Diglyceride prodrug strategy for enhancing the bioavailability of norfloxacin

Article abstract of DOI:10.1016/j.chemphyslip.2011.03.006

Prodrug approach using diglyceride as a promoiety is a promising strategy to improve bioavailability of poorly absorbed drugs and the same was explored in the present work to improve oral bioavailability of norfloxacin; a second generation fluoroquinolone antibacterial. The prodrug was synthesized by standard procedures using dipalmitine as a carrier and the structure was confirmed by spectral analysis. Higher Log P indicated improved lipophilicity. The ester linkage between norfloxacin and dipalmitine would be susceptible to hydrolysis by lipases to release the parent drug and carrier in the body. In vivo kinetic studies in rats indicated 53% release of norfloxacin in plasma at the end of 8 h. The prodrug exhibited improved pharmacological profile than the parent compound at equimolar dose that indirectly indicated improved bioavailability.

Full text of DOI:10.1016/j.chemphyslip.2011.03.006

Chemistry and Physics of Lipids 164 (2011) 307–313
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Diglyceride prodrug strategy for enhancing the bioavailability of norfloxacin
Suneela Dhaneshwara,∗, Kunal Tewaria, Sonali Joshib, Dhanashree Godboleb, Pinaki Ghoshc
a Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Paud Road, Pune 411038, Maharashtra, India
b Department of Biotechnology, Fergusson College, Pune, India
c Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Pune 411038, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Prodrug approach using diglyceride as a promoiety is a promising strategy to improve bioavailability of
poorly absorbed drugs and the same was explored in the present work to improve oral bioavailability of
norfloxacin; a second generation fluoroquinolone antibacterial. The prodrug was synthesized by standard
procedures using dipalmitine as a carrier and the structure was confirmed by spectral analysis. Higher
Log P indicated improved lipophilicity. The ester linkage between norfloxacin and dipalmitine would be
susceptible to hydrolysis by lipases to release the parent drug and carrier in the body. In vivo kinetic
studies in rats indicated 53% release of norfloxacin in plasma at the end of 8 h. The prodrug exhibited
improved pharmacological profile than the parent compound at equimolar dose that indirectly indicated
improved bioavailability.
Received 1 January 2011
Received in revised form 25 March 2011
Accepted 28 March 2011
Available online 6 April 2011
Keywords:
Prodrug
Bioavailability
Norfloxacin
Diglyceride
© 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
passage (Notari, 1987). Drugs such as LK-A, l-dopa, bupranolol,
phenytoin and closantel have been conjugated with diglyceride
Norfloxacin (NFX) is a second generation fluoroquinolone. The
mechanism of entry into cells is by diffusion (Chu and Fernandes,
1991). Because of low lipophilicity, it very slowly diffuses through
cell membrane and is unable to attain therapeutic concentration
at the site of infection. NFX faces a major shortcoming of low
bioavailability (30–40%), single oral dose of 400 mg giving peak
serum level of about 1.5 ␮g/ml. Due to limited plasma protein bind-
ing (5–15%), NFX is efficiently excreted via renal route that results
in its serum half life of approximately 3 h (Coessens et al., 1997).
NFX has limited clinical use in the treatment of urinary tract infec-
tions, although it is active in vitro against many Gram-positive
and Gram-negative bacteria because its urine concentration is
100–300 times higher than that in serum which largely exceeds its
minimal inhibitory concentration (MIC) and minimal bactericidal
concentration (MBC) (Siporin, 1989). Enhancing NFX penetration
into the infection site and slowing down renal filtration would
improve the antibacterial efficiency of this antibiotic. One of the
approaches in improving the bioavailability and hence the antibac-
terial efficiency is the prodrug design/drug latentiation. Prodrugs
are considered as drugs containing specialized non-toxic protective
groups utilized in a transient manner to alter or eliminate the unde-
sirable properties of the parent drug molecule. This approach is
widely applicable in enhancing bioavailability and bio-membrane
for improved bioavailability (Sugihara and Furuuchi, 1988; Garzon-
Aburbeh et al., 1986; Mantelli et al., 1985; Scriba, 1993a,b; Scriba
et al., 1995; Lambert et al., 1995; Poupaert et al., 1995). Review
of literature reveals that efforts are mainly focused on derivati-
zation of piperazine nitrogen of NFX for designing its prodrugs
with improved pharmaceutical, pharmacokinetic and pharmaco-
dynamic properties. Mannich bases (Abuo-Rahma et al., 2009)
and N-masked prodrugs (Hirosato et al., 1989) of NFX have been
reported for enhancing its lipophilicity, bioavalability and in vivo
activity. (Acyloxy) alkyl carbamate esters of NFX were designed
to mask its bitter taste (Alexander et al., 1991). Macromolecular
dextran prodrug of NFX with improved water solubility has been
developed for its intravenous administration (Veerle and Etienne,
1997). However there are no reported attempts where carboxylic
group of NFX has been derivatized for designing its prodrug. The
present work explores dipalmitin as a promoiety to improve oral
bioavailability of NFX.
