Bioorganic & Medicinal Chemistry Letters 21 (2011) 4404–4408

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused2-aminopyrimidines as antitubercular and antidiabetic agents

Nimisha Singh a, Sarvesh Kumar Pandey a, Namrata Anand a, Richa Dwivedi b, Shyam Singh c,Sudhir Kumar Sinha c, Vinita Chaturvedi b, Natasa Jaiswal c, Arvind Kumar Srivastava c, Priyanka Shah d,M. Imran Siddiqui d, Rama Pati Tripathi a,⇑a Medicinal and Process Chemistry Division, Central Drug Research Institute, CSIR, Lucknow 226 001, Indiab Division of Drug Target Discovery and Development, Central Drug Research Institute, CSIR, Lucknow 226 001, Indiac Biochemistry, Central Drug Research Institute, CSIR, Lucknow 226 001, Indiad Molecular and Structure Biology Division, Central Drug Research Institute, CSIR, Lucknow 226 001, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 March 2011Revised 8 June 2011Accepted 10 June 2011Available online 17 June 2011

Keywords:Cycloalkyl fused 2-aminopyrimidinesbis-Benzylidenecycloalkanonesa-Glucosidase inhibitorGlycogen phosphorylase inhibitorAntitubercular agentsDHFR inhibitor

0960-894X/$ - see front matter � 2011 Elsevier Ltd.doi:10.1016/j.bmcl.2011.06.040

⇑ Corresponding author. Tel.: +91 522 212411 418x2623938/2629504.

E-mail address: rp_tripathi@cdri.res.in (R.P. Tripat

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylam-ines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence ofNaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tubercu-losis H37Rv strain and the a-glucosidase and glycogen phosphorylase enzymes. Few of the compoundshave shown interesting in vitro activity with MIC up to 3.12 lg/mL against M. tuberculosis and very goodinhibition of a-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound40 exhibited about 58% ex vivo activity at MIC of 3.12 lg/mL. The present study opens a new gate to syn-thesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterialdihydrofolate reductase is the possible target of these compounds.

� 2011 Elsevier Ltd. All rights reserved.

Aminopyrimidines constitute a common structural motif in alarge number of naturally occurring and biologically active com-pounds.1 In fact aminopyrimidines with substitutents either at N-or at 4th-position are present in many drug-like scaffolds withgreat chemotherapeutic potential. They are associated with anti-fungal,2 pesticidal3 and enzyme inhibitory activities. They inhibitseveral kinases, such as Bcr-Abl kinase,4 rho-associated proteinkinase5 and glycogen synthase kinase (GSK3).6 One of suchcompounds, imatinib, a 2-aminopyrimidine is a highly selectiveBcr-Abl kinase inhibitor and has been successfully used for thetreatment of chronic myeloid leukemia.4 Several 2-substitutedquinazolines and 2-aminopyrimidines are known as broad spec-trum antibacterial agents.7 Quinazolines are known to inhibit thecyclin-dependent kinases (Cdks), especially Cdk4, associated withthe cell cycle response to growth signals and the initiation of celldivision; thus they have potential to be exploited as anticanceragent also.8 A number of compounds with pteridine, quinazolineand pyrimidine rings are potent inhibitors of dihydrofolate reduc-tase (DHFR) enzyme, required for de novo biosynthesis of purines

All rights reserved.

4462; fax: +91 522 2623405/

hi).

and thymidylic acid.9 The well known antitubercular drug INHhas recently been reported to inhibit this enzyme.10 The aminopyr-imidines have recently been shown as potent inhibitors of DHFR inMycobacterium tuberculosis and malarial parasite.11 On the otherhand, amino quinazolines and tetrahydro-aminoquinazolines areconsidered to be derivatives of aminopyrimidines and possessanalgesic, narcotic, diuretic, antihypertensive, antimalarial, seda-tive, hypoglycemic, antibiotic and antitumor activities12 besidestheir agricultural and industrial applications.13 An association be-tween diabetes mellitus (DM) and tuberculosis has long been ob-served by the clinicians.14 Patients with both diabetes and TBtake a longer time to respond to antituberculosis treatment, andare more susceptible for multi-drug resistant TB (MDR-TB).15

Hence, future research demands an aim to control the global epi-demic of diabetes which also affects the control and preventionof tuberculosis.

