Effects of skeletal modifications of ciprofloxacin on ... - ResearchGate

188 JOURNAL <strong>of</strong> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007 

ARTICLES 

<strong>Effects</strong> <strong>of</strong> <strong>skeletal</strong> <strong>modifications</strong> <strong>of</strong> cipr<strong>of</strong>loxacin on antibacterial, 

antifungal and cytotoxic activities 

Rakhshinda Siddiqui 1 

, Najma Sultana 1 

, Khalid Mohammed Khan 2 

, Nosheen Akbar 2 

, Muhammad Ali 2 

, Saeed 

Arayne 1 

[ Abstract] A series <strong>of</strong> cipr<strong>of</strong>loxacin <strong>skeletal</strong> variants have been prepared and screened for different antifungal, 

antimicrobial and cytotoxicity to monitor the variation in the activity.The structures <strong>of</strong> these derivatives were 

confirmed by using spectroscopic techniques such as IR,1H唱NMR and EIMS.The antibacterial activity <strong>of</strong> these de唱 

rivatives were carried out in comparison with its parent analogue i.e.cipr<strong>of</strong>loxacin while these derivatives were 

foundtobelessactive.Further more two activities,antifungal and cytotoxic activities <strong>of</strong> these derivatives were also 

carried out in order to establish the effect <strong>of</strong> new substituent on the biological activity <strong>of</strong> the cipr<strong>of</strong>loxacin. 

[ Key words] antimicrobial;antifungal;cytotoxic;cipr<strong>of</strong>loxacin 

INTRODUCTION 

Cipr<strong>of</strong>loxacin hydrochloride belongs to the second唱gen唱 

eration quinolone antibiotic.Quinolone antibacterial act 

by the inhibition <strong>of</strong> bacterial topoisomerase Ⅱ(DNA 

gyrase) and probably topoisomerase Ⅳ in Gram唱posi唱 

tive species,thus inhibiting tertiary negative super coi唱 

[1 ~5] 

ling <strong>of</strong> bacterial DNA .This effect perhaps is asso唱 

ciated with binding <strong>of</strong> quinolones with DNA唱gyrase and 

[6 ] 

the complex formed rapidly becomes bactericidal .It 

is the most widely used agent among this class,due to 

its broad spectrum and excellent oral bioavailability [7] 

. 

It is effective in the treatment <strong>of</strong> urinary tract 

[8 ] 

infections, prostatitis , shigellosis [9] 

[ 10] 

, gonorrhea , 

continuous ambulatory peritoneal dialysis infec唱 

[11 ,12 ] 

tions , some half <strong>of</strong> the <strong>of</strong> diabetic foot infec唱 

[13 ] 

tions , also effective against Bacillus anthrax [14] 

and 

as the prophylactic drug for meningococcal infec唱 

[15 ~17 ] 

tions .It has also been found to show anti唱tumor 

[17 ] 

activity against P388 leukemia .Generally it is well 

tolerated but should be discontinued because <strong>of</strong> an ad唱 

1 Department <strong>of</strong> Pharmaceutical Chemisty,Faculty <strong>of</strong> Pharmacy,University <strong>of</strong> Karachi,Karachi 75270,Pakistan 

verse event observed in 3畅5% <strong>of</strong> patients treated,pri唱 

marily involving the gastrointestinal (1畅5%), skin 

(0畅6%) and central nervous systems (0畅4%). 

In the light <strong>of</strong> excellent antibacterial activities <strong>of</strong> 

cipr<strong>of</strong>loxacin and its severe side effects, we undertook 

the present study with the aim that if the hydrogen <strong>of</strong> 

piperazine moiety present at position seven <strong>of</strong> the 

parent molecule will be replaced with different acyl 

groups in order to check the influence <strong>of</strong> newly intro唱 

duced residue on the antibacterial and cytotoxic proper唱 

ties <strong>of</strong> the cipr<strong>of</strong>loxacin.During the course <strong>of</strong> bioassays 

we also randomly screened the same compounds for 

their antifungal potential. 

Generally,cipr<strong>of</strong>loxacin in THF was reacted with 

respective acid chloride in the presence <strong>of</strong> triethyl 

amine as a base to afford the acylated derivatives in 

moderate to excellent yields.The structure variants <strong>of</strong> 

cipr<strong>of</strong>loxacin were characterized by using the state唱<strong>of</strong>唱 

the唱art spectroscopic techniques like 1H唱NMR,IR and 

EI mass spectrometry. 

