Galactosyl prodrug of ketorolac: Synthesis, stability, and pharmacological and pharmacokinetic evaluations

Article abstract of DOI:10.1021/jm900051r

Although ketorolac is one of the most potent anti-inflammatory and analgesic drugs, its use has been strongly limited owing to the high incidence of adverse effects reported, particularly in the gastrointestinal tract. Using the prodrug approach, which al

Full text of DOI:10.1021/jm900051r

3794
J. Med. Chem. 2009, 52, 3794–3800
Galactosyl Prodrug of Ketorolac: Synthesis, Stability, and Pharmacological and
Pharmacokinetic Evaluations
Annalisa Curcio,‡,| Oscar Sasso,§,| Daniela Melisi,*,‡ Maria Nieddu,# Giovanna La Rana,§ Roberto Russo,§ Elisabetta Gavini,†
Gianpiero Boatto,# Enrico Abignente,‡ Antonio Calignano,§ and Maria Grazia Rimoli‡
Department of Pharmaceutical and Toxicological Chemistry and Department of Experimental Pharmacology, Faculty of Pharmacy, Federico II
UniVersity of Naples, Via Domenico Montesano 49, 80131, Naples, Italy, and Department of Drug, Chemistry and Toxicology and Department
of Drug Sciences, Faculty of Pharmacy, UniVersity of Sassari, Via Muroni 23/a, 07100, Sassari, Italy
ReceiVed January 15, 2009
Although ketorolac is one of the most potent anti-inflammatory and analgesic drugs, its use has been strongly
limited owing to the high incidence of adverse effects reported, particularly in the gastrointestinal tract.
Using the prodrug approach, which allows the reduction of toxicological features of the parent drug without
altering its pharmacological properties, we synthesized an orally administrable prodrug of ketorolac by means
of its reversible conjugation to D-galactose (ketogal). In a single dose study, its pharmacokinetic profile was
compared with that of ketorolac. Moreover, we found that this prodrug was able to maintain the anti-
inflammatory and the analgesic activity of the drug without giving rise to gastric ulcer formation. Thus,
these results indicate that ketogal is a highly effective and valid therapeutic alternative to ketorolac itself.
Introduction
gastric mucosa while preserving satisfactory pharmacological
activity, some scientists have created esteric and amidic deriva-
tives bound to polymeric carriers or amino acids to mask the
carboxylic acid group.8,9
Since metabolizable carbohydrates, especially hexoses, are
gastroprotective agents,10 being components of the gastric
mucus,11,12 we decided to harness the synergic action of sugar
release and the prodrug approach.
On the basis of our past experience in the synthesis of
prodrugs,13–16 we chose D-galactose to design a novel
ketorolac prodrug. In this paper, we report the synthesis of
a ketorolac-galactose conjugate (ketogal). Both its stability
and its pharmacokinetic profile were evaluated in a single
dose study. Moreover, in vivo experiments were performed
to assess its pharmacological activities and, more important,
its potential capability to reduce ulcerogenic activity in
comparison with its parent drug ketorolac.
Ketorolac, a racemic mixture of pyrrolizine carboxylic acids,
is one of the most effective nonsteroidal anti-inflammatory drugs
(NSAIDsa). Because its analgesic efficacy is comparable to that
of morphine, it is often administrated in postoperative pain
treatment.1 As indicated in a recent study on NSAIDs, ketorolac
is among those drugs that present the highest risk of toxic
reactions, a feature that has drastically limited its use despite
its analgesic and anti-inflammatory efficacy.2 Accordingly,
nowadays its administration is limited to very short periods of
time in the treatment of renal colic and moderate and severe
postoperative pain.3 Mild and severe gastrointestinal adverse
events are ascribable to the drug’s direct impact on the
gastrointestinal mucosa and to its indirect effect, via blood
circulation, following absorption.4–6
In general, a prodrug approach consists of designing phar-
macologically inactive derivatives that undergo biotransforma-
tion before exhibiting their pharmacological effects following
the release of the active drug. Such a technique has proven to
be useful to overcome pharmaceutical and pharmacokinetic
barriers in clinical drug application, including toxicity, chemical
instability, low oral absorption, lack of site specificity, and poor
patient acceptance.7
Results
General Procedure for the Synthesis of Ketogal. Esterifi-
cation of ketorolac (1) with 1,2,3,4-di-O-isopropylidene-D-R-
galactopyranose (DIPG) in the presence of N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC) and
4-(dimethylamino)pyridine (DMAP), dissolved in dry dichlo-
romethane, yielded 75% of 1a. Ketals were completely removed
with trifluoroacetic acid (TFA) dissolved in dry dichloromethane
to obtain 65% of ketogal (Scheme 1).