We are involved in design and development of new antituber-cular16 and antidiabetic agents17 for quite some time. In a recentstudy we have shown the anti-tubercular activity of bis-benzylid-enecycloalkanones.16a In continuation to these efforts, we werefurther prompted to synthesize a series of hybrid molecules, con-taining both the aminopyrimidine and benzylidene cycloalka-nones, with both antitubercular and antidiabetic potentials. The

N. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4404–4408 4405

compounds were evaluated in vitro against M. tuberculosis H37Rvstrain and also against a-glucosidase and glycogen phosphorylase,the target enzymes in diabetes. The protocol for the synthesis ofcompounds is very simple and economical. The compounds syn-thesized having dual activities may offer new leads to developnew antitubercular drugs for the treatment of tuberculosis in dia-betic patients.

The synthesis of the targeted compounds is very simple andstraight forward involving reaction of different bis-benzylidenecyclohexanones (1–11) and bis-benzylidene cycloheptanones (12–20)16a with guanidine hydrochloride in presence of NaH in DMFas solvent to give the respective 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydroquinazolin-2-ylamines (21–31) and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines(32–40) in moderate to good yields (Scheme 1). The startingbis-benzylidene cyloalkanones were very recently synthesized byus. Unlike earlier such reports18 for the synthesis of 2-aminopyrim-idines from chalcones and guanidine hydrochloride where anoxidizing agent is required to convert dihydropyrimidines topyrimidines, but in the present study the aromatization took placespontaneously with simple aerial oxidation.

All the compounds were purified by column chromatographyand were characterized on the basis of their spectroscopic (IR,ESMS, NMR and 13C NMR) data and microanalyses (Supplementarydata). In general, the IR spectra of the compounds exhibited char-acteristic strong absorbance in the range of 3400–3100 cm�1 for

+Reference 16a

1-20

Ethanolic KOH, RT

O

nArH

OO

n

Ar

Compounds 21-31 (n = 1)21. Ar = 2-Furfuryl, 22. Ar = Phenyl, 23. Ar = 4-F-phAr = 4-Cl-phenyl, 26. Ar = 4-OMe-phenyl, 27. Ar

28. Ar = 3,4,5 tri-OMe-phenyl, 29. Ar = 4-Benzyloxy

Compound 32-40 (n = 2)32. Ar = 2-Furfuryl, 33. Ar = Phenyl, 34. Ar = 4-Br-p36. Ar = 4-OMe-phenyl, 37. Ar = 3,4 di-OMe-pheny39. Ar = 1-Naphthyl, 40. Ar = 2-Naphthyl

Scheme 1. Synthesis of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydroquinazolin-2-ylam2-ylamines.

+Reference 16a

Ethanolic KOH, RT

O

ArH

OO

Ar

Scheme 2. Unsuccessful attempt for the synthesis of cyclo

O

e12

63

4

5e34

5

(I)steric clash(II

Figure 1. Conformers of cyclopentanone, cyclohexanone and

the –NH2 group. Further, in the 1H NMR spectra of aminopyrim-dines a two proton singlets of NH2 group were observed at aroundd 5.0–4.0 ppm were exchangeable with D2O. The only olefinic pro-ton, CH@C appeared as a singlet in the range of d 8.0–6.9 ppm. Thealicyclic and aromatic protons were observed at their usual chem-ical shifts. 13C NMR spectra of the compounds were also in accor-dance with their proposed structures.

However, reaction of bis-benzylidene cyclopentanones withguanidine hydrochloride to get the cyclopentyl fused aminopyrim-idines under the above reaction was unsuccessful as we were un-able to isolate the required products (Scheme 2).

It may be explained in terms of the conformations of bis-benzyl-idene cyclopentanones (Fig. 1). Cyclopentanone ring (I) is energet-ically most stable in the twist boat conformation. The 1,4 additionand cyclization of the guanidine at the position-5 in I is hindereddue to the steric clash provided by the methylene group at C-4 inthe twist boat conformation. Whereas the trans configuration ofthe –C@O group to the C2-substituted benzylidene prevents the1,4 addition at this position. However, in the case of bis-benzyli-dene cyclohexanones(II) and cycloheptanones(III) both the benzyl-idene groups although occupy the equatorial positions, remain inthe cis configuration to the –C@O group and facilitate the 1,4 addi-tion reaction.16a

A plausible reaction mechanism for the reaction to get amino-pyrimidines is proposed and depicted in Figure 2. It involves con-jugate Michael addition of guanidine (A) on one of the two

