Rational drug design, synthesis and biological evaluation of dihydrofolate reductase inhibitors as...

FutureMedicinalChemistry

Preliminary Communication

part of

Rational drug design, synthesis and biological evaluation of dihydrofolate reductase inhibitors as antituberculosis agents

Nilesh R Tawari1, Seema Bag1, Archana Raju1, Arundhati C Lele1, Ranjeet Bairwa1, Mukti Kanta Ray2, MGR Rajan2, Laxman U Nawale3, Dhiman Sarkar3 & Mariam S Degani*,1

1Department of Pharmaceutical Sciences

& Technology, Institute of Chemical

Technology, Matunga (E),

Mumbai 400019, India 2Radiation Medicine Center,

Bhabha Atomic Research Centre,

Tata Memorial Hospital Annex, Parel,

Mumbai 400012, India 3Combichem-Bioresource Center,

Organic Chemistry Division,

CSIR-National Chemical Laboratory,

Pune 411008, India

*Author for correspondence:

Tel.: +91 22 33612213

Fax: +91 22 33611020

[email protected]

979Future Med. Chem. (2015) 7(8), 979–988 ISSN 1756-891910.4155/FMC.15.48 © 2015 Future Science Ltd

Future Med. Chem.

Preliminary Communication 2015/04/28

7

8

2015

Background: A series of 2,4-diamino-s-triazines was designed, with potential for activity against Mycobacterium tuberculosis (Mtb) dihydrofolate reductase enzyme, on the basis of virtual screening results and structure-based drug design. Results: The compounds were evaluated against Mtb (H37Rv) and their cytotoxicity was assessed using VERO cell lines. Of particular note, two compounds were found to have the most promising antituberculosis activity (6b minimum inhibitory concentration: 1.76 μM and 6i minimum inhibitory concentration: 1.57 μM) along with low cytotoxicity (CC50: >300 μM). The enzyme assay results of these two indicated significant inhibition of Mtb dihydrofolate reductase along with selectivity. Selected derivatives were tested against dormant tubercle bacilli in vivo and ex vivo indicating potential inhibition. Conclusion: This study provides promising antituberculosis dihydrofolate reductase inhibitors that can act as potential leads for further development.

Tuberculosis (TB), caused by the deadly obligate pathogen Mycobacterium tuberculosis (Mtb), remains the most fatal infectious dis-ease worldwide [1]. In the year 2012, WHO has estimated 8.6 million people developed TB and 1.3 million died of the disease [2]. The unusual cell wall barrier, ability to remain dormant and emergence of multi-drug (MDR) as well as extensively drug resistant (XDR) Mtb strains have made TB treatment difficult [3]. Hence, newer, more active anti-TB agents, having minimum cross resistance with existing drugs, are needed to combat TB. Dihydrofolate reductase (DHFR), a key enzyme of the folate path-way, plays an important role in cell growth and proliferation [4,5] and is found in many infective microorganisms including Mtb. Inhibitors of DHFR are successful agents for the treatment of various infectious diseases as well as for cancer (Methotrexate- MTX). However, very few classes of compounds are reported as Mtb DHFR inhibitors.

Gerum et al. developed an efficient, simple strategy for Mtb DHFR inhibitor screening, using an engineered strain of Saccharomy-ces cerevisiae, which was dependent on Mtb

DHFR for its growth. Using this system, they found that analogs of WR99210 are also potent inhibitors of the Mtb DHFR [6] Li et al. [7]. have reported three-dimensional structure of Mtb DHFR. In an attempt to synthesize specific Mtb DHFR inhibi-tors, a series of 2,4-diaminopyrimidines designed with a two-carbon tether between a glycerol-mimicking triol and the 6-posi-tion of the heterocycle and aryl substituents at the 5-position has been reported [8]. Reyn-olds et al. [9]. have reported synthesis of two boron-containing, ortho-icosahedral carbo-rane lipophilic antifolates. Siddiqui et al. [10]. have suggested a virtual screening workflow for the prioritization of compounds against Mtb DHFR. Rao et al. [11]. have reported structure-based design of a novel class of potent and selective small peptide inhibitor of Mtb DHFR.

