts per million (ppm). Mass spectrometry data were obtained on a Xevo G2QTof mass spectrometer in ESI + mode. Melting points were determined in open-end capillary tubes and are uncorrected. Bovine pancreatic RNase A, yeast tRNA, 2’,3’-cCMP, 3’-CMP, and human serum albumin (HSA) were purchased commercially. UV/Vis measurements were made using a UV/Vis spectrophotometer (Model Lambda 25). Concentrations of the solutions were estimated spectrophotometrically using the following data: e278.5 = 9800 m1 cm1 (RNase A)[46] and e268 = 8500 m1 cm1 (2’,3’-cCMP).[43] The purity of the two most active RNase A inhibitors 14 and 26 in this series was analyzed on an Enable C18H reversed-phase column (15 cm  4.0 mm) at a flow rate of 1.0 mL min1, l = 254 nm. The mobile phase was a mixture of a buffer consisting of Na2HPO4, NaH2PO4, tetrabutylammonium sulfate, pH 3.0 (adjusted  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

1-[5’-O-Trityl-2’-deoxy-2’-S-(methoxycarbonylmethyl)thio-b-d-xylofuranosyl]uracil 3 and 1-[5’-O-trityl-3’-deoxy-3’-S-(methoxycarbonylmethyl)thio-b-d-arabinofuranosyl]uracil 4: To a suspension of NaH (60 % in dispersed in mineral oil, 0.19 g, 8 mmol) in DMF (5 mL), methyl 2-thioacetate (1.45 mL, 16 mmol) was added slowly at 0 8C under argon. The suspension was allowed to warm at room temperature while the solution turned yellow. The reaction mixture was cooled to 0 8C and epoxide 2[39] (1.5 g, 3.2 mmol) in DMF (5 mL) was added. After 8 h, satd aq NH4Cl solution (50 mL) was added to the reaction mixture, and the solution was partitioned and washed with EtOAc (2  50 mL). Combined organic layers were washed with brine, separated, dried over anhyd Na2SO4 and filtered. The filtrate was evaporated under reduced pressure to afford a mixture of compounds 3 and 4. The mixture was separated and purified over silica gel column to afford compound 3 (eluent: 30–45 % EtOAc in PE) as minor isomer (0.55 g, 30 %). White solid, mp: 76–82 8C; 1H NMR (CDCl3, 25 8C, TMS): d = 3.43–3.51 (m, 2 H), 3.65–3.84 (m, 7 H), 4.00 (bs, 1 H), 4.24 (bs, 1 H), 4.41–4.43 (m, 1 H), 5.46 (d, J = 8.0 Hz, 1 H), 6.06 (s, 1 H), 7.24–7.33 (m, 9 H), 7.43– 7.51 (m, 6 H), 7.67 (d, J = 8.0 Hz, 1 H), 9.89 ppm (s, 1 H); 13C NMR, 50 MHz (CDCl3, 25 8C, TMS): d = 32.6 (CH2), 53.0, 56.2, 62.3 (CH2), 75.6, 83.6, 87.4, 91.6, 100.9, 127.4, 128.1, 128.8, 141.4, 143.6 (C), 150.3 (C), 164.5 (C), 171.2 ppm (C); HRMS (ESI + ), m/z calcd for [M + Na] + C31H30N2O7SNa: 597.1671, found: 595.1686. Compound 4 was obtained as the major isomer (0.96 g, 52 %) (eluent: 50–60 % EtOAc in PE). White solid, mp: 60–64 8C; 1H NMR (CDCl3, 25 8C, TMS): d = 3.37 (d, J = 15.2 Hz, 1 H), 3.54–3.59 (m, 3 H), 3.64–3.68 (m, 4 H), 3.84–3.88 (m, 1 H), 4.61–4.65 (m, 1 H), 4.87–4.88 (m, 1 H), 5.31 (d, J = 8.0 Hz, 1 H), 6.15 (d, J = 5.6 Hz, 1 H), 7.25–7.35 (m, 9 H), 7.44–7.46 (m, 6 H), 8.06 (d, J = 8.0 Hz, 1 H), 10.00 ppm (s, 1 H); 13C NMR, 50 MHz (CDCl3, 25 8C, TMS): d = 32.8 (CH2), 47.6, 52.8, 61.9 (CH2), 78.3, 80.8, 85.4, 87.6, 101.5, 127.5, 128.2, 128.9, 142.1, 143.4 (C), 151.3 (C), 164.6 (C), 170.9 ppm (C); HRMS (ESI + ), m/z calcd for [M + Na] + C31H30N2O7SNa: 597.1671, found: 595.1652. 