Arch. Pharm. Res.

14(1), 48-51 (1991)

Nalidixic Acid Prodrugs: Amides from amino acid

ester and nalidixic acid

Ashish Taori, Rajesh Nema, D. V. Kohli and R. K. Uppadhyay

Department of Pharmaceutica/ Science, Dr. H. S. Gout Vikhwavidya/ya, Sagar (I~P.), India
(Received November 26, 1990)

Abstraet [] Amides from amino acid ester and nalidixic acid were synthesized. The
solubility characteristics and partition coefficient of the compounds were studied. The
hydrolysis of the compounds was studied in the simulated gastric fluid and simulated
intestinal fluid. Some compounds showed better antibacterial activity than nalidixic acid.

Keywords[]Nalidixic acid, amide, amino acid ester, partition coefficient

Nalidixic acid (1-ethyl-7-methyl-4-oxo-l,4-dihydro- stirred under reflux for 4 hours. The solvent was
1,8-napthyridine-3-carboxylic acid) is an antimicro- removed under reduced pressure and the residue
bial agent. Most widely used against infections due was triturated with several 20 ml portions of cold
to gram negative organisms and specially in the ether at 0~ The crude product was recrystallized
urinary tract infections J). It has a high melting point from hot methanol (25 m/) by slow addition of ether
(225-231~ and has a relatively poor water and lipid (150-200 m/) followed by cooling at 0~ L-alanine
solubility. methyl ester hydrochloride (79% yield, mp. 153-155 ~
The present work aims at the preparation and L-phenylalanine methyl ester hydrochloride (81%
evaluation of some amide derivatives of nalidixic yield, rap. 158-163 ~ DL-glycine methyl ester hydro-
acid which would be non-irritable, readily absorb- chloride (83% yield, mp. 162=164~ and DL-trypto-
able, rapidly showing its effects and has got a grea- phan methyl ester hydrochloride (76% yield mp.
ter half life in body. 201~ ~) were thus synthesized.
The objective of this study was to investigate: a)
whether amide derivatives of nalidixic acid would Nalidiric acid chloride
behave as prodrugs, b) to what extent the physical Nalidixic acid 40g (0.17 mol) was dissolved in 300
properties of the prodrugs vary with structure, c) ml of chloroform and then the solution was added
to what degree the in vitro cleavage rates vary with to 23.8 g (0.2 moo of thionyl chloride. The contents
structure and d) to seek prodrug derivatives of na- were refluxed for 4 hours at 60~176 The solvent
lidixic acid which would be non-irritant, readily ab- and excess of thionyl chloride were distilled off un-
sorbed and rapidly hydrolyzed to release free drug. der reduced pressure to give the pure nalidixic acid
chloride in the solid form.
EXPERIMENTAL METHODS
Amides from amino acid ester hydrochloride and nalidixic
General procedure for the synthesis acid chloride
The compounds were synthesized in the following The procedure based on modified Scholten-Bau-
steps: Methyl ester hydrochloride of amino acid uti- men reaction3) was adapted. Amino acid methyl es-
lizing the process of Ronald 2), methyl ester hydroch- ter hydrochloride (0.025 mol) was added slowly to
lorides of L-alanine, L-phenylalanine and trypto- 50ml ice cold potassium carbonate solution (10%),
phan were synthesized. Amino acid (0.05 mol) was and the reaction mixture was stirred for 30 minutes
added to a solution of thionyl chloride (0.05 mol at room temperature. To this solution nalidixic acid
in methanol) and the reaction mixture was then chloride (0.025 tool) was added gradually and finally

48
 Na//ktbdc Acl~ Prodrugs 49

Table I. Characteristics of amide derivatives of nalidixie acid

C2H,
I
H3C "-,,r/~N . ' 2 ~ N ~ .