Our research group has been involved in the search for DHFR inhibitors. Toward this goal we have reported molecular mod-eling studies for DHFR inhibitors and also synthesized several classes of novel, diverse inhibitors of DHFR for opportunistic patho-gens, including Mycobacterium avium [12–16].

For reprint orders, please contact [email protected]

Figure 1. Some of the hits obtained from the virtual screening with 2,4-diamino group substituted on s-triazine moiety.

H2N

NH2

N

N

N Cl

Cl

H2N

NH2

N

N

N

O

N

N

N

NH2

H2N

O

Cl

Cl

NH2

H2N

N

N

N

O

O

ZINC00002645 ZINC00004396 ZINC00011433ZINC00011427

980 Future Med. Chem. (2015) 7(8) future science group

Preliminary Communication Tawari, Bag, Raju et al.

Recently, we have carried out optimization studies for isolation of Mtb DHFR enzyme from recombinant Saccharomyces cerevisiae [17] strains and its purification using a novel affinity column [18].

Availability of high resolution crystal structures of Mtb DHFR [7] provides a good starting point for struc-ture-based drug design. In this light, the current work aims at structure-based drug design of potential Mtb DHFR inhibitors.

Computational aspectsThe structure of Mtb DHFR was obtained from Pro-tein Data Bank (PDB ID: 1DF7 with a resolution of 1.7 A°). Crystal structure was prepared by pro-tein preparation wizard in Schrödinger, LLC, New York, NY [19]. Structures of the reported ligands and the designed molecules were optimized and energy minimized using LigPrep module, Schrödinger [20]. Docking studies were performed using Glide molecu-lar docking protocol of Schrödinger [21]. The virtual screening of publicly available ZINC database [22] was carried out to identify hits as potential Mtb DHFR inhibitors (Figure 1).

SynthesisMelting points were recorded on Thermomix Comp-bell electronics, having oil-heating system and were uncorrected. The microwave reactions were carried out using CEM Focused Microwave System in mono-mode, Model Discover. Analytical thin-layer chroma-tography (TLC) was carried out on precoated plates SiO

2 (Silica gel 60, F 254, Merck). SiO

2 (Silica gel

420, Merck) was used for column chromatography using CombiFlash® RETRIEVE® system. FTIR spec-tra were recorded on ‘Buck scientific infrared spec-troscopy M500 spectrophotometer’ using KBr pellets. NMR spectra were recorded on JEOL AL 300 MHz spectrometer with DMSO-d6 as solvent. The mass

spectrum was recorded on a Waters Q/TOF Micro-mass spectrometer. All the reagents and chemicals used were of ‘reagent grade.’

The synthesis of the designed derivatives, 6a-r, was carried out according to the reactions depicted in Figure 2.

The designed compounds 6s-z, were synthesized using the synthetic route shown Figure 3.

General procedure for synthesis of 2,4-diamino-s-triazineSynthesis of substituted alcohol intermediatesThe methanolic solution (20 ml) of substituted alde-hyde or ketone (1, 0.021 mol), was treated with NaBH

4

(0.8 g, 0.021 mol) at room temperature. After comple-tion of the reaction (monitored by TLC), the reaction mixture was concentrated under reduced pressure and the solid residue obtained was partitioned between 25 ml water and 25 ml EtOAc. The organic layer was separated and the aqueous layer was extracted further with EtOAc (2 × 10 ml). The combined organic lay-ers were washed with brine (25 ml), dried over anhy-drous NaSO

4, and concentrated in vacuo, to yield

corresponding alcohol, 2.

Synthesis of substituted benzyl halidesPBr

3 (3.8 g, 0.014 mol) was added drop wise over a

period of 20 min to the clear solution of substituted alcohol, 2 (0.014 mol) in CCl

4 (5 ml), pyridine (1.65 g,

0.021 mol) and the reaction mixture was stirred for 2 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was poured in ice-cold water and extracted with dichlorometh-ane (2x10 ml). Dichloromethane layer was dried over anhydrous Na

2SO

4 and concentrated under reduced

pressure to yield crude alkyl bromide, 3, which was used for the next reaction without further purification.