2 HEJ Research Institute <strong>of</strong> Chemistry,International Center for Chemical and Biological Sciences,Unviersity <strong>of</strong> Karachi,Karachi 75270,Pakistan 

Correspondence to Khalid Mohammed khan,HEJ Research Institute <strong>of</strong> Chemistry,International Center for Chemical and Biological Sciences,University <strong>of</strong> 

Karachi,Karachi 75270,Pakistan 

E 唱mail:hassaan2@super.net.pk

JOURNAL <strong>of</strong> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007 

MATERIALS AND METHODS 

Tabross Pharmaceuticals, Karachi, gifted cipr<strong>of</strong>loxacin 

ydrochloride and the microorganisms for antimicrobial 

activity were obtained from ESSA laboratory, Karachi 

Pakistan. 

IR spectra ( KBr) were recorded on a Bruker FT 

-IR IFS48 spectrophotometer.EI mass spectra were 

measured with various MAT 711 (70 eV ) spectrome唱 

ters and data are tabulated as m /z.1H唱NMR spectra 

were recorded in DMSO唱d6 using Bruker AC300 (300 

MHz) spectrophotometer.Splitting patterns were as 

follows: s, singlet; d, doublet; dd, double doublets; t, 

triplet;m, multiplet.Chemical shifts are reported in δ 

( ppm) and coupling constants are given in Hz.The 

progress <strong>of</strong> all reactions was monitored by TLC, which 

was performed on 2畅0 cm ×5.0 cm aluminum sheets 

pre唱coated with silica gel 60F254 to a thickness <strong>of</strong> 

0畅25 mm ( Merck).The chromatograms were visualized 

under ultraviolet light ( 254 ~366 nm ) or iodine 

vapours.Melting points were determined in glass capil唱 

laries on a Büchi 434 melting point apparatus and are 

uncorrected.The title compounds were synthesized and 

characterized successfully and satisfactorily. 

Regeneration<strong>of</strong> Free Cipr<strong>of</strong>loxacin 

A solution <strong>of</strong> cipr<strong>of</strong>loxacin hydrochloride (10 g ) in 

water (50 ml) was treated with an excess <strong>of</strong> 5% aque唱 

ous sodium bicarbonate solution resulting in the forma唱 

tion <strong>of</strong> white precipitates which were filtered through 

suction filtration and left to dry as a neutral cipr<strong>of</strong>loxa唱 

cin (1,8.3 g,83%) . 

These precipitates were pure enough and used as 

starting material for all the reactions without purifica唱 

tion. 

General Procedure for the Cipr<strong>of</strong>loxacin Deriva唱 

tives 

Cipr<strong>of</strong>loxacin(1) 

Cipr<strong>of</strong>loxacin(1畅0 g,3.01 mmol) was dissolved in 30 

ml <strong>of</strong> anhydrous THF.To this solution,triethylamine (1 

molar equivalent,0畅629 ml) and one molar equivalent 

<strong>of</strong> appropriate acid chloride was added and the result唱 

ant mixture was refluxed until completion <strong>of</strong> the reac唱 

tion ( TLC analysis).The resultant mixture was cooled 

189 

to room temperature, excess <strong>of</strong> solvent was removed in 

vaccuo and the residue was suspended in brine and ex唱 

tracted with dichloromethane (3 ×50).The organic 

phase was dried over Na2 SO4 , filtered and evaporated 

at reduced pressure to afford the solid product in pure 

form. 

7-(4-Acetyl唱1唱piperazinyl) -6唱fluoro -1 唱cyclo唱 

propyl -4唱oxo -1,4唱dihydro唱3唱quinoline carboxylic 

acid(2) 

Yield:0畅48 g (73%);M.P.= 261℃ ~263 ℃;IR nmax 

cm -1 

: 3421 , 3046, 2921, 1711, 1627, 1470, 1337, 

1217; 1 

H唱NMR (300 MHz唱DMSO -d 6 ):d 15畅16 ( s, 

1 H,H -14),8畅6 (s,1H,H -2) ,7畅8 (d,1H,J =7畅8 

Hz,H -5),7畅5(d,1 H,J =4 .3 Hz, H -8 ) ,3畅8 (s, 

1 H,H -11),3畅6 (s,4H,H -16,20),2畅4(s,4H,H 

-17,19) ,2畅0 (s,3 H,H -2 ′),1畅3(d,2H,J =3 畅7 

Hz,H -13) ,1畅1 (s,2 H,H -12);EIMS m /z (% rel. 

abund.):373 (M +1 

,18),329 (68 ),301 (6 ),257 

(10),231 (8). 

