key: cord-260604-lz1qd69t
authors: Singh, Ramendra K.; Yadav, Dipti; Rai, Diwakar; Kumari, Garima; Pannecouque, C.; De Clercq, Erik
title: Synthesis, structure–activity relationship and antiviral activity of 3′-N,N-dimethylamino-2′,3′-dideoxythymidine and its prodrugs
date: 2010-09-30
journal: European Journal of Medicinal Chemistry
DOI: 10.1016/j.ejmech.2010.05.028
sha: 
doc_id: 260604
cord_uid: lz1qd69t

Abstract A probable NRTI molecule, viz. 3′-N,N-dimethylamino-2′,3′-dideoxythymidine (4) and its 5′-O-carboxyl ester prodrugs – 5′-(N-α-BOC-l-phenylalanyl)-3′-N,N-dimethylamino-2′,3′-dideoxythymidine (5), 5′-l-phenylalanyl-3′-N,N-dimethylamino-2′,3′-dideoxythymidine (6) and 5′-decanoyl-3′-N,N-dimethylamino-2′,3′-dideoxythymidine (7) have been synthesized and screened against HIV, HSV-1 and 2, parainfluenza-3, vesicular stomatitis and several other viruses. The compound 6 showed good antiviral activity with EC50 value 0.03μM (SI=8) against VSV in Hela and HEL cell lines. However, the lead compound 4 and its derivatives 5, 6 and 7 showed no remarkable activity against HIV-1 and other viruses. Molecular docking studies with HIV-1 RT using DS 2.5 and pymol softwares have shown marked differences in the interaction patterns between the lead compound 4 and AZT.

HIV-1 reverse transcriptase (HIV-1 RT), a primary target for developing anti-HIV drugs, is the enzyme responsible for generating linear dsDNA from ssRNA e the genetic material of HIV-1. This dsDNA acts as a provirus and leads to viral expression through integration in the host genome, transcription and translation processes.

There are several drugs that inhibit HIV-1 RT and they are classified as nucleos(t)ide RT inhibitors (N(t)RTIs) and non-nucleoside RT inhibitors (NNRTIs). The NRTIs have emerged as important therapeutic agents for the development of anti-HIV drugs [1e4] . The NRTIs after intracellular phosphorylation to their active triphosphate form by cellular kinases [5] , act as competitive inhibitors with respect to the dNTP substrates. These molecules when incorporated by HIV-1 RT in the growing template/primer, cause chain termination [6, 7] since they lack 3 0 -OH group, which is necessary for chain elongation and thus, they inhibit HIV-1 RT [8, 9] . Despite their tremendous utility, nucleoside analogues may cause serious cellular toxicity by interacting with human DNA polymerase g [10] , present in mitochondria, which results in mitochondrial dysfunctions. Thus NRTIs, show many side effects [11e13] . In addition to this, four more significant obstacles currently limiting the clinical application of nucleoside analogues are oral absorption, cellular uptake, short halflife and viral resistance [14] . So, there is a need for new nucleoside mimics and their prodrugs to overcome the limitations. We have already reported several nucleosidic [15, 16] and non-nucleosidic molecules [17] with significant antiviral activities.

The NRTIs, by virtue of acting as chain terminators of (À) strand DNA synthesis during reverse transcription, have been developed as potential drugs against HIV/AIDS. In order to explore such new inhibitors, we report here the synthesis of 3 0 -N,N-dimethylamino-2 0 ,3 0 -dideoxythymidine (DMAT) and its 5 0 -O-ester prodrugs with amino and fatty acids. The lead molecule, DMAT, was designed keeping AZT, an approved NRTI drug against HIV/AIDS, in mind, where azido group has been replaced by dimethylamino function, Fig. 1 . Thus, this molecule being a dideoxynucleoside is expected to act as NRTI against HIV-1 RT enzyme. Similarly, the prodrugs, having biodegradable ester linkages, are supposed to enhance the cellular uptake due to their high lipophilic nature.

