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188 JOURNAL
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188 JOURNAL <str<strong>on</strong>g>of</str<strong>on</strong>g> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007<br />
ARTICLES<br />
<str<strong>on</strong>g>Effects</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>skeletal</str<strong>on</strong>g> <str<strong>on</strong>g>modificati<strong>on</strong>s</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin <strong>on</strong> antibacterial,<br />
antifungal and cytotoxic activities<br />
Rakhshinda Siddiqui 1<br />
, Najma Sultana 1<br />
, Khalid Mohammed Khan 2<br />
, Nosheen Akbar 2<br />
, Muhammad Ali 2<br />
, Saeed<br />
Arayne 1<br />
[ Abstract] A series <str<strong>on</strong>g>of</str<strong>on</strong>g> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin <str<strong>on</strong>g>skeletal</str<strong>on</strong>g> variants have been prepared and screened for different antifungal,<br />
antimicrobial and cytotoxicity to m<strong>on</strong>itor the variati<strong>on</strong> in the activity.The structures <str<strong>on</strong>g>of</str<strong>on</strong>g> these derivatives were<br />
c<strong>on</strong>firmed by using spectroscopic techniques such as IR,1H唱NMR and EIMS.The antibacterial activity <str<strong>on</strong>g>of</str<strong>on</strong>g> these de唱<br />
rivatives were carried out in comparis<strong>on</strong> with its parent analogue i.e.cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin while these derivatives were<br />
foundtobelessactive.Further more two activities,antifungal and cytotoxic activities <str<strong>on</strong>g>of</str<strong>on</strong>g> these derivatives were also<br />
carried out in order to establish the effect <str<strong>on</strong>g>of</str<strong>on</strong>g> new substituent <strong>on</strong> the biological activity <str<strong>on</strong>g>of</str<strong>on</strong>g> the cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin.<br />
[ Key words] antimicrobial;antifungal;cytotoxic;cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin<br />
INTRODUCTION<br />
Cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin hydrochloride bel<strong>on</strong>gs to the sec<strong>on</strong>d唱gen唱<br />
erati<strong>on</strong> quinol<strong>on</strong>e antibiotic.Quinol<strong>on</strong>e antibacterial act<br />
by the inhibiti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> bacterial topoisomerase Ⅱ(DNA<br />
gyrase) and probably topoisomerase Ⅳ in Gram唱posi唱<br />
tive species,thus inhibiting tertiary negative super coi唱<br />
[1 ~5]<br />
ling <str<strong>on</strong>g>of</str<strong>on</strong>g> bacterial DNA .This effect perhaps is asso唱<br />
ciated with binding <str<strong>on</strong>g>of</str<strong>on</strong>g> quinol<strong>on</strong>es with DNA唱gyrase and<br />
[6 ]<br />
the complex formed rapidly becomes bactericidal .It<br />
is the most widely used agent am<strong>on</strong>g this class,due to<br />
its broad spectrum and excellent oral bioavailability [7]<br />
.<br />
It is effective in the treatment <str<strong>on</strong>g>of</str<strong>on</strong>g> urinary tract<br />
[8 ]<br />
infecti<strong>on</strong>s, prostatitis , shigellosis [9]<br />
[ 10]<br />
, g<strong>on</strong>orrhea ,<br />
c<strong>on</strong>tinuous ambulatory perit<strong>on</strong>eal dialysis infec唱<br />
[11 ,12 ]<br />
ti<strong>on</strong>s , some half <str<strong>on</strong>g>of</str<strong>on</strong>g> the <str<strong>on</strong>g>of</str<strong>on</strong>g> diabetic foot infec唱<br />
[13 ]<br />
ti<strong>on</strong>s , also effective against Bacillus anthrax [14]<br />
and<br />
as the prophylactic drug for meningococcal infec唱<br />
[15 ~17 ]<br />
ti<strong>on</strong>s .It has also been found to show anti唱tumor<br />
[17 ]<br />
activity against P388 leukemia .Generally it is well<br />
tolerated but should be disc<strong>on</strong>tinued because <str<strong>on</strong>g>of</str<strong>on</strong>g> an ad唱<br />
1 Department <str<strong>on</strong>g>of</str<strong>on</strong>g> Pharmaceutical Chemisty,Faculty <str<strong>on</strong>g>of</str<strong>on</strong>g> Pharmacy,University <str<strong>on</strong>g>of</str<strong>on</strong>g> Karachi,Karachi 75270,Pakistan<br />
verse event observed in 3畅5% <str<strong>on</strong>g>of</str<strong>on</strong>g> patients treated,pri唱<br />
marily involving the gastrointestinal (1畅5%), skin<br />
(0畅6%) and central nervous systems (0畅4%).<br />
