Aminocarbonyloxymethyl ester prodrugs of flufenamic acid and diclofenac: Suppressing the rearrangement pathway in aqueous media

Article abstract of DOI:10.1002/ardp.200600145

Aminocarbonyloxymethyl ester prodrugs are known to undergo rearrangement in aqueous solutions to form the corresponding N-acylamine side product via an O → N intramolecular acyl transfer from the carbamate conjugate base. Novel aminocarbonyloxymethyl esters of diclofenac and flufenamic acid containing amino acid amide carriers were synthesized and evaluated as potential prodrugs displaying less ability to undergo rearrangement. These compounds were prepared in reasonable yield by a four-step synthetic method that uses the appropriate N-Boc-protected amino acid N-hydroxysuccinimide ester and secondary amine and chloromethyl chloroformate as key reactants. Their reactivity in pH 7.4 buffer and 80% human plasma at 37°C was assessed by RP-HPLC. The aminocarbonyloxymethyl esters containing a secondary carbamate group derived from amino acids such as glycine or phenylalanine were hydrolyzed quantitatively to the parent drug both in non-enzymatic and enzymatic conditions, with no rearrangement product being detected. The oral bioavailability in rats was determined for selected diclofenac derivatives. These derivatives displayed a bioavailability of 25 to 68% relative to that of diclofenac, probably due to their poor aqueous solubility and lipophilicity. These results suggest that further optimization of aminocarbonyloxymethyl esters as potential prodrugs for non-steroidal anti-inflammatory drugs require the use of amino acid carriers with ionizable groups to improve aqueous solubility.

Full text of DOI:10.1002/ardp.200600145

32
Arch. Pharm. Chem. Life Sci. 2007, 340, 32–40
Full Paper
Aminocarbonyloxymethyl Ester Prodrugs of Flufenamic Acid
and Diclofenac: Suppressing the Rearrangement Pathway in
Aqueous Media
Lina Ribeiro1, Nuno Silva1, Jim Iley2, Jarkko Rautio4, Tomi Jꢀrvinen4, Hꢁlder Mota-Filipe3,
Rui Moreira1, and Eduarda Mendes1
1 CECF, Faculdade de Farmꢀcia da Universidade de Lisboa, Lisboa Codex, Portugal
2 Chemistry Department, The Open University, Walton Hall, Milton Keynes, U. K.
3 UFF, Faculdade Farmꢀcia da Universidade de Lisboa, Lisboa Codex, Portugal
4 Department of Pharmaceutical Chemistry, University of Kuopio, Kuopio, Finland
Aminocarbonyloxymethyl ester prodrugs are known to undergo rearrangement in aqueous solu-
tions to form the corresponding N-acylamine side product via an O fi N intramolecular acyl
transfer from the carbamate conjugate base. Novel aminocarbonyloxymethyl esters of diclofenac
and flufenamic acid containing amino acid amide carriers were synthesized and evaluated as
potential prodrugs displaying less ability to undergo rearrangement. These compounds were
prepared in reasonable yield by a four-step synthetic method that uses the appropriate N-Boc-
protected amino acid N-hydroxysuccinimide ester and secondary amine and chloromethyl chlo-
roformate as key reactants. Their reactivity in pH 7.4 buffer and 80% human plasma at 378C was
assessed by RP-HPLC. The aminocarbonyloxymethyl esters containing a secondary carbamate
group derived from amino acids such as glycine or phenylalanine were hydrolyzed quantitati-
vely to the parent drug both in non-enzymatic and enzymatic conditions, with no rearrange-
ment product being detected. The oral bioavailability in rats was determined for selected diclofe-
nac derivatives. These derivatives displayed a bioavailability of 25 to 68% relative to that of diclo-
fenac, probably due to their poor aqueous solubility and lipophilicity. These results suggest that
further optimization of aminocarbonyloxymethyl esters as potential prodrugs for non-steroidal
anti-inflammatory drugs require the use of amino acid carriers with ionizable groups to
improve aqueous solubility.