Synthesis of substituted ether intermediatesThe mixture of p-hydroxybenzaldehyde (Figure 2) or 4-hydroxy-3-methoxybenzaldehyde or 3-hydroxy-4-methoxybenzaldehyde (Figure 3) (0.0163 mol), the

Key term

Dihydrofolate reductase: Enzyme of the folate pathway essential for cell survival.

Figure 2. Synthetic route for the designed s-triazine derivatives. 6a-r; (i) NaBH4, MeOH, RT.; (ii) PBr3, Pyridine, RT; (iii) K2CO3, CH3CN, Reflux; (iv) I2, liq. ammonia, Tetrahydrofuran, RT; (v) dicyandiamide, KOH, 2-methoxyethanol, 100°C. RT: Room temperature.

O

R1 R1 R1 R2

R2 R2

R2

R1

OH Br4-hydroxybenzaldehyde

OHC O

R2

R1

N

N

N

NH2

H2N

O

ONC

R1R2

(i) (ii) (iii)

(v)

(iv)

1 2 3

4 a-p

5 a-p6 a-r

6a: R1 = H, R2 = H;6c: R1 = 4-F, R2 = H;6e: R1 = 4-OMe, R2 = H;6g: R1 = 2, 4di-CI, R2 = H;6i: R1 = 3-CI, R2 = H;6k: R1 = H, R2 = Me;6m: R1 = H, R2 = Ph;6o: R1 = 4-F, R2 = Me;6q: R1 = 2,4-diCI, R2 = Me;

6b: R1 = 4-CI, R2 = H;6d: R1 = 4-SMe, R2 = H;6f: R1 = 4-Me, R2 = H;6h: R1 = 2-CI, R2 = H;6j: R1 = 3-OMe, R2 = H;6l: R1 = H, R2 = Et;6n: R1 = 4-CI, R2 = Me;6p: R1 = 4-Br, R2 = Me;6r: Cycloentyl instead of benzyl

www.future-science.com 981future science group

Diamino triazines as Mtb DHFR inhibitors Preliminary Communication

alkyl bromide, 3, (0.0163 mol) and K2CO

3 (3.39 g,

0.024 mol) in acetonitrile (20 ml) was refluxed for 12–24 h. After completion of reaction (monitored by TLC), the reaction mixture was concentrated to dry-ness under reduced pressure. The obtained residue was partitioned between 25 ml water and 25 ml EtOAc. The organic layer was separated, and the aqueous layer was extracted further with EtOAc (2 × 10 ml). The combined organic layers were concentrated under reduced pressure to obtain crude product which on column chromatography yielded the corresponding ether derivative, 4.

Synthesis of substituted nitrile intermediatesTo the solution of, 4 (0.0040 mol) in THF (5 ml); liq. ammonia (10 ml) was added at room temperature and the reaction mixture was stirred for 30 min, to obtain a white colored suspension. Iodine crystals (1.13 g, 0.0044 mol) were added to the above suspension, which resulted in dark brown reaction mixture, which was stirred further at room temperature. Within 30 min of stirring at room temperature, decolorization of the reac-tion mixture was observed. Stirring was continued for additional 1 h. The reaction mixture was then treated with saturated solution of sodium thiosulphate (20 ml) and 25 ml EtOAc was added subsequently. The organic layer was separated and the aqueous layer was extracted further with EtOAc (2 × 10 ml). The combined organic

layers were washed with brine (25 ml), dried over anhy-drous NaSO

4, and concentrated in vacuo, to yield the

corresponding nitrile derivative, 5.

Synthesis of substituted s-triazine derivativesDicyandiamide (0.24g, 0.0029 mol), KOH (0.20 g, 0.0036 mol) and water (0.5 ml) were added to solu-tion of, 5, (0.0024 mol) in 2-methoxyethanol (5 ml). The resultant reaction mixture was heated in an oil bath at 100°C for 5–6 h. After completion of reaction, water was added to the reaction mixture that resulted in precipitation of solid, which was filtered, washed with hot water (50 ml) and dried in oven to afford the corresponding s-triazine derivatives, 6.