Stability Tests of Ketogal and Ketorolac. Data concerning
chemical stability at pH 1 (hydrochloric acid, 0.1 N) and at pH
7.4 and pH 8.0 (buffer solutions) and enzymatic stability in
plasma of both ketogal and ketorolac are reported in Table 1.
When we evaluated chemical stability of both compounds, the
prodrug appeared quite stable at both pH 7.4 and pH 8.0 and
less stable at pH 1. To determine whether ketogal was suitable
for enzymatic hydrolysis and thus apt to regenerate the parent
drug, we incubated the compound in rat plasma. The half-life
of enzymatic stability was lower than the one in buffer solutions.
On the contrary, ketorolac demonstrated to be extremely stable
under all conditions.
With respect to ketorolac, many efforts have been made to
synthesize new prodrugs. For instance, to avert damage to the
* To whom correspondence should be addressed. Phone: +39081678612.
Fax: +39081678612. E-mail: melisi@unina.it.
‡
Department of Pharmaceutical and Toxicological Chemistry, Federico
II University of Naples.
|
These authors contributed equally to this study.
Department of Experimental Pharmacology, Federico II University of
§
Naples.
#
Department of Drug, Chemistry and Toxicology, University of Sassari.
Department of Drug Sciences, University of Sassari.
†
a Abbreviations: AUC, area under the curve; AUMC, area under the first
moment curve; CL, clearance; CMC, carboxymethyl cellulose; DIPG,
1,2,3,4-di-O-isopropylidene-D-R-galactopyranose; DMAP, 4-(dimethylami-
no)pyridine; EDC, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hy-
drochloride; ip, intraperitoneal; MRT, mean residence time; NMR, nuclear
magnetic resonance; NSAIDs, nonsteroidal anti-inflammatory drugs; rpm,
revolutions per minute; TLC, thin layer chromatography; TFA, trifluoro-
acetic acid; TMS, tetramethylsilane.
10.1021/jm900051r CCC: $40.75
2009 American Chemical Society
Published on Web 05/21/2009

Galactosyl Prodrug of Ketorolac
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3795
Scheme 1. Ketogal Synthesisa
a (a) EDC, DMAP/DCM dry, room temp, 12 h; (b) TFA/DCM dry, room temp, 48 h.
Table 1. Chemical and Enzymatic Stability of Ketogala
t1/2 (h)
compd
pH 1
pH 7.4
pH 8.0
plasma
ketogal
ketorolac
2.1 ( 0.15
>4
>4
>4
>4
>4
1.5 ( 0.80
>4
a Data are reported as the mean ( SEM.
Effect of Ketogal on Acetic Acid Induced and Mag-
nesium Sulfate Induced Writhes. The ip administration of an
irritating dose of 0.6% acetic acid solution produced a robust
visceral pain, which peaked 5-20 min after injection of the
acid. The nocifensive behavior was attenuated in a dose-
dependent manner by oral administration of ketorolac (0.01-10
mg/kg) (Figure 1A). In particular, the effect was significant at
doses of 1 and 10 mg/kg (p < 0.01). Synthetic derivative ketogal
showed, at equimolecular doses of ketorolac (0.0163-16.3 mg/
kg), a dose-dependent analgesic effect (Figure 1A) stronger than
that produced by ketorolac. In fact, 0.163 mg/kg ketogal
produced a significant (p < 0.01) analgesic effect, whereas the
equimolecular dose of ketorolac (0.1 mg/kg) had no effect
(Figure 1A).
Acetic acid-evoked writhing responses are accompanied by
a profound, often lethal peritoneal inflammation. To determine
whether ketorolac and ketogal inhibit visceral pain, we tested
the effects of these compounds on magnesium sulfate-evoked
writhing in experimental models that exhibit a less marked
inflammatory component.