DMF, NaH, 100 oC,8-12 h

Guanidine hydrochloride

21-40

Ar

n

Ar

NN

Ar

NH2

enyl, 24. Ar = 4-Br-phenyl, 25.= 3,4 di-OMe-phenyl,phenyl, 30. Ar = 1-Naphthyl, 31. Ar = 2-Naphthyl

henyl, 35. Ar = 4-Cl-phenyl,l, 38. Ar = 3,4,5-tri-OMe-phenyl,

ines and of 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-

DMF, NaH, 100 ºC,

8-10 h

Guanidine hydrochlorideAr

Ar

NN

Ar

NH2

pentane fused 2-aminopyrimidines resulting failure.

O

e

eO

e

e

1

2

2

1

4

5

3 6

7

) (III)

cycloheptanone rings of bis-benzylidene cycloalkanones.

Ar

Oδ

-

+

ArAr

O

Ar

δ

H2N NH.HCl

NH2 NaH

H2N NH

NH

NHHN

NH2

Ar

O

Ar

N

Ar

N

NH

HN

Ar

N

NH

H

Ar

HO

H

Ar

-H2ON

Ar

N

NH2

H

H

Ar

[O]

N

Ar

N

NH2

Ar

nn

n

nnn

n

(A)

(B)

(C)

(D)(E)(F)

(G)

-ΝaCl-H2 1,4−addition

cyclization/condensation

Figure 2. The plausible reaction mechanism for the formation of 2-aminopyrimidines.

Table 1In silico docking score and in vitro inhibitory activities against the M. tuberculosisH37Rv strain and a-glucosidase and glycogen phosphorylase inhibitory activity ofcompounds 21–40

Compound Finaldockingenergy(kcal/mol)

C Log Pa MICH37Rv(lg/mL)

% Inhibition

a-Glucosidase

Glycogenphosphorylase

21 �13.346 3.691 >12.5 �17.3 �25.222 �13.415 5.129 6.25 �20.1 �6.1023 �16.665 5.424 12.5 �25.7 �21.124 �14.605 6.864 12.5 �71.8 �33.925 �12.729 6.564 >12.5 �72.8 �18.326 �11.911 5.094 >12.5 �25.3 �10.027 �11.336 4.541 >12.5 �29.3 �11.328 7.847 3.881 >12.5 �14.3 �30.429 �14.644 4.541 >12.5 �53.1 �0.8730 �13.304 7.477 12.5 +13.7 +11.3031 15.755 7.447 6.25 �6.0 �5.032 �9.700 4.250 >12.5 �4.87 +34.333 �14.017 5.668 >12.5 �0.07 �11.334 �12.828 7.423 >12.5 �5.86 �43.035 �12.580 7.123 >12.5 11.4 �11.336 �7.303 5.653 >12.5 �63.2 �33.937 �13.900 5.100 6.25 �6.4 �14.838 �9.624 4.440 >12.5 �19.6 �43.039 �16.486 8.034 >12.5 �26.9 �37.440 �21.392 8.036 3.12b �19.7 �37.7Isoniazid – �0.668 0.75 �39.0 –Ethambutol – �0.1188 3.25 – �39.0

a C Log P was determined by OSIRIS Property Explorer Programme, available athttp://www.organic-chemistry.org/prog/peo/.

b Nontoxic at a concentration of 10� MIC (31.2 lg/mL) to VERO cell line andmouse bone marrow derived macrophages.

4406 N. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4404–4408

olefinic bonds of enone moiety (B) to give the intermediate (C) fol-lowed by reaction between the amine group and the keto group ofenone to give cyclic product (D), on dehydration leads to a dihydro-pyrimidine skeleton (E). The latter on tautomerization gives anintermediate (F) which aromatizes itself by the aerial oxidationinto the respective 2-aminopyrimidine nucleus (G). The aromatiza-tion in dihydropyrimidines reported earlier in such reaction gener-ally requires K3Fe(CN)6 for the oxidation in an additional step ofaromatization.18 In the case of NaH in DMF, the most acidic tertiaryproton get abstracted very easily and facilitates the aerial oxidationto give required product.