1 -Cyclopropyl -7 -[ 4 -( ethoxycarbonyl) -1唱 

piperazinyl] -6唱fluoro唱4唱oxo唱1,4唱dihydro唱3唱quino唱 

line carboxylic acid (3 ) 

Yield:0畅87 g (87%);M.P.=161℃ -163℃;IR nmax 

cm -1 

: 3453 , 3097, 2977, 2830, 1736, 1626, 1476, 

1386,1342 ,1210 ,1098;1H唱NMR (300 MHz唱DMSO - 

d 6 ):d 15畅2 (s,1H,H -14 ) ,8畅6(s,1H,H -2),7畅9 

( d,1H,J =7.9 Hz,H -5) ,7畅5 (d,1 H,J =4畅4 Hz, 

H -8),4畅15 ( s,1H, H -11),4畅14 ( d,2H,J =5 畅7 

Hz,H -4′) ,4畅10 ( s,1H, H -1 ′),4畅0 (s,4 H,H - 

16,20) ,3畅2 (s,4H, H -17,19 ),1畅8 (s,3H, H - 

5′),1畅1(s,2H,H -13) ,1畅0 (s,2H,H -12 ) ; EIMS 

m /z (% rel.abund.):417 (M +,25) ,373 (11) ,343 

(100),299 (7),230 (6). 

1 -Cyclopropyl -7 -[ 4 -( 2,2唱dichloroacetyl) -1唱 

piperazinyl] -6唱fluoro唱4唱oxo唱1,4唱dihydro唱3唱quino唱 


Yield:0畅51 g (51%);M.P.=219℃ ~221℃ ( de唱 

composed); IRnm ax cm -1 

: 3424, 3013, 2958, 2926, 

1719,1625, 1493, 1444, 1249, 805; 1 

H -NMR (300 

MHz -DMSO -d6 ):d 15畅2 (s,1 H,H -14),8畅6(s, 

1 H,H -2) ,7畅7 (d,1H,J =9 .8 Hz,H -5),7畅5 (d, 

1 H,J =5畅3 Hz,H -8),5畅7(s,1H,H -2 ′),4畅1(s, 

2 H,H -11),3畅7 (s,4H,H -16,20),2畅4(s,4H,H 

-17,19) ,1畅1 (d,2H,J =3畅7 Hz,H -13 ) ,0畅8 (s,


2 H,H -12);EIMS m /z (% rel.abund.) :441 ( M + 

, 

44) ,397 (12) ,329 (2) ,231 (12畅4),203 (23). 

1 -Cyclopropyl唱7唱[ 4 唱( 2, 2唱dimethylpropanoyl )唱1唱 

piperazinyl ]唱6唱fluoro唱4 唱oxo唱1 , 4唱dihydro唱3唱quino唱 


Yield:0畅80 g (80%);M.P.= 28 0 ℃ ~28 2 ℃ ; IR nmax 

cm -1 

: 3520 , 3467, 3093, 2960, 1722, 1669, 1441, 

1385,1256,1013; 1 

H -NMR (300 MHz -DMSO唱d 6 ): 

d 8畅6 (s,1 H,H -2 ) , 7畅9 (d,1H, J =2畅6 Hz, H - 

5),7畅5(d,1 H,J =5畅3 Hz,H -8),4畅3(s,1H,H - 

11) ,3畅7 (s,4H,H -16,20),2畅4(s,4H, H -1 7 , H 

-19) ,1畅2 (d,2H,J =3畅7 Hz,H -13) ,1畅0 (s,2H, 

H -12) ,1畅04 (s,9H,H -3 ′,4′,5′);EIMS m /z (% 

rel.abund.):414 (M + 

,14),370 (32),335 (85), 

330 (2畅8),314 (32),288 (33),270 (11),256 

(13),148 (8),56 (100). 

1唱Cyclopropyl唱6唱fluoro唱(4唱octanoyl唱1唱pipera唱zinyl)唱 

4唱oxo唱1,4唱dihydro唱3唱quinoline carboxylic acid (6) 


cm -1 

: 3461 , 3094, 2957, 1726, 1629, 1468, 1264, 

1124; 1 

H唱NMR (300 MHz唱DMSO唱d 6 ):d 8畅6 (s,1H, 

H -2),7畅9(d,1H,J =7畅8 Hz,H -5),7畅5(d,1H,J 

=2畅9 Hz,H -8),4畅1(t,1H, J =7 畅4 Hz,H -2 ′), 

3畅8 (s,1H, H -11),3畅4 (s,4H, H -16,20),2畅4 

( s,4H,H -17,19),1畅29 -1畅21 ( m,10H, H -3 ′, 

4′,5 ′,6′,7′) ,0畅87 ( s,2H, H -13 ) .0畅86 ( s,2H,H 

-12),0畅84 (d,2H,J =7 畅3 Hz,H -8 ′);EIMS m /z 

(% rel.abund.):457 ( M + 

,10),437 (2畅5),413 

(2畅4) ,370 (2) ,342 ( 13) ,286 (12) ,231 (100 ) ,56 

(90畅5). 