Temporary masking of carboxylic group can also protect NFX
from forming chelates with polyvalent metal ions which would
indirectly help to enhance its bioavailability. The ester linkage
between NFX and diglyceride would be hydrolyzed by lipases to
release the parent drug and carrier in the body.
2. Chemistry (Scheme 1)
∗
Dipalmitate of 1,3-dihydroxyacetone (3) was synthesized by
stirring dihydroxyacetone (1) and palmitoyl chloride (2) in chlo-
Corresponding author. Tel.: +91 202 543 7237.
E-mail address: suneeladhaneshwar@rediffmail.com (Suneela Dhaneshwar).
0009-3084/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2011.03.006

308
S. Dhaneshwar et al. / Chemistry and Physics of Lipids 164 (2011) 307–313
Scheme 1.
roform in presence of anhydrous pyridine for 3 h and was then
reduced to 1,3-dipalmitin (4) with the help of sodium borohy-
dride at 5 ◦C by external cooling (Bentley and McCrae, 1970).
The piperazine –NH of NFX (5) was BOC-protected by stirring
it with Boc2O, 1 M NaOH and THF overnight at room temper-
ature (Tanaka et al., 2008). The BOC-protected NFX (6) was
converted into its acid chloride (7) by heating it with PCl5
for 30 min in a water bath (Furniss et al., 1989). Compounds
(4) and (7) were coupled by stirring the solution for 40 h at
room temperature to give BOC-protected prodrug (8) which was
recrystallized from petroleum ether (Khan and Akhter, 2005).
Deprotection was done by adding TMSBr in one portion to stirring
solution of (8) in CH2Cl2 to get prodrug NFXDG (norfloxacin-
1,3-dipalmitin ester or 1-ethyl-6-fluoro-4-oxo-7-piperazin-1-yl-
1,4-dihydro-quinoline-3-carboxylate of 1,3-dipalmitoyl-glycerol).
It was further purified by preparative TLC using solvent system ben-
zene:ethyl acetate:methanol:glacial acetic acid (60:60:15:30 drop,
v/v).
a pump (Jasco LC Net II/ADC, Serial No. B224461095), with sampler
programmed at 20 ␮l capacity per injection and a UV/VIS detector
(Jasco UV 2075). Data were integrated using Jasco Borwin version
1.5. The column used was HiQ Sil C18HS (4.6 mm I.D. × 250 mm
L; Batch: #080253; Column Number: 0HS00422) in the reversed
phase partition chromatographic condition. Particle size of the
column was 5 ␮m. The system was used in an air-conditioned
HPLC laboratory atmosphere (20 1 ◦C). Before analysis, the mobile
phase was degassed using sonicator and filtered through a 0.45 ␮m
membrane filter. Sample solutions were also filtered through the
same. The system was equilibrated before making an injection. The
column was monitored for UV absorbance at a detection wave-
length selected after considering the overlay spectra of all the
components. All the kinetic studies were carried out in tripli-
cate. The K values from the plots were calculated separately and
average K and S.D. value was determined. The half lives were cal-
culated using the software ‘PCP Disso’ developed by Department of
Pharmaceutics, Poona College of Pharmacy, Pune. The process was
validated as per U.S.P. XXIV edition using different parameters like
accuracy, selectivity, sensitivity and reproducibility.
3. Experimental
HCl buffer (100 ml) was taken in a 250 ml beaker kept on a mag-
netic stirrer at a temperature of 37 ◦C with continuous stirring.
NFXDG (13.6 mg) was added to this buffer solution. Immediately
0.1 ml of sample was withdrawn (0 min reading) from beaker
with help of 1 ml pipette whose end was tied with muslin cloth
and simultaneously 0.1 ml of buffer was replaced in the beaker.
