Indian Journal of Chemistry Vol. 428 , February 2003, pp. 372-381

Azole compounds designed by molecular modelling show antifungal activity as predicted

Rajeshri G Karki, Vijay M Gokhale, Prashant S Kharkar & Vithal M Kulkarni*

Pharmaceutical Division, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India Telephone: 91-22-4145616; Fax: 91-22-4145614; E-mail : vi thaI @biogate.com

Received 13 February 2002; accepted (revised) 8 August 2002

Rational approaches involving drug discovery technologies such as computational and combinatorial chemistry and high throughput screening have been useful tools to design and discover new drugs more effi ciently. The interplay among structure-acti vity relationships, computer modelling, chemical synthesis and pharmacological testing can lead to better products for a particular therapeutic purpose. The work presented in thi s paper reports an example of successfu l application of computer-aided drug design method to find new azole antifungal agents. The designed compounds have been synthesized in the laboratory and tested for anti fungal activity against Candida albicans ATCC 24433 in vitro. Two compounds exhibit good ac tivity ill vitro , which can be optimized for better activity .

With the continuing increase in clinically important fungal di sease, primarily seen in immuno­compromised patients, the need for new and improved systemically active antifungal agents continues l

.

There are substantially fewer antifungal agents. Since the launching of fluconazole 1 and

itraconazole 2 for the treatment of systemic mycoses a decade ago, the race to market a new azole continues2

.

The azole derivatives are completely synthetic, and many new compounds in this class have become available. In general, the broad antifungal activity of azoles includes most yeasts and filamentous fungi 3

.

The azoles act primarily on ergosterol biosynthesis at the C-14 demethy lation step, a cytochrome P450-dependent reaction. The resulting ergosterol depletion and accumulation of methylated sterols, interferes with the "bulk" functions of ergosterol4

• The structure of the plasma membrane is altered, making it more vulnerable to further damage and altering the activity of several membrane bound enzymes, such as those for nutrient transport and chitin synthesis. The overall

effect is fungistatic rather than fungicidal , limiting the utility of these drugs. Nevertheless, azoles are being used more frequently in the treatment of systemic mycoses, even in severely immunocompromised patients.

Though fluconazole, in particular, has excellent pharmacokinetic properties5 the emergence of resis­tant strains has made development of new azole antifungal agent a necessity. Development of resistance to azole antifungals particularly fluconazole involves development of energy driven efflux pumps which pump out azole antifungal from cell, or mutation in the active site of the enzyme6

. In this paper, we report the design, synthesis of new azoles and their antifungal activity.

Design strategy Docking analysis of a series of known azole

antifungal agents was performed earlier using structure-based drug design method.7 The amino ac id residues of cytochrome P450 14a-demethylase

oN, ~~H N~N r N \ -1 N:-I F N-

rN

N ;J C1:©rCI

'N 0 CH

l , .. ,"... 'c~CH~H3 "><0 0 / - /'"

0-1

F

0, J.,,,H -0-" 1\ -0-" '1-~ --~O N N N~N - '-I -

Fluconazole (1 ) Itraconazole (2)

KARKI el al.: AZOLE ANTIFUNGAL AGENTS 373

(Cyt-P45014aDM) involved in the interaction with azoles have been identified in our laboratory. This is depicted in Figure 1. The 2,4-difluorophenyl ring of voricona­zole is involved in interactions with hydrophobic resi­dues Leu244, Va1247, Phe98 and Tyr96. The 5-tluoro-

HYDROPHOBIC ACCESS CHANNEL

Leu 244

pyrimidine which forms the variable substituent in voriconazole is located in the hydrophobic access channel made of amino acid residues Tyr96, ThrlOl, Phe87 and Pro86. The N4 nitrogen of triazole forms a

co-ordinate bond with the heme iron of Cyt-P45014aDM.