1-[2’-Deoxy-2’-S-(methoxycarbonylmethyl)thio-b-d-xylofuranosyl]uracil 5: Compound 3 (0.2 g, 0.35 mmol) was stirred with TFA in CH2Cl2 (20 %, 15 mL) at room temperature. After 0.5 h, the volatile components were evaporated to dryness under reduced pressure and residual liquid was co-evaporated with toluene (2  5 mL). The residue thus obtained was dissolved in CH3OH (2 mL), loaded onto silica gel column and purified to obtain compound 5 (0.085 g, 72 %) (eluent: 5–20 % CH3OH in CHCl3). Hygroscopic solid; 1H NMR ([D6]DMSO, 25 8C): d = 3.39 (bs, 1 H), 3.56–3.74 (m, 7 H), 3.99–4.02 (m, 5 H), 4.07–4.08 (m, 1 H), 4.75–4.78 (m, 1 H), 5.66–5.69 (m, 1 H), 5.78 (d, J = 3.6 Hz, 1 H), 5.86 (d, J = 2.8 Hz, 1 H), 7.77 (d, J = 8.4 Hz, 1 H), 11.33 ppm (s, 1 H); 13C NMR ([D6]DMSO, 25 8C): d = 33.0 (CH2), 52.9, 56.2, 59.8 (CH2), 74.6, 84.5, 89.7, 102.3, 141.3, 151.1 (C), 163.8 (C), 170.7 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C12H16N2O7SNa: 355.0576, found: 355.0586. 1-[2’-Deoxy-2’-S-(carboxymethyl)thio-b-d-xylofuranosyl]uracil 6: LiOH·H2O (0.02 g, 0.51 mmol) and H2O (4 mL) were added to a solution of compound 3 (0.1 g, 0.17 mmol) in THF (16 mL). The reaction mixture was stirred for 1 h and volatile components were evaporated under reduced pressure. Solid NaHSO4·H2O (0.1 g) was added in portions to the residue and the mixture was stirred until pH 7. The residue was diluted with H2O (10 mL) and the solution thus ChemMedChem 2014, 9, 2138 – 2149

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CHEMMEDCHEM FULL PAPERS obtained was washed with EtOAc (3  15 mL). Combined organic layers were separated, dried over anhyd Na2SO4 and filtered. The filtrate was concentrated under reduced pressure. The crude solid thus obtained was stirred with TFA in CH2Cl2 (5 mL, 20 %) at room temperature. After 0.5 h, the volatile components were evaporated to dryness under reduced pressure and residual liquid was coevaporated with the mixture of toluene and carbon tetrachloride. The residue was triturated well with chilled Et2O to obtain compound 6 (0.04 g, 73 %). Hygroscopic solid; 1H NMR ([D6]DMSO, 25 8C): d = 3.41–3.57 (m, 3 H), 3.62–3.73 (m, 2 H), 3.87 (s, 1 H), 4.00– 4.03 (m, 1 H), 4.08–4.09 (m, 1 H), 5.66 (d, J = 8.0 Hz, 1 H), 5.87 (d, J = 2.0 Hz, 1 H), 7.77 (d, J = 7.6 Hz, 1 H), 11.32 ppm (s, 1 H); 13C NMR ([D6]DMSO, 25 8C): d = 33.1 (CH2), 55.8, 59.5 (CH2), 74.3, 84.3, 89.6, 102.0, 141.1, 150.7 (C), 163.5 (C), 171.2 ppm (C); HRMS (ESI + ), m/z calcd for [M + Na] + C11H14N2O7SNa: 341.0419, found: 341.0432. 1-[5’-O-Trityl-2’-deoxy-2’-S-(methoxycarbonylmethyl)sulfonyl-bd-xylofuranosyl]uracil 7: MMPP (0.25 g, 0.50 mmol) was added to a solution of 3 (0.25 g, 0.26 mmol) in anhyd CH3OH (20 mL) and the mixture was stirred at room temperature under N2 atmosphere. After 8 h, the reaction mixture was concentrated, treated with satd aq NaHCO3 and the solution was washed with EtOAc (2  25 mL). Organic layers were separated, dried over anhyd Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue thus obtained was purified over silica gel column to afford compound 7 (0.21 g, 80 %). Eluent for column chromatography: 40–60 % EtOAc in PE. White solid, mp: 92–98 8C; 1H NMR (CDCl3, 25 8C, TMS): d = 3.47–3.50 (m, 1 H), 3.63–3.67 (m, 1 H), 3.74 (s, 3 H), 4.11 (d, J = 15.6 Hz, 1 H), 4.39–4.44 (m, 2 H), 4.80 (d, J = 15.2 Hz, 1 H), 4.94 (d, J = 3.2 Hz, 1 H), 5.63 (d, J = 8.0 Hz, 1 H), 6.46 (d, J = 2.4 Hz, 1 H), 7.23–7.33 (m, 9 H), 7.46–7.48 (m, 6 H), 7.65 (d, J = 7.6 Hz, 1 H), 10.06 ppm (s, 1 H); 13C NMR (CDCl3, 25 8C, TMS): d = 53.7, 57.3 (CH2), 61.7 (CH2), 70.7, 73.9, 84.2, 84.9, 102.7, 127.4, 128.1, 128.7, 140.8, 143.4 (C), 150.8 (C), 163.9 (C), 164.1 ppm (C); HRMS (ESI + ), m/z calcd for [M + Na] + C31H30N2O9SNa: 629.1570, found: 629.1558. 