Representative synthetic protocol for s-triazines6-(4-(benzyloxy)phenyl)-1,3,5-triazine-2,4-diamine (6a): To a clear solution of p-hydroxybenzaldehyde (1 g, 0.082 mol) in acetonitrile; K

2CO

3 (1.69 g,

0.0122 mol) and benzyl chloride (1.03 g, 0.082 mol) was added according to of general procedure to yield 1.44 g, (yield: 83%) of 4-(benzyloxy)benzaldehyde. To the clear solution of 4-(benzyloxy)benzaldehyde (1.2 g, 0.0056 mol) in THF (5 ml); liq. ammonia (10 ml) and iodine crystals (1.6 g, 0.0062 mol) were added according to of general procedure to yield 1.05 g (yield: 89%) of 4-(benzyloxy)benzonitrile. 4-(benzy-

Figure 3. Synthetic route for the designed s-triazine derivatives. 6s-z; (iv) I2, liq. ammonia, Tetrahydrofuran, room temperature; (v) dicyandiamide, KOH, 2-methoxyethanol, 100°C.

Br

R1 R2

OOHC

O R2R1

O

O R2

NC

R1

N

N

N

NH2

O

O

R2H2N

H2N

R1

O

O

R2

OHCR1

O

O

R2

NCR1

R1

N

N

N

NH2

O

OR2

(iii)

(iv)

(iv) (v)

(v)

3

4 s-v

4 w-z 5 w-z6 w-z

5 s-v6 s-v

6s: R1 = 3-CI, R2 = H;6t: R1 = 4-F, R2 = H;6u: R1 = 4-OMe, R2 = H;6v: R1 = 4-CI, R2 = H

6w: R1 = 3-CI, R2 = H;6x: R1 = 4-F, R2 = H;6y: R1 = 4-OMe, R2 = H;6z: R1 = 4-CI, R2 = H

4-hydroxy-3-methoxybenzaldehyde

3-hydroxy-4-methoxybenzaldehyde



loxy)benzonitrile (1.0 g, 0.0048 mol) was dissolved in 2-methoxyethanol (5 ml). To this clear solution; dicyandiamide (0.44 g, 0.0052 mol), KOH (0.53 g, 0.0095 mol) and water (0.5 ml) were added according to of general procedure to get 1.23 g (yield: 88%) of 6-(4-(benzyloxy)phenyl)-1,3,5-triazine-2,4-diamine (6a). M.P. 172–175°C; IR (KBr): V

max cm [-1]3486,

3367, 3169, 3113, 1654, 1623, 1384, 1250 [1]; H-NMR (300MHz, DMSO): δ 8.20 (d, 1H), 7.78–7.88 (d, 2H, 1H, D

2O exch.), 7.30–7.50 (m, 5H), 7.20 (bs, 1H,

D2O exch.), 7.18–7.20 (bs, 1H, D

2O exch.), 7.00–7.18

(m, 2H), 6.60 (bs, 1H, D2O exch.), 5.20 (s, 2H) [13];

C-NMR (60 MHz, DMSO): δ 167.36, 160.78, 160.62, 136.70, 129.71, 129.64, 129.31, 128.69, 128.41, 128. 26, 128.03, 127.88, 127.71, 126.71, 114.18, 69.3; M/S 294.08 [M + 1] [+]

The details of synthesis and characteriza-tion of all the derivatives have been given in the supplementary information.

Biological evaluationAll the synthesized compounds were screened against Mtb H

37Rv using two fold dilution technique, in

order to determine the actual minimum inhibitory concentration (MIC) using resazurin microtiter assay (REMA) [23,24]. MIC is the lowest concentration at which complete inhibition was observed and was determined by visual inspection (color change from blue to pink in case of growth)(protocol details in sup-plementary information) (Table 1). Isoniazid (INH), methotrexate (MTX) and trimethoprim (TMP) were used as reference drugs. The compounds were also

evaluated for toxicity on mammalian VERO cell line (C1008) (African monkey Kidney epithelial cells) using 96-well microtitre plate and the CC

50 values

were determined [25].Representative compounds in the range of most

active (<2 μM- 6b, 6i), active (∼5 μM 6d, 6z), moderately active (1–20 μM 6x, 6w) and relatively inactive (20–100 μM 6l, 6q) were selected for fur-ther screening against dormant tubercle bacilli. Com-pounds with activity >100 μM were considered as inactive and not considered for further studies.