Figure I- Amino acid residues involved in interaction with voriconazol Green: Vori conazole: Red : Heme; Blue: Amino ac id residues of Cyt-P4S0 14 DM

374 INDIAN J. CHEM., SEC B, FEBRUARY 2003

Imidazole or triazole and dihalophenyl ring systems constitute recognition moieties for azole antifungals while variable fragment portion is responsible for imparting properties such as meta­bolism, resistance development to the molecules etc. It was observed that the conformation adapted by the variable side chain must be linear since it is required for binding in the hydrophobic access channel. Here linearity stands for the straight conformation of the variable portion which requires that the torsion angle values around the bond (Figure 2) lie near 180°. The straight conformation of the variable portion places the four atoms defining the torsion angle (Figure 2) in one plane. Such binding is essential for proper positioning of the nitrogen on the heterocycle to form a co-ordinate bond with the heme iron of Cyt­P4S014aDM. Subsequently pharmacophore model was generated for azole antifungals using APEX-3D8

program which has helped in determining the minimum structural requirements for inhibition of Cyt-P4S014aDM. The alignment of the azole antifungals based on the pharmacophore model generated has been used in our laboratory as an input for Comparative Molecular Field Analysis (CoMFA), a 3D-QSAR method9

. Both the approachess.9 have supported the hypothesis of conformational rigidity and requirement of straight conformation for Cyt­P4S014aDM inhibition. Continuing in this area of research, we have recently reported a feature based pharmacophore for Candida albicans MyristoylCoA: Protein N-Myristoyltransferase (NMT) inhibitors lO

and synthesis of some inhibitors of NMT as potential 'f I II anti unga agents . Based on the combined results of these studies, we

designed a novel series of azoles by retaining both the difluorophenyl and the triazole ring while the variable fragment was selected such that it had linear conformation. The molecular modelling was performed using QUANTA software with CHARMm

~N ..... \' N N.=.1

F

Figure 2 - General structure showing the bond (thick line) about which the torsion angle is defined. The four atoms defining the

torsion angle are shown by the asterisk.

forcefield 12 running on Silicon Graphics IRIS 40/20 workstation 13.

Experimental Section All chemicals and solvents were obtained from

commercial sources and purified using standard procedure whenever required. The melting points were recorded as usual and are uncorrected. The structures of the compounds were confirmed by IR and I H NMR spectra. IR spectra were recorded on a Buck Scientific M-SOO spectrometer and IH NMR spectra on a Varian 360ML 60 MHz and Bruker AM 500 MHz FT-NMR with tetramethylsilane (TMS) as an internal standard. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. TLC wa~ performed on aluminium sheets pre-coated with Silica Gel 60 F254 . The purity of the compounds was determined by high­performance liquid chromatography (HPLC) method using Jasco PU 980 with UV /VIS detector. Data integration was performed using Borwin (version 1.21) software program.

Synthesis of 1-(lH-l,2,4-triazol-l-yl)-2-(2,4-difl­uorophenyl)-3-(2,6-dimethylmorpholin-4-yl)-pro­pan-2-ol 9. Compound 9 was synthesized as depicted in Scheme I. In the first step 1,3-difluorobenzene 3

V-F

F

3

CICH~OCI 1 AlCI3 .. Dichloromethane

:/ F 9=CI

:::.....1

F

4

CI9-

0

F ,N~ K,cO, N:-~N~F 1 + HN 1 --=~::..:.~ 1

:::..... '=N PTC :::.....

F F

4 5 6

Scheme I

KARKI et al.: AZOLE ANTIFUNGAL AGENTS 375

was subjected to Friedel-Crafts acylation by treatment with chloroacetyl chloride in presence of anhyd. alu­minium chloride to get 2,4-difluorophenacyl chloride 4. This was then reacted with 1,2,4-triazole 5 to get the ketone 6. This ketone was then reacted with di­methylsulfoxonium methylide obtained from tri­methylsulfoxonium iodide and sodium hydroxide in presence of a phase transfer catalyst, tetrabutylammo­nium bromide, to get a yellow oil of ox irane 7. The oxirane 7 was later treated with dimethyl morpholine 8 in presence of potassium carbonate to get the re­quired compound 9.