1-[2’-Deoxy-2’-S-(methoxycarbonylmethyl)sulfonyl-b-d-xylofuranosyl]uracil 8: Compound 7 (0.2 g, 0.33 mmol) was stirred with TFA in CH2Cl2 (15 mL, 20 %) at room temperature. After 0.5 h, the volatile components were evaporated to dryness under reduced pressure and residual liquid was co-evaporated with a mixture of toluene and carbon tetrachloride. The residue was triturated well with chilled Et2O to obtain compound 8 (0.09 g, 72 %) as white hygroscopic solid. 1H NMR, 200 MHz ([D6]DMSO, 25 8C): d = 3.59–3.80 (m, 5 H), 3.91–3.95 (m, 1 H), 4.23 (d, J = 3.4 Hz, 1 H), 4.62 (s, 2 H), 4.72 (bs, 1 H), 5.81 (d, J = 8.0 Hz, 1 H), 6.39 (d, J = 3.6 Hz, 1 H), 7.71 (d, J = 8.0 Hz, 1 H), 11.51 ppm (s, 1 H); 13C NMR, 50 MHz ([D6]DMSO, 25 8C): d = 53.5, 57.3 (CH2), 59.2 (CH2), 69.7, 73.9, 82.7, 85.3, 103.5, 140.8, 150.9 (C), 163.4 (C), 163.5 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C12H17N2O9S: 365.0655, found: 365.0648. 1-[2’-Deoxy-2’-S-(carboxymethyl)sulfonyl-b-d-xylofuranosyl]uracil 9: A suspension of compound 7 (0.2 g, 0.33 mmol) and TMTOH (0.30 g, 1.65 mmol) was heated at 70 8C for 4 h in 1,2-dichloroethane (DCE). All the volatile components were then evaporated under reduced pressure. The obtained crude mass was stirred with 10 % TFA in CH2Cl2 (5 mL) at room temperature. After 3 h, the volatile components were evaporated to dryness under reduced pressure, and residual liquid was co-evaporated with a mixture of toluene and carbon tetrachloride. The residue was washed with CHCl3 first and then triturated well with chilled Et2O to obtain compound 9 (0.087 g, 75 %). White solid, mp: 149–155 8C; 1H NMR ([D6]DMSO, 25 8C): d = 3.60–3.64 (m, 1 H), 3.70–3.74 (m, 1 H), 3.90–3.94 (m, 1 H), 4.21 (d, J = 3.2 Hz, 1 H), 4.40 (d, J = 15.2 Hz, 1 H), 4.48 (d, J = 15.2 Hz, 1 H), 4.70 (d, J = 2.8 Hz, 1 H), 5.8 (dd, J = 1.6, 4.8 Hz, 1 H), 6.30 (bs,

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org 1 H), 6.36 (d, J = 3.6 Hz, 1 H), 7.68 (d, J = 8.0 Hz, 1 H), 11.51 (s, 1 H), 13.68 ppm (bs, 1 H); 13C NMR, 50 MHz ([D6]DMSO, 25 8C): d = 57.1 (CH2), 58.7 (CH2), 69.1, 73.2, 82.3, 84.9, 103.1, 140.2, 150.5 (C), 162.8 (C), 163.9 ppm (C); HRMS (ESI + ), m/z calcd for [M + Na] + C11H14N2O9SNa: 373.0318, found: 373.0301. 1-[3’-Deoxy-3’-S-(methoxycarbonylmethyl)thio-b-d-arabinofuranosyl]uracil 10: Compound 4 (0.2 g, 0.35 mmol) was converted into compound 10 (0.08 g, 68 %) following the method described for compound 5. Eluent for column chromatography: 5–25 % CH3OH in CHCl3. Hygroscopic solid; 1H NMR ([D6]DMSO, 25 8C): d = 3.17–3.21 (m, 1 H), 3.55–3.56 (m, 2 H), 3.60–3.69 (m, 5 H), 3.72–3.75 (m, 1 H), 4.26–4.29 (m, 1 H), 5.16–5.19 (m, 1 H), 5.57 (dd, J = 1.6, 8.0 Hz, 1 H), 5.86 (d, J = 5.6 Hz, 1 H), 6.01 (d, J = 5.6 Hz, 1 H), 7.75 (d, J = 8.0 Hz, 1 H), 11.25 ppm (s, 1 H); 13C NMR ([D6]DMSO, 25 8C): d = 32.6 (CH2), 48.8, 52.6, 60.6 (CH2), 75.8, 82.1, 84.4, 100.6, 142.5, 150.9 (C), 163.7 (C), 170.6 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C12H17N2O7S: 333.0756, found: 333.0773. 