Magnesium sulfate produced a reversible nocifensive response
when injected intraperitoneally in mice. The administration of
magnesium sulfate 120 mg/kg, ip caused a mean of 6 ( 0.9
writhing episodes in mice (n ) 6). Ketorolac (0.01-10 mg/kg)
and ketogal (equimolecular doses of ketorolac), administered
1 h before magnesium sulfate, inhibited this response in a dose-
dependent manner (Figure 1B).
Effect of Ketogal on Carrageenan-Induced Paw
Edema. As already reported in a previous characterization of
this model,17 mouse paw edema developed in two distinct
phases: an acute first phase peaking at 6 h and a second phase
peaking at 72 h after carrageenan challenge. Ketogal treatment
(0.163, 1.63, or 16.3 mg/kg), 1 h before carrageenan, markedly
reduced paw edema in a time- and dose-dependent manner
(Figure 2B). During the first phase (0-6 h), all doses of ketogal
Figure 1. Analgesic effect on acetic acid-induced (A) or magnesium
sulfate-induced (B) writhings. Control animals (open bar) received oral
doses of CMC, ketorolac (closed bar, 0.01-10 mg/kg), or ketogal
(straight bar, 0.0163-16.3 mg/kg) 1 h before ip administration of
chemical agents. Data are expressed as mean values ( SEM of six
animals for each group: (**) p < 0.01 versus control group.
significantly (p < 0.001) inhibited edema formation (Figure 2B).
In the second phase (24-96 h), ketogal remained significantly
effective (p < 0.01 for 1.63 and 16.3 mg/kg) until 48 h after
carrageenan (Figure 2B). Furthermore, at the highest dose (16.3
mg/kg), it was still significantly effective even 72 h after
carrageenan challenge (Figure 2B).
To compare the efficacy of ketogal with that of ketorolac,
the latter was tested under the same experimental conditions at
a doses of 0.1, 1.0, and 10 mg/kg (equimolecular doses of

3796 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Curcio et al.
Figure 2. Anti-inflammatory effect of oral administration of ketorolac (A) (0.1-10 mg/kg) and ketogal (B) (0.163-16.3 mg/kg) on mouse carrageenan
paw edema. Control animals were treated with CMC, ketorolac, or ketogal 1 h before subplantar injection of carrageenan. Data are expressed as
mean values ( SEM of six animals for each group: (***) p < 0.001; (**) p < 0.01 versus control group.
Table 2. Ulcerogenic Activity
oral dose
(mg/kg)
gastric
lesion score
drug
no. of ulcersa
control (CMC, 0.5%)
ketorolac
ketogal
0
0.1
0.163
4 ( 0.4
1 ( 0.4
10
1
a Data are reported as the mean ( SEM.
ketogal) (Figure 2A). The lowest dose did not reduce paw
edema, whereas 1.0 and 10 mg doses elicited a time- and dose-
dependent reduction. During the second phase, only the highest
dose (10 mg) was active until 48 h (p < 0.001 at 24 h; p < 0.01
at 48 h) (Figure 2A).
Figure 3. Plasma concentration time profile of ketorolac upon oral
administration of ketorolac itself (0.100 mg/kg) and of ketogal (0.163
mg/kg). Data are expressed as mean values ( SD (n ) 3).
Effect of Ketogal on Ulcerogenic Activity. Ketogal remark-
ably decreased ulcerogenic activity compared to ketorolac.
Noteworthy, ketogal caused only irritation without ulceration
(Table 2). Thus, our results indicate that ketogal remarkably
reduced ulcerogenicity compared to ketorolac itself.
Pharmacokinetic Studies. The pharmacokinetic profile of
ketogal was compared to that of ketorolac according to plasma,
stomach, kidney, and liver concentrations after oral administra-
tion of ketorolac (0.100 mg/kg) and ketogal (0.163 mg/kg).
Ketorolac plasma concentration profile versus time is shown
in Figure 3, and pharmacokinetic parameters are reported in
Table 3. A different pharmacokinetic profile was observed for
the ketorolac, resulting from prodrug hydrolysis and the
ketorolac itself. A single dose of ketorolac, orally administrated,
was rapidly absorbed and eliminated within 6 h (Figure 3).