Preparation of 2,4-difluorophenacyl chloride 4. In a 250 rnL three-necked flask equipped with a dou­ble surface condenser, a mechanical stirrer, a drop­ping funnel and a heating mantle, dichloromethane (15 rnL) was added followed by anhyd. aluminium chloride (7.0 g, 0.053 mole). The temperature of the reaction was maintained at 10 °C using ice-bath. The mixture was stirred for 30 mjn and then chloroacetyl chloride (4.2 mL, 0.053 mole) in dichloromethane (10 rnL) was added dropwise during 45 min. The reaction mixture was stirred for 30 min after complete addi­tion. To this 1,3-difluorobenzene (4.29 rnL, 0.043 mole) in dichloromethane (5 rnL) was added over a period of 1 hr. The reaction mixture was refluxed for 3 hr. The progress of the reaction was monitored by TLC. The reaction mixture was cooled to room tem­perature and then poured in a thin stream onto a mix­ture of crushed ice and 3 N HCI (300 rnL) with stir­ring. The organic layer was separated and the aq. layer was extracted with dichloromethane (3 x 100 rnL). The combined organic layer was washed with sodium bicarbonate and water. The solvent was evaporated under reduced pressure to give viscous oily liquid, which solidified into colourless crystals after cooling, yield 8.0 g (95%); m. p. 52-54°C.

Preparation of 2'-(lH-l,2,4-triazol-l-yl)-2,4-difluoroacetophenone 6. In a 100 rnL two-necked flask equipped with a double surface condenser, a thermometer pocket, and a magnetic stirrer, compound 4 (5 g, 0.026 mole) in acetone (30 rnL) was added to a mixture sodium bromjde (1.33 g, 0.013 mole) and of tetrabutylammonium iodide (0.5 g, 10%) in acetone (50 rnL). The reaction mixture was refluxed for 2 hr and then filtered in vacuo. The filtrate was added dropwise to a stirred solution of 1,2,4-triazo\e 5 (l.99 g, 0.028 mole) and anhyd. potassium carbonate (5.38 g, 0.039 mole) in acetone (50 rnL) taken in a 3-necked flask maintained at room temperature. The reaction mixture was stirred for 30

min and acetone was evaporated. The residue was dissolved in ethyl acetate (50 rnL). The organic layer was washed with brine, dried over anhyd. sodium sulphate and concentrated under reduced pressure. The product was purified by acid-base treatment to get cream coloured solid, yield 2.9 g (50%); m. p. 94-96°C; TLC:Mobile phase = hexane:ethyl acetate (3:2), Rr = 0.46.

Preparation of 2-[(lH-l,2,4-triazol-l-yl)methyIJ-2-(2,4-difluorophenyl)oxirane 7 14

• In a single­necked flask fitted with a reflux condenser 2'-(1 H-1,2,4-triazol-1-yl)-2,4-difluoroacetophenone 6 (2 g, 0.009 mole) was dissolved in toluene (20 rnL) with stirring at room temperature. To this, cetrimide (0.2 g, 10%), trimethylsulfoxonium iodide (2.18 g, 0.0099 mole) and sodium hydroxide (0.4 g, 0.01 mole) dissolved in water (3 rnL) were added. The reaction mixture was heated to 60°C for 4 hr and poured over crushed ice after cooling. The organic layer was separated and the aq. layer was extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhyd. sodium sulphate and concentrated. The evaporation of solvent under reduced pressure gave a dark brown oil, which was used without further purification to avoid degradation of oxirane during purification, yield l.5 g (66%); TLC: Mobile phase = hexane:ethyl acetate (3:2), Rr= 0.54.

Preparation of 1-(lH-l,2,4-triazol-l-yl)-2-(2,4-difluorophenyl)-3-(2,6-dimethylmorpholin-4-yl)-pro­pan-2-ol 9. A mixture of oxirane 7 (l.5 g, 0.0063 mole), 2,6-dimethylmorpholine 8 (0.87 g, 0.0076 mole) and anhyd. potassium carbonate (l.28 g, 0.0093 mole) in toluene (25 rnL) was heated at 90°C for 9 hr. The organic layer was cooled and washed with brine. It was dried using anhyd. sodium sulphate and concentrated under reduced pressure to get a brown coloured semi-solid compound. The product was purified by chromatography on a silica gel column, eluting with a gradient of 5-10% hexane/ethy I acetate (v/v) to get a white coloured solid, yield l.3 g (45%); m. p. 133-134°C; TLC: Mobile phase = hexane:ethyl acetate (4:2), Rr = 0.68.

Synthesis of 1-(2' -chloro )-2,4-difluoropropiophe­none 10. Compound 10 was synthesized by a procedure similar to the synthesis of compound 4. The product was isolated by distillation at 110 °C under reduced pressure to get a colourless liquid, yield 6.9 g (67%); TLC: Mobile phase = hexane: ethyl acetate (5:0.4), Rr = 0.78.