20 ␮l of this sample was injected in the C18 column and eluted
with the mobile phase phosphate buffer 0.05 M (pH 3.0 adjusted
3.1. In vitro hydrolysis kinetics
A new HPLC method was developed for estimation of NFX
released from NFXDG in HCl buffer (pH 1.2), phosphate buffer
(pH 7.4), stomach and intestinal homogenates (Rainsford, 1975;
Morris et al., 1989; Nielson and Bundgaard, 1988; Jung et al., 2001;
Scheline, 1973). The HPLC system used for this purpose consisted of

S. Dhaneshwar et al. / Chemistry and Physics of Lipids 164 (2011) 307–313
309
with o-phosphoric acid):acetonitrile (25:75, v/v) at a flow rate of
1.2 ml/min and elute was monitored at wavelength of 279 nm and
chromatograms of all the components were taken by measuring
the absorption with a sensitivity of AUFS 0.01. Similarly samples
were withdrawn after every 15 min till 1 hand then on an hourly
basis till 6 h and injected in the column.
were then vortexed for 2 min and again centrifuged at 5000 rpm for
10 min at 0–5 ◦C. The samples were analyzed by HPLC, using same
procedure as mentioned above for in vivo release in blood. The con-
centration of NFX in rat urine and feces was calculated using the
equations Y = 32072x + 14879 and Y = 26713x + 40315 respectively
which were generated from calibration curves in rat urine and feces.
Same procedure as described earlier was followed for phos-
phate buffer. The kinetics was monitored by the increase in NFX
concentration with time.
3.3. Antimicrobial screening (Cheng et al., 2009; Kim et al., 2010;
Desboisa et al., 2010)
To study the release of NFX from NFXDG in stomach
homogenate, a male Wistar rat was anesthetized by ether and mid-
line incision was made. Sections of stomach were collected and
washed to remove their contents, homogenized and diluted to half
concentration with isotonic HCl buffer (pH 1.2). NFXDG (13.6 mg)
was dissolved in minimum amount of DMF (1–2 ml) and volume
was made up to 10 ml with HCl buffer (pH 1.2) (1360 ␮g/ml). 5 ml
of this solution was added to 10 ml volumetric flask and volume was
made up to 10 ml with HCl buffer (pH 1.2) (680 ␮g/ml). This was
considered as the stock solution. To each eppendorf tube, 0.8 ml of
the stock solution of prodrug and 0.2 ml of stomach homogenate
was added and kept in incubator at 37 ◦C. The eppendorf tubes
were taken out at different time intervals and centrifuged at
5000 rpm at 4 ◦C for 10 min. The supernatant (0.1 ml) was taken
in fresh eppendorf tube and 0.9 ml of methanol was added to it
and centrifuged again at 5000 rpm and 4 ◦C for 10 min. The sample
(20 ␮l) was injected in the C18 column and eluted with the mobile
phase phosphate buffer 0.05 M (pH 3.0 adjusted with o-phosphoric
acid):acetonitrile (25:75, v/v) at a flow rate of 1.2 ml/min and elute
was monitored at wavelength of 279 nm. Similar procedure was
followed for small intestinal homogenate and release was studied
till 10 h.
NFXDG was screened for in vivo antimicrobial activity in Swiss
albino mice infected with S. aureus in a mouse renal abscess model
described by Kim et al. (2010) and the results were compared with
the standard i.e. NFX. The bacterial strain S. aureus (NCIM 2079) was
procured from National Collection of Industrial Microorganisms
(NCIM), Pune, India which was grown in nutrient broth (100 ml)
culture medium at 37 ◦C for 18–20 h. Growth of colonies of bac-
teria was monitored on mannitol salt agar base. Nutrient broth
and mannitol salt agar were procured from HIMEDIA, India. Aver-
age of six reading was calculated and all data were expressed as
mean S.E.M. Statistical differences between the groups were cal-
culated by two-way ANOVA followed by Bonferroni’s test.
Suspension of microorganism (S. aureus) was prepared by
adding one loopful of bacterial culture into 100 ml of sterilized
nutrient broth. It was incubated for 24 h at 37 ◦C in incubator shaker.
Optical density (OD) was measured at 600 nm and the solution was
diluted until absorbance was equal to 0.5 which corresponded to
1 × 107 CFU/ml. Mice were weighed, marked and segregated into
various treatment groups. Tail vein of mouse was dilated with
absolute alcohol and then inoculated by injecting 1 ml of S. aureus
suspension using a 30 gauge needle and syringe. After 12 h of infec-
tion, they were orally administered with 3 doses of NFX (5.5 mg/kg)
and NFXDG (14.98 mg/kg) at the intervals of 6 h. On 4th day of
infection, animals were sacrificed by cervical dislocation. A midline
incision was made and kidneys were identified and dissected out.