Keywords: Aminocarbonyloxymethyl Esters / Bioavailability / NSAIDs / Prodrugs /
Received: September 11, 2006; accepted: October 4, 2006
DOI 10.1002/ardp.200600145
the activity of cyclooxygenases (COXs) [2]. The COX
Introduction
enzyme exists in two isoforms: a constitutive isoform,
COX-1, found in most tissues including stomach, kidney,
and platelets, and an inducible isoform, COX-2, expressed
at the site of inflammation [3, 4]. Classical NSAIDs, such
as ibuprofen, flufenamic acid, diclofenac, and aspirin,
preferentially inhibit COX-1 [5, 6], thus suppressing the
biosynthesis of prostaglandins that maintain gastric
mucosal integrity and leading to gastrointestinal (GI)
side effects [6], including ulceration and hemorrhage.
Moreover, the carboxylic acid function present in most
classical NSAIDs can also impart local GI irritation, called
direct contact effect [7–9]. Since the introduction of spe-
Non-steroidal anti-inflammatory drugs (NSAIDs) are
widely used in the treatment of rheumatic and inflam-
matory conditions [1]. The pharmacological activity of
NSAIDs is related to the blocking of prostaglandin H2
(PGH2) biosynthesis from arachidonic acid by inhibiting
Correspondence: Eduarda Mendes, CECF, Faculty of Pharmacy, Uni-
versity of Lisbon, Av. Forꢁas Armadas, 1600-083 Lisboa, Portugal
E-mail: ermendes@ff.ul.pt
Fax: +351 21 794-6470
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Arch. Pharm. Chem. Life Sci. 2007, 340, 32–40
Prodrugs of Diclofenac and Flufenamic Acid
33
Scheme 1. Rearrangements of several prodrugs 2 in pH, 7.4 buf-
fer to form the corresponding N-acylamines.
Figure 1. Molecular structures of acyloxymethyl-type deriv-
atives 1 and aminocarbonyloxymethyl derivatives 2 and 3.
cific COX-2 inhibitors, which are less harmful to the GI
tract [6–10], the use of conventional NSAIDs has
declined. However, the safety profile of COX-2 inhibitors
has been questioned due to the risk of ulcer complica-
tions in high-risk individuals [11, 12] and to cardiovascu-
lar adverse effects that, ultimately, lead to high drop-out
rates [13–15]. Thus, the need for NSAIDs with improved
GI tolerability still exists.
One approach that has been used to decrease NSAID-
induced GI toxicity without adversely affecting their
anti-inflammatory activity is to mask the carboxylic acid
group by synthesizing the corresponding ester prodrugs
[7–9]. A major requisite for these prodrugs is that they
must be readily hydrolyzed, enzymatically or chemically,
after oral absorption to quantitatively release the parent
drug [16]. In addition, the pro-moiety should be non-toxic
and readily excreted. Since they offer enormous potential
in terms of low toxicity, amino acids have been consid-
ered the ideal carriers for the development of prodrugs.
An obvious approach for carboxylic acids prodrugs that
contain an a-amino acid carrier is to prepare acyloxy-
methyl-type derivatives, e.g. 1 (Fig. 1) [17]. The presence of
the basic a-amino group has been reported to improve
the water-solubility of prodrugs 1, but unfortunately,
usually leads to chemically unstable compounds [17, 18].
This is due to the strong electron-withdrawing effect of
the protonated amino group at physiological pH and to
the ability of the unprotonated amino group to enhance
hydrolysis via nucleophilic or general-base catalysis [16]
and to undergo intramolecular rearrangement to form
an amide [17].
Scheme 2. Preparation of aminocarbonyloxymethyl derivatives
3.
Prodrugs 2 are readily hydrolyzed to the parent com-
pound in human plasma while displaying reasonable sta-
bility in aqueous solutions. However, several of the pro-
drugs 2 that contain a secondary carbamate group, parti-
cularly those containing glycine (2, R1 = R2 = H), undergo
rearrangement in pH 7.4 buffer, to form the correspond-
ing N-acylamine most probably via an O fi N intramole-
cular acyl transfer from the carbamate conjugate base
(Scheme 1) [19]. This rearrangement pathway is signifi-
cantly reduced or suppressed when sterically-hindered
aminoacids (e.g. Phe or Val and N-methylglycine (sarco-
sine)) are used as carriers. To further evaluate the poten-
tial of aminocarbonyloxymethyl esters as prodrugs for
NSAIDs, we have prepared a series of aminocarbonyloxy-
methyl derivatives 3 of flufenamic acid and diclofenac,
in which amino acid amides are used as carriers
(Scheme 2). The replacement of the amino acid ester by
Recently, we synthesized a series of aminocarbonyloxy-
methyl esters, 2, (Fig. 1) designed as carboxylic acid pro-
drugs [19]. Conceptually, this approach is based on the
low chemical reactivity of carbamates in neutral aqueous
media [20] and corresponds to an inversion of the amino
acid position in the potentially unstable counterparts 1.