Instead, although plasma concentrations of the drug released
from ketogal were lower than those of ketorolac during the first
2 h, they nonetheless remained higher than that of the drug itself
throughout the remaining time of the experiments. Therefore,
ketogal revealed the typical profile of a controlled release drug
system. The pharmacokinetic parameters related to these curves
were calculated and are listed in Table 3. The areas under the
curve from time 0 to 6 h, AUC0-6, obtained from both curves,
were very similar, thus indicating that the prodrug did not
modify the bioavailability of ketorolac. On the other hand, the
pharmacokinetic parameters pinpointed that the pharmacokinetic
behavior of the drug released from ketogal and the drug itself
was different, thereby suggesting that the prodrug was respon-
sible for a sustained release of the drug and its longer half-life.
Therefore, the introduction of a galactosyl promoiety on
ketorolac elicited a slow and sustained release of ketorolac, thus
prolonging its activity. Regression analysis was performed, and

Galactosyl Prodrug of Ketorolac
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3797
Table 3. Pharmacokinetic Parametersa
AUC0-6
(µg·h/mL)
AUMC0-6
(µg·h/mL) MRTb (h) CL(area) c(mL/h)
drug
ketorolac
ketorolac released 5.23 ( 0.09
by ketogal
t1/2 (h)
tmax (h) Cmax (µg/mL)
t1/2 d(h)
t1/2 e(h)
1.65 ( 0.05
1 ( 0
1 ( 0
0.40 ( 0.05
0.24 ( 0.03
0.8550 ( 0.08 3.3 ( 0.14 3.1 ( 0.1
0.9350 ( 0.03 5.6 ( 0.75 4.5 ( 0.37
94.23 ( 6.08
80.36 ( 4.34
1.99 ( 0.01 0.39 ( 0.41
2.4 ( 0.32 1.68 ( 0.03
a Data are reported as the mean ( SEM. b MRT: mean residence time (time for 63.2% of administered dose to be eliminated). c CL: clearance. Systemic
clearance based on observed data points: AUC0-6
analysis, 1-2 h).
.
d Elimination phase (terminal phase regression analysis, 4-6 h). e Absorption phase (first residual regression
Figure 5. Stomach concentration of ketogal vs time after oral
administration (0.163 mg/kg). Data are expressed as mean values (
SD (n ) 3).
Figure 4. Ketorolac distribution vs time in stomach, liver, and kidney
after oral administration of ketorolac itself (0.100 mg/kg) (A) and of
ketogal (0.163 mg/kg) (B).
pharmacokinetic parameters of absorption and elimination phase
were calculated by plotting experimental data as semilog
concentration vs time. Our data demonstrated that the t1/2
absorption phase of ketorolac released from ketogal was higher
than that of the drug alone (Table 3), a finding that demonstrates
a slower absorption of the drug when administered as a prodrug.
Finally, the values of MRT, CL, and t1/2 elimination phase
demonstrated that the elimination rate of ketorolac released from
the prodrug was slower than that from the drug itself (Table 3).
The following data are based on the determination of drug
distribution in stomach, liver, and kidney after oral administra-
tion of both compounds (Figure 4). Six hours following oral
administration of ketorolac, initial peak concentrations were
followed by a progressive decrease in drug amount in all three
organs analyzed (Figure 4A). By contrast, oral doses of ketogal
were slowly hydrolyzed in the stomach, an event that reduced
the drug concentration but prolonged its release for up to 2 h
after administration. These effects were due to its stability at
acidic pH (about 2 h) and its t1/2 absorption phase, which was
remarkably higher than the value found for ketorolac (Table
3). Remarkably, such behavior was consistent with the scarce
ulcerogenic effect of the prodrug. Moreover, the lower absorp-
tion rate of the prodrug determined, in turn, a lower concentra-
tion of the drug in the plasma (Figure 3) and its accumulation
in the stomach (Figure 4B). Figure 5 also illustrates ketogal
concentration in the stomach throughout the entire experimental
procedure. The presence of ketogal in the liver (Figure 6) and
the distribution profile of ketorolac from the prodrug may also
indicate that the prodrug was absorbed and accumulated in the
liver. Indeed, the rapid and significant hydrolysis of the prodrug
in the liver caused an increase in ketorolac levels (Figure 4B).