All of the above aminopyrimidine derivatives (21–40) wereevaluated for their antitubercular activity against the virulentstrain of M. tuberculosis H37Rv using Agar microdilution method.19

INH and ethambutol were used as standard drugs. As evident fromTable 1, compounds 22, 23, 24, 30, 31, 37 and 40 exhibited moder-ate to potent antitubercular activity against M. tuberculosis, H37Rvwith MIC ranging from 12.5 to 3.12 lg/mL. It is evident that inthese compounds the change in antitubercular activity profilesby and large does not correlate with the substitutions on the arylring. However, compounds 30, 31 and 40 with naphthalene ringshow moderate to very good in vitro activity with MIC 12.5, 6.25and 3.12 lg/mL, respectively. Compounds 22, 23, 24 and 37 whichhave either unsubstituted or substituted phenyl groups also showgood activity with MIC values ranging between 6.25 and 12.5 lg/mL. Subsequent cytotoxicity evaluation of compound 40 was car-ried out against a mammalian cell line (VERO)20 and mouse bonemarrow derived macrophages21 showing no toxicity. Due to its po-tential activity and non toxic nature, compound 40 was furtherevaluated for their potential effect to kill intracellular (ex vivo) M.tuberculosis H37Rv in mouse bone marrow derived macrophages.22

Compound 40 led to the inhibition of 58% growth of intracellular

Figure 3. (a) Compound 23, (b) compound 31, (c) compound 39, and (d) compound 40 in the inhibitor binding site of M. tuberculosis DHFR (PDB code: IDF7). The proteinmolecule is shown in the surface representation, whereas docked compounds are illustrated in the stick representation. (e) Residues involved in interaction with compound40 in the docked model. (f) Simultaneous utilization of NADP and MTX binding sites by compound 40 in the docked model.

N. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4404–4408 4407

bacterium at its MIC of 3.12 lg/mL as compared to standard drugINH with P99% reduction in the growth of the bacterium. Molec-ular modeling was also carried out for all the synthesized com-pounds against M. tuberculosis DHFR.

The above aminopyrimidines (21–40) were also screenedin vitro for their inhibitory potential against a-glucosidase and gly-cogen phosphorylase enzymes in order to see their antidiabetic po-tential as shown in Table 1. The enzyme inhibitory activities weredetermined using the earlier reported protocols.23,24 Except com-pounds 31, 34, 35 and 37 other compounds of the series exhibitedmoderate to good inhibition of a-glucosidase and glycogen phos-phorylase at the concentration of 100 lM. The range of inhibitionfor a-glucosidase and glycogen phosphorylase is 13–72% and

10–40%, respectively. A closure look into the enzyme inhibitoryactivities revealed that among all these compounds the substitu-tion of the phenyl rings of arylidene moieties results in the betterinhibition of a-glucosidase as compared to other compounds withthe unsubstituted aromatic rings. Thus compounds with 4-bromo-(24), 4-chloro- (25), 4-benzyloxy- (29) and 4-methoxy (36) groupsin the phenyl ring show very good inhibition against a-glucosidasewith 71.8%, 72.8%, 53.1% and 63.2% inhibitions, respectively. Simi-larly compounds with 4-substituted phenyl rings in the aboveamino pyrimidines were better inhibitors of the glycogen phos-phorylase as compared to those with unsubstituted phenyl ring.The most potent anti-TB molecule, compound 40 with 2-naphthylsubstituent showed inhibition of both the enzymes, a-glucosidase

4408 N. Singh et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4404–4408

and glycogen phosphorylase. It was also observed that these 2-aminopyrimidine derivatives with fused cyclohexyl moiety showbetter a-glucosidase inhibitory activities than their cycloheptylanalogues.

In order to gain further insight into the antitubercular action ofthese compounds, molecular docking studies of M. tuberculosisDHFR (PDB 1DF7) were carried out and docking energies are shownin Table 1. Interestingly, docking energy obtained through molecu-lar modeling studies correlate well with the experimentally deter-mined MIC values. For instance, compounds 23 (MIC 12.5 lg/mL),31 (MIC 6.25 lg/mL) and 40 (MIC 3.12 lg/mL) display good bind-ing affinities with docking energies of �16.665, �15.755 and21.392 kcal/mol, respectively. Interestingly, the most potent com-pound 40 has the most favored docking energy and suggested thatcompounds with naphthalene ring are likely to exhibit greaterinhibition. This further adds to the confidence level of the presentanalysis.