In the light <str<strong>on</strong>g>of</str<strong>on</strong>g> excellent antibacterial activities <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin and its severe side effects, we undertook<br />
the present study with the aim that if the hydrogen <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
piperazine moiety present at positi<strong>on</strong> seven <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
parent molecule will be replaced with different acyl<br />
groups in order to check the influence <str<strong>on</strong>g>of</str<strong>on</strong>g> newly intro唱<br />
duced residue <strong>on</strong> the antibacterial and cytotoxic proper唱<br />
ties <str<strong>on</strong>g>of</str<strong>on</strong>g> the cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin.During the course <str<strong>on</strong>g>of</str<strong>on</strong>g> bioassays<br />
we also randomly screened the same compounds for<br />
their antifungal potential.<br />
Generally,cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin in THF was reacted with<br />
respective acid chloride in the presence <str<strong>on</strong>g>of</str<strong>on</strong>g> triethyl<br />
amine as a base to afford the acylated derivatives in<br />
moderate to excellent yields.The structure variants <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin were characterized by using the state唱<str<strong>on</strong>g>of</str<strong>on</strong>g>唱<br />
the唱art spectroscopic techniques like 1H唱NMR,IR and<br />
EI mass spectrometry.<br />
2 HEJ Research Institute <str<strong>on</strong>g>of</str<strong>on</strong>g> Chemistry,Internati<strong>on</strong>al Center for Chemical and Biological Sciences,Unviersity <str<strong>on</strong>g>of</str<strong>on</strong>g> Karachi,Karachi 75270,Pakistan<br />
Corresp<strong>on</strong>dence to Khalid Mohammed khan,HEJ Research Institute <str<strong>on</strong>g>of</str<strong>on</strong>g> Chemistry,Internati<strong>on</strong>al Center for Chemical and Biological Sciences,University <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
Karachi,Karachi 75270,Pakistan<br />
E 唱mail:hassaan2@super.net.pk
JOURNAL <str<strong>on</strong>g>of</str<strong>on</strong>g> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007<br />
MATERIALS AND METHODS<br />
Tabross Pharmaceuticals, Karachi, gifted cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin<br />
ydrochloride and the microorganisms for antimicrobial<br />
activity were obtained from ESSA laboratory, Karachi<br />
Pakistan.<br />
IR spectra ( KBr) were recorded <strong>on</strong> a Bruker FT<br />
-IR IFS48 spectrophotometer.EI mass spectra were<br />
measured with various MAT 711 (70 eV ) spectrome唱<br />
ters and data are tabulated as m /z.1H唱NMR spectra<br />
were recorded in DMSO唱d6 using Bruker AC300 (300<br />
MHz) spectrophotometer.Splitting patterns were as<br />
follows: s, singlet; d, doublet; dd, double doublets; t,<br />
triplet;m, multiplet.Chemical shifts are reported in δ<br />
( ppm) and coupling c<strong>on</strong>stants are given in Hz.The<br />
progress <str<strong>on</strong>g>of</str<strong>on</strong>g> all reacti<strong>on</strong>s was m<strong>on</strong>itored by TLC, which<br />
was performed <strong>on</strong> 2畅0 cm ×5.0 cm aluminum sheets<br />
pre唱coated with silica gel 60F254 to a thickness <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
0畅25 mm ( Merck).The chromatograms were visualized<br />
under ultraviolet light ( 254 ~366 nm ) or iodine<br />
vapours.Melting points were determined in glass capil唱<br />
laries <strong>on</strong> a Büchi 434 melting point apparatus and are<br />
uncorrected.The title compounds were synthesized and<br />
characterized successfully and satisfactorily.<br />
Regenerati<strong>on</strong><str<strong>on</strong>g>of</str<strong>on</strong>g> Free Cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin<br />
A soluti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin hydrochloride (10 g ) in<br />
water (50 ml) was treated with an excess <str<strong>on</strong>g>of</str<strong>on</strong>g> 5% aque唱<br />
ous sodium bicarb<strong>on</strong>ate soluti<strong>on</strong> resulting in the forma唱<br />
ti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> white precipitates which were filtered through<br />
sucti<strong>on</strong> filtrati<strong>on</strong> and left to dry as a neutral cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxa唱<br />
cin (1,8.3 g,83%) .<br />