376 INDI AN J. CHEM .. SEC B. FEBRUARY 2003

General procedure for synthesis of benzyloxy substituted azoles 11-13. The substituted and un­substituted benzy loxy azole compounds (compounds 11, 12 and 13 in Table I) were synthesized by procedure outl ined in Scheme II. In the first step the (u n)substi tuted benzy l alcohol was converted to ketone 11a-13a, by reacting with ei ther 2,4-difl uoro­phenacy l chloride 4 or 1-(2' -chloro)-2,4-difluoropro­piophenone 10. The ketone was then reacted with dimethylsul fo ni um methylide obtained from

(lrF Y

F

3

CH3 I

CICHCOCI / AlCI3

Di chlorom e thane •

CH3

O~C--LCI

¢rF F

10

Table I-Structures and spectral data of the synthesized compounds

Compe: RI No.

9 H

11 H < ) CH, O-

12 CH.\

C!-0-CH' O-

( }--CH,CHl - -

'L-Y

15 n

16 H F G>--CH,CH,-

34 i7.6,2900.9, 1605.8, 1429.6, 1259.3.

3369.8, i6 17.S, 1501.3, i427.4, 1274.6, 11 50.2. 852.9.

3369.8, 162 1, 1520. 1434.6, [27:.4. 1154.8, R64. 1

3362.3, 2952 .6, !603.4. 1493.6, 1260.2. 85 1.3

3380.2,2950.4, 1602 .2, 1458.6, 772.8

3348.6,2905.4. 1624.2.1211.6, 826.4

I H NMR (8 ppm)

1.0 (t, 6H, CH :1); 1.5-3 .0 (m, 611 of d imethyl morpholine ri ng); 3-4 (m, 4H, CH2); 4 .5 (s, I H, OH, D20 exchange­able); 6.4-7.5 (m, 3H, aromatic); 7.7 (s, IH , tri azole): 8.0 (s, I H, triazole)

1.3 (5, 21-1 , CH2): 1.9 (5, 2H, CH2): 4.2 (5, I H, 01-1 , 0 20 exchangeab le): 5.2 (2H, CH2); 6.4-8.0 (m. 8H. aromatic): 8. 1 (5. I H, triazole); 8.3 (5. I H. tri azole).

0.9 (d. 31-1, CH3): J.6 (2 1-/ , CH2): 3.5 (s. I H, OH. D20 exchangt::able); 4.9 (s. 2H, CH2); 5.2 (q, I H, CH); 6.6-7.8 (m, SI-i . aromatic); 7.9 (5, I H, triazole); 8.2 (5, I H, tri azole)

1.2 (d, 3H, CHJ): 2.80 (q, I H, CH); 3.70-5 .00 (m. 5H, CH2 and OH merged; IH D20 exchangeabie); 6.3-7 .3 (171, 7H, aromatic); 7.4 (s. I H, triazolc); 7.7 (5, I H, triazo!e).

2. 1 - 2.5 (m, 4H, CH2); 4 .2 ( I H, S, OH, 0 20 exchange­ab:c); 4.6 - 5.0 (5, 2H. CH2); 6.7 - 7.4 (m, 8H, aromatic); 7.6 (s, In, tri:lzole); 7.8 (s, I H, triazole) .

1.9 - 2.5 (41'1, m, CHz); 4. 1 (I H, S, 0 11 D20 exchange­able); 4.2 - 4.5 (s, 2H, CH~): 6.5 - 7.1 (Ill, 7H, 3romatic); 7.4 (s, I H, triazole); 7.6 (s , 11-1 , triazolc).

KARKI el al.: AZOLE ANTIFUNGAL AGENTS 377

F aq . NaOH , PTC ..

Toluene F

11a R1 =H=R2 12a R1 = CH3 R2 = H 13a R1=CH3 R2=CI

I (CH3bS+ I ·, PTC / base DMSO

t

R2frO F

NaH, DMF

F

11 RFH=R2

12 R1 = CH3 R2 = H 13 R1 = CH3 R2 = CI

F

11b R1= H=R2 12b R1 = CH3 R2 = H 13b R1 =CH3R2=CI

Scheme II

trimethylsulfonium iodide and sodi um hydride to get a yellow oil of oxirane llb-13b. The oxirane was later treated with sodium tri azolide to get the required compound 11-13.