Finally, the distribution profile of the drug and prodrug in the
kidney (Figure 4A and Figure 4B) demonstrated that the amount
of ketorolac released from the prodrug was eliminated at a
Figure 6. Liver concentration of ketogal vs time after oral administra-
tion (0.163 mg/kg). Data are expressed as mean values ( SD (n ) 3).
slower rate than the drug alone, as demonstrated by the
elimination parameters (Table 3). Indeed, concerning ketogal,
the drug was found in the kidney after 4 h while ketorolac
concentration was significantly high only within the first 2 h.
No trace of the prodrug was detected in kidney samples.
Discussion
Several NSAIDs drugs, including ketorolac, cause a wide
range of gastrointestinal adverse events. Although ketorolac
exerts an excellent therapeutic activity, its chronic use has long
been limited owing to the high incidence of these adverse effects.
To overcome these undesired drug reactions, new strategies have
been investigated to create suitable pharmaceutical formulations
that would preserve the powerful anti-inflammatory and anal-
gesic activity of the parent drug while minimizing its negative
effects.18–20
In this paper, we synthesized for the first time a galactosyl
derivative of ketorolac, which we named ketogal. Drug stability
experiments were carried out to verify the handling of this new
prodrug and its ability to release the parent drug both enzymati-
cally and nonenzymatically in plasma and under physiological
conditions (pH 7.4), respectively. As this study entailed oral
administration of the drugs, stability analyses were also
performed under acidic and basic conditions to mimick gastric
and intestinal pH. Under the same experimental conditions,
ketorolac showed high stability, thus demonstrating it to be the
active drug.

3798 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Curcio et al.
12H, ketals); 2.80, 2.90 (m, 2H, 2-H); 3.30 (m, 1H, 1-H); 4.05 (m,
1H, 4′-H); 4.15 (m, 1H, 5′-H); 4.20 (m, 2H, 6′-H); 4.40 (m, 1H,
2′-H); 4.50 (m, 1H, 3′-H); 4.60 (m, 2H, 3-H); 5.35 (m, 1H, 1′-H);
5.90 (d, 1H, 7-H); 6.80 (d, 1H, 6-H); 7.45 (m, 2H, 2,6-Ph); 7.55
(m, 1H, 4-Ph); 7.85 (m, 2H, 3,5-Ph). 13C NMR (CDCl3): δ 20 and
22 (4CH3-ketals); 30 (C-2); 44 (C-3); 48 (C-1); 65 (C-6′); 66.7
(C-4′); 71 (C-5′); 71.5 (C-2′); 72 (C-3′); 97 (C-1′); 105 (C-7); 109
and 111 (C-ketals); 125 (C-6); 127 (C-5); 129 (3,5-Ph); 130 (2,6-
Ph); 133 (4-Ph); 140 (C-8); 142 (1-Ph); 175 (CO ketone); 185 (CO
ester). m/z: 498 (M + H)+. Anal. (C32H41NO8) C, H, N.
Synthesis of Ketorolac-D-galactos-6′-yl Ester (Ketogal). Two
milliliters of trifluoroacetic acid (TFA) was added to a solution of
1a (1.45 g, 2.9 mmol) in dry dichloromethane (10 mL). The mixture
was continuously stirred at room temperature for 48 h. The solvents
were then evaporated and purified by flash chromatography on silica
gel. Elution with CHCl3 in a gradient of CH3OH gave 800 mg of
ketogal as a white solid (65% yield). Mp: 195 °C. 1H NMR
(CD3OD): δ 2.85 (m, 2H, 2-H); 3.50 (m, 1H, 1-H); 3.75 (m, 1H,
4′-H); 3.85 (m, 1H, 5′-H); 4.20 (m, 2H, 6′-H); 4.30 (m, 1H, 2′-H);
4.35 (m, 1H, 3′-H); 4.50 (m, 2H, 3-H); 5.15 (m, 1H, 1′-H); 6.15
(d, 1H, 7-H); 6.80 (d, 1H, 6-H); 7.45 (m, 2H, 2,6-Ph); 7.55 (m,
1H, 4-Ph); 7.75 (m, 2H, 3,5-Ph). 13C NMR (CD3OD): δ 30 (C-2);
44 (C-3); 48 (C-1); 65.5 (C-6′); 68 (C-4′); 70 (C-5′); 73.1 (C-2′);
74.9 (C-3′); 94 and 99 (C-1′); 105 (C-7); 125 (C-6); 127 (C-5);
129 (3,5-Ph); 130 (2,6-Ph); 133 (4-Ph); 140 (C-8); 142 (1-Ph); 175
(CO ketone); 185 (CO ester). m/z: 418 (M + H)+. Anal.