Four top scorer compounds from docking studies, 23, 31, 39 and40 were further analyzed for their interactions with the protein insilico. These compounds occupy a long groove, largely lined byhydrophobic residues, adjacent to the NADP binding site of the pro-tein (Fig. 3a–d). In addition to making several hydrophobic interac-tions, compounds utilize their amino head group to interact withthe protein through some hydrogen bonds also (Fig. 3e). Interac-tions involving Phe31 seem to be particularly important. The resi-due, in most of the cases, is involved in the p–p interactions withthe aromatic ring of the compound. The groove already has beenshown to provide interaction site to methotrexate (MTX), anotherknown inhibitor for the mycobacterial DHFR.25 The compound 40simultaneously occupies the NADP binding site and the MTX bind-ing site (Fig. 3f). This simultaneous utilization, presumably, leads tothe greater in vitro inhibition by the molecule. Although compound39 shows the comparable docking energy with compounds 32 and31, it exhibits weaker inhibition in in vitro studies. The reason be-hind this anomaly becomes evident when the docked structure isanalyzed. The molecule, on account of substitution at second posi-tion, does not remain in the extended conformation as the com-pounds with substitution at the first position. Hence the moleculefails to occupy both the sites simultaneously and results in higherMIC value. Comparative analysis of docking data and in vitro studiesindicate that the data can be used for the design of novel inhibitorswith high degree of confidence.

In conclusion, we have synthesized cycloalkyl fused aminopyr-imidines with potent antitubercular, a-glucosidase and glycogenphosphorylase inhibitory activities. The docking studies withmycobacterial DHFR show very strong correlation with theirin vitro inhibition results. Thus the synthesized compounds withdual antitubercular and antidiabetic may offer new prototypes todevelop drugs for diabetic tuberculosis.

Acknowledgments

The authors thank DRDO New Delhi, CSIR New Delhi and DBTNew Delhi, for financial assistance. N.S. and N.A. are thankful toCSIR New Delhi for SRF and S.K.P. is thankful to CSIR New Delhifor RA fellowship. The authors sincerely thank SAIF Division, CDRI

Lucknow, for providing the spectral data. This is CDRI Communica-tion No. 7862

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bmcl.2011.06.040.

References and notes

1. Lagoja, I. M. Chem. Biodivers. 2005, 2, 1.2. Ackermann, P.; Stierli, D.; Jung, P. M. J.; Maienfisch, P.; Cederbaum, F. E. M.;

Wenger, J. F. Int. Pat. Appl. WO 03/047347 A1, 2003.3. Bretschneider, T.; Es-Sayed, M.; Fischer, R.; Maurer, F.; Erdelen, C.; L}osel, P. Int.

Pat. Appl. WO 02/067684 A1, 2002.4. Capdeville, R.; Buchdunger, E.; Zimmermann, J.; Matter, A. Nat. Rev. Drug

Discov. 2002, 1, 493.5. Sehon, C. A.; Lee, D.; Goodman, K. B.; Wang, G. Z.; Viet, A. Q. Int. Pat. Appl. WO

2006/009889 A1, 2006.6. Goff, D. A.; Harrison, S. D.; Nuss, J. M.; Ring, D. B.; Zhou, X. A. U.S. Pat. 6,417,185

B1, 2002.7. Kung, Pei-Pei; Casper, M. D.; Cook, K. L.; Wilson-Lingardo, L.; Risen, L. M.;

Vickers, T. A.; Ranken, R.; Blyn, L. B.; Wyatt, J. R.; Cook, P. D.; Ecker, D. J. J. Med.Chem. 1999, 42, 4705.

8. (a) Fry, D. W.; Garrett, M. D. Curr. Opin. Oncol. Endocr. Met. Invest. Drugs 2000, 2,40; (b) Garrett, M. D.; Fattaey, A. Curr. Opin. Genet. Dev. 1999, 9, 104.

9. (a) Agarwal, K. C.; Sharma, V.; Shakya, N.; Gupta, S. Bioorg. Med. Chem. Lett.2009, 19, 5474; (b) Chen, M. J.; Shimada, T.; Moulton, A. D.; Harrison, M.;Nienhuis, A. W. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 7435.

10. Argyrou, A.; Vetting, M. W.; Aladegbami, B.; Blanchard, J. S. Nat. Struct. Mol. Biol.2006, 13, 408.

11. (a) Kumar, A.; Siddiqui, M. I. J. Mol. Graph. Model. 2008, 27, 476; (b) Hawser, S.;Lociuro, S.; Islam, K. Biochem. Pharmacol. 2006, 71, 941.