Synthesis of ketone 11a-13a. In a three-necked f1a~k equipped with a dropping funnel, an air conden­ser and maintained at 25°C, compound 4 (or 10) (0.03 mole) was dissolved in toluene (30 mL). To this (un)substituted benzyl alcohol (0.03 mole) , tetrabutyl­ammonium bromide (10%) were added and stirred for 30 min. To this aq. sodium hydroxide solution (50%) (30 mL) was added dropwise and the reaction mixture was stilTed for I hr. The organic layer was separated and the aq. layer was extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhyd. sodium sulphate and concentrated. The evaporation of solvent under redu(:ed pressure gave an orange coloured semi-solid compound. The product was purified by chromatography on a silica gel column, eluting with a gradient of 5-10% hexane/ethyl acetate (v/v) to get a colourless liquid product.

Synthesis of oxirane llb-13b. A solution of ylide was prepared under nitrogen using trimethylsulfonium

iodide (0.88 g, 0.0043 mole), sodium hydride (0.260 g, 0.0108 mole, 55%) and dimethyl sulfoxide (10 mL). A solution of ketone 11a-13a (0.0036 mole) in dimethyl su lfoxide (10 mL) was added with stilTing and the reaction mixture was stirred at room temperature for 30 min . The reaction was quenched in ice-cold water. The product was isolated by extracting the aq. layer with ethyl acetate (2x50 mL). The organic layer was washed with water and dried over anhyd. sodium su lfate. Evaporation of the solvent under reduced pressure gave an oi ly oxirane llb-13b which was used without further purification in the next step.

Synthesis of (un)substituted benzyloxyazoles 11-13. 1,2,4-Triazole (0.26 g, 0.0038 mole) was added slowly to a suspensi on of sodium hydride (0.183 g, 0.0076 mole) (55% mineral oil dispers ion , washed with n-hexane) in DMF (IS mL) under stirring at O°C. When the hydrogen gas ceased to evolve, a soluti on of the crude oxirane llb-13b (0.0025 mole) in DMF (10 mL) was added. The mixture was then sti rred at 90°C for 2 hr. The reaction mixture was then poured onto crushed ice. The aq. layer was extracted with ethyl acetate

378 INDIAN J. CHEM .. SEC B, FEBRUARY 2003

(2x25 mL). The organic layer was washed with brine and dried over anhyd. sodium sulfate. The evaporation of the sol vent under reduced pressure gave a crude product. The product was purified by chromatography on a silica gel column, eluting with a gradient of 5-10% hexane/ethyl acetate (v/v).

The structures and the spectral data of the compounds synthesized are summarized in Table I.

General procedure for synthesis of phenethyl substituted azoles. The substituted and unsubstituted phenethyl azole compounds (compounds 15 and 16 in Table I) were synthesized by procedure outlined in Scheme III. In the first step the (un)substituted benzal­dehyde was reacted with 2,4-difluoroacetophenone 14 in presence of aq. sodium hydroxide and ethanol to get the corresponding chalcone 15a and 16a. The chalcone on selective reduction with tributylamine - formic acid and palladium on carbon catalyst gave the correspond­ing difluorophenyl ketone 15b and 16b'5 . The ketone was then reacted with dimethysulfoxonium methyl ide obtained from trimethylsulfoxonium iodide and sodium hydride to get corresponding oxirane 15c and 16c which on treatment with sodium triazolide in dry DMF resulted in compound 15 and 16 respectively.

Synthesis of chalcone 15a and 16a. In a three­necked flask equipped with a reflux condenser and a magnetic stirrer, 2,4-difluoroacetophenone (3.0 g, 0.019 mole) was dissolved in ethanol (30 mL) and cooled to 20°C. To this aq. sodium hydroxide solution (10%) (20 mL) was added with stirring followed by (un)substituted benzaldehyde (0.019 mole). The reac­tion mixture was maintained at 20°C overnight. The precipitated product was filtered , dried and recrystal·· I ized from ethanol to get 0.012 mole of the product.

Synthesis of ketone 15b and 16b. In a three­necked flask equipped with a reflux condenser and a magnetic stirrer, formic acid (98%) (0.45g, 0.0098 mole) was added slowly to a reaction mixture containing chalcone (0.01 mole) 15a or 16a, tribu­tylamine (2.3 g, 0.0125 mole) and of 10% palladium adsorbed on carbon (0.3 g). The reaction mixture was heated to 100 °C with stirring for 10 hr. It was then cooled to room temperature and filtered. The filtrate was extracted with dichloromethane. The organic layer was washed with 10% hydrochloric acid solu­tion followed by water and dried over anhyd. sodium sulfate. Evaporation of the organic solvent gave 0.004 mole of the product 15b and 16b.