1唱Cyclopropyl唱6唱fluoro唱7唱( 4唱nonanoyl唱1唱piperazinyl )唱 

4唱oxo唱1,4唱dihydro唱3唱quinoline carboxylic acid (7) 

Yield:0畅73 g (73%);M.P.= 151℃ ~153℃; 

IR nm ax cm -1 

: 3467,3358, 3093,2923, 1722,1628, 

1469,1384,1340,1257,1023; 1 

H唱NMR (300 MHz唱 

DMSO唱d 6 ):d 8畅6 (s,1H, H -2),7畅9 (d,1H, J = 

7畅8 Hz,H -5),7畅5 (d,1H , J =2 畅9 Hz, H -8), 

4畅1 (t,1H, J =7 畅4 Hz, H -2 ′) ,3畅8 (s,1H, H - 

11),3畅4(s,4H ,H -16,20),2畅4 (s,4 H, H -1 7 , 

19),1畅31 -1畅71 ( m,12H , H -3 ′,4′,5′,6′,7′, 

8′),0畅87 (s,2 H,H -13 ) , 0畅86 ( s,2H, H -12), 

0畅84 ( d,2H, J =7 畅3 Hz, H -8 ′);EIMS m /z (% 

rel.abund.): 471 ( M + 

, 2 ), 384 ( 1畅6), 345 

(11畅7),231 (35),71 (12),56 (100) . 

1 唱Cyclopropyl唱7 唱(4唱decanoyl唱1唱piperazinyl) 7唱6 唱flu唱 

oro唱4唱oxo唱1, 4 唱dihydro唱3唱quinoline carboxylic acid 

(8 ) 

Yield:0畅68 g (68%);M.P.= 10 7 ℃ ~10 9 ℃ ; IR nmax 

cm -1 

: 3466 , 3093, 3011, 2923, 1723, 1628, 1469, 

1385,1340,1257; 1 

H -NMR (300 MHz -DMSO - 

d 6 ):d 8畅6 (s,1H,H -2),7畅9(d,1H,J =7畅8 Hz,H 

-5 ) ,7 畅5(d,1H,J =2畅9 Hz,H -8) ,4畅1 (t,1H,J = 

7畅4 Hz,H -2 ′),3畅8(s,1H,H -11),3畅4(s,4H,H 

-16,20) ,2畅4 (s,4H,H -17,19),1畅29 -1畅21 ( m, 

10H, H -3 ′,4′,5′,6′,7′),0畅87 (s,2H, H -13). 

0畅86 (s,2H,H -1 2 ) , 0畅85 ( t,3H,J =7 畅3 Hz,H - 

10′);EIMS m /z (% rel.abund.) :485 (M + 

,16 ),384 

(11) ,330 (10) ,311 ( 29) ,301 (34) ,289 (44) ,275 

(46) ,257 (36) ,245 ( 52) ,231 (83) ,56 (100) . 

7 唱( 4唱Benzoyl唱1唱piperazinyl )唱1唱cyclopropyl唱6唱fluo唱 

ro唱4唱oxo唱1, 4唱dihydro唱3唱quinoline carboxylic acid 

(9 ) 


cm -1 

: 3408 , 3056, 2919, 1720, 1622, 1529, 1486, 

1387,1330,1251,1008; 1 

H -NMR (300 MHz -DM唱 

SO -d6 ):d 8畅6 (s,1 H,H -2 ) , 7畅9 (d,1H, J =7 畅8 

Hz,H -5),7畅5(d,1H,J =2畅9 Hz, H -8),7畅5- 

7畅4 (m,1H, H -3 ′,4′,5 ′,6′,7′),3畅8(s,1 H,H - 

11),3畅3 (s,4H, H -16,20),3畅2 (s,4H, H -1 7 , 

19),2畅1 (s,2H,H -13) ,1畅3 (s,2 H,H -12 ) ; EIMS 

m /z (% rel.abund.) : 391 ( M + 

,44畅3),286 (1), 

257 (11),231 (5),177 (3),159 (1),56 (70). 