All these molecules have been evaluated for their activity against various viruses, like HIV-1, vesicular stomatitis, HSV-1 & 2, reovirus, parainfluenza virus and many others.

q Work presented in Nucleic Acids Symposium (Series No. 52) held in Japan (Ref. [37] ).

A new thymidine analogue, viz. 3 0 -N,N-dimethylamino-2 0 ,3 0dideoxythymidine has been synthesized from thymidine as shown in Scheme 1. 5 0 -O-(4,4 0 -Dimethoxytrityl)-3 0 -mesylthymidine, obtained after 5 0 -OH selective protection with DMTCl followed by mesylation with MsCl, when reacted with potassium phthalimide witnessed the introduction of nucleophile (phthalimide ion) into the sugar moiety [18] . During conversion of 1e2, the elimination of C3 0 -mesyloxy group was effected through intramolecular displacement by attack of carbonyl group of thymidine at C2, which resulted in formation of 2,3 0 -anhydronucleoside intermediate. This result was well in accordance with the literature data. It is suggested that under this condition, phthalimide ion is introduced into the 'down' (ribo) configuration [19] . The 3 0 -phthalimide derivative 2 was treated with methanolic methylamine, which through methanolysis and aminolysis resulted in formation of compound 3 [20] . Reaction of 3 with formic acid and aqueous formaldehyde (40%) yielded 4. In this reaction, formaldehyde used was the source of methyl groups, while formic acid supplied the hydrogens involved in the reduction. This treatment also removed the DMT group from compound 3. As expected, the UV absorption spectra of all intermediates were similar to that of thymidine, while dissimilar R f values showed that all these changes took place in the sugar moiety and not in the aglycon part [19] .

All compounds 4e7 have been screened for their antiviral potential against HIV and several other viruses and the results are shown in Table 1 . The lead compound DMAT and its prodrugs were found inactive against HIV-1 ROD and HIV-1 IIIB strains and showed toxicity. Compounds 4 and 5 have showed similar values of CC 50 and EC 50 against all viruses studied and thus no activity was observed, while 5 0 -L-phenylalanyl derivative 6, having free amino function was potentially active at an EC 50 of 0.03 mM against VSV in HeLa and HEL cell cultures. The EC 50 value of compound 6 has been found to be eightfold lower than its CC 50 value (SI ¼ 8). Besides this, it has also showed moderate activity at an EC 50 of around 0.05 mM against HSV-1 & 2, coxsackie B4, and RSV in HeLa cell culture. At the same EC 50 value, it was found active against VV, TK HSV, in HEL cell lines; PIV-3, RV-1, SV and PTV in Vero cell culture and ECV (FIPV) and FHV in CRFK cell culture. The 5 0 -decanoyl derivative 7 has shown some activity against FCV (FIPV) and FHV in CRFK cell culture.

All compounds were also tested against influenza A H1N1/H3N2 subtype and Influenza B in MDCK cell lines. However, none of these compounds was able to inhibit cytopathic effects of Influenza A or B virus at subtoxic concentrations or the highest concentration tested.

It was observed that compounds 6 and 7 only, showed antiviral properties against VSV (EC 50 0.03) and FCV (EC 50 0.05), respectively. However, the antiviral potency of the prodrug 6 was still significantly lower than that of established antiviral drugs e (S)-DHPA, BVDU and ribavirine against VSV as was evident from its CC 50 and EC 50 values shown in Table 1 .

Since nucleosides exhibit only modest bioavailability [21] , we prepared the prodrug molecules using amino and fatty acids to improve their bioavailability [22, 23] and chemical stability. This was done to ensure enhanced cellular uptake and sustained release of drug molecule without compromising its properties [24e27].