These precipitates were pure enough and used as<br />
starting material for all the reacti<strong>on</strong>s without purifica唱<br />
ti<strong>on</strong>.<br />
General Procedure for the Cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin Deriva唱<br />
tives<br />
Cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin(1)<br />
Cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin(1畅0 g,3.01 mmol) was dissolved in 30<br />
ml <str<strong>on</strong>g>of</str<strong>on</strong>g> anhydrous THF.To this soluti<strong>on</strong>,triethylamine (1<br />
molar equivalent,0畅629 ml) and <strong>on</strong>e molar equivalent<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> appropriate acid chloride was added and the result唱<br />
ant mixture was refluxed until completi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the reac唱<br />
ti<strong>on</strong> ( TLC analysis).The resultant mixture was cooled<br />
189<br />
to room temperature, excess <str<strong>on</strong>g>of</str<strong>on</strong>g> solvent was removed in<br />
vaccuo and the residue was suspended in brine and ex唱<br />
tracted with dichloromethane (3 ×50).The organic<br />
phase was dried over Na2 SO4 , filtered and evaporated<br />
at reduced pressure to afford the solid product in pure<br />
form.<br />
7-(4-Acetyl唱1唱piperazinyl) -6唱fluoro -1 唱cyclo唱<br />
propyl -4唱oxo -1,4唱dihydro唱3唱quinoline carboxylic<br />
acid(2)<br />
Yield:0畅48 g (73%);M.P.= 261℃ ~263 ℃;IR nmax<br />
cm -1<br />
: 3421 , 3046, 2921, 1711, 1627, 1470, 1337,<br />
1217; 1<br />
H唱NMR (300 MHz唱DMSO -d 6 ):d 15畅16 ( s,<br />
1 H,H -14),8畅6 (s,1H,H -2) ,7畅8 (d,1H,J =7畅8<br />
Hz,H -5),7畅5(d,1 H,J =4 .3 Hz, H -8 ) ,3畅8 (s,<br />
1 H,H -11),3畅6 (s,4H,H -16,20),2畅4(s,4H,H<br />
-17,19) ,2畅0 (s,3 H,H -2 ′),1畅3(d,2H,J =3 畅7<br />
Hz,H -13) ,1畅1 (s,2 H,H -12);EIMS m /z (% rel.<br />
abund.):373 (M +1<br />
,18),329 (68 ),301 (6 ),257<br />
(10),231 (8).<br />
1 -Cyclopropyl -7 -[ 4 -( ethoxycarb<strong>on</strong>yl) -1唱<br />
piperazinyl] -6唱fluoro唱4唱oxo唱1,4唱dihydro唱3唱quino唱<br />
line carboxylic acid (3 )<br />
Yield:0畅87 g (87%);M.P.=161℃ -163℃;IR nmax<br />
cm -1<br />
: 3453 , 3097, 2977, 2830, 1736, 1626, 1476,<br />
1386,1342 ,1210 ,1098;1H唱NMR (300 MHz唱DMSO -<br />
d 6 ):d 15畅2 (s,1H,H -14 ) ,8畅6(s,1H,H -2),7畅9<br />
( d,1H,J =7.9 Hz,H -5) ,7畅5 (d,1 H,J =4畅4 Hz,<br />
H -8),4畅15 ( s,1H, H -11),4畅14 ( d,2H,J =5 畅7<br />
Hz,H -4′) ,4畅10 ( s,1H, H -1 ′),4畅0 (s,4 H,H -<br />
16,20) ,3畅2 (s,4H, H -17,19 ),1畅8 (s,3H, H -<br />
5′),1畅1(s,2H,H -13) ,1畅0 (s,2H,H -12 ) ; EIMS<br />
m /z (% rel.abund.):417 (M +,25) ,373 (11) ,343<br />
(100),299 (7),230 (6).<br />
1 -Cyclopropyl -7 -[ 4 -( 2,2唱dichloroacetyl) -1唱<br />
piperazinyl] -6唱fluoro唱4唱oxo唱1,4唱dihydro唱3唱quino唱<br />
line carboxylic acid (4 )<br />
Yield:0畅51 g (51%);M.P.=219℃ ~221℃ ( de唱<br />
composed); IRnm ax cm -1<br />
: 3424, 3013, 2958, 2926,<br />
1719,1625, 1493, 1444, 1249, 805; 1<br />
H -NMR (300<br />
MHz -DMSO -d6 ):d 15畅2 (s,1 H,H -14),8畅6(s,<br />
1 H,H -2) ,7畅7 (d,1H,J =9 .8 Hz,H -5),7畅5 (d,<br />
1 H,J =5畅3 Hz,H -8),5畅7(s,1H,H -2 ′),4畅1(s,<br />
2 H,H -11),3畅7 (s,4H,H -16,20),2畅4(s,4H,H<br />
-17,19) ,1畅1 (d,2H,J =3畅7 Hz,H -13 ) ,0畅8 (s,
190 JOURNAL <str<strong>on</strong>g>of</str<strong>on</strong>g> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007<br />
2 H,H -12);EIMS m /z (% rel.abund.) :441 ( M +<br />
,<br />
44) ,397 (12) ,329 (2) ,231 (12畅4),203 (23).<br />
1 -Cyclopropyl唱7唱[ 4 唱( 2, 2唱dimethylpropanoyl )唱1唱<br />
piperazinyl ]唱6唱fluoro唱4 唱oxo唱1 , 4唱dihydro唱3唱quino唱<br />
line carboxylic acid (5 )<br />
Yield:0畅80 g (80%);M.P.= 28 0 ℃ ~28 2 ℃ ; IR nmax<br />
cm -1<br />
: 3520 , 3467, 3093, 2960, 1722, 1669, 1441,<br />
1385,1256,1013; 1<br />
H -NMR (300 MHz -DMSO唱d 6 ):<br />
d 8畅6 (s,1 H,H -2 ) , 7畅9 (d,1H, J =2畅6 Hz, H -<br />
5),7畅5(d,1 H,J =5畅3 Hz,H -8),4畅3(s,1H,H -<br />
11) ,3畅7 (s,4H,H -16,20),2畅4(s,4H, H -1 7 , H<br />
-19) ,1畅2 (d,2H,J =3畅7 Hz,H -13) ,1畅0 (s,2H,<br />
H -12) ,1畅04 (s,9H,H -3 ′,4′,5′);EIMS m /z (%<br />
rel.abund.):414 (M +<br />
,14),370 (32),335 (85),<br />
330 (2畅8),314 (32),288 (33),270 (11),256<br />
(13),148 (8),56 (100).<br />
1唱Cyclopropyl唱6唱fluoro唱(4唱octanoyl唱1唱pipera唱zinyl)唱<br />
4唱oxo唱1,4唱dihydro唱3唱quinoline carboxylic acid (6)<br />
Yield:0畅48 g (43%);M.P.= 229℃ ~231 ℃;IR nmax<br />
cm -1<br />
: 3461 , 3094, 2957, 1726, 1629, 1468, 1264,<br />
1124; 1<br />
H唱NMR (300 MHz唱DMSO唱d 6 ):d 8畅6 (s,1H,<br />
H -2),7畅9(d,1H,J =7畅8 Hz,H -5),7畅5(d,1H,J<br />