They were washed with sterilized phosphate buffer saline (PBS) or
sterile water for injection (SWFI) to remove blood. Adrenal glands
were carefully removed without damaging the kidney. One kidney
was fixed in 10% formalin and sent for histopathological studies.
5 ␮m sections of kidney were taken and stained with haematoxylin
and eosin. The other kidney was quickly transferred to a sterilized
eppendorf tube containing 500 ␮l of sterilized PBS or SWFI. It was
homogenized in aseptic conditions using sterilized pestles. 1 ml of
homogenate was added to 9 ml of sterile distilled water in a test
tube (dilution 1:10) and subsequently serially diluted till dilution
of 1:108 was achieved. One ml of each dilution was mixed with
15 ml of sterile nutrient agar medium (cooled to 45 ◦C) and poured
in a sterile Petri dish. All the Petri-dishes were incubated at 37 ◦C for
24 h. Incubator was pre-sterilized by fumigation. S. aureus colonies
showing golden yellow pigmentation were counted and docu-
mented for each plate. Colonies were counted by a blinded observer
who was unaware of experimental protocols. The results were
recorded as CFU g−1 of tissue and are expressed as Log10 (CFU) g−1
of kidney tissue.
3.2. In vivo hydrolysis kinetics (Scheline, 1973; Nakamura et al.,
1992)
For in vivo kinetic studies, a new HPLC method was developed for
simultaneous estimation of NFXDG and NFX in rat plasma, feces and
urine of Wistar rats. Same HPLC instrument with its accessories was
used for this study as described in Section 3.1. Human plasma used
for method development was purchased from Serological Institute
of India, Navi Peth, Pune, India.
A male Wistar rat was starved for 24 h prior to use. The reading
obtained prior to drug treatment was considered as zero min read-
ing. An oral dose of NFXDG (36.84 mg/kg) was administered and
blood samples were collected by retro-orbital puncture from both
the eyes of the rat in EDTA-coated tubes at an interval of 15 min for
the next 1 h. Then subsequently blood was collected on an hourly
basis till 12 h and finally at 24th hour. The tubes were centrifuged at
5000 rpm at 0–5 ◦C for 10 min. Supernatant solution (0.1 ml) of cen-
trifuged blood was added to eppendorf tube and 0.9 ml methanol
was added to it for plasma protein precipitation. All the solutions
were then vortexed for 2 min and again centrifuged at 5000 rpm for
10 min at 0–5 ◦C. Then these samples were analyzed by HPLC, using
same procedure as mentioned in Section 3.1. The concentration of
NFX released from NFXDG in plasma was calculated using the equa-
tion Y = 189902x − 104132 which was generated from calibration
curve in plasma.
In order to study the release of NFX in urine and feces, male
Wistar rats were starved for 24 h prior to use but had free access to
water. The animals were then placed in metabolic cages and NFXDG
(36.84 mg/kg) was orally administered. The pooled samples of urine
and feces were collected at 24 h and were diluted with isotonic
phosphate buffer solution (pH 7.4) by 10-fold and then centrifuged
at 5000 rpm at 0–5 ◦C for 10 min. The supernatant solutions (0.1 ml)
were added to another set of eppendorf tubes (1 ml capacity) and
0.9 ml methanol was added to each one of them. The solutions
Histopathologicalstudies ofthe kidneysofmicewere carriedout
at Dhande Laboratory, Pune, India. Photomicrographs of the same
were taken on Leica Microscope attached with a camera (10×/20×),
provided by Department of Histopathology, Ruby Hall Clinic, Pune.
3.4. Evaluation of survival rate in mice (Ikeda et al., 2000)
Swiss albino mice were divided in 3 groups (n = 10). All the ani-
mals were infected by S. aureus. Group I (negative control) did not
receive any treatment, whereas groups II and III received 3 doses
of NFX (5.5 mg/kg) and NFXDG (14.98 mg/kg) respectively at a time
interval of 6 h. No further treatment was given to the animals after-

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S. Dhaneshwar et al. / Chemistry and Physics of Lipids 164 (2011) 307–313
wards. All the three groups were monitored over the next seven
days for mortality and % survival was calculated for each day.