7 唱[4唱(2唱Chlorobenzoyl)唱1 唱piperazinyl]唱1唱cyclopro唱 

pyl唱6 唱fluoro唱4 唱oxo唱1,4唱dihydro唱3唱quinoline carbox唱 

ylic acid (10 ) 

Yield:0畅80 g (80%);M.P.=191℃ ~193℃;IR nmax 

cm -1 

: 3468 , 3058, 2959, 1717, 1627, 1464, 1399, 

1340,1292; 1 

H -NMR (300 MHz -DMSO -d 6 ):d 

15畅0 (s,1H,H -14 ),8畅6 (s,1H,H -20 ) ,7畅9 (d, 

1 H,J =7畅8 Hz,H -5 ) ,7 畅5(d,1 H,J =5 畅5 Hz, H - 

8),7畅6(dd,J =8畅5 Hz,H -3 ′),7畅4(ddd, J =8 畅5 

Hz,H -4 ′),7畅3 (dd, J =8畅5 Hz, H -6 ′), 7畅22 

( ddd,J =8畅5 Hz,H -5 ′),3畅8(d,1H,J =1畅2 Hz,H 

-11) ,3畅4 (s,4 H,H -16,20 ) ,2畅4 (s,4H,H -1 7 , 

19),1畅1 (d,2H,J =2畅3 Hz,H -13),0畅9(s,2H,H 

-12);EIMS m /z (% rel.abund.):560 (M + 

,17), 

516 (49),432 (2),390 (4),314 (10),288 (12), 

257 (16),7 (22),230 (61),2 (19),148 (20),4


(61),56 (100). 

1 唱Cyclopropyl唱6 唱fluoro唱7 唱[ 4唱( 4唱methoxybenzoyl )唱 

1 唱piperazinyl]唱4唱oxo唱1, 4唱dihydro唱3唱quinoline car唱 

boxylic acid (11) 

Yield:0畅79 g (79%);M.P.=170℃ ~172℃;IR nmax 

cm -1 

: 3412 , 3016, 2930, 1719, 1602, 1467, 1433, 

1255,1167; 1 

H -NMR ( 300 MHz -DMSO -d6):d 

8畅6 (s,H,H -2 ) , 7畅9(d,1 H, J =6畅6 Hz,H -5), 

7畅6 (d,1H,J =5畅0 Hz.1,H -8 ) , 7畅4 (d,2H, J = 

6畅3 Hz,H -3 ′,7′),7畅1(d,2 H,J =6 畅5 Hz, H -4 ′, 

6′),3畅8(s,2H, H -11),3畅7 (s,3H, H -8 ′),3畅3 

( s,4H, H -16,20),2畅4 (s,4H, H -17,19),1畅3 

( d,2H,J =4畅9 Hz, H -13),0畅8(s,2H, H -12); 

EIMS m /z (% rel.abund.):465 (M + 

,2畅6),420 

(4),289 (3),271 (2),230 (3畅6),201 (2),151 

(33),135 (74),92 (33),77 (47),56 (50). 

1 唱Cyclopropyl唱6 唱fluoro唱4 唱oxo唱7 唱[ 4 唱( 3, 4, 5唱trime唱 

thoxybenzoyl )唱1唱piperazinyl ]唱1, 4唱dihydro唱3唱quin唱 

oline carboxylic acid (12) 


cm -1 

: 3048 , 3056, 2919, 2855, 1720, 1298, 1622, 

1486,1251, 1210; 1 


d 6 ):d 15畅1 (s,1H,H -14) ,8畅6 (s,1H,H -2),7畅8 

( d,1H,J =4畅7 Hz,H -5),7畅5 (d,1H,J =4畅7 Hz,H 

-7),7畅6(d,2H, J =2 畅2 Hz H -3 ′,7′),4畅1(s, 

1 H,H -11),3畅7 (s,9H, H -8 ′,9′,10′),3畅6 (s, 

4 H,H -16,20),2畅3 (s,4H, H -17,19),1畅9 (s, 

2 H,H -13),1畅2 (s,4H, H -12);EIMS m /z (% 

rel.abund.):391 (M + 

,6),279 (27),197 (2畅5), 

167 (53) ,149 ( 100) . 