=2畅9 Hz,H -8),4畅1(t,1H, J =7 畅4 Hz,H -2 ′),<br />
3畅8 (s,1H, H -11),3畅4 (s,4H, H -16,20),2畅4<br />
( s,4H,H -17,19),1畅29 -1畅21 ( m,10H, H -3 ′,<br />
4′,5 ′,6′,7′) ,0畅87 ( s,2H, H -13 ) .0畅86 ( s,2H,H<br />
-12),0畅84 (d,2H,J =7 畅3 Hz,H -8 ′);EIMS m /z<br />
(% rel.abund.):457 ( M +<br />
,10),437 (2畅5),413<br />
(2畅4) ,370 (2) ,342 ( 13) ,286 (12) ,231 (100 ) ,56<br />
(90畅5).<br />
1唱Cyclopropyl唱6唱fluoro唱7唱( 4唱n<strong>on</strong>anoyl唱1唱piperazinyl )唱<br />
4唱oxo唱1,4唱dihydro唱3唱quinoline carboxylic acid (7)<br />
Yield:0畅73 g (73%);M.P.= 151℃ ~153℃;<br />
IR nm ax cm -1<br />
: 3467,3358, 3093,2923, 1722,1628,<br />
1469,1384,1340,1257,1023; 1<br />
H唱NMR (300 MHz唱<br />
DMSO唱d 6 ):d 8畅6 (s,1H, H -2),7畅9 (d,1H, J =<br />
7畅8 Hz,H -5),7畅5 (d,1H , J =2 畅9 Hz, H -8),<br />
4畅1 (t,1H, J =7 畅4 Hz, H -2 ′) ,3畅8 (s,1H, H -<br />
11),3畅4(s,4H ,H -16,20),2畅4 (s,4 H, H -1 7 ,<br />
19),1畅31 -1畅71 ( m,12H , H -3 ′,4′,5′,6′,7′,<br />
8′),0畅87 (s,2 H,H -13 ) , 0畅86 ( s,2H, H -12),<br />
0畅84 ( d,2H, J =7 畅3 Hz, H -8 ′);EIMS m /z (%<br />
rel.abund.): 471 ( M +<br />
, 2 ), 384 ( 1畅6), 345<br />
(11畅7),231 (35),71 (12),56 (100) .<br />
1 唱Cyclopropyl唱7 唱(4唱decanoyl唱1唱piperazinyl) 7唱6 唱flu唱<br />
oro唱4唱oxo唱1, 4 唱dihydro唱3唱quinoline carboxylic acid<br />
(8 )<br />
Yield:0畅68 g (68%);M.P.= 10 7 ℃ ~10 9 ℃ ; IR nmax<br />
cm -1<br />
: 3466 , 3093, 3011, 2923, 1723, 1628, 1469,<br />
1385,1340,1257; 1<br />
H -NMR (300 MHz -DMSO -<br />
d 6 ):d 8畅6 (s,1H,H -2),7畅9(d,1H,J =7畅8 Hz,H<br />
-5 ) ,7 畅5(d,1H,J =2畅9 Hz,H -8) ,4畅1 (t,1H,J =<br />
7畅4 Hz,H -2 ′),3畅8(s,1H,H -11),3畅4(s,4H,H<br />
-16,20) ,2畅4 (s,4H,H -17,19),1畅29 -1畅21 ( m,<br />
10H, H -3 ′,4′,5′,6′,7′),0畅87 (s,2H, H -13).<br />
0畅86 (s,2H,H -1 2 ) , 0畅85 ( t,3H,J =7 畅3 Hz,H -<br />
10′);EIMS m /z (% rel.abund.) :485 (M +<br />
,16 ),384<br />
(11) ,330 (10) ,311 ( 29) ,301 (34) ,289 (44) ,275<br />
(46) ,257 (36) ,245 ( 52) ,231 (83) ,56 (100) .<br />
7 唱( 4唱Benzoyl唱1唱piperazinyl )唱1唱cyclopropyl唱6唱fluo唱<br />
ro唱4唱oxo唱1, 4唱dihydro唱3唱quinoline carboxylic acid<br />
(9 )<br />
Yield:0畅53 g (53%);M.P.= 273℃ ~275 ℃;IR nmax<br />
cm -1<br />
: 3408 , 3056, 2919, 1720, 1622, 1529, 1486,<br />
1387,1330,1251,1008; 1<br />
H -NMR (300 MHz -DM唱<br />
SO -d6 ):d 8畅6 (s,1 H,H -2 ) , 7畅9 (d,1H, J =7 畅8<br />
Hz,H -5),7畅5(d,1H,J =2畅9 Hz, H -8),7畅5-<br />
7畅4 (m,1H, H -3 ′,4′,5 ′,6′,7′),3畅8(s,1 H,H -<br />
11),3畅3 (s,4H, H -16,20),3畅2 (s,4H, H -1 7 ,<br />
19),2畅1 (s,2H,H -13) ,1畅3 (s,2 H,H -12 ) ; EIMS<br />
m /z (% rel.abund.) : 391 ( M +<br />
,44畅3),286 (1),<br />
257 (11),231 (5),177 (3),159 (1),56 (70).<br />
7 唱[4唱(2唱Chlorobenzoyl)唱1 唱piperazinyl]唱1唱cyclopro唱<br />
pyl唱6 唱fluoro唱4 唱oxo唱1,4唱dihydro唱3唱quinoline carbox唱<br />
ylic acid (10 )<br />
Yield:0畅80 g (80%);M.P.=191℃ ~193℃;IR nmax<br />
cm -1<br />
: 3468 , 3058, 2959, 1717, 1627, 1464, 1399,<br />
1340,1292; 1<br />
H -NMR (300 MHz -DMSO -d 6 ):d<br />
15畅0 (s,1H,H -14 ),8畅6 (s,1H,H -20 ) ,7畅9 (d,<br />
1 H,J =7畅8 Hz,H -5 ) ,7 畅5(d,1 H,J =5 畅5 Hz, H -<br />
8),7畅6(dd,J =8畅5 Hz,H -3 ′),7畅4(ddd, J =8 畅5<br />
Hz,H -4 ′),7畅3 (dd, J =8畅5 Hz, H -6 ′), 7畅22<br />
( ddd,J =8畅5 Hz,H -5 ′),3畅8(d,1H,J =1畅2 Hz,H<br />
-11) ,3畅4 (s,4 H,H -16,20 ) ,2畅4 (s,4H,H -1 7 ,<br />
19),1畅1 (d,2H,J =2畅3 Hz,H -13),0畅9(s,2H,H<br />
-12);EIMS m /z (% rel.abund.):560 (M +<br />
,17),<br />
516 (49),432 (2),390 (4),314 (10),288 (12),<br />
257 (16),7 (22),230 (61),2 (19),148 (20),4
JOURNAL <str<strong>on</strong>g>of</str<strong>on</strong>g> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007<br />
(61),56 (100).<br />
1 唱Cyclopropyl唱6 唱fluoro唱7 唱[ 4唱( 4唱methoxybenzoyl )唱<br />
1 唱piperazinyl]唱4唱oxo唱1, 4唱dihydro唱3唱quinoline car唱<br />
boxylic acid (11)<br />
Yield:0畅79 g (79%);M.P.=170℃ ~172℃;IR nmax<br />
cm -1<br />
: 3412 , 3016, 2930, 1719, 1602, 1467, 1433,<br />
1255,1167; 1<br />
H -NMR ( 300 MHz -DMSO -d6):d<br />
8畅6 (s,H,H -2 ) , 7畅9(d,1 H, J =6畅6 Hz,H -5),<br />
7畅6 (d,1H,J =5畅0 Hz.1,H -8 ) , 7畅4 (d,2H, J =<br />
6畅3 Hz,H -3 ′,7′),7畅1(d,2 H,J =6 畅5 Hz, H -4 ′,<br />
6′),3畅8(s,2H, H -11),3畅7 (s,3H, H -8 ′),3畅3<br />
( s,4H, H -16,20),2畅4 (s,4H, H -17,19),1畅3<br />
( d,2H,J =4畅9 Hz, H -13),0畅8(s,2H, H -12);<br />
EIMS m /z (% rel.abund.):465 (M +<br />
,2畅6),420<br />
(4),289 (3),271 (2),230 (3畅6),201 (2),151<br />
(33),135 (74),92 (33),77 (47),56 (50).<br />
1 唱Cyclopropyl唱6 唱fluoro唱4 唱oxo唱7 唱[ 4 唱( 3, 4, 5唱trime唱<br />
thoxybenzoyl )唱1唱piperazinyl ]唱1, 4唱dihydro唱3唱quin唱<br />
oline carboxylic acid (12)<br />
Yield:0畅43 g (43%);M.P.= 280℃ ~282 ℃;IR nmax<br />
cm -1<br />