The above selected compounds were tested for their in vitro and ex vivo effects against dormant tubercle bacilli M. tuberculosis H

37Ra ATCC 25177

which is susceptible to control drugs (Rifampicin, Isoniazid, Ethambutol and Pyrazinamide). Com-pounds were screened for their inhibitory effect on Mtb by in vitro according to standard XTT Reduc-tion Menadione Assay (XRMA) protocol as described previously [26,27]. The MIC was recorded as the low-est concentration/highest dilution of the compounds/control drugs that completely inhibited the growth of Mycobacterium cultures. Results are described in Table 2.

The ex vivo effects of compounds against dormant state M. tuberculosis H

37Ra was done according to stan-

dard NR Assay protocol as described previously [28]. (Protocol described in Supplementary Information).

Enzyme assayThe two most active derivatives were subjected to enzyme assay. DHFR activity was determined



spectrophotometrically [29] by monitoring the oxi-dation of NADPH for 2 min at 340 nm and 37°C. Standard assay mixture contained 50 mM potas-sium phosphate (pH 7.4), 5 mM DTT, and 60 μM NADPH and the reaction was started after 3 min of incubation by the addition of 45 μM DHF. A unit of the enzyme activity is defined as the amount of the enzyme converting 1.0 μmol of DHF and NADPH to THF and NADP [+] per min. This is calculated from the change in absorbance at 340 nm under spec-ified assay condition [30]. The assay was carried out in 96-well microtiter plates and analyzed using the micro plate reader with Gen5 software from Synergy H1 BioTek in the kinetic mode.

Results & discussionsDesign of molecules was carried out using rational design approach, wherein initially the reported inhibi-tors of Mtb DHFR were studied using molecular dock-ing to understand key interactions; followed by virtual screening to identify potential hits. The obtained vir-tual hits were modified manually to incorporate groups that improve interactions with the enzyme active site and allow series expansion. The designed compounds were synthesized and evaluated biologically, using in vitro whole cell assay against Mtb followed by enzyme assay, for the most active derivatives, against Mtb and human DHFR. Cytotoxicity evaluation was carried out using MTT assay in VERO cell line. Selected com-

Table 1. In vitro antituberculosis activity and the cytotoxicity of s-triazine derivatives 6a-r.

NH2

H2N

N

N

N

O

R2

R1 R1

NH2

H2N

N

N

N

O

R2

O

NH2

N

N

N

H2NO

R2O

R1

6 a-r 6 s-v 6 w-z

Code R1 R2 MIC H37Rv (μM) CC50VERO cell line (μM)6a H H 87.36 463.656b 4-Cl H 1.76 332.556c 4-F H 3.95 337.286d 4-SMe H 5.98 456.676e 4-OMe H 3.37 368.026f 4-Me H 29.45 494.556g 2,4-diCl H 66.07 364.436h 2-Cl H 10.71 341.716i 3-Cl H 1.57 387.476j 3-OMe H 38.66 504.106k H Me 2049.78 315.606l H Et 40.86 323.616m H Ph 18.87 416.876n 4-Cl Me 8.57 354.016o 4-F Me 67.81 344.266p 4-Br Me 56.47 282.206q 2,4-diCl Me 89.36 273.766r -cyclopentyl† - 244.62 405.436s 3-Cl H 6.26 293.466t 4-F H 3.39 359.956u 4-OMe H 3.91 387.696v 4-Cl H 3.63 408.066w 3-Cl H 18.45 285.066x 4-F H 10.25 439.446y 4-OMe H 12.00 399.016z 4-Cl H 4.92 335.39TMP - - 28.9 NDMTX - - 0.97 NDINH - - 1.88 > 500†Instead of benzyl. INH: Isoniazid; MTX: Methotrexate; ND: Not determined; TMP: Trimethoprim.



pounds were tested against dormant tubercle bacilli in vitro and ex vivo.