1 -Cyclopropyl -6唱fluoro唱7唱[ 4唱( 1唱naphthoyl )唱1唱 

piperazinyl ]唱4唱oxo唱1, 4 唱dihydro唱3唱quinoline car唱 

boxylic acid 


cm -1 

: 3480 , 3055, 2931, 1725, 1625, 1478, 1435, 

1386,1249, 1204; 1 


d 6 ):d 15畅2 (s,1H,H -14) ,8畅6 (s,1H,H -2),8畅0 

( d,1H,J =6畅3 Hz,H -5 ) , 7畅5(d,1H,J =5畅0 Hz, 

H -8),7畅8(t,1H,J =8畅4 Hz,H -8 ′) ,7畅7 (d,1H, 

J =8畅4 Hz,H -6 ′),7畅66 ( d,1H, J =8畅4 Hz, H - 

9′),7畅61 (d,1H,J =8畅3 Hz,H -5 ′),7畅5(d,1H,J 

=8畅3 Hz, H -3 ′),7畅4(t,1H, J =8 畅4 Hz, H -7 ′) 

7畅1 (t,1H, J =8畅3 Hz, H -3′),3畅8 (s,1H, H - 

11) ,3畅5 (s,4H, H -16,20),2畅4 (s,4H, H -1 7 , 

19),1畅2 (s,2H,H -13) ,0畅9 (s,2 H,H -12 ) ; EIMS 

m /z (% rel.abund.):485 (M + 

,0畅39),441 (19), 

286 ( 13 ) , 257 ( 36 ) , 245 ( 2畅8),55 (56),127 

(61),101 (3),56 (100). 

Antibacterial Studies ( In vitro) 

191 

The synthesized compounds were screened for antibac唱 

terial activity against the number <strong>of</strong> Gram -negative 

organisms including Klebsiella pneumoniae,Escherichi唱 

a coli, Proteus mirabilis, Shigella dysenteriae, Pseudo唱 

monas aeruginosa, Salmonella typhi, Salmonella typhi 

para,A Salmonella typhi para B and Gram -positive 

organisms like Staphylococcus aureus and Bacillus sub唱 

tilis by using literature protocol [18] 

.In the agar well dif唱 

fusion method,wells were duged in the media with the 

help <strong>of</strong> a sterile metallic borer with contres at least 24 

mm part.Two to eight hours old bacterial inoculums 

containing approximately 104 ~106 colony forming 

units ( CFU) /ml were spread on the surface <strong>of</strong> nutrient 

agar with the help <strong>of</strong> a sterile cotton swab.Cotton swab 

was rotated firmly against the upper inside well <strong>of</strong> the 

tube to express excess fluid.Entire agar surface <strong>of</strong> the 

plate was streaked with the swab three times turning 

the plate 60°between each streaking.Recommended 

concentration <strong>of</strong> the test sample (2 mg /ml <strong>of</strong> DMSO) 

was then added in their respective wells.Other wells 

supplemented with DMSO and reference antibacterial 

drugs serving as negative and positive controls,respec唱 

tively.The plates were incubated immediately at 37 ℃ 

for 14 ~19 horlater,if necessary.Antibacterial activity 

was determined by measuring the diameter <strong>of</strong> zones 

showing complete inhibition ( mm).Growth inhibition 

was calculated with reference to positive control. 

Fungicidal Bioassay ( In vitro) 

The synthesized compounds were tested for antifungal 

activity against the fungi including Aspergillus flavus, 

Trichophyton longifusis,Candida albicans,Microsporum 

canis,Fusraium solani and Candida glaberata.Ampho唱 

tericin B and miconazol were used as standard drugs. 

For tube diffusion test 12 mg <strong>of</strong> sample was dissolved in 

1 ml <strong>of</strong> DMSO serving as stock solution.Sabouraud 

dextrose agar ( Oxoid, Hampshire, England ) was 

seeded with 105 CFU /ml fungal spore suspensions and


transferred to Petri plates.Discs soaked in 20 ml (10 

mg /ml in DMSO) <strong>of</strong> all compounds were placed at 

different positions on the agar surface.The plates were 

incubated at 32 ℃ for seven days.The results were 

recorded as zones <strong>of</strong> inhibition in mm [19] 

. 

Cytotoxicity Bioassay ( In vitro) 

Twenty mg <strong>of</strong> the test sample was dissolved in the re唱 

spective organic solvent.From this stock solution 5,50, 

500 μl were transferred to vials (3 vials /concentra唱 

tion).The final concentration was 10,100,1000 μg / 

ml respectively, and the solvent was evaporated over 

night. 

Brine shrimp ( Artemia salina leach ) eggs were 

hatched and after two days, when shrimp larvae were 

ready,1 ml <strong>of</strong> seawater and 10 shrimps were added to 

each vial (30 shrimps /dilution) and the volume was 

adjusted with seawater to 5 ml per vial.After 24 hthe 

[ 20] 

number <strong>of</strong> survivors was counted and data were ana唱 

lyzed by Finney computer program to determine the 

[21 ] 

LD50 . 