: 3048 , 3056, 2919, 2855, 1720, 1298, 1622,<br />
1486,1251, 1210; 1<br />
H -NMR (300 MHz -DMSO -<br />
d 6 ):d 15畅1 (s,1H,H -14) ,8畅6 (s,1H,H -2),7畅8<br />
( d,1H,J =4畅7 Hz,H -5),7畅5 (d,1H,J =4畅7 Hz,H<br />
-7),7畅6(d,2H, J =2 畅2 Hz H -3 ′,7′),4畅1(s,<br />
1 H,H -11),3畅7 (s,9H, H -8 ′,9′,10′),3畅6 (s,<br />
4 H,H -16,20),2畅3 (s,4H, H -17,19),1畅9 (s,<br />
2 H,H -13),1畅2 (s,4H, H -12);EIMS m /z (%<br />
rel.abund.):391 (M +<br />
,6),279 (27),197 (2畅5),<br />
167 (53) ,149 ( 100) .<br />
1 -Cyclopropyl -6唱fluoro唱7唱[ 4唱( 1唱naphthoyl )唱1唱<br />
piperazinyl ]唱4唱oxo唱1, 4 唱dihydro唱3唱quinoline car唱<br />
boxylic acid<br />
Yield:0畅70 g (70%);M.P.= 283℃ ~285 ℃;IR nmax<br />
cm -1<br />
: 3480 , 3055, 2931, 1725, 1625, 1478, 1435,<br />
1386,1249, 1204; 1<br />
H -NMR (300 MHz -DMSO -<br />
d 6 ):d 15畅2 (s,1H,H -14) ,8畅6 (s,1H,H -2),8畅0<br />
( d,1H,J =6畅3 Hz,H -5 ) , 7畅5(d,1H,J =5畅0 Hz,<br />
H -8),7畅8(t,1H,J =8畅4 Hz,H -8 ′) ,7畅7 (d,1H,<br />
J =8畅4 Hz,H -6 ′),7畅66 ( d,1H, J =8畅4 Hz, H -<br />
9′),7畅61 (d,1H,J =8畅3 Hz,H -5 ′),7畅5(d,1H,J<br />
=8畅3 Hz, H -3 ′),7畅4(t,1H, J =8 畅4 Hz, H -7 ′)<br />
7畅1 (t,1H, J =8畅3 Hz, H -3′),3畅8 (s,1H, H -<br />
11) ,3畅5 (s,4H, H -16,20),2畅4 (s,4H, H -1 7 ,<br />
19),1畅2 (s,2H,H -13) ,0畅9 (s,2 H,H -12 ) ; EIMS<br />
m /z (% rel.abund.):485 (M +<br />
,0畅39),441 (19),<br />
286 ( 13 ) , 257 ( 36 ) , 245 ( 2畅8),55 (56),127<br />
(61),101 (3),56 (100).<br />
Antibacterial Studies ( In vitro)<br />
191<br />
The synthesized compounds were screened for antibac唱<br />
terial activity against the number <str<strong>on</strong>g>of</str<strong>on</strong>g> Gram -negative<br />
organisms including Klebsiella pneum<strong>on</strong>iae,Escherichi唱<br />
a coli, Proteus mirabilis, Shigella dysenteriae, Pseudo唱<br />
m<strong>on</strong>as aeruginosa, Salm<strong>on</strong>ella typhi, Salm<strong>on</strong>ella typhi<br />
para,A Salm<strong>on</strong>ella typhi para B and Gram -positive<br />
organisms like Staphylococcus aureus and Bacillus sub唱<br />
tilis by using literature protocol [18]<br />
.In the agar well dif唱<br />
fusi<strong>on</strong> method,wells were duged in the media with the<br />
help <str<strong>on</strong>g>of</str<strong>on</strong>g> a sterile metallic borer with c<strong>on</strong>tres at least 24<br />
mm part.Two to eight hours old bacterial inoculums<br />
c<strong>on</strong>taining approximately 104 ~106 col<strong>on</strong>y forming<br />
units ( CFU) /ml were spread <strong>on</strong> the surface <str<strong>on</strong>g>of</str<strong>on</strong>g> nutrient<br />
agar with the help <str<strong>on</strong>g>of</str<strong>on</strong>g> a sterile cott<strong>on</strong> swab.Cott<strong>on</strong> swab<br />
was rotated firmly against the upper inside well <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
tube to express excess fluid.Entire agar surface <str<strong>on</strong>g>of</str<strong>on</strong>g> the<br />
plate was streaked with the swab three times turning<br />
the plate 60°between each streaking.Recommended<br />
c<strong>on</strong>centrati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the test sample (2 mg /ml <str<strong>on</strong>g>of</str<strong>on</strong>g> DMSO)<br />
was then added in their respective wells.Other wells<br />
supplemented with DMSO and reference antibacterial<br />
drugs serving as negative and positive c<strong>on</strong>trols,respec唱<br />
tively.The plates were incubated immediately at 37 ℃<br />
for 14 ~19 horlater,if necessary.Antibacterial activity<br />
was determined by measuring the diameter <str<strong>on</strong>g>of</str<strong>on</strong>g> z<strong>on</strong>es<br />
showing complete inhibiti<strong>on</strong> ( mm).Growth inhibiti<strong>on</strong><br />
was calculated with reference to positive c<strong>on</strong>trol.<br />
Fungicidal Bioassay ( In vitro)<br />
The synthesized compounds were tested for antifungal<br />
activity against the fungi including Aspergillus flavus,<br />
Trichophyt<strong>on</strong> l<strong>on</strong>gifusis,Candida albicans,Microsporum<br />
canis,Fusraium solani and Candida glaberata.Ampho唱<br />
tericin B and mic<strong>on</strong>azol were used as standard drugs.<br />
For tube diffusi<strong>on</strong> test 12 mg <str<strong>on</strong>g>of</str<strong>on</strong>g> sample was dissolved in<br />
1 ml <str<strong>on</strong>g>of</str<strong>on</strong>g> DMSO serving as stock soluti<strong>on</strong>.Sabouraud<br />
dextrose agar ( Oxoid, Hampshire, England ) was<br />