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an amino acid amide is expected to decrease the carba- acid derivatives were detected during the hydrolysis of
mate pKa by ca. 1.2 units [21] and thus suppress the O fi N secondary carbamates 3a, b, d–h. Thus, compounds 3 do
rearrangements compared with 2. We have also carried not undergo the rearrangement involving an O fi N
out a kinetic study that evaluates the influence of the intramolecular acyl transfer from the carbamate conju-
amino acid amide pro-moiety on the chemical reactivity gate base (Scheme 1) that can be observed for compounds
and stability of aminocarbonyloxymethyl prodrugs of 2. Not surprisingly, the hydrolysis of tertiary carbamates
flufenamic acid and diclofenac 3 in human plasma. Since 3c, d, which lack an ionisable carbamate NH, also pro-
the oral bioavailability of diclofenac is only 50% [22], we ceeded with quantitative formation of the parent car-
have compared the bioavailability of several diclofenac boxylic acid.
derivatives 2 and 3 with diclofenac itself.
The hydrolysis of compounds 3a–h was studied in iso-
tonic pH 7.4 phosphate buffer at 378C, and the corre-
sponding half-lives are given in Table 1. Inspection of
Table 1 shows that compounds 3 are very stable in pH 7.4
buffer, hydrolyzing with half-lives ranging between 10 h
and 16 d. This represents a major increase in chemical
Results and discussion
Synthesis
The target prodrugs 3 were synthesised via the corre- stability over their ester counterparts 2. For example, the
sponding amino acid amide derivatives 6 (Scheme 2). The diclofenac N,N-diethylamide derivative 3g is hydrolyzed
N-Boc protected N-hydroxysuccinimidyl amino acid ester about 20 times more slowly than its ethyl ester counter-
4 reacted with the appropriate amine to afford the corre- part 2a. A similar effect is observed for the flufenamic
sponding N-Boc protected derivatives 5 in yields of 70–
acid derivative 3e, which is hydrolyzed approx. nine
90%. These latter derivatives, 5a–e, upon treatment with times more slowly than the corresponding ethyl ester
trifluoroacetic acid (TFA) to remove the Boc-protecting prodrug [19]. The large differences in chemical reactivity
group, gave as oils the deprotected amino acid deriv- between the amino acid ester 2 and amide 3 derivatives,
atives 6 isolated in 90–95% yields. Compounds 6 reacted can be ascribed to differences both in the electronic (i.e.
with chloromethyl chloroformate in dichloromethane pKa) and steric properties of the terminal amino acid
(DCM), in the presence of the phase-transfer catalyst tetra- amide and ester moieties, which are likely to affect both
butylammonium (Bu4NBr, TBAB) and triethylamine, to the rearrangement and hydrolysis pathways. For exam-
form the corresponding chloromethyl intermediates 7 in ple, the carbamate group of compound 3g is expected to
about 50% yield (Scheme 2). Finally, reaction of 7 with be about 2 pKa units less acidic than its counterpart 2a,
the appropriate sodium salt of diclofenac or flufenamic based on the inductive effects of the substituent on the
acid in DCM formed the target prodrug 3 in variable yield carbamate NH group [21; see section 3.8]. The rearrange-
ranging from 20% to 50%. The structure of compounds ment pathway finds some parallel to the E1cb elimina-
3a–h follows from their spectroscopic and analytical tion-addition mechanism operating in the hydrolysis of
data. In particular, the 1H-NMR spectra of the derivatives secondary carbamates in that it also involves the forma-
3a, 3b, 3f, and 3h revealed by the diastereotopic nature of tion of the carbamate conjugate base [20]. However, the
the CH2Ph protons which resonate as doublet of doublets effect exerted by the N-substituents of secondary carba-
at approx. 3.3 ppm. The diastereotopic nature of the mates, e.g. 8 (Fig. 2), on the rate of alkaline hydrolysis is
OCH2O was also evident for compounds 3b and 3h as not large, as indicated by the Hammett q value of 0.64
revealed by the presence of a pair of doublets at approx. [20], thus suggesting that the estimated drop in the pKa of
6.0 ppm. In the proline derivative 3c the OCH2O signal 3 relatively to that of 2 should reduce the rate of rearran-
appears as two pairs of doublets, reflecting the diastereo- gement; however, it is unlikely this effect could be
topic nature of the methylene protons as well as the pre- enough to suppress completely this pathway.