RESULTS AND DISCUSSION 

Our synthetic pathway was based on the preparation <strong>of</strong> 

different derivatives <strong>of</strong> cipr<strong>of</strong>loxacin [1] 

by treating it 

with respective acid chloride in tetrahydr<strong>of</strong>uran ( THF) 

in the presence <strong>of</strong> triethylamine ( TEA) as a base.The 

resultant mixture was refluxed and the completion <strong>of</strong> 

the reaction was monitored by TLC analysis.The struc唱 

[2 ~13] 

tures <strong>of</strong> all the synthesized derivatives were con唱 

firmed by using the sophisticated spectroscopic tech唱 

niques like 1H唱NMR, IR and EI mass spectrometry 

( Scheme 1,Table 1). 

Antimicrobial Activity <strong>of</strong> Cipr<strong>of</strong>loxacin Derivatives 

[ 2 ~13] 

All the synthesized compounds were screened for 

their antimicrobial affects against 8 Gram唱negative in唱 

cluding Klebsiella pneumoniae, Escherichia coli, Pro唱 

teus mirabilis, Shigella dysenteriae, Pseudomonas 

aeruginosa,Salmonella typhi, Salmonella typhi para A 

and Salmonella typhi para B as well as two Gram唱posi唱 

tive organisms namely Staphylococcus aureus and Ba唱 

cillus subtilis.The results <strong>of</strong> in vitro antibacterial activi唱 

ty are collected in Table 2. 

Table 1 The Structures <strong>of</strong> All the Synthesized Dervatives 

S.No. R Yield (%) 

2 -CH 3 75 

3 -OC 2 H 5 87 

4 -CHCl 2 51 

5 -C( CH 3 ) 3 80 

6 -C 7 H 15 44 

7 -C 8 H 17 73 

8 -C 9 H 19 68 

9 53 

10 

Cl 

11 OMe 79 

12 

OM e 

OM e 

OMe 

13 70 

80 

24


HClHN 

F 

N 

O 

N 

THF,TEA,RCOX 

70 ℃ 

X =Cl 

COOH 

R 

5%aq.NaHCO 3 

room temp erature. 

O 

N 

F 

N 

Scheme 1 

Table 2 In vitro Antimicrobial Activity Results <strong>of</strong> Cipr<strong>of</strong>loxacin (1) and Its Derivatives (2 -13) against Gram唱negative and 

Gram唱positive Bacteria ( zone <strong>of</strong> inhibition in mm) 

Gram唱negative 

HN 

2-13 

O 

N 

F 

N 

1 

COOH 

O 

N 

COOH 

Compound No. 1 2 3 4 5 6 7 8 9 10 11 12 13 

Escherichia coli 28 19 6 10 6 6 13 16 26 8 7 20 9 

Pseudomonas aeruginosa 23 17 13 10 17 8 9 8 11 8 8 16 8 

Salmonella typhi 28 19 7 18 13 12 18 12 22 6 7 18 6 

Salmonella typhi para A 30 22 11 24 15 13 15 13 20 7 8 20 7 

Salmonella typhi para B 32 16 12 18 13 10 10 8 18 8 6 17 11 

Proteus mirabilis 20 11 8 10 5 11 11 9 14 8 8 13 9 

Klebsiella pneumoniae 25 22 10 20 12 9 12 8 24 6 7 15 8 

Shigella dysenteriae 26 13 13 19 13 8 8 10 10 9 10 9 10 

Gram唱positive 

Bacillus subtilis 27 25 10 16 8 7 14 10 17 10 9 14 10 

Staphylococcus aureus 30 20 6 8 9 14 12 11 23 7 7 22 6 

It was found that the structural analogues <strong>of</strong> cipro唱 

floxacin (1) showed varying degree <strong>of</strong> antimicrobial 

activity against all the tested bacterial strains.Com唱 

pounds 9 and 2 showed good activities against Esche唱 

richia coli,however,compound 2 exhibited comparable 

activity against Bacillus subtilis.Good activities <strong>of</strong> com唱 

pounds 2,9,and 12 were observed against Staphylococ唱 

cus aureus,while compound 9 showed good activity a唱 

gainst Salmonella typhi.Four derivatives 2,4,9,and 12 

exhibited good activities against Salmonella typhi para 

A,where compound 2,4,and 9 were found to be active 

against Klebsiella pneumoniae.Compound 9 in the case 

193 

<strong>of</strong> Klebsiella pneumoniae showed almost equivalent de唱 

gree <strong>of</strong> activity compared to the cipr<strong>of</strong>loxacin [1] 

. 