CONHR
II
O

S. No, Compound R Molecular Yield % Melting

formula* point ~

1 1 -CH-CH2 ~ C22H>N~O4 85.12 161-163

I N_=/--
COOCH3
2 2 -CH2 CIsHlTN304 85.90 185-187
/
COOCH3 H

3 3 -CH-CH~ 1~_12 C24Hz4N404 86.40 164-167

/
COOCH3
-CH-CH3
4 4 I ClsHlgN304 87.23 184-187
COOCH3
*All compounds were analysed for C,H and N. All values were within -+ 0.4% of the theoretical value. The IR
spectra in KBr phase showed the presence of amide linkage in all compounds. The m.p. was determined in
open capillaries and are uncorrected.

Table H. Physical data for amide derivatives of nalidixie acid

Compound Solubility in Partition coeffi- Fractional Availavility

(prodmg) 0.1 N HC1 benzene cient benzene/ distribution factor
mg/ml mg/m/ 0.1 N HCI f.d.
1 0.7 12.5 17.85 0.9469 16.84
2 I. 1 27.0 24.54 0.9608 34.83
3 0.9 32.0 35.55 0.9726 30.23
4 1.4 45.0 32.14 0.9698 42.77
Fractional distribution (f.d.) of the compound in benzene/water system calculated by dividing the benzene solubility
by the sum of the benzene and water solubility.
Availability factor=f.d.• aqueous solubility • 104

the mixture was stirred vigorously for 2 hours at 37~ were determined in 0.1 N HC1 and benzene
10~ The compounds thus obtained were washed (spectral grade) in rotating bottles using an assem-
with 0.5% cold sodium hydroxide solution and recry- bly reported earlier4). HC1 (0.1 N) was employed in
stallized from chloroform. The physical data of order to ascertain that the compounds remained
these compounds are listed in Table I and II. completely unionized in the aqueous medium and
also to reduce the degree of hydrolysis of the com-
EVALUATION OF PRODRUGS pounds during equilibration. The aqueous samples
were rotated for 6 hours and the benzene samples
Solubility studies for 16 hours. Excess compound was removed by
The saturation solubilities of the compunds at filtration through a sintered glass funnel and sam-
50 As/wsh Toori Rojosh Nero& D.~ Koh/i ond H.l( Uppodhyay

Table III. In vitro hydrolysis of prodrugs in simulated gastric and intestinal fluid

S.No. Time % Release of % Release of % Release of % Release of

prodrug 1 prodrug 2 prodrug 3 prodrug 4
S.G.F. I,F. S.G.F. I.F. S.G.F. I.F. S.G.F. I.F.
l 0.5 12.6 16.8 8.5 14.5 12.4 12.5 11.0 15.0
2 1.0 23.0 30.0 21.5 31.0 19.6 26.2 21.2 29.5
3 1.5 33.28 40.0 29.06 39.5 27.0 40.8 38.2 43.0
4 2.0 39.5 52.4 34.5 48.4 34.2 50.2 36.8 54.6
5 2.5 45.1 59.0 39.24 61.6 40.0 56.0 44.0 64,0
6 3.0 50.0 66.2 44.26 68.0 47.2 61.0 51.0 70.8
7 3.5 55.5 75.5 48.52 71.0 52.4 62.8 57.8 76,4
8 4.0 59.2 77.0 50.5 72.6 54.0 64.2 60,4 78,8
S.G.F.=Simulated gastric fluid, I . F . : Intestinal fluid.

pies were analysed. in compound 1.