The lead compound 4 and its prodrug 5 did not show any antiviral activity, whereas the prodrug 7 showed activity, albeit, too little against FCV and FHV. However, the prodrug 6 showed good activity against VSV with SI value equal to 8. The enhanced reactivity of free amino acid ester prodrug may be attributed to its electronic activation by the positively charged amino terminus at physiological pH, which derives support from the fact that L-amino acid esters enhance nucleoside absorption via human peptide transporters (hPEPT1) of active transport system [28, 29] . Furthermore, since bone marrow progenitor cells lack this active transport system for amino acid, prodrug 6 is expected to show reduced toxicity at bone marrow level. Therefore, compound 6 has potential to be developed as an effective antiviral agent against VSV.

The lead compound 4, being a 2 0 ,3 0 -dideoxynucleoside, was expected to act as chain terminator and thus, interfere with viral cell metabolism and its prodrugs were designed and synthesized to enhance its cellular uptake. However, this molecule showed almost no anti-HIV activity and rather proved cytotoxic. This might be DMAT :

R 1 = tyrosynyl, lysinyl, phenylalanyl, valyl etc. R 1 = decanoyl, myristoyl, pamitoyl etc. expected because of introduction of dimethylamino group at 3 0 -C of ribose moiety, which did not show any interaction with HIV RT during molecular docking experiments. The lead molecule 4 was designed because of its similarity with AZT, but it completely failed in fulfilling the objectives. It was surprising to see that replacement of azido group by dimethylamino group turned the molecule highly toxic.

The fact that the lead molecule DMAT (and its prodrugs) being a dideoxynucleoside and having structural features similar to AZT, did not show any activity against HIV and rather proved toxic, prompted us to study its interaction with HIV-1 RT. 1 We performed elaborate molecular computational studies using the software Discovery Studio (DS) 2.5. This molecule showed some striking differences with AZT e the approved anti-HIV drug as shown in Table 2 . The physiochemical studies suggested that both the molecules e AZT and DMAT, followed Lipinski 0 s rule of five but they have marked differences in their molecular volume, total polar surface area (TPSA) and log P values. Similarly, the docking experiment also showed quite different patterns of their interaction with HIV-1 RT (Figs. 2e4) . Although DMAT formed one more H-bond with HIV-1 RT than AZT, the crucial interactions as shown by AZT were missing in the case of DMAT. The azido function on AZT formed three H-bonds e one with Arg72 and two with Asp113 (the amino acid constituting the dNTP-binding site), whereas the dimethylamino function on DMAT formed no H-bonds at all. Similarly, the H-bond formed by phosphate moiety of AZT (and TTP as well) with Val111 was missing in the case of DMAT. However, the aglycan part in both the molecules formed two H-bonds with Arg72 through oxo group and "O" atom in the sugar ring. The stability of DMATTPeRT complex was also less than that of AZTTPeRT or TTPeRT complexes as is evident from their minimization energies. A comparative account of interaction of TTP, AZTTP and DMATTP and stability of their respective complexes with HIV-1 RT has been shown in Table 3 . Thus, the physiochemical and docking studies suggested the possible reasons for inactivity of the lead molecule and its prodrugs.

In conclusion, we have synthesized 3 0 -N,N-dimethylamino-2 0 ,3 0 -dideoxythymidine 4 and its 5 0 -O-carboxyl ester prodrug derivatives 5e7 and evaluated their antiviral activity against HIV-1 (III B and ROD strains), HSV-1/2, vesicular stomatitis, parainfluenza-3 virus and many others. It was observed that compounds 6 and 7 only, showed moderate antiviral properties against VSV (EC 50 0.03) and FCV (EC 50 0.05), respectively. Further mechanistic studies on molecules 6 and 7 are in progress against vesicular stomatitis, feline corona and feline herpes viruses.

The inference drawn from the present studies that for a nucleoside analogue to be an effective NRTI, being a dideoxy derivative is not sufficient enough criterion and rather it must possess the characteristic features too for requisite interactions at the dNTPbinding site of HIV-1 RT enzyme shall definitely help in designing new NRTI molecules.