3.5.3. Synthesis of BOC-NFX (6)
A mixture of the norfloxacin (5) (1.56 mmol, 0.5 g), Boc2O
(1.64 mmol, 0.35 g) and 3.3 ml of 1 M NaOH solution in 15.5 ml of
THF was stirred at room temperature overnight. After the removal
of the organic solvent, the residue was neutralized with saturated
aqueous ammonium chloride solution. The mixture was extracted
with ethyl acetate (3× 15 ml) and dried over anhydrous sodium
sulfate. The combined organics were concentrated in vacuum to
provide (6).
3.5. General procedure
All chemicals used in the synthesis were of AR grade. Norfloxacin
was obtained as gift sample from Emcure Pharmaceutical Pvt.
Ltd., Pune, India and dihydroxyacetone was purchased from Merck
(Germany). Palmitoyl chloride was purchased from SAS Chemicals
and Co., Mumbai, India. The reactions were monitored by TLC on
pre-coated silica gel plates-60 F264 (Merck) using solvent system of
benzene:ethyl acetate:methanol:glacial acetic acid (2:2:0.5:1 drop,
v/v) and iodine vapours/UV light as detecting agents. The IR spec-
trum of the synthesized compound was recorded on JASCO, V-530
FTIR in potassium bromide (anhydrous, IR grade). NMR spectra
of the intermediate and synthesized compound were recorded in
CDCl3 using 1H NMR Varian Mercury 300 MHz with superconduct-
ing magnet using TMS as internal standard while 13C NMR spectra
were recorded on Bruker Instruments Model WM 250 NMR spec-
trometer at Department of Chemistry, University of Pune, Pune,
India. Chemical shifts are reported in ppm downfield on ı scale.
Elemental analysis was carried out on Hereaus Vario EL apparatus
at CDRI, Lucknow, India. The ꢀmax of synthesized compound was
determined on JASCO V530, UV–visible double beam spectropho-
tometer in hydrochloric acid buffer (pH 1.2), phosphate buffer (pH
7.4) and chloroform. Partition coefficient was determined in chlo-
roform/phosphate buffer (pH 7.4). Pharmacological screening of
the synthesized compound was carried out in the Department of
Pharmacology, Poona College of Pharmacy and its animal facility is
approved by Committee for the Purpose of Control and Supervision
of Experimental Animals (CPCSEA). The experimental protocols for
the same were approved by the Institutional Animal Ethical Com-
mittee.
Mp: 240–245 ◦C, Rf = 0.87 in glacial acetic acid:acetone:n-
butanol:toluene:water (1:1:0.5:1:1, v/v), % yield: 94.2.
3.5.4. Synthesis of BOC-NFX acid chloride (7)
In a round bottom flask (6) (3.3 mmol, 1 g) and pure PCl5
(3.3 mmol, 0.7 g) were mixed. Flask was fitted with CaCl2 guard-
tube and connected latter to gas absorption device. Flask was
heated on water bath, with occasional shaking, until reaction com-
menced and then for further 30 min or until vigorous evolution of
HCl had almost ceased.
Mp: 265–270 ◦C, Rf = 0.65 in benzene:ethyl acetate:
methanol:glacial acetic acid (2:2:0.5:1 drop, v/v), % yield: 95.
3.5.5. Synthesis of BOC-NFXDG (8)
Freshly distilled dichloromethane (35 ml) was used to dis-
solve (4) (2.6 mmol, 1.5 g). Dry pyridine (3.2 mmol, 0.3 ml) and (7)
(2.96 mmol, 1.0 g) were added at once, the contents were stirred for
40 h at room temperature and then diluted with 50 ml of water fol-
lowed by extraction with 2× 15 ml of CH2Cl2. The CH2Cl2 extracts
were combined, washed with 1% HCl, dried over anhydrous sodium
sulfate and evaporated to dryness. Solid mass so obtained was crys-
tallized from petroleum ether.
Mp: 325–330 ◦C, Rf = 0.80 in benzene:ethyl acetate:
methanol:glacial acetic acid (2:2:0.5:1 drop, v/v), % yield: 75,
IR (KBr; cm−1) 3080 olefenic and aromatic C–H stretching, 1733
3.5.1. Synthesis of dipalmitate of 1,3-dihydroxyacetone (3)
Dihydroxyacetone (22 mmol, 2.0 g) was stirred in chloroform
(85 ml) under nitrogen at room temperature. To this was added
palmitoyl chloride (45.5 mmol, 13.9 ml) followed by anhydrous
pyridine (4.3 ml). The heterogeneous mixture was stirred for 3 h
and diluted with water and the chloroform layer was separated.