seeded with 105 CFU /ml fungal spore suspensi<strong>on</strong>s and
192 JOURNAL <str<strong>on</strong>g>of</str<strong>on</strong>g> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007<br />
transferred to Petri plates.Discs soaked in 20 ml (10<br />
mg /ml in DMSO) <str<strong>on</strong>g>of</str<strong>on</strong>g> all compounds were placed at<br />
different positi<strong>on</strong>s <strong>on</strong> the agar surface.The plates were<br />
incubated at 32 ℃ for seven days.The results were<br />
recorded as z<strong>on</strong>es <str<strong>on</strong>g>of</str<strong>on</strong>g> inhibiti<strong>on</strong> in mm [19]<br />
.<br />
Cytotoxicity Bioassay ( In vitro)<br />
Twenty mg <str<strong>on</strong>g>of</str<strong>on</strong>g> the test sample was dissolved in the re唱<br />
spective organic solvent.From this stock soluti<strong>on</strong> 5,50,<br />
500 μl were transferred to vials (3 vials /c<strong>on</strong>centra唱<br />
ti<strong>on</strong>).The final c<strong>on</strong>centrati<strong>on</strong> was 10,100,1000 μg /<br />
ml respectively, and the solvent was evaporated over<br />
night.<br />
Brine shrimp ( Artemia salina leach ) eggs were<br />
hatched and after two days, when shrimp larvae were<br />
ready,1 ml <str<strong>on</strong>g>of</str<strong>on</strong>g> seawater and 10 shrimps were added to<br />
each vial (30 shrimps /diluti<strong>on</strong>) and the volume was<br />
adjusted with seawater to 5 ml per vial.After 24 hthe<br />
[ 20]<br />
number <str<strong>on</strong>g>of</str<strong>on</strong>g> survivors was counted and data were ana唱<br />
lyzed by Finney computer program to determine the<br />
[21 ]<br />
LD50 .<br />
RESULTS AND DISCUSSION<br />
Our synthetic pathway was based <strong>on</strong> the preparati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
different derivatives <str<strong>on</strong>g>of</str<strong>on</strong>g> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin [1]<br />
by treating it<br />
with respective acid chloride in tetrahydr<str<strong>on</strong>g>of</str<strong>on</strong>g>uran ( THF)<br />
in the presence <str<strong>on</strong>g>of</str<strong>on</strong>g> triethylamine ( TEA) as a base.The<br />
resultant mixture was refluxed and the completi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g><br />
the reacti<strong>on</strong> was m<strong>on</strong>itored by TLC analysis.The struc唱<br />
[2 ~13]<br />
tures <str<strong>on</strong>g>of</str<strong>on</strong>g> all the synthesized derivatives were c<strong>on</strong>唱<br />
firmed by using the sophisticated spectroscopic tech唱<br />
niques like 1H唱NMR, IR and EI mass spectrometry<br />
( Scheme 1,Table 1).<br />
Antimicrobial Activity <str<strong>on</strong>g>of</str<strong>on</strong>g> Cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin Derivatives<br />
[ 2 ~13]<br />
All the synthesized compounds were screened for<br />
their antimicrobial affects against 8 Gram唱negative in唱<br />
cluding Klebsiella pneum<strong>on</strong>iae, Escherichia coli, Pro唱<br />
teus mirabilis, Shigella dysenteriae, Pseudom<strong>on</strong>as<br />
aeruginosa,Salm<strong>on</strong>ella typhi, Salm<strong>on</strong>ella typhi para A<br />
and Salm<strong>on</strong>ella typhi para B as well as two Gram唱posi唱<br />
tive organisms namely Staphylococcus aureus and Ba唱<br />
cillus subtilis.The results <str<strong>on</strong>g>of</str<strong>on</strong>g> in vitro antibacterial activi唱<br />
ty are collected in Table 2.<br />
Table 1 The Structures <str<strong>on</strong>g>of</str<strong>on</strong>g> All the Synthesized Dervatives<br />
S.No. R Yield (%)<br />
2 -CH 3 75<br />
3 -OC 2 H 5 87<br />
4 -CHCl 2 51<br />
5 -C( CH 3 ) 3 80<br />
6 -C 7 H 15 44<br />
7 -C 8 H 17 73<br />
8 -C 9 H 19 68<br />
9 53<br />
10<br />
Cl<br />
11 OMe 79<br />
12<br />
OM e<br />
OM e<br />
OMe<br />
13 70<br />
80<br />
24
JOURNAL <str<strong>on</strong>g>of</str<strong>on</strong>g> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007<br />
HClHN<br />
F<br />
N<br />
O<br />
N<br />
THF,TEA,RCOX<br />
70 ℃<br />
X =Cl<br />
COOH<br />
R<br />
5%aq.NaHCO 3<br />
room temp erature.<br />
O<br />
N<br />
F<br />
N<br />
Scheme 1<br />
Table 2 In vitro Antimicrobial Activity Results <str<strong>on</strong>g>of</str<strong>on</strong>g> Cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin (1) and Its Derivatives (2 -13) against Gram唱negative and<br />