Computational aspectsThe Glide docking protocol was validated, using flex-ible docking protocol, by reproducing the observed crystallographic structure of the native ligand, Metho-trexate, with an RMSD value of 1.85 A°.

Molecular docking was carried out in the active site of Mtb DHFR for inhibitors reported in the literature, to understand the forms of interaction of the inhibitors and predict their binding orientation. These studies as well as referred literature indicated the necessity of 2,4-diamino substituted nitrogen containing hetero-cycle for Mtb DHFR inhibition. Further, the distal steric/hydrophobic part was found to be important for potency and selectivity. The bridge joining these two parts was found to govern the orientation of the mol-ecule in the active site. The bridge, hence has a direct impact on activity and selectivity by governing the alignment of the two parts in the active site (Figure 4).

Thereafter, virtual screening using public data-base was carried out, using Virtual Screening Work-flow implemented in Maestro, which returned several hits. In majority of the virtual hits, the 2,4-diamino groups were substituted on pyrimidine, dihydrotri-azine, quinazolie or pteridine nucleus, which have been explored extensively for DHFR inhibitory activity in various microorganisms. Interestingly, few virtual hits (Figure 3) had 2,4-diamino groups substituted on s-tri-azine ring system. As the 2,4-diamino-s-triazine ring system has not been largely explored for DHFR inhibitory activity, this feature was taken as starting point for design of inhibitors.

Design of molecules (6a-z)Based on structures of virtual hits and gained insight about the inhibitory potential by molecular docking of reported inhibitors, a scaffold was designed, using 6-phenyl-s-triazine-2,4-diamine as the basic struc-tural feature. Hydrophobic groups were added on the phenyl ring for favorable hydrophobic interactions in the receptor active site. Figure 4 depicts the designed scaffold along with anticipated interactions in receptor active site.

The compounds 6a-r were designed with R = H (Figure 4) and various substituents on the terminal aro-matic ring in order to have structural diversity using Topliss approach [31]. Compounds 6s-6z were designed to check the effect of additional methoxy group on the phenyl ring directly attached to the triazine ring.

The designed molecules were studied by molecular docking studies. Figure 5 depicts the docked pose of compound, 6a, in the active site of Mtb DHFR. The molecular docking studies revealed that as anticipated the 2,4-diamino-s-triazine ring binds at the bottom of active site forming hydrogen bonds with Ile94, Ile5 and Asp27. The 6-phenyl ring forms hydrophobic interac-tions with Ile20 and Phe31 and the distal phenyl ring forms favorable interactions with Pro51, Leu50, Leu57 and Phe31.

Based on the favorable in silico interactions of the designed derivatives with receptor active site, these analogs were considered for synthesis and biological evaluation.

Synthesis of moleculesThe designed compounds 6a-r were synthesized using the synthetic route shown Figure 2. The substituted ben-zyl bromides, 3, required for the synthesis of target com-pounds were prepared by reduction of corresponding substituted aldehyde/ketone, 1, using NaBH

4 in metha-

nol. The reduced products, 2, were then converted to

Table 2. In vitro antituberculosis activity of s-triazine derivatives against dormant tubercle bacilli in vitro and in infectious macrophage model.

Code MIC against Mtb (H37Ra) Dormant (μM) MIC against Infection Model of Mtb (H37Ra) Dormant (μM)

6b 48.51 71.79

6d 218.91 285.79

6i 131.86 137.75

6l 74.15 76.67

6q 26.26 34.55

6w 47.61 57.69

6x 282.15 283.33

6z 59.45 33.09

Rifampicin 0.061 0.058

Key term

2,4-Diamino-s-triazine: Class of nitrogen heterocyclic compounds.

Figure 4. Designed scaffold and with anticipated interactions with the active site residues of the hits obtained from the virtual screening with 2,4-diamino group substituted on s-triazine moiety.