~R2 R2

0:' R2Q-CHO qh y F

~I

F

14

.. NaOH I Methanol

N~ N b' N

F

15 R2= H 16 R2= F

F Pd/ C /'" .. ~ I HCOOH I (8u)3N

F

15a R2= H 16a R2= F

NaH I DMF

F

15b R2= H 16b R2= F

I (CH~~O+r, PTC t DMSO

F

15c R2 = H

16c R2= F

Scheme III

KARKI et at.: AZOLE ANTIFUNGAL AGENTS 379

Synthesis of oxirane ISc and 16c. A solution of ylide was prepared under nitrogen from trimethyl­sulfoxonium iodide (0.5 g, 0.0021 mole), sodium hydride (0.120 g, 0.005 mole, 55%) and of dimethyl sulfoxide (10 mL). A solution of ketone ISb or 16b (0.0021 mole) dissolved in dry DMSO was added with stirring and the reaction mixture was heated at 60°C for 30 min. The reaction was cooled to room temperature and quenched in ice-cold water. The product was isolated by extracting the aq. layer with ethyl acetate (2x50 mL). The organic layer was washed with water and dried over anhyd. sodium sulfate. Evaporation of the solvent under reduced pressure gave an oily oxirane ISc and 16c (0.0019 mole). As the oxirane was unstable it was used without further purification in the next step.

Synthesis of phenethyl substituted azoles 15 and 16. 1,2,4-Triazole (170 mg, 0 .0025 mole) was added slowly to a suspension of sodium hydride (110 mg, 0.0046 mole, 55% mineral oil dispersion, washed with n-hexane) in DMF (15 mL) with stirring at O°c. When the hydrogen gas ceased to evolve, a solution of the crude oxirane (0.0017 mole) in DMF (10 mL) was added. The mixture was then stirred at 90°C for 2 hr. The reaction mixture was then poured onto crushed ice. The aq. layer was extracted with ethyl acetate (2x25 mL). The organic layer was washed with brine and dried over anhyd. sodium sulfate. The evaporation of the solvent under reduced pressure gave crude product. The product was purified by chromatography on a silica gel column, eluting with a gradient of 5-10% hexane/ethyl acetate (v/v).

The structures and the spectral data of the compounds synthesized are summarized in Table I.

Biological screeningl6,17

The activity of antimicrobials may be demonstrated under suitable conditions by their inhibitory effect on microorganisms. The antifungal activity of the synthesized compounds was carried out using tube dilution method and the mInimum inhibitory concentration was determined by visual comparison with the positive and negative control tubes. A stock solution of the compound was prepared using dimethyl sulfoxide. To 2 mL of sterile Sabouraud 's dextrose broth taken in a test tube, 20 III to 80 III of the stock solution was added followed by a loopful of an authentic culture of Candida aLbicans A TCC 24433 (NCIM 3557) (corresponding to 5x105

CFU/mL). This corresponds to concentration range of

50, 100, 150 and 200 Ilg/mL of the compounds. The tests were carried out in duplicate. The tubes were incubated at 37°C and observed for growth at the end of 24 and 48 hr. A set of negative and positive control of growth was also kept for incubation along with the sample tubes. In the tube for negative growth, the highest volume of dimethyl sulfoxide used was added and no culture was added while in the tube representing maximum growth (positive control) the highest volume of dimethyl sulfoxide used was added followed by a loopful of culture. Minimum inhibitory concentration was taken as the minimum con­centration of the compound at which the clarity of the medium in the sample tube was the same as the negative control indicating complete inhibition of growth. Fluconazole was taken as the reference standard. If the compounds were found to exhibit complete inhibition at 50 Ilg/mL, further screening was done at concentrations as low as 0.01 Ilg/mL.

The antifungal activity data is summarized in Table II.