1-[3’-Deoxy-3’-S-(carboxymethyl)thio-b-d-arabinofuranosyl]uracil 11: Compound 4 (0.2 g, 0.35 mmol) was converted into compound 11 (0.07 g, 65 %) following the method described for compound 6. Hygroscopic solid; 1H NMR ([D6]DMSO, 25 8C): d = 3.15–3.18 (m, 1 H), 3.45 (s, 2 H), 3.58–3.75 (m, 4 H), 4.25–4.28 (m, 1 H), 5.54–5.57 (m, 1 H), 5.84 (bs, 1 H), 5.99 (d, J = 5.2 Hz, 1 H), 7.72 (d, J = 8.0 Hz, 1 H), 11.23 ppm (s, 1 H); 13C NMR, 50 MHz ([D6]DMSO, 25 8C): d = 32.8 (CH2), 48.4, 60.5 (CH2), 75.3, 82.0, 84.1, 100.2, 142.1, 150.5 (C), 163.3 (C), 171.1 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C11H15N2O7S: 319.0600, found: 319.0606. 1-[5’-O-Trityl-3’-deoxy-3’-S-(methoxycarbonylmethyl)sulfonyl-bd-arabinofuranosyl]uracil 12: Compound 4 (0.5 g, 0.52 mmol) was converted into compound 12 (0.44 g, 84 %) following the method described for compound 7. Eluent for column chromatography: 60–80 % EtOAc in PE. White solid, mp: 99–104 8C; 1H NMR (CDCl3, 25 8C, TMS): d = 3.47–3.50 (m, 1 H), 3.64–3.66 (m, 1 H), 3.74 (s, 3 H), 4.17 (d, J = 15.2 Hz, 1 H), 4.61–4.65 (m, 3 H), 5.15 (bs, 1 H), 5.26 (d, J = 8.2 Hz, 1 H), 6.06–6.08 (m, 1 H), 7.19–7.32 (m, 9 H), 7.47–7.49 (m, 6 H), 7.88 (d, J = 8.2 Hz, 1 H), 10.84 ppm (s, 1 H); 13C NMR, 50 MHz (CDCl3, 25 8C, TMS): d = 53.5, 57.2 (CH2), 63.5 (CH2), 65.9, 72.2, 74.6, 86.3, 87.6, 101.3, 127.5, 128.2, 128.9, 142.7, 143.5 (C), 150.8 (C), 163.4 (C), 165.5 ppm (C); HRMS (ESI + ), m/z calcd for [M + Na] + C31H30N2O9SNa: 629.1570, found: 629.1547. 1-[3’-Deoxy-3’-S-(methoxycarbonylmethyl)sulfonyl-b-d-arabinofuranosyl]uracil 13: Compound 12 (0.2 g, 0.33 mmol) was converted into compound 13 (0.08 g, 65 %) following the method described for compound 8. Hygroscopic solid; 1H NMR ([D6]DMSO, 25 8C): d = 3.60–3.64 (m, 1 H), 3.70–3.73 (m, 4 H), 4.06–4.09 (m, 1 H), 4.30–4.33 (m, 1 H), 4.54–4.63 (m, 2 H), 4.68–4.70 (m, 1 H), 5.56–5.59 (m, 1 H), 5.91 (d, J = 4.4 Hz, 1 H), 7.70 (d, J = 8.0 Hz, 1 H), 11.32 ppm (s, 1 H); 13C NMR, 50 MHz ([D6]DMSO, 25 8C): d = 53.5, 57.0 (CH2), 62.2 (CH2), 68.0, 71.0, 76.4, 85.9, 100.9, 142.7, 150.0 (C), 163.7 (C), 163.8 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C12H17N2O9S: 365.0655, found: 365.0642. 1-[3’-Deoxy-3’-S-(carboxymethyl)sulfonyl-b-d-arabinofuranosyl]uracil 14: Compound 12 (0.2 g, 0.33 mmol) was converted into compound 14 (0.09 g, 78 %) following the method described for compound 6. White hygroscopic solid. 1H NMR ([D6]DMSO, 25 8C): d = 3.62–3.66 (m, 1 H), 3.71–3.75 (m, 1 H), 4.10–4.12 (m, 1 H), 4.32– 4.34 (m, 1 H), 4.40–4.48 (m, 2 H), 4.68–4.70 (m, 1 H), 5.59 (d, J = 8.0 Hz, 1 H), 5.93 (d, J = 4.8 Hz, 1 H), 7.20 (d, J = 8.0 Hz, 1 H), 11.35 ppm (s, 1 H); 13C NMR, 50 MHz ([D6]DMSO, 25 8C): d = 57.3 (CH2), 62.2 (CH2), 67.7, 70.8, 76.4, 85.8, 100.7, 142.6, 150.9 (C), 163.7

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CHEMMEDCHEM FULL PAPERS (C), 164.4 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C11H15N2O9S: 351.0498, found: 351.0493. 1-[5’-O-Trityl-3’-deoxy-3’-S-(methoxycarbonylmethyl)sulfonyl-2’acetoxy-b-d-arabinofuranosyl]uracil 15: Compound 12 (1.31 g, 2.16 mmol) was dissolved in dry pyridine (20 mL) and cooled at 0 8C. To this ice-cold solution acetic anhydride (0.31 mL, 3.24 mmol) was added slowly. The mixture was stirred at room temperature for 8 h; satd NaHCO3 solution was added, and the aqueous layer was washed with EtOAc (3  50 mL). Organic layer was separated, dried over anhyd Na2SO4, filtered and the filtrated was concentrated under reduced pressure. The crude mass thus obtained was purified by column chromatography (eluent: 30–45 % EtOAc in PE) to afford compound 15 (1.1 g, 78 %). White solid, mp: 