The aqueous layer was extracted with two 20 ml portions of chlo-
roform and the chloroform solutions were combined and washed
once with water. Concentration of the chloroform to small volume
resulted in the precipitation of a crystalline solid which was recrys-
tallized from methylene chloride–ether (20:20) to give (3) as small
plates.
C O stretching (ester), 1626 C O stretching (pyridone), 1474
C–C and C–N stretching (quinoline), 1249 C–F stretching. 1H NMR
(CDCl3; ı ppm): 8.76 [s; 1H] – C2 proton, 7.69 [d; JH–F = 12.6 Hz;
1H] – C5 proton, 7.15 [d; JH–F = 7.12 Hz; 1H] – C8 proton, 4.49
[q; 2H] –N–CH2CH3, 2.2–2.3 [q; 4H] – CH2–NH–CH2, 1.58 [t; 3H]
–N–CH2CH3, 1.40 [s; 9H] [–C(CH3)3]. 1.25 [s; 56H] – (CH2)14 × 2,
1.03 [m; 6H] – CH3 × 2. 13C NMR (CDCl3; ı ppm): fluoroquinolone
backbone: 177.5 (C4), 169.9 (–COO), 149.5 (C2), 108.2 (C3), 121.5
(C4a), 113.1 (C5), 134.6 (C6), 146.1 (C7), 106.9 (C8), 138.6 (C8a),
51.1 CH3–CH2–N–, 14.9 CH3–CH2–N–, 47.67 C˛, 44.7 Cˇ, 27.7
[–C(CH3)3], 66.8 [–C(CH3)3], 150.7 (N–COO) dipalmitin backbone:
64.3 (–CH2–CH–CH2–), 69.9 (–CH2–CH–CH2–), 168.2 (2× –OCO–),
33.6–23.1 (2 × 14 –CH2), 14.2 (2× –CH3).
Mp: 70–75 ◦C, Rf = 0.81 in hexane:ethyl acetate (6:1, v/v), % yield:
58.
3.5.6. Synthesis of 1-ethyl-6-fluoro-4-oxo-7-piperazin-1-yl-1,4-
dihydro-quinoline-3-carboxylic acid
3.5.2. Synthesis of 1,3-dipalmitin (4)
A mixture of tetrahydrofuran (75 ml) and benzene (15 ml) was
used to dissolve (3) (5.3 mmol, 3.0 g). Water (4.5 ml) was slowly
added to this solution with stirring and the temperature of the mix-
ture was reduced to approximately 5 ◦C by external cooling in an
ice bath; a milky-white suspension resulted. Sodium borohydride
(7.9 mmol, 0.3 g) was added to this heterogeneous mixture; after
30 min, excess borohydride was destroyed by drop-wise addition
of glacial acetic acid (0.5 ml) and the solution was diluted with chlo-
roform (30–40 ml) and transferred to a separating funnel, washed
with water, aqueous sodium bicarbonate followed by water again
and the chloroform layer dried over magnesium sulfate to give (4)
as a waxy white solid, which was recrystallized from chloroform.
Thin layer chromatography did not show any traces of the 1, 2
isomer.
2-hexadecanoyloxy-1-hexadecanoyloxymethyl-ethyl ester
(NFXDG)
TMSBr (15.2 mmol, 2.0 ml) was added in one portion to a stirring
solution of (8) (1.0 mmol, 1.0 g) in CH2Cl2. After 18 h, the solvent
was removed at reduced pressure. The solid thus obtained was re-
suspended in water and the pH adjusted to 7.4 by addition of NaOH.
Product obtained was filtered, dried and purified by preparative
TLC using solvent system benzene:ethyl acetate:methanol:glacial
acetic acid (60:60:15:30 drops, v/v).
Mp: 295–300 ◦C, Rf = 0.76 in benzene:ethyl acetate:
methanol:glacial acetic acid (2:2:0.5:1 drop, v/v), % yield 95.
IR (KBr; cm−1) 3501 –NH stretching (piperazine), 3082 olefenic
and aromatic C–H stretching, 1731 C O stretching (ester). 1H NMR
(CDCl3; ı ppm): 8.88 [s; 1H] – C2 proton, 7.87 [d; JH–F = 13.0 Hz;
1H] – C5 proton, 7.2 [d; JH–F = 7.0 Hz; 1H] – C8 proton, 4.55 [q; 2H]
–N–CH2CH3, 2.298–2.370 [q; 4H] – CH2–NH–CH2, 2.0 [s; 1H] – NH
Mp: 78–80 ◦C, Rf = 0.75 in hexane:ethyl acetate (6:1, v/v), % yield:
65.