Gram唱positive Bacteria ( z<strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> inhibiti<strong>on</strong> in mm)<br />
Gram唱negative<br />
HN<br />
2-13<br />
O<br />
N<br />
F<br />
N<br />
1<br />
COOH<br />
O<br />
N<br />
COOH<br />
Compound No. 1 2 3 4 5 6 7 8 9 10 11 12 13<br />
Escherichia coli 28 19 6 10 6 6 13 16 26 8 7 20 9<br />
Pseudom<strong>on</strong>as aeruginosa 23 17 13 10 17 8 9 8 11 8 8 16 8<br />
Salm<strong>on</strong>ella typhi 28 19 7 18 13 12 18 12 22 6 7 18 6<br />
Salm<strong>on</strong>ella typhi para A 30 22 11 24 15 13 15 13 20 7 8 20 7<br />
Salm<strong>on</strong>ella typhi para B 32 16 12 18 13 10 10 8 18 8 6 17 11<br />
Proteus mirabilis 20 11 8 10 5 11 11 9 14 8 8 13 9<br />
Klebsiella pneum<strong>on</strong>iae 25 22 10 20 12 9 12 8 24 6 7 15 8<br />
Shigella dysenteriae 26 13 13 19 13 8 8 10 10 9 10 9 10<br />
Gram唱positive<br />
Bacillus subtilis 27 25 10 16 8 7 14 10 17 10 9 14 10<br />
Staphylococcus aureus 30 20 6 8 9 14 12 11 23 7 7 22 6<br />
It was found that the structural analogues <str<strong>on</strong>g>of</str<strong>on</strong>g> cipro唱<br />
floxacin (1) showed varying degree <str<strong>on</strong>g>of</str<strong>on</strong>g> antimicrobial<br />
activity against all the tested bacterial strains.Com唱<br />
pounds 9 and 2 showed good activities against Esche唱<br />
richia coli,however,compound 2 exhibited comparable<br />
activity against Bacillus subtilis.Good activities <str<strong>on</strong>g>of</str<strong>on</strong>g> com唱<br />
pounds 2,9,and 12 were observed against Staphylococ唱<br />
cus aureus,while compound 9 showed good activity a唱<br />
gainst Salm<strong>on</strong>ella typhi.Four derivatives 2,4,9,and 12<br />
exhibited good activities against Salm<strong>on</strong>ella typhi para<br />
A,where compound 2,4,and 9 were found to be active<br />
against Klebsiella pneum<strong>on</strong>iae.Compound 9 in the case<br />
193<br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> Klebsiella pneum<strong>on</strong>iae showed almost equivalent de唱<br />
gree <str<strong>on</strong>g>of</str<strong>on</strong>g> activity compared to the cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin [1]<br />
.<br />
In general,the compound 2,4,9,and 12 showed<br />
good activities against Escherichia coli, Bacillus subti唱<br />
lis, Staphylococcus aureus,Salm<strong>on</strong>ella typhi,Salm<strong>on</strong>el唱<br />
la typhi para A and Klebsiella pneum<strong>on</strong>iae.Comparis<strong>on</strong><br />
<str<strong>on</strong>g>of</str<strong>on</strong>g> the activities <str<strong>on</strong>g>of</str<strong>on</strong>g> different analogues <str<strong>on</strong>g>of</str<strong>on</strong>g> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin<br />
(1) indicates that the amidic linkage at piperazine<br />
moiety may be resp<strong>on</strong>sible for the changes in the anti唱<br />
microbial properties <str<strong>on</strong>g>of</str<strong>on</strong>g> the parent molecule.A careful<br />
selecti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> the substituent at the nitrogen <str<strong>on</strong>g>of</str<strong>on</strong>g> piperazine<br />
nucleus may result into a more active antibiotic based
194 JOURNAL <str<strong>on</strong>g>of</str<strong>on</strong>g> CHINESE CLINICAL MEDICINE VOLUME 2|NUMBER 4|April 2007<br />
Table 3 Results <str<strong>on</strong>g>of</str<strong>on</strong>g> Screening Cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin (1) and its Derivatives (2 -13) for their Antifungal Activity in vitro ( z<strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> in唱<br />
hibiti<strong>on</strong> in mm)<br />
Test fungi<br />
Compound No. A B 1 2 3 4 5 6 7 8 9 10 11 12 13<br />
Trichophyt<strong>on</strong>l<strong>on</strong>gifusus 100 0 0 0 0 0 0 0 60 60 0 25 0 0 0<br />
Candida albicans 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0<br />
Aspergillus flavus 0 100 0 0 0 0 0 0 60 0 0 45 0 0 0<br />
Microsporum canis 100 0 0 0 0 0 0 0 55 55 0 50 0 50 0<br />
Fusarium solani 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0<br />
Candida glaberata 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0<br />
Note:A =Mic<strong>on</strong>azole;B =Amphotericine B<br />
<strong>on</strong> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin (1).These preliminary studies provide<br />
us lead compounds like 2,4,9,and 12 for further modi唱<br />
ficati<strong>on</strong>s at nitrogen <str<strong>on</strong>g>of</str<strong>on</strong>g> piperazine or other active posi唱<br />