76–80 8C; 1 H NMR, (CDCl3, 25 8C, TMS): d = 2.10 (s, 3 H), 3.48–3.50 (m, 1 H), 3.62–3.64 (m, 1 H), 3.75 (s, 3 H), 4.25 (d, J = 16.0 Hz, 1 H), 4.76–4.80 (m, 2 H), 4.96 (d, J = 16.0 Hz, 1 H), 5.59 (d, J = 8.4 Hz, 1 H), 5.81 (d, J = 4.4 Hz, 1 H), 6.18 (d, J = 4.0 Hz, 1 H), 7.26–7.37 (m, 9 H), 7.50–7.52 (m, 6 H), 7.69 (d, J = 8.0 Hz, 1 H), 10.58 ppm (s, 1 H); 13C NMR, (CDCl3, 25 8C, TMS): d = 20.9, 53.3, 56.7 (CH2), 63.5 (CH2), 65.6, 72.5, 73.9, 85.0, 87.2, 96.2 (C), 101.2, 127.5, 128.2, 128.7, 141.2, 143.5 (C), 149.7 (C), 163.7 (C), 164.6 (C), 170.1 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C33H33N2O10S: 649.1856, found: 649.1885. 4-(1,2,3-Triazol-1-yl)-1-[b-d-5-O-trityl-3-deoxy-3-S-(methoxycarbonylmethyl)sulfonyl-2-acetoxyarabinofuranosyl]pyrimidin2(1 H)-one 16: Compound 15 (0.642 g, 0.99 mmol) was dissolved in dry CH2Cl2 and cooled at 0 8C. To this ice cold solution 1H-1,2,4triazole (0.77 g, 11.2 mmol) was added. The turbid solution formed, became clear after addition of triethylamine (TEA; 1.55 mL, 11.2 mmol). To this ice-cold solution POCl3 (0.24 mL, 2.56 mmol) was added slowly and the reaction mixture was stirred for 3 h at room temperature. Cold H2O was added, and the aqueous layer was washed with CH2Cl2 (2  50 mL). Organic layer was separated, dried over anhyd CaCl2 and concentrated under reduced pressure. The yellow crude mass thus obtained was purified by column chromatography (eluent: 40–60 % EtOAc in PE) to afford compound 16 (0.55 g, 79 %). White solid, mp: 74–78 8C; 1H NMR, (CDCl3, 25 8C, TMS): d = 1.92 (s, 3 H), 3.50–3.54 (m, 1 H), 3.62–3.66 (m, 1 H), 3.75– 3.77 (m, 3 H), 4.10 (d, J = 16.0 Hz, 1 H), 4.77–4.81 (m, 2 H), 4.85–4.88 (m, 2 H), 5.95–5.97 (m, 1 H), 6.18 (d, J = 4.4 Hz, 1 H), 6.97 (d, J = 7.6 Hz, 1 H), 7.26–7.47 (m, 15 H), 8.14 (s, 1 H), 8.26 (d, J = 7.6 Hz, 1 H), 9.26 ppm (s, 1 H); 13C NMR, (CDCl3, 25 8C, TMS): d = 20.7, 53.5, 57.0 (CH2), 63.2 (CH2), 65.7, 72.5, 75.2, 87.1, 87.4 (C), 94.3, 127.7, 128.2, 128.8, 143.4, 143.6 (C), 147.5, 153.8, 154.3 (C), 159.8 (C), 163.5 (C), 169.6 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C35H34N5O9S: 700.2077, found: 700.2051. 4-(1,2,3-Triazol-1-yl)-1-[5’-O-trityl-2’,3’-anhydro-b-d-lyxofuranosyl]uracil 18: Compound 2 (2.0 g, 4.27 mmol) was converted into compound 18 (1.65 g, 74 %) following the method described for compound 16. Eluent for column chromatography: 40–70 % EtOAc in PE. White solid, mp: 64–68 8C; 1H NMR, 200 MHz (CDCl3, 25 8C, TMS): d = 3.42–3.59 (m, 2 H), 3.93–3.94 (m, 1 H), 4.19–4.20 (m, 1 H), 4.30–4.35 (m, 1 H), 6.28 (s, 1 H), 6.95 (d, J = 7.2 Hz, 1 H), 7.25–7.52 (m, 15 H), 8.07 (d, J = 7.2 Hz, 1 H), 8.12 (s, 1 H), 9.27 ppm (s, 1 H); 13 C NMR, 50 MHz (CDCl3, 25 8C, TMS): d = 56.1, 57.4, 62.4 (CH2), 77.9, 84.6, 87.3 (C), 94.7, 127.5, 128.1, 128.7, 141.4, 143.5 (C), 148.1, 154.2, 154.6 (C), 159.6 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C30H26N5O4 : 520.1985, found: 520.1986. 1-(5’-O-Trityl-2’,3’-anhydro-b-d-lyxofuranosyl)cytosine 19:[39] To a mixture of compound 18 (1.38 g, 2.66 mmol) in THF (15 mL) aq NH4OH (1.0 mL) was added and stirred for 4 h. The volatile components were evaporated under reduced pressure and the semi-solid