S. Dhaneshwar et al. / Chemistry and Physics of Lipids 164 (2011) 307–313
311
Fig. 1. In vitro kinetic studies: % release of NFX in HCl buffer (pH 1.2).
Fig. 3. In vitro kinetic studies: % release of NFX in stomach homogenate.
piperazine, 1.58 [t; 3H] –N–CH2CH3, 1.254 [s; 56H] – (CH2)14 × 2,
1.05 [m; 6H] – CH3 × 2. 13C NMR (CDCl3; ı ppm): fluoroquinolone
backbone: 177.5 (C4), 169.9 (–COO), 149.5 (C2), 108.2 (C3), 121.5
(C4a), 113.1 (C5), 134.6 (C6), 146.1 (C7), 106.9 (C8), 138.6 (C8a), 51.1
CH3–CH2–N–, 14.9 CH3–CH2–N–, 47.67 C˛, 44.7 Cˇ, dipalmitin
backbone: 64.3 (–CH2–CH–CH2–), 69.9 (–CH2–CH–CH2–), 168.2
(2× –OCO–), 33.6–23.1 (2 × 14 –CH2), 14.2 (2× –CH3) Anal. calcu-
lated for C51H84FN3O7: C, 70.39; H, 9.73; N, 4.83; found: C, 70.35;
H, 9.75; N, 4.82.
4. Results and discussion
The structure of NFXDG was confirmed by IR, 1H NMR, 13C
NMR and C, H, N analysis and was found to be in accordance
with the anticipated structure. Protection of NFXDG with Boc was
confirmed by disappearance of NH piperazine peak at 3501 cm−1
in the IR spectrum and its reappearance after final deprotec-
tion of Boc-NFXDG. 1H NMR and 13C NMR also confirmed that
protection–deprotection was achieved successfully. Melting point
of NFXDG was found to be 295–300 ◦C (uncorrected). Partition coef-
ficient of NFXDG was determined in chloroform: phosphate buffer
(pH 7.4) and was found to be 5.25 which was 2.7 times higher as
compared to NFX (1.94). This enhanced lipophilicity would improve
the ability of NFXDG to cross the bacterial membrane in a positive
manner because it has been reported that partition coefficient is
an important parameter for biological activity of fluoroquinolones.
In vitro kinetic studies were performed in HCl buffer (pH 1.2) and
phosphate buffer (pH 7.4). Release studies were also carried out in
tissue homogenates of stomach and small intestine of rat. The kinet-
Fig. 4. In vitro kinetic studies: % release of NFX in intestinal homogenate.
ics was monitored by increase in concentration of NFX released
from NFXDG at 385 nm in HCl buffer (pH 1.2) and at 415 nm in phos-
phate buffer (pH 7.4). Percent release of NFX from NFXDG in HCl
buffer (pH 1.2) and stomach homogenates is shown in Figs. 1 and 3
respectively. The studies revealed that hydrolysis of the prodrug
started after 2 h and released negligible amounts (3.4% and 5%)
of NFX in HCl buffer (pH 1.2) and stomach homogenates respec-
tively over a period of 6 h. However in phosphate buffer (pH 7.4;
Fig. 2) and intestinal homogenates (Fig. 4), the activation of the
prodrug started early (after 1 h) furnishing 21% and 32% release of
NFX respectively over a period of 10 h. The release of NFX from
Fig. 2. In vitro kinetic studies: % release of NFX in phosphate buffer (pH 7.4).
Fig. 5. In vivo kinetic studies: % release of NFX after oral administration in rat.