Chemicals were obtained from E. Merck India Ltd, India and SigmaeAldrich Chemical Company, USA. Melting points were determined on electrothermal apparatus and are uncorrected. Silica gel for TLC and column chromatography (60e120 mesh) was obtained from E. Merck India Ltd. UV measurements were carried out on Hitachi 220S spectrophotometer. HPLC analyses were performed using 250 Â 4.6 mm 2 C18 column and solvent system MeOH:H 2 O (6:4) at a flow rate of 1 ml/min on 6 AD Binary Gradient Shimadzu HPLC system. 1 H NMR spectra were recorded on DRX 300 MHz instrument using DMSO-d 6 as solvent and TMS as an internal standard. 13 C NMR spectra were recorded in CDCl 3 on a Varian XL-300 spectrometer operating at 75 MHz. Mass spectra were obtained using a Thermofinnigan TRACE-DSQ Electrospray Ionization (ESI) mass spectrometer. Elemental analyses were carried out on a PerkineElmer 240-C analyzer. All solvents were dried and distilled prior to use.

Thymidine (2.415 g, 10 mmol) was suspended in dry pyridine (w100 ml) in a 250 mL round bottomed flask and added DMAP (61 mg, 0.05 equivalent), TEA (1.9 ml, 1.4 equivalent) and DMTCl (4.1 g, 1.2 equivalent) and stirred the reaction mixture for 2.5 h. TLC at this point showed complete disappearance of thymidine. The contents were cooled to 0 C and methanesulfonyl chloride (1.23 ml, 10.89 mmol) was added dropwise. Stirring was continued further for 2 h and the reaction mixture was allowed to warm up to 25 C. After stirring the reaction mixture for an additional 30 min, the contents were poured into crushed ice/water and the viscous mass so obtained was extracted in chloroform (50 ml) and washed with water (2 Â 10 ml). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated on a rotary evaporator to obtain the crude product. Silica gel column purification with a linear gradient up to 10% ethyl acetate in hexane, afforded the pure compound, 5 0 -O-(4,4 0 -dimethoxytrityl)-3 0mesyl-2 0 ,3 0 -dideoxythymidine, which was refluxed for 12 h with potassium phthalimide (9.0 g) in dimethylformamide (450 ml). The solvent was removed under reduced pressure and the residue extracted with ethyl acetate. The ethyl acetate layer was finally washed with water (3 Â 10 ml), dried over anhydrous Na 2 SO 4 and concentrated to get the title compound in crude form (4.72 g, 70%).

The compound 2 (4.34 g) was stirred with methylamine (16 ml) in methanol at 105 C for 20 h. The reaction mixture was evaporated to an oily residue, washed with water, dissolved in ethanol (100 ml) and treated with hydrochloric acid to neutralize the residual methylamine. The acidic solution was concentrated under reduced pressure, which resulted in crystallization. The crystals were separated and the residual water and acids were removed by co-evaporation with benzene under reduced pressure from the mother liquor, which was further subjected to crystallization. The crystalline residue was triturated with ethanol and ether. The title compound was obtained as colorless crystals (3.04 g, 87%). Dicyclohexylcarbodiimide (515 mg, 2.5 mmol) was added to a stirred solution consisting of 3 0 -N,N-dimethylamino-2 0 ,3 0dideoxythymidine (269 mg, 1 mmol), DMAP (183.25 mg, 1.5 mmol,) and N-a-t-boc-L-phenylalanine (397.95 mg, 1.5 mmol) in pyridine (10 ml) under anhydrous condition. The progress of reaction was monitored by TLC. After the reaction was complete (70e72 h), the separated DCU was filtered off. The filtrate was evaporated to dryness under reduced pressure and the title compound was purified by column chromatography over silica gel using a mixture of ethyl acetate/hexane (8.5:1.5) as eluent. Yield: (269 mg, 62%); mp 97 C; R f : 0.46 (DCM:MeOH 9.5:0.5); UV (EtOAc) l max 265 nm. C18 HPLC (265 nm) t R ¼ 2.24 min; 1 H NMR (DMSO-d 6 ); d 1.23 (s, 9H, t- 