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org mass thus obtained was purified by column chromatography (eluent: 2–7 % CH3OH in CHCl3) to afford tritylated cytidine epoxide 19 (1.08 g, 88 %). White solid, mp: 145–150 8C; 1H NMR, 200 MHz (CDCl3, 25 8C, TMS): d = 3.31–3.49 (m, 2 H), 3.82–3.83 (m, 1 H), 3.97– 3.99 (m, 1 H), 4.10–4.20 (m, 1 H), 5.67 (d, J = 7.6 Hz, 1 H), 6.24 (s, 1 H), 7.21–7.53 ppm (m, 16 H); 13C NMR, 50 MHz (CDCl3, 25 8C, TMS): d = 56.4, 56.6, 62.5 (CH2), 76.9, 83.0, 87.2 (C), 94.8, 127.4, 128.1, 128.8, 142.5, 143.7 (C), 156.1 (C), 166.1 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C28H26N3O4 : 468.1923, found: 468.1901. N6,N6-Bis(tert-butoxycarbonyl)-1-(5’-O-trityl-2’,3’-anhydro-b-dlyxofuranosyl)cytosine 20: To a well-stirred solution of epoxide 19 (1.0 g, 2.14 mmol) in dry THF (20 mL), 4-dimethylaminopyridine (DMAP; 0.627 g, 5.14 mmol) was added. To this mixture, di-tert-butyldicarbonate (Boc2O; 1.12 mL, 5.14 mmol) was added slowly and kept at room temperature for 2 h. Volatile components were evaporated under reduced pressure, and the crude mass thus obtained was purified by column chromatography (eluent: 20–40 % EtOAc in PE) to afford compound 20 (1.21 g, 85 %) as a transparent gum; 1H NMR, 200 MHz (CDCl3, 25 8C, TMS): d = 1.55 (s, 18 H), 3.34– 3.52 (m, 2 H), 3.77–3.79 (m, 1 H), 4.03–4.04 (m, 1 H), 4.21–4.24 (m, 1 H), 6.19 (s, 1 H), 6.97 (d, J = 7.6 Hz, 1 H), 7.23–7.49 (m, 15 H), 7.75 ppm (d, J = 7.6 Hz, 1 H); 13C NMR, 50 MHz (CDCl3, 25 8C, TMS): d = 27.6, 55.9, 56.8, 62.3 (CH2), 77.3, 83.8, 84.8 (C), 87.0 (C), 96.0, 127.2, 127.9, 128.6, 143.5 (C), 144.8, 149.4 (C), 154.3 (C), 162.5 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C38H42N3O8 : 668.2972, found: 668.2968. N6-(tert-Butoxycarbonyl)-1-[5’-O-trityl-3’-deoxy-3’-S-(methoxycarbonylmethyl)thio-b-d-xylofuranosyl]cytosine 21 and N6-(tert-Butoxycarbonyl)-1-[5’-O-trityl-3’-deoxy-3’-S-(methoxycarbonylmethyl)thio-b-d-arabinofuranosyl]cytosine 22: Compound 20 (0.89 g, 1.33 mmol) was converted into compounds 21 and 22 by following the method described for compounds 3 and 4. The mixture was separated by column chromatography (eluent: 30–60 % EtOAc in PE). Compound 21 was isolated as a hygroscopic solid (0.22 g, 25 %); 1H NMR (CDCl3, 25 8C, TMS): d = 1.52 (s, 9 H), 3.49– 3.55 (m, 2 H), 3.59–3.71 (m, 5 H), 3.86 (s, 1 H), 3.95 (d, J = 16 Hz, 1 H), 4.24 (d, J = 2.8 Hz, 1 H), 4.50–4.53 (m, 1 H), 5.98 (s, 1 H), 7.07 (d, J = 7.6 Hz, 1 H), 7.24–7.50 (m, 15 H), 7.94 ppm (d, J = 7.6 Hz, 1 H); 13 C NMR, (CDCl3, 25 8C, TMS): d = 28.1, 33.4 (CH2), 52.7, 56.5, 62.4 (CH2), 75.9, 82.6 (C), 84.3, 87.3 (C), 93.6, 94.3, 127.3, 128.0, 128.7, 143.5 (C), 144.7, 151.4 (C), 155.4 (C), 163.0 (C), 171.1 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C36H40N3O8S: 674.2536, found: 674.2522. Compound 22 was obtained as a hygroscopic solid (0.45 g, 50 %); 1H NMR (CDCl3, 25 8C, TMS): d = 1.51 (s, 9 H), 3.30 (d, J = 15.6 Hz, 1 H), 3.51–3.61 (m, 5 H), 3.64 (s, 3 H), 3.90–3.94 (m, 1 H), 4.68–4.71 (m, 1 H), 6.19 (d, J = 5.2 Hz, 1 H), 7.00 (d, J = 7.2 Hz, 1 H), 7.25–7.47 (m, 15 H), 8.29 ppm (d, J = 7.6 Hz, 1 H); 13C NMR, 50 MHz (CDCl3, 25 8C, TMS): d = 28.2, 32.7 (CH2), 47.9, 52.8, 62.3 (CH2), 78.4, 81.2, 82.7 (C), 86.9, 87.5 (C), 95.0, 127.5, 128.2, 128.9, 143.5 (C), 145.6, 151.3 (C), 156.3 (C), 162.9 (C), 170.8 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C36H40N3O8S: 674.2536, found: 674.2532. N6-(tert-Butoxycarbonyl)-1-[5’-O-trityl-3’-deoxy-3’-S-(methoxycarbonylmethyl)sulfonyl-b-d-xylofuranosyl]cytosine 23: A solution of compound 21 (0.5 g, 0.74 mmol) in CH2Cl2 (11 mL) was added