Results and Discussion Over the past few years there have been remarkable

advances made in azole antifungals. The key development has been the transition from topically active imidazole derivatives to orally bioavailable triazole compounds. In an attempt to develop new molecules against fungal infections and C. aLbicans in particular, in our laboratory the structural require­ments for C. aLbicans inhibition has been determined by docking analysis7 and pharmacophore modd . Making use of this information, new molecules have been designed, synthesized and screened for antifungal activity in vitro.

The designed compounds had the difluorophenyl nucleus and the triazole ring. These constitute the recognition groups for binding the Cyt-P45014aDM. The variable substituent was selected such that it had

Table II - Anti funga l activity of the synthesized compounds

Compd m.p. Yi eld % MIC (0C) (%) Purity

9 133-134 45 98.45 Inact ive at 128 I-lglmL

11 118-120 55 90.43 Inac tive at 128 I-lg/mL

12 128- 130 25 92.0 12.51-lg/mL

13 >230 60 97.11 < 6.25 I-lg/mL

15 11 2- 114 32 90.00 50 I-lg/mL

16 132-135 40 90.00 0.021-lg/mL

380 INDIAN J. CI-IEM ., SEC B, FEBRUARY 2003

a linear conformation. The molecules were constructed using molecular modelling tools in Quanta (version 4.0) and docked into the active site of Cyt-P45014aDM. It was observed from computational studies that In voriconazole 14, the variable substituent was linear (torsion angle 178°) and exhibited good interaction with the hydrophobic access channel.

Yoriconazole is more active than fluconazole and itraconazole and has better activity profile2

. This may probably be due to the increased interaction with the hydrophobic access channel. To confirm the results of our computational findings, two com­pounds with bent conformation (compound 9 and compound 11; torsion angle 131.6° and 143.0° re­spectively) and four compounds with linear orienta­tion (compounds 12 and 13 with torsion angle i 79° and compounds 15 and 16 with torsion angle 168°) were designed and synthesized. Compounds were synthesized under standard reaction conditions as depicted in Schemes I-III. The molecules were iso­lated as racemic mixtures and tested as such. The biological activity data supports our computational results that linear conformation of the variable sub­sti tuent is important for the activity of the com­pound. Compounds 12, 13, 15 and 16 are more ac·· tive than compounds 9 and 11. Compound 12 is more active than compound 11. The only difference between compounds 11 and 12 is that compound 12 possesses an additional methyl group. In case of compound 12, introduction of CH3 for RI makes the variable substituent linear (torsion ang le 179°) while in case of compounds 15 and 16, introduction of ad­ditional methylene group between R2 and carbon bearing the 01-1 group is responsible for linearity and also increased interaction of the variable substituent R2 with the hydrophobic access channel. Compound 13 with 4-chloro substituent is more active than compound 12 and compound 16 with a 4-tluoro sub­stituent is more active than compound 15, indicating that increasing the lipophilicity will increase the ac­tivity of the compound. Compound 16 has the high­est ClopP value (data not shown) . The increased lipophilicity may also be responsible for better pene­tration.

The 4-halogenated aromatic rings (compounds 13 and 16) provide additional interaction with the hydrophobic environment formed by the residues Phe87, Tyr96, Val295 and Val296 (Figure 1) in the access channel. These highly electronegative groups

(CI, F) do not involve any electronic interaction with the receptor as seen from the docking studi es.

The binding with the hydrophobic access channel is essential for proper positioning of the nitrogen on the heterocycle to form a co-ordinate bond with the

heme iron of Cyt-P45014aDM. Thus the synthetic and the activity test results are consistent with our computational findings.

Note added in Proof: While this work was in pro­gress, we noticed the disclosure of compound 9 in the literature1s. The antifungal activity of this compound was not reported.

Conclusion Molecular modelling studies were integrated into

the design of potential new antifungal agents. Novel (lIn)sllbstituted benzyloxy azoles anrl (un)substituted phenethyl azoles have been designed and synthe­sized and these compounds were screened for anti­fungal activ ity against C. albicans ATCC 24433 in vitro. The activity data supports our computational findings that linear conformation of the variable sub­stituent increases the interaction with the hydropho­bic access channel and helps in proper placement of the triazole nitrogen for co-ordination with the heme iron of the Cytochrome P-4S0 i4a demethylase. Fur­ther optimization of activity may yie ld more active compounds.

Acknowledgement

The authors thank University Grants Commission (UGC), New Delhi, DSA and COSIST Programmes and All India Council for Technical Education (AICTE), New Delhi for the research grant~.

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