All the compounds had a melting point lower
Determination of partition coefficient than nalidixic acid (mp. 225-231~ The higher mel-
The partition coefficient of each compound be- ting point of nalidixic acid may probably be ascri-
tween benzene and 0.1 N HC1 was determined us- bed to intramolecular hydrogen bonding 8). By intro-
ing a method reported earlier5). The partition co- ducing the amide linkage such hydrogen bonding
efficients can be regulated of the unionized com- is no longer possible which helps to decrease the
pounds as HC1 (0.1N) was used gs the aque- crystal lattice forces present in the acid. Partition
ous phase. coefficients and lipid solubility of the compounds
was higher as compounds to the parent drug.
Availability factors
The availability factors which are products of the DISCUSSION
molar aqueous solubilities and a factor related to
the lipid/water distribution characteristics of the com- The results of antibacterial activity are shown in
pounds were determined 6). Table IV, all the compounds showed antibacterial
activity at 0.1% w/v concentration. The antibacterial
In vitro hydrolysis rates activity at 0.1% w/v concentration is significant. The
Half lives for hydrolysis of the compounds at 37~ increase in the concentration of the active com-
in 0.1 N phosphate buffer with trypsin (0.1%) and pounds increase the activity. Compound 3 was
in simulated gastric fluid (pH 1.2) were determined. found to be more potent than nalidixic acid, where
The results are shown in Table III. as other three compounds showed less activity
than nalidixic acid.
Anti-bacterial activity This study was concerned primarily with establi-
All the compounds were studied for their antibac- shing the principles rather than a determination of
terial activity against E. coli and S. typhi using agar synthesized compoounds. The aminde linkage
diffusion with cup method 71. The results are shown which was employed to activity profiles of create
in Table IV. derivative of nalidixic acid improved the physico-
chemical parameters of the compounds. It seems
RESULTS fair to assume that the compounds reported here
would behave as nalidixic acid prodmgs.
The amide derivatives of nalidixic acid were less
soluble in 0.1 N HC1 and have greater solubility ACKNOWLEDGEMENT
in benzene. Compounds 2, 3 and 4 were found
to have greater solubility in 0.1 N HC1 than 1 show- Authors are thankful to Dr. N. K. Jain, Professor
ing the presence of highest crystal lattice energy and Head, Department of Pharmaceutical Sciences,
 Na/idixic Acid Prodrugs 51

Table IV. Anfibaterial activity

Micro- Average diameter of zone of inhibition (ram)

organism Compound 1 Compound 2 Compound 3 Compound 4
a b c a b c a b c a b c
tvphi 10.5 13.0 12.5 10.0 12.0 13.0 12.0 14.0 13.0 11.0 12.5 13.0
E. coli 11.0 13.5 13.0 - 11.5 12.5 13.0 15.0 13.0 10.5 12.5 13.0
a: 0.1% w/v solution of compound in DMF
b: 0,2% w/v solution of compound in DMF
c: 0.2% w/v solution of nalidixic acid

Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.) for 4. Sounder, J. C. and Ellenbogen, W. C.: Control
providing necessary facilities to carry out this work. of damphetamine sulphate sustained release cap-
We are also thankful to Dr. Meena Tripathi, Resear- sules, Drug Std. 26, 77 (1958).
ch Associate, Department of Pharmaceutical Scien- 5. Dittert, L. W., Irwin, G. M., Chong, C. W. and
ces, Dr. H. S. Gour Vishwavidyalaya, Sagar (M.P) Swintosky, J. V.: Acetaminophen prodrugs. II-En-
for valuable suggestion. zymic and non-enzymic hydrolysis of carbonate
esters, J. Pharm. Sci 57, 780 (1968).
LITERATURE CITED 6. Ditterk L. W., Caldwell, H. C., Adams, H. J., Ir-
win, G. M. and Scointosky, J. V.: Acetaminophen
1. Raynold, J.E.F. (ed.).: Martindale-The Extra Phar- prodrugs. I-Synthesis, physicochemical properties
macopoeia, 28th ed., The Pharmaceutical Press, and analgesic activity, J. Pharma. Sci 57, 774
London, pp. 1047-1051 (1908). (1968).
2. Webb, Ronald, Haskell, M. W. and Stammer, 7. Rawlin's E. A.: Bentley's Test Book of Pharmaceu-
C. H.: A nuclear magnetic resonance method for tics, 8th ed., English Language Book Society, Lon-
distinguishing a-amino acids from B & Y iso- don, p 447 (1977).
mers, J. Org. Chem. 34, 576 (1969). 8. Bundgaard, H., Mork, N. and Heolgarrd, A.:
3. Morrison, R. T. and Boyd, R. N.: Organic Chemis- Enhanced delivery of nalidixic acid through hu-
try, 4th ed., Allyn and Bacon, Inc. Boston, p. 821 man skin via acyloxymethyl ester prodrugs. Int.
(1983). J Pharm. 55, 91 (1989).