to MMPP (0.73 g, 1.48 mmol) moistened with H2O (0.15 mL). The reaction mixture was stirred for 3 h and then filtered though a Celite bed. The filtrate was dried over anhydrous CaCl2, filtered and the filtrate was concentrated under reduced pressure. The crude mass thus obtained was purified by flash column chromatography (eluent: 2–5 % CH3OH in CH2Cl2) to afford compound 23 (0.39 g, 75 %). Hygroscopic solid; 1H NMR (CDCl3, 25 8C, TMS): d = 1.49–1.52 (s, 9 H), 3.52–3.56 (m, 1 H), 3.64–3.70 (m, 4 H), 3.78 (s, 1 H), 4.15 (d,

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CHEMMEDCHEM FULL PAPERS J = 15.6 Hz, 1 H), 4.46 (s, 1 H), 4.49–4.51 (m, 1 H), 4.97 (d, J = 3.2 Hz, 1 H), 5.05 (d, J = 15.6 Hz, 1 H), 6.39 (d, J = 1.6 Hz, 1 H), 7.22–7.34 (m, 10 H), 7.44–7.47 (m, 6 H), 7.96 ppm (d, J = 7.6 Hz, 1 H); 13C NMR (CDCl3, 25 8C, TMS): d = 27.5, 53.7, 57.4 (CH2), 61.7 (CH2), 70.8, 73.9, 84.2, 85.0, 85.3 (C), 87.6 (C), 95.1, 127.4, 128.1, 128.7, 140.8, 143.4 (C), 146.9 (C), 150.8 (C), 163.9 (C), 164.1 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C36H40N3O10S: 706.2434, found: 706.2444. 1-[2’-Deoxy-2’-S-(carboxymethyl)sulfonyl-b-d-xylofuranosyl]cytosine 24: Compound 23 (0.1 g, 0.14 mmol) was converted into compound 24 (0.03 g, 60 %) following the method described for compound 6. White solid, mp: 160–165 8C; 1H NMR ([D6]DMSO, 25 8C): d = 3.58–3.63 (m, 1 H), 3.69–3.73 (m, 1 H), 3.88–3.90 (m, 1 H), 4.12– 4.16 (m, 1 H), 4.27–4.30 (m, 1 H), 4.37–4.38 (m, 1 H), 4.64–4.67 (m, 1 H), 5.78 (d, J = 8.4 Hz, 1 H), 6.35–6.36 (m, 1 H), 7.66–7.75 ppm (m, 3 H); 13C NMR, 50 MHz ([D6]DMSO, 25 8C): d = 57.6 (CH2), 59.1 (CH2), 69.6, 73.7, 82.8, 85.3, 95.6, 140.7, 151.0 (C), 163.3 (C), 164.4 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C11H16N3O8S: 350.0658, found: 350.0650. N6-(tert-Butoxycarbonyl)-1-[5’-O-trityl-3’-deoxy-3’-S-(methoxycarbonylmethyl)sulfonyl-b-d-arabinofuranosyl]cytosine 25: Compound 22 (0.5 g, 0.74 mmol) was converted into compound 25 (0.44 g, 84 %) following the method described for compound 23. Eluent for column chromatography: 3–5 % CH3OH in CHCl3. Hygroscopic solid; 1H NMR (CDCl3, 25 8C, TMS): d = 1.49–1.53 (bs, 9 H), 3.52 (d, J = 10.4 Hz, 1 H), 3.72 (bs, 4 H), 4.08 (d, J = 14.8 Hz, 1 H), 4.54–4.59 (m, 1 H), 4.63–4.69 (m, 1 H), 5.18 (bs, 1 H), 6.13 (d, J = 4.8 Hz, 1 H), 6.96 (d, J = 7.2 Hz, 1 H), 7.24–7.51 (m, 15 H), 8.34 ppm (d, J = 7.6 Hz, 1 H); 13C NMR, 50 MHz (CDCl3, 25 8C, TMS): d = 27.4, 53.4, 57.0 (CH2), 63.5 (CH2), 65.9, 72.0, 74.5, 85.2 (C), 86.4, 87.5 (C), 96.4, 127.3, 128.0, 128.8, 142.5, 143.4 (C), 146.8 (C), 150.6 (C), 163.3 (C), 165.4 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C36H40N3O10S: 706.2434, found: 706.2454. 1-[3’-Deoxy-3’-S-(carboxymethyl)sulfonyl-b-d-arabinofuranosyl]cytosine 26: Compound 25 (0.1 g, 0.14 mmol) was converted into compound 26 (0.032 g, 65 %) following the method described for compound 6. Hygroscopic solid; 1H NMR ([D6]DMSO, 25 8C): d = 3.60–3.65 (m, 1 H), 3.69–3.73 (m, 1 H), 4.11–4.12 (m, 1 H), 4.30–4.36 (m, 3 H), 4.66–4.68 (m, 1 H), 5.57 (d, J = 8.0 Hz, 1 H), 5.9 (d, J = 4.8 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1 H), 8.58 (bs, 1 H), 8.69 ppm (bs, 1 H); 13C NMR, 50 MHz ([D6]DMSO, 25 8C): d = 56.4 (CH2), 61.2 (CH2), 66.8, 69.9, 75.4, 84.8, 96.7, 141.6, 149.9 (C), 162.7 (C), 163.4 ppm (C); HRMS (ESI + ), m/z calcd for [M + H] + C11H16N3O8S: 350.0658, found: 350.0660.