ti<strong>on</strong>s like carboxylic acid residue or the ket<strong>on</strong>ic carb<strong>on</strong>唱<br />
yl functi<strong>on</strong>.It may be envisi<strong>on</strong>ed that the <str<strong>on</strong>g>modificati<strong>on</strong>s</str<strong>on</strong>g><br />
at nitrogen <str<strong>on</strong>g>of</str<strong>on</strong>g> piperazine residue and other two seem唱<br />
ingly active positi<strong>on</strong>s may result into a better antibiotic<br />
with the minimizati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> its side effects.<br />
Antifungal Activity<br />
An interesting feature <str<strong>on</strong>g>of</str<strong>on</strong>g> the present study is that when<br />
all synthesized compounds (1 ~12) al<strong>on</strong>g with cipro唱<br />
floxacin (1) were randomly screened for 6 fungal<br />
strains i.e.Trichophyt<strong>on</strong> l<strong>on</strong>gifusus,Candida albicans,<br />
Aspergillus flavus,Microsporumcanis,Fusarium solani<br />
and Candida glaberata,it is revealed that cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin<br />
(1 ) was inactive against any <str<strong>on</strong>g>of</str<strong>on</strong>g> the fungal strains<br />
used.Nevertheless,compound 7 showed good activities<br />
against Trichophyt<strong>on</strong> l<strong>on</strong>gifusus,Aspergillus flavus and<br />
Microsporumcanis.Compound 8 was found to be active<br />
against Trichophyt<strong>on</strong> l<strong>on</strong>gifusus and Microsporum canis<br />
strains.Compound 10 showed moderate activities a唱<br />
gainst Aspergillus flavus and Microsporum canis,while<br />
compound 12 was found to be moderately active against<br />
Microsporumcanis. Antifungal activity in cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin analogues may<br />
open new avenues for designing antifungal agents based<br />
<strong>on</strong> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin (1) skelet<strong>on</strong>.For enhancing the anti唱<br />
fungal strength <str<strong>on</strong>g>of</str<strong>on</strong>g> the lead molecules 7,8,10,and 12,<br />
either modificati<strong>on</strong> in the alkyl side chain <str<strong>on</strong>g>of</str<strong>on</strong>g> compound<br />
6 and 7 or by varying the substituti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> phenyl ring in<br />
compound 10 and 12.In additi<strong>on</strong>,the synthetic modifi唱<br />
cati<strong>on</strong>s <strong>on</strong> carboxyl group or ket<strong>on</strong>ic carb<strong>on</strong>yl functi<strong>on</strong><br />
may enhance the antifungal activity.<br />
Cytotoxic Activity<br />
Bioactive compounds are <str<strong>on</strong>g>of</str<strong>on</strong>g>ten toxic to Artemia salina<br />
( leach, brine shrimp ) larvae.The bioassay method<br />
adopted is rapid and inexpensive which has been devel唱<br />
oped for screening and m<strong>on</strong>itoring <str<strong>on</strong>g>of</str<strong>on</strong>g> physiologically<br />
active compound.<br />
During this experiment etoposide is used as stan唱<br />
dared drug and LD50 is found to be 0畅178 μg /ml while<br />
it has been found that neither cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin nor its de唱<br />
rivatives is cytotoxic.<br />
CONCLUSION<br />
The <str<strong>on</strong>g>skeletal</str<strong>on</strong>g> <str<strong>on</strong>g>modificati<strong>on</strong>s</str<strong>on</strong>g> <str<strong>on</strong>g>of</str<strong>on</strong>g> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin (1) showed<br />
a varying degree <str<strong>on</strong>g>of</str<strong>on</strong>g> antimicrobial activities particularly<br />
compound 2,4,9,and 12 exhibited promising antimi唱<br />
crobial activities devoid <str<strong>on</strong>g>of</str<strong>on</strong>g> any toxicity and may be<br />
used as lead compounds for further investigati<strong>on</strong>s.Ad唱<br />
diti<strong>on</strong>ally compound 7,8,10,and 12 exhibited encoura唱<br />
ging antifungal activities and provided a base to explore<br />
the antifungal activities <str<strong>on</strong>g>of</str<strong>on</strong>g> cipr<str<strong>on</strong>g>of</str<strong>on</strong>g>loxacin (1) skelet<strong>on</strong><br />
c<strong>on</strong>taining molecules.Further research based <strong>on</strong> over<br />
preliminary results may lead to a new class <str<strong>on</strong>g>of</str<strong>on</strong>g> com唱<br />
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( Editor LEE)