In general,the compound 2,4,9,and 12 showed 

good activities against Escherichia coli, Bacillus subti唱 

lis, Staphylococcus aureus,Salmonella typhi,Salmonel唱 

la typhi para A and Klebsiella pneumoniae.Comparison 

<strong>of</strong> the activities <strong>of</strong> different analogues <strong>of</strong> cipr<strong>of</strong>loxacin 

(1) indicates that the amidic linkage at piperazine 

moiety may be responsible for the changes in the anti唱 

microbial properties <strong>of</strong> the parent molecule.A careful 

selection <strong>of</strong> the substituent at the nitrogen <strong>of</strong> piperazine 

nucleus may result into a more active antibiotic based


Table 3 Results <strong>of</strong> Screening Cipr<strong>of</strong>loxacin (1) and its Derivatives (2 -13) for their Antifungal Activity in vitro ( zone <strong>of</strong> in唱 

hibition in mm) 

Test fungi 

Compound No. A B 1 2 3 4 5 6 7 8 9 10 11 12 13 

Trichophytonlongifusus 100 0 0 0 0 0 0 0 60 60 0 25 0 0 0 

Candida albicans 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Aspergillus flavus 0 100 0 0 0 0 0 0 60 0 0 45 0 0 0 

Microsporum canis 100 0 0 0 0 0 0 0 55 55 0 50 0 50 0 

Fusarium solani 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Candida glaberata 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Note:A =Miconazole;B =Amphotericine B 

on cipr<strong>of</strong>loxacin (1).These preliminary studies provide 

us lead compounds like 2,4,9,and 12 for further modi唱 

fications at nitrogen <strong>of</strong> piperazine or other active posi唱 

tions like carboxylic acid residue or the ketonic carbon唱 

yl function.It may be envisioned that the <strong>modifications</strong> 

at nitrogen <strong>of</strong> piperazine residue and other two seem唱 

ingly active positions may result into a better antibiotic 

with the minimization <strong>of</strong> its side effects. 

Antifungal Activity 

An interesting feature <strong>of</strong> the present study is that when 

all synthesized compounds (1 ~12) along with cipro唱 

floxacin (1) were randomly screened for 6 fungal 

strains i.e.Trichophyton longifusus,Candida albicans, 

Aspergillus flavus,Microsporumcanis,Fusarium solani 

and Candida glaberata,it is revealed that cipr<strong>of</strong>loxacin 

(1 ) was inactive against any <strong>of</strong> the fungal strains 

used.Nevertheless,compound 7 showed good activities 

against Trichophyton longifusus,Aspergillus flavus and 

Microsporumcanis.Compound 8 was found to be active 

against Trichophyton longifusus and Microsporum canis 

strains.Compound 10 showed moderate activities a唱 

gainst Aspergillus flavus and Microsporum canis,while 

compound 12 was found to be moderately active against 

Microsporumcanis. Antifungal activity in cipr<strong>of</strong>loxacin analogues may 

open new avenues for designing antifungal agents based 

on cipr<strong>of</strong>loxacin (1) skeleton.For enhancing the anti唱 

fungal strength <strong>of</strong> the lead molecules 7,8,10,and 12, 

either modification in the alkyl side chain <strong>of</strong> compound 

6 and 7 or by varying the substitution <strong>of</strong> phenyl ring in 

compound 10 and 12.In addition,the synthetic modifi唱 

cations on carboxyl group or ketonic carbonyl function 

may enhance the antifungal activity. 

Cytotoxic Activity 

Bioactive compounds are <strong>of</strong>ten toxic to Artemia salina 

( leach, brine shrimp ) larvae.The bioassay method 

adopted is rapid and inexpensive which has been devel唱 

oped for screening and monitoring <strong>of</strong> physiologically 

active compound. 

During this experiment etoposide is used as stan唱 

dared drug and LD50 is found to be 0畅178 μg /ml while 

it has been found that neither cipr<strong>of</strong>loxacin nor its de唱 

rivatives is cytotoxic. 

CONCLUSION 

The <strong>skeletal</strong> <strong>modifications</strong> <strong>of</strong> cipr<strong>of</strong>loxacin (1) showed 

a varying degree <strong>of</strong> antimicrobial activities particularly 

compound 2,4,9,and 12 exhibited promising antimi唱 

crobial activities devoid <strong>of</strong> any toxicity and may be 

used as lead compounds for further investigations.Ad唱 

ditionally compound 7,8,10,and 12 exhibited encoura唱 

ging antifungal activities and provided a base to explore 

the antifungal activities <strong>of</strong> cipr<strong>of</strong>loxacin (1) skeleton 

containing molecules.Further research based on over 

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195 

( Editor LEE)

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