Favorable interactionwith Phe 31

Necessary H-bonding interaction at the bottom of active site lle 94, lle 5 and Asp 27

Potency salt bridgewith Asp 27

Hy/Ar = Hydrophobic aromaticR = -H/ -OCH3

Hydrophobic interactionsPro 51, Leu 50,Leu 57, Phe 31

NN

N

O

R

H2N

NH2

Hy/Ar

Figure 5. Docked pose of 6a in the active site of Mycobacterium tuberculosis dihydrofolate reductase.

LEU 50LEU 57

ILE 20

PHE 31

ALA 7

TRP 6

ILE 5

ILE 94

ASP 27

GLN 28

2.7742.144

UNK 900

PRO 51



corresponding substituted benzyl bromides, 3, using PBr3

and pyridine. The isolated benzyl bromides, 3, which were highly lacrymatic in nature, were used directly for further etherification reaction with 4-hydroxybenzalde-hyde, in presence of K

2CO

3 using acetonitrile as solvent,

to yield corresponding intermediate aldehyde deriva-tives, 4a-p. The intermediate, 4r, required for synthesis of, 6r, was synthesized by using cyclopentyl bromide instead of benzyl halide in the etherification reaction. The aldehyde intermediates, 4a-r, were converted to corresponding nitriles, 5a-r, in presence of iodine and aqueous ammonia. The nitriles, 5a-r, were converted to target compounds, 6a-r, using dicyandiamide and KOH with 2-methoxyethanol as solvent.

For the synthesis of derivatives 6s-z, the alkyl bro-mide intermediates, 3, obtained by the synthetic route shown in Figure 2, were reacted with 4-hydroxy-3-me-thoxybenzaldehyde or 3-hydroxy-4-methoxybenz-aldehyde, in presence of K

2CO

3 using acetonitrile as

solvent, to yield corresponding intermediate aldehyde derivatives, 4s-v and 4w-z, respectively (Figure 3). The aldehyde intermediates, 4s-z, were converted to cor-responding nitriles, 5s-z, in presence of iodine, aque-ous ammonia. The nitriles, 5s-z, were converted to target compounds, 6a-z, using dicyandiamide, KOH

and 2-methoxyethanol as solvent. The synthesized compounds, 6a-z, were suitably characterized.

Biological evaluationThe in vitro whole cell activity evaluation against Mtb H

37Rv indicated that the unsubstituted parent com-

pound, 6a, has activity ∼87 μM, giving a good start point for further modifications. Replacement of distal benzyl substitution with pentyl ring led to decrease in



the activity thus indicating the importance of terminal aromatic nucleus. Among the terminal aromatic substi-tuted analogues, mono-substituted terminal aromatic ring derivatives were more potent than di-substituted analogues. Methoxy group was incorporated to probe the space available in the active site. It was hypothe-sized that adding electronegative bulky group would fit in the space available in active site more tightly. Fur-thermore, this group will help to orient the phenyl ring for proper binding. Addition of this methoxy group on the 6-phenyl ring of the s-triazine, led to derivatives with decreased activity, exceptions being 6t and 6u which retained the activity of the corresponding par-ent derivatives. All derivatives with the terminal benzy-loxy group at meta position to the s-triazine ring have shown decreased activity as compared with the corre-sponding para substituted analogues. The substitution of the benzyl group by pentyl group showed decreased activity due to the loss of strong aromatic interactions in the distal part of active site. Addition of methoxy group at the meta position or shifting the terminal benzyl oxy group to the meta position of the s-triazine ring resulted in decreased activity due to increased bulk at the meta position which added unfavorable inter-actions. Of particular note, two compounds, 6b and 6i, have shown excellent activity, better than first-line anti-TB agent Isoniazid and known, selective DHFR inhibitor Trimethoprim.

These compounds were also evaluated for their cyto-toxicity against VERO cell line. The values are given in Table 1. All active compounds were relatively nontoxic with selectivity index (CC

50/MIC) of > 200.

Testing against dormant tubercle bacilliThe in-vitro biological testing in Mtb H

37Ra dormant

cells and ex vivo testing using infected model of macro-phages THP1 monocytic cell line of the selected deriv-atives indicated that few compounds show potential inhibition as seen in Table 2.