12. Jhone, S.; Jucker, E. Birkauser Verlag: Basel 1982, 26, 259.13. Brown, D. J. The Chemistry of Heterocyclic Compounds; John Wiley and Sons:

New York, 1996.14. (a) Nichols, G. P. Am. Rev. Tuberc. 1957, 76, 1016; (b) Silwer, H.; Oscarsson, P. N.

Acta Med. Scand. Suppl. 1958, 335, 1; (c) Patel, J. C. Ind. J. Med. Sci. 1989, 43, 177.15. Edsall, J.; Collins, J.; Gray, J. Am. Rev. Respir. Dis. 1970, 102, 725.16. (a) Singh, N.; Pandey, J.; Yadav, A.; Chaturvedi, V.; Bhatnagar, S.; Gaikwad, A.

N.; Sinha, S. K.; Kumar, A.; Shukla, P. K.; Tripathi, R. P. Eur. J. Med. Chem. 2009,44, 1705; (b) Tripathi, R. P.; Pandey, J.; Kukshal, V.; Ajay, A.; Mishra, M.; Dube,D.; Chopra, D.; Dwivedi, R.; Chaturvedi, V.; Ramchandran, R. Med. Chem.Commun. 2011. doi:10.1039/c0md00246a; (c) Bisht, S. S.; Dwivedi, N.;Chaturvedi, V.; Anand, N.; Misra, M.; Sharma, R.; Kumar, B.; Dwivedi, R.;Singh, S.; Sinha, S. K.; Gupta, V.; Mishra, P. R.; Dwivedi, A. K.; Tripathi, R. P. Eur.J. Med. Chem. 2010, 46, 5965; (d) Ajay, A.; Singh, V.; Singh, S.; Pandey, S.;Gunjan, S.; Dubey, D.; Sinha, S. K.; Singh, B. N.; Chaturvedi, V.; Tripathi, R.;Ramachandran, R.; Tripathi, R. P. Bioorg. Med. Chem. 2010, 18, 8289.

17. (a) Tewari, N.; Tiwari, V. K.; Mishra, R. C.; Tripathi, R. P.; Srivastava, A. K.;Ahmad, R.; Srivastava, R.; Srivastava, B. S. Bioorg. Med. Chem. 2003, 11, 2911; (b)Bisht, S. S.; Fatima, S.; Tamrakar, A. K.; Rahuja, N.; Jaiswal, N.; Srivastva, A. K.;Tripathi, R. P. Bioorg. Med. Chem. Lett. 2009, 19, 2699.

18. Yu, M.; Pochapsky, S. S.; Snider, B. B. J. Org. Chem. 2008, 73, 9065.19. (a) Saito, H.; Tomioka, H.; Sato, K.; Emori, M.; Yamane, T.; Yamashita, K.

Antimicrob. Agents Chemother. 1991, 35, 542; (b) McClatchy, J. K. Lab. Med. 1978,9, 47.

20. Cory, A. H.; Owen, T. C.; Barltrop, J. A.; Cory, J. G. Cancer Commun. 1991, 3, 207.21. Mosmann, T. J. Immunol. Methods 1983, 65, 55.22. Skinner, P. S.; Furney, S. K.; Kleinest, D. A.; Orme, I. M. M. Antimicrob. Agents

Chemother. 1995, 39, 750.23. (a) Cogoli, A.; Mosimann, H.; Vock, C.; Balthazar, A. K. V.; Semenza, G. Eur. J.

Biochem. 1972, 30, 7; (b) Matsui, T.; Yoshimoto, S.; Osajima, K.; Oki, T.; Osajima,Y. Biosci. Biotech. Biochem. 1996, 60, 2019; (c) Hubscher, G.; West, G. R. Nature1965, 205, 799.

24. (a) Rall, T. W.; Sutherland, E. W.; Barthet, J. J. Biol. Chem. 1957, 224, 463; (b) VanTeeffelen, J. W.; Brands, J.; Stroes, E. S.; Vink, H. Trends Cardiovasc. Med. 2007,17, 101; (c) Nieuwdorp, M. J. Appl. Physiol. 2008, 104, 845.

25. Rongbao, L.; Sirawaraporn, R.; Chitnumsub, P.; Sirawaraporn, W.; Wooden, J.;Athappilly, F.; Turley, S.; Hol, W. G. J. J. Mol. Biol. 2000, 295, 307.