312
S. Dhaneshwar et al. / Chemistry and Physics of Lipids 164 (2011) 307–313
Norfloxacin is used widely in the treatment of urinary tract
infection as it acts against Escherichia coli and S. aureus. There-
fore NFXDG was screened for in vivo antimicrobial activity and the
extent of protection provided by NFXDG against S. aureus-induced
renal abscess was studied using mouse renal abscess model and
the results were compared with the standard i.e. norfloxacin The
number of colonies/200 mg of kidney tissue for NFX treated group
were 85 22.74 which were not significantly low as compared to
the negative control group (117 47.81). However the number of
colonies was significantly reduced to as low as 10 5.01 in NFXDG
treated group. The number of colonies/200 mg of kidney tissue was
also expressed as logarithm of colony forming units per gram of
kidney tissue [Log10 (CFU) g−1 of kidney; Fig. 6]. At an oral dose of
5.5 mg/kg of NFX thrice a day (at 6 h intervals), the decrease in the
Log10 (CFU) g−1 of kidney was not statistically significant (7.62) in
comparison to control group (7.767). Interestingly, equimolar oral
doses of NFXDG (14.98 mg/kg; thrice a day) provided statistically
significant protection (6.698). Number of microorganisms/g of kid-
ney tissue were also calculated for each group. The control group
that did not receive any drug treatment showed extensive growth
of 58,500,000 microorganisms while NFX treated group showed
42,500,000 microorganisms at 105 dilution per gram of kidney tis-
sue. NFXDG treated group significantly brought down the number
to 5,000,000 (12 times lower than the control). These promising
results indicate improved bioavailability of NFXDG compared to
NFX as the activities were carried out at equimolar doses.
Fig. 6. Log10 (CFU) g−1 of kidney tissue.
NFXDG followed first order kinetics in aqueous buffers and tis-
sue homogenates. Rate of hydrolysis of NFXDG in small intestinal
homogenate was more as compared to phosphate buffer (pH 7.4)
owing to presence of lipase in GIT.
The histopathology of infected mice kidneys of the nega-
tive control group revealed marked inflammatory cell infiltration
with microabscess formation in the interstitial tissue. Neutrophils
were seen in tubules while some glomeruli appeared inflamed.
This histopathology was suggestive of acute superactive intersti-
tial nephritis of kidney with microabscess and focal glomerulitis.
NFX-treated kidney exhibited mild, acute interstitial nephritis
characterized by inflammatory cell infiltration with few microab-
scess formations while the glomeruli and tubules appeared
unremarkable. NFXDG was able to restore the normal morphol-
ogy of kidney without any signs of inflammatory cell infiltration,
interstitial nephritis, glomerulitis or abscess at the equimolar
dose. The results are depicted in Fig. 7. Statistical differences
between the groups were calculated by two-way ANOVA followed
by Bonfferoni’s test. All data are expressed as means SD. Dif-
ferences were considered at a P value of <0.001 in relation to
control.
In vivo release kinetics was investigated in blood (Fig. 5), feces
and urine of Wistar rats. After oral administration, NFX was not
detected in the plasma for first 2 h indicating that NFXDG was not
hydrolyzed in stomach during that time span. This finding is in
accordance with the results of in vitro release studies in stomach
homogenates where it was observed that the release started after
2 h. NFX was detected in the plasma at 3rd hour and after that,
its concentration consistently and slowly increased during 3–6 h
attaining a maximum release of 54% at the end of 8 h. The con-
centration of NFX declined over the period of time and became
negligible at the end of 12 h. The release followed zero order kinet-
ics. From the in vitro and in vivo release studies it can be concluded
that minimum hydrolysis (21–32%) occurred in the lumen of upper
GIT while maximum concentration of NFXDG that remained intact
might have been absorbed into blood owing to its high lipophilic-
ity followed by its hydrolysis in blood corresponding to a total of
54% release of NFX at 8th hour. The slow rate of hydrolysis might be
useful in giving longer duration of action. Traces of NFX were found
in urine and feces indicating its renal and intestinal excretion.
Survival rate studies were also performed on albino mice
infected with S. aureus to compare the effects of NFXDG with NFX
and negative control over a period of 7 days (Fig. 8). Survival rate
in negative control of mice challenged with S. aureus infection was
only 10% which was brought to 40% by NFX. NFXDG-treated mice
exhibited maximum rate of survival (60%) at equimolar dose to
Fig. 7. Photomicrographs of kidney sections infected with S. aureus. (A) Negative control showing acute super-active interstitial nephritis with marked inflammatory
infiltration/microabccess formation and focal glomerulitis. (B) Infected kidney section treated with NFX showing mild acute interstitial nephritis with interstitial inflammatory
cell infiltration/few microabccesses, glomeruli with unremarkable tubules. (C) Infected kidney section treated with NFXDG showing unremarkable glomeruli, tubules,
interstitial tissue and blood vessels without evidence of inflammation. Overall histopathology of kidney is unremarkable.

S. Dhaneshwar et al. / Chemistry and Physics of Lipids 164 (2011) 307–313
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The authors thank Emcure Pharmaceuticals Limited, Pune,
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