Biophysical studies Agarose gel electrophoresis assays: Qualitative analysis of RNase A inhibition was determined by the relative degradation of tRNA in an agarose gel. In this method, 20 mL RNase A (2.0 mm) was mixed with 20 (0.05 mm), 20 (0.10 mm) and 20 mL (0.15 mm) compounds 6, 9, 11, 14, 24, and 26 to a final volume of 100 mL, and the resulting solutions were incubated for 3 h; 20 mL aliquots of the incubated mixtures were then mixed with 20 mL of tRNA solution (5.0 mg mL1 tRNA, freshly dissolved in RNase-free water) and incubated for another 30 min. To this mixture, 10 mL sample buffer (containing 10 % glycerol and 0.025 % bromophenol blue) was added; 15 mL from each solution was taken and loaded into a 1.1 % agarose gel. The gel was run using 0.04 m Tris/acetic acid/ EDTA (TAE) buffer (pH 8.0). Finally, the residual tRNA was visualized by ethidium bromide staining under UV light.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org Precipitation assays: Inhibition of the ribonuclease activity of RNase A was analyzed by the precipitation assay as described by Bond.[47] In this method 10 mL RNase A (1.30 mm) was mixed with 40 mL compounds (0.34 mm) individually to a final volume of 100 mL and incubated for 2 h at 37 8C; 20 mL of the resulting solutions from the incubated mixtures were then mixed with 40 mL of tRNA (5 mg mL1 tRNA freshly dissolved in RNase A-free water), 40 mL Tris·HCl buffer (pH 7.5) containing 5 mm EDTA and 0.5 mg mL1 HSA. After incubation of the reaction mixture at 25 8C for 30 min, 200 mL ice-cold 1.14 m perchloric acid containing 6 mm uranyl acetate was added to quench the reaction. The solution was then kept on ice for another 30 min and centrifuged at 4 8C at 12 000 rpm (8855 g) for 5 min; 50 mL of the supernatant was taken and diluted to 1 mL. The decrease in absorbance at 260 nm was measured and compared with a control set. Inhibition kinetics: To determine the inhibition constants and mode of inhibition of RNase A by compounds 6, 9, 11, 14, 24, and 26, a spectrophotometric method was used as described by Anderson et al.[43] The assay was performed in 0.1 m Mes·NaOH buffer, pH 6.0 containing 0.1 m NaCl using 2’,3’-cCMP as substrate. The inhibition constants were calculated from initial velocity data by Lineweaver– Burk plot. For the Lineweaver–Burk plot the reciprocal of initial velocity was plotted against the reciprocal of substrate concentration at a constant inhibitor concentration according to Equation (1):   1 K ½I 1 1 ¼ m 1þ þ v Vmax Ki ½S Vmax

ð1Þ

Kinetics experiments were performed with two fixed inhibitor concentrations and another in the absence of inhibitor with varying substrate (2’,3’-cCMP) concentrations. The slopes from the doublereciprocal plot were again plotted against the corresponding inhibitor concentrations to get inhibition constants (Ki).

Determination of partition coefficients (log P) The shake-flask method: The shake-flask method was followed with slight modification from the reported procedure.[48] Accurately weighed (0.25, 0.50, 1.00 and 2.00 mg) portions of 3’-CMP, inhibitor 14 and 26 were individually dissolved in 10.0 mL 0.01 m phosphate buffer (pH 7.4) in volumetric flasks. The UV absorption of each solution was recorded from 600 to 200 nm with a UV/Vis spectrophotometer. The absorbance values of these solutions at the absorption maximum were plotted against their concentrations. A linear relationship ensured that the molar absorptivity at the maximum (emax) was obtained from complete solutions and that Beer’s Law was followed. The emax of both inhibitors and 3’-CMP at ~ 260 nm were calculated. Similarly, the emax of the inhibitors and 3’-CMP in 1-octanol were also determined. From the freshly prepared buffer solutions containing inhibitors and 3’-CMP, 5.00 mL of each solution (which produced the linear relationship) were transferred to individual centrifuge cones. To each cone 5.00 mL octanol was added and the tube capped tightly. The mixtures were shaken 40 times and vortexed for 5 min followed by centrifugation at 2000 rpm (246 g) for 1 h. The aqueous and the organic phases were separated, and the UV trace of each recorded. The inhibitor concentration in each phase was calculated from its respective absorbance. The P value of the nucleoside was determined by Equation (2). Consequently the log P values were also determined and termed as SFlog Poctanol/water. P ¼ coctanol =cwater

ð2Þ

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CHEMMEDCHEM FULL PAPERS Computational Methods Calculated partition coefficients (log P): Experimentally obtained log P values were correlated by computational methods. Two methods were used: 1) The QuipProp (QikProp, version 3.8, Schrçdinger LLC, New York, NY, 2013) and 2) ALOGPS (VCCLAB, Virtual Computational Chemistry Laboratory).[49] Before running the automated programs, the corresponding Mol2 files of inhibitors 14, 26 and 3’-CMP were generated in Sybyl using MMFF94 force field and charges. The log P values corresponding to computational methods 1 and 2 are designated as QPlog Poctanol/water and Alog Poctanol/water, respectively. Docking studies: The crystal structure of RNase A (PDB ID: 1FS3) was downloaded from the Protein Data Bank[50] and used as the PDB file for docking only after subtraction of water molecules and other ions present in it. The 3D structures of compounds 6, 9, 11, 14, 24, and 26 were generated in Sybyl 6.92 (Tripos Inc., St. Louis, MO, USA) and their energy-minimized conformations were obtained with the help of the MMFF94 force field using MMFF94 charges with a gradient of 0.005 kcal mol1 by 1000 iterations with all other default parameters. The FlexX software as part of the Sybyl suite was used for docking of the ligands to the protein. The ranking of the generated solutions was performed using a scoring function that estimates the free binding energy (DG) of the protein–ligand complex considering various types of molecular interactions as described by Rarey et al.[51] Each docked conformation is looked upon as a “suggestion” of how the ligand may bind with the protein. PyMOL[52] was used for visualization of the docked conformations.

Acknowledgements The authors thank the Department of Biotechnology, Ministry of Science and Technology, New Delhi for funding. D.D. thanks the Council for Scientific and Industrial Research, New Delhi for a fellowship, and the Department of Chemistry and Central Research Facility, IIT Kharagpur for access to instrumental facilities. Keywords: carboxymethylsulfonyls · docking · inhibitors · modified nucleosides · pyrimidines · RNase A

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