Decanoyl chloride (0.20 ml, 1 mmol) was added dropwise to an ice-cold stirred solution consisting of the lead compound 4 (269 mg, 1 mmol), DMAP (0.32 g, 2.6 mmol) in pyridine (25 ml) and the reaction mixture stirred overnight at room temperature under anhydrous condition. Reaction mixture was concentrated under reduced pressure and partitioned between ethyl acetate and water. The organic fraction was concentrated to a residue and the product was purified by column chromatography over silica gel using ethyl acetate/hexane (9.5:0.5) as eluent. Yield: (200 mg, 59%) mp 160 C; 10.04 (s, 1H, NH, Thy); 13 

Cell cultures were prepared in microtiter trays and inoculated with 100 CCID50 (1 CCID50 corresponding to the virus stock dilution that proved infective for 50% of cell cultures). After 1 h virus adsorption to the cells at 37 C, the residual virus was replaced by cell culture medium (Eagle minimum essential medium supplemented with 3% fetal calf serum) and various concentrations of the test compounds. Virus cytopathogenicity was recorded as it reached completion in the untreated virus-infected cell cultures, i. e., at 1e2 days for vesicular stomatitis virus; 2 days for coxsackie; 2e3 days for vaccinia, herpes simplex type 1 and 2 and sindbis; 4 days for respiratory syncytial virus and 6e7 days for reo and parainfluenza viruses [33, 34] . The antiviral activity of compounds is expressed as EC 50 (the concentration (mM) required to inhibit virusinduced cytopathogenicity by 50%).

Cytotoxicity of all compounds was assessed on the basis of two parameters: (i) alteration of normal cell morphology, and (ii) inhibition of macromolecule (DNA, RNA and protein) synthesis. Cytotoxicity (CC 50 ) of compounds was examined by trypan blue exclusion test. To evaluate cytotoxicity, uninfected confluent cell cultures treated with various concentrations of test compounds were incubated in parallel with virus-infected cell cultures prepared in plastic trays containing 24 wells (16 mm diameter; Falcon plastics). After 2 days of incubation at 37 C in a CO 2 incubator, when the cell cultures were confluent, culture medium was removed from each well and 1 ml of maintenance medium containing serial concentrations of the test compounds was added. For cell control, 1 ml of maintenance medium without compound was added. All cultures were incubated at 37 C, and after 2 and 7 days of incubation, compounds were withdrawn and the viability of the cells was determined by the trypan blue exclusion method.

Antiviral screening against HIV-1 (IIIB and ROD strains) was monitored by the efficiency of test compounds to inhibit syncytia formation after HIV infection of MT-4 cells following the MTT method 

Molecular docking of compound 4 and its prodrugs 5, 6 and 7 into the dNTP-binding site of HIV-1 RT was carried out using DS 2.5 software and PDB code 3E01 of HIV-1 RT. CDOCKER was used and flexible docking was done. Stimulated annealing was done to get the energy-minimized structures of the molecules. The energy-minimized forms were then docked into HIV-1 RT. Visualization was done using PyMol software. All molecular modeling studies were performed on an Intel Pentium 2.99 GHz processor, 1.99 GB RAM with Windows XP professional version 2002 operating system.

and references therein

Proceedings of the 4th International Workshop on HIV Drug Resistance & Treatment Strategies

Drugs, microbes, host e the elements of chemotherapy

and references therein

Financial assistance from the Department of Biotechnology (DBT) and Indian Council of Medical Research (ICMR), New Delhi, Government of India for carrying out this work is sincerely acknowledged.