(C21H23NO8) C, H, N.
Stability Tests of Ketogal and Ketorolac. Prodrug solutions
(100 µg/mL), maintained at 37 °C, were prepared by dissolving
ketogal in pH 7.4 and pH 8.0 phosphate buffers, in hydrochloric
acid 0.1 N (pH 1) or in plasma. Aliquots (20 µL) withdrawn hourly
during the 24 h incubation period were analyzed by HPLC. To
determine enzymatic stability, an aliquot of plasma was extracted
with acetonitrile (1:2) every hour and the solutions were vortexed
and centrifuged at 3000 rpm (1000g) for 10 min. The supernatant
(20 µL) was analyzed by HPLC. The same procedure was applied
to ketorolac by preparing equimolecular ketorolac tris salt solutions
(90.5 µg/mL).
The hydrolysis of the compound was followed by HPLC diode
array detection, which will be described later. Pseudo-first-order
half-lives (t1/2) for the chemical and enzymatic hydrolyses were
calculated from the linear slopes of logarithm plots of remaining
esters over time.
HPLC System. Chromatographic separations were performed
using a 1090 L liquid chromatograph (Hewlett-Packard, Palo Alto,
CA) equipped with a diode array detector HP 1040A. All separa-
tions were accomplished on a Phenomenex Luna C 18 (250 mm ×
4.6 mm, particle size 5 µm). The selected wavelength was 313 nm.
The mobile phase used in the separation consisted of acetonitrile
and aqueous 1 mM phosphoric acid, pH 3 [68:32]. The flow rate
was 1 mL/min with an injection volume of 20 µL. All reagents
and solvents were analytical grade. Deionized and distilled water
was purified through a Milli Q system (Millipore). Retention times
were 6.7 min for ketorolac and 3.8 min for ketogal.
Animals. Male Swiss mice weighing 20-25 g were purchased
from Harlan Italy (San Pietro al Natisone, UD, Italy). Animals were
housed in groups of 5-6 per cage (24 cm × 72 cm × 12 cm).
Food and water were given ad libitum. Housing conditions were
thermostatically maintained at 24 ( 1 °C with a constant humidity
(65%) and a 12:12 light/dark cycle.
All animal experiments complied with the Italian D.L. No. 116
of January 27, 1992, and with associated guidelines in the European
Communities Council Directive of November 24, 1986, 86/609/
ECC. All surgical procedures were reviewed and approved by the
Ministero della Ricerca Scientifica and conformed to the guidelines
of the International Association for the Study of Pain.21
Analgesic Activity. Acute analgesia produced by the drugs was
assessed by the acetic acid- and magnesium sulfate-induced writhing
methods in mice.22,23 Animals were fasted overnight prior to
treatment and received free access to water during the experiments.
Pharmacological investigations of the synthesized prodrug
were done for anti-inflammatory, analgesic, and ulcerogenic
activities. The range of ketogal doses (0.0163-16.3 mg/kg)
administered was equimolar to the range of doses of ketorolac
(0.01-10 mg/kg). Our results clearly showed that ketogal
administration minimized the gastrointestinal toxicity while
preserving the anti-inflammatory and analgesic activity. Note-
worthy, ketogal effectively reduced acetic acid-induced writh-
ings and carrageenan-induced paw edema up to 6 h after
administration. Furthermore, the ulcerogenic index of ketogal
was significantly lower than the one exhibited by the parent
drug. Intriguingly, the reduction in adverse events obtained with
the ketorolac-galactose conjugate was most likely attributable
to its ability to inhibit the direct contact of carboxyl group of
ketorolac with the gastric mucosa, a circumstance that eventually
leads to gastric damage.