Enzyme assayThe two most potent derivatives were tested for their ability to inhibit Mtb DHFR enzyme. Further, to

evaluate the selectivity of these compounds toward Mtb DHFR over human DHFR, enzyme assay using human DHFR was also carried out. MTX and TMP were used as antifolate standards. The enzyme assay results have been shown in Table 3.

MTX, a very potent DHFR inhibitor, is non-selec-tive and therefore toxic. The IC

50 values of the two

most potent compounds, 6b and 6i, indicate that they are less potent than MTX. However, they are 65- to 80-times more selective than MTX. TMP is less potent than MTX but is five-times more selective toward the pathogenic enzyme. Compounds 6b and 6i are three-times more potent and selective than TMP. Hence, the synthesized compounds may be considered as selective and potent Mtb DHFR inhibitors.

ConclusionBased on the virtual hits obtained as well as earlier lit-erature, a series of molecules was designed, incorpo-rating 2,4-diaminosubstituted heterocycle as the core moiety. Of particular note, two compounds, 6b and 6i have potent whole cell activity against Mtb (MIC: 1.76 and 1.57 μM) along with low cytotoxicity (CC

50:

>300 μM). The IC50

values of these compounds indi-cate that the compounds are potent and selective Mtb DHFR inhibitors. Additionally, a few of the molecules showed some inhibition of the dormant form of the bacilli both in vitro and ex vivo in infected macro-phages. Thus, this study has provided promising anti-TB DHFR inhibitors which can act as potential leads for further development.

Future perspectiveFolate inhibitors are an important class of compounds. The clinically useful drugs of this class are reported as inhibitors of the enzyme DHFR. This strategy has been proved to be a special tool, by taking advantage of the basic different features of the microbial and human folate metabolism and therefore allows target-ing the pathway at different steps. Due to increasing resistance development to the current drugs available for TB infection, increased efforts in anti-infective drug discovery are urgently needed. The future will

Table 3. Enzyme activity against of most active s-triazine derivatives against Mycobacterium tuberculosis and human dihydrofolate reductase.

Code IC50Mtb DHFR (μM) IC50 Human DHFR (μM) Selectivity

6b 5.175 ±0.195 83.3 ±2.4 ∼16

6i 5.62 ±0.11 72.49 ±0.66 ∼13

MTX 0.00825 ±0.00025 0.0016 ±0.0003 ∼0.2

TMP 16.09 ±0.32 83.46 ±3.46 ∼5

MTX: Methotrexate; TMP: Trimethoprim.



see some molecular studies of DHFR being combined with computer aided drug design to produce novel anti-TB agents. Also, we intend to explore the effec-tiveness of the activity against resistant strains of Mtb with our collaborators. The enzymes of the relatively unexplored folate pathway will provide a promising platform for developing selective and potent drugs that do not exhibit cross resistance.

Supplementary dataTo view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/full/10.4155/FMC.15.48

Financial & competing interests disclosureN Tawari is thankful to Department of Biotechnology, New

Delhi (BT/CR7858/MED/141142/2006), S Bag, R Bairwa are

thankful to University Grants Commission, New Delhi, A Raju

is thankful to Indian Council of Medicinal Research, New Delhi

(PHA-BMS-45/94/2012) and A Lele is thankful to Department

of Biotechnology, New Delhi, (BT/PR5441/BID/07/101/2004).

The authors have no other relevant affiliations or financial in-

volvement with any organization or entity with a financial inter-

est in or financial conflict with the subject matter or materials

discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this

manuscript.

Executive summary

• Design: A series of 2,4-diamino-s-triazines was designed as dihydrofolate reductase inhibitors against Mycobacterium tuberculosis.

• Synthesis and biological evaluation: Two of the synthesized compounds showed promising antituberculosis activity against Mtb H37Rv. The compounds were found to be relatively non-toxic by testing on VERO Cell line. Some of the molecules showed potential inhibition of the dormant form of the bacilli in vitro and in infected macrophages.

• Enzyme assay: In vitro enzyme assay against dihydrofolate reductase indicated potency and selectivity.• Conclusion: This study provides potential leads for further development as antituberculosis agents.

ReferencesPapers of special note have been highlighted as: • of interest; •• of considerable interest.

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