Altogether, our pharmacokinetic studies of ketogal have
demonstrated that this prodrug is a potential candidate for a
slower and sustained release form of ketorolac. Equally
important is the fact that we were able to demonstrate the
capability of this new prodrug to reduce ulcerogenicity while
preserving the high pharmacological efficacy of its parent drug,
as demonstrated by the fact that the drug reached therapeutic
but not toxic plasma concentrations.
On the basis of these results, this prodrug approach may
constitute a possible pharmacological solution to minimize
gastrointestinal toxicity while preserving the powerful anti-
inflammatory and analgesic activities of the ketorolac.
Experimental Section
Drugs and Chemicals. All chemicals used for the synthesis of
ketogal were purchased from commercial sources (Sigma-Aldrich).
The commercially available ketorolac tris salt was dissolved in
water, acidified with an aqueous solution of HCl until a pH level
of 4.5 was attained, and extracted with CHCl3. To obtain ketorolac
free acid 1, the organic layer was dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo. All reagents and solvents
used for stability tests were analytical grade. Deionized and distilled
water was purified through a Milli Q system (Millipore). For
enzymatic stability, mouse plasma (male Swiss mice) was supplied
by the Department of Experimental Pharmacology, Faculty of
Pharmacy, “Federico II” University of Naples, Italy.
Chemistry. The course of reactions and purity of products were
monitored by TLC (silica gel 60F254s, Merck), and spots were
detected by UV radiation and after iodine exposition. Flash
chromatography was performed on Merck silica gel (0.040-0.063
mm). Melting points (mp) were determined with a Buchi B-540
hot stage microscope. Elemental analyses, indicated by the symbols
of the elements, were performed on a Perkin-Elmer model 240
elemental analyzer and were within (0.4% of theoretical values.
ESI mass spectra were recorded with an Applied Biosystems API
2000 spectrometer. Proton and carbon-13 nuclear magnetic reso-
nance (NMR) spectra were recorded on a Varian Mercury 400
spectrometer operating at 400 MHz. Chemical shift values are
reported in δ units (ppm) relative to TMS, which was used as the
internal standard.
Synthesis of Diacetone 6′-O-Ketorolac-D-galactopyranoside
(1a). Ketorolac 1 (1.0 g, 3.9 mmol), 1,2,3,4-di-O-isopropylidene-
D-R-galactopyranose (DIPG) (1.01 g, 3.9 mmol), N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC) (748 mg,
3.9 mmol), and 4-(dimethylamino)pyridine (DMAP) (24 mg, 0.19
mmol) were dissolved in dry dichloromethane (10 mL). The reaction
mixture was kept under continuous stirring at room temperature
for 12 h. The organic layer was washed several times with water,
dried over anhydrous sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by column chromatography on
silica gel and eluted with CHCl3, giving 1.45 g of 1a as a white
solid (75% yield). 1H NMR (CDCl3): δ 1.30, 1.31, 1.40, 1.41 (4s,

Galactosyl Prodrug of Ketorolac
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3799
One hour after oral administration of ketorolac free acid or
ketogal, all mice, divided into groups of 6 per cage, received
intraperitoneal (ip) injections of acetic acid 0.6%, 0.5 mL of saline,
and 120 mg/kg magnesium sulfate, dissolved in 10 mL of saline.
For acetic acid, the total number of writhes, a parameter of
chemically induced pain detectable by constriction of abdomen,
turning of trunk, and extension of hind legs, was counted for 20
min starting 5 min after the administration of the irritant agent; for
magnesium sulfate, writhes were counted for 5 min. The analgesic
effect was expressed as the number of writhes in comparison with
control.
Acknowledgment. The authors thank Dr. Paola Merolla for
English editorial revision.
Supporting Information Available: HPLC chromatograms and
mass spectrum of ketogal. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Extraction Procedure from Biological Matrices for
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mL were prepared and analyzed using the above procedure.
Tolmetine was used as internal standard (retention time of 9.3 min
under HPLC conditions described above). Sensitivity limits were
0.1 µg/mL for ketogal and 0.05 µg/mL for ketorolac.

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