sence of syn- and anti-rotamers. The spectroscopic detec-
Another major difference between amino acid amides
tion of rotamers has been reported for other prolyl carba- 3 and their amino acid ester counterparts 2 is the size of
mate frameworks, similar to the barrier of acyclic ter- the amino acid C-terminal substituent. In the absence of
tiary amides [23, 24].
volume descriptors for the CONR3R4 amide in 3, we have
determined the molecular refractivity (MR) and molar
volume (MV) for CH3CONR3R4 using the ACDlabs ChemS-
Products and kinetics of chemical hydrolysis
The hydrolysis of N-aminocarbonyloxymethyl ester pro- ketch 8.0 freeware [25], and compared it with that of
drugs 3 proceeds with quantitative formation of the par- CH3CO2CH2CH3. The calculated MR and MV values are
ent carboxylic acid at pH 7.4. As we had anticipated, and 35.83 and 128.7 cm3 for the piperidine amide, 33.58 and
in contrast to their ester counterparts 2, no N-acyl amino 131.9 cm3 for the N,N-diethylamide, and 22.35 and
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Arch. Pharm. Chem. Life Sci. 2007, 340, 32–40
Prodrugs of Diclofenac and Flufenamic Acid
35
Table 1. Kinetic data for the hydrolysis of compounds 3a–3h and the corresponding ester 2a in pH 7.4 isotonic phosphate buffer
and in 80% human plasma at 378C, together with their apparent partition coefficients at pH 7.4 (Log Papp; mean l SD, n = 3).
Compound
NR3R4
R3CO2H
pH 7.4 phosphate buffer
80% Human plasma
t1/2 (min)
LogPapp
t1/2 (h)
3a
164
434
4.22 l 0.01
3b
3c
3d
160
385
124
301
462
26
4.06€ 0.34
4.00 l 0.29
3.65 l 0.07
a)
3e
3f
160
157
16.6
126
3.79 l 0.16
3g
3h
13.3
22.6
3.77
26.9
3.84 l 0.05
a)
2a
0.98
4.17
4.16 l 0.15
a)
Not determined.
98.0 cm3 for the ethyl ester. These results imply that the
Quite interestingly, the glycine derivative 3e, which is
amide groups in 3 are significantly bulkier than the ethyl a secondary carbamate, hydrolyzes essentially at the
ester moiety in 2, thus suggesting that this effect might same rate as its tertiary carbamate (sarcosine) counter-
significantly contribute to the suppression of the rear- part 3d. This contrasts with the observed differences
rangement pathway for prodrugs 3.
between the rates of alkaline hydrolysis of secondary
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Arch. Pharm. Chem. Life Sci. 2007, 340, 32–40
Table 2. Pharmacokinetic parameters of diclofenac after oral
administration of diclofenac sodium and prodrugs 2a, 3g, and 3h
in rats. (Mean l SE, n = 5).
(E1cb mechanism) and tertiary carbamates (classical
addition-elimination mechanism). For example, blocking
the carbamate acidic NH proton, e.g. by methyl substitu-
tion, usually leads to dramatic reductions in the rate of
alkaline hydrolysis, typically by a factor of 103 to 108 [26].
Therefore, the absence of any reduction in reactivity of
the sarcosine derivative 3d when compared to its glycine
counterpart 3e is also consistent with the absence of the
rearrangement pathway.
Compound
Cmax of diclofenac
AUC0 – 4h
(lg/mL)
(lg.h/mL)
Diclofenac
3.374 € 0.63
2.330 € 0.27
1.012 € 0.22
2.257 € 0.30
9.069
6.143
2.219
5.552
2a
3g
3h
The rates of hydrolysis for the flufenamic acid deriv-
atives at pH 7.4 are seven to twelve times smaller than
those of the diclofenac counterparts (e.g. 3e versus 3g),
reflecting the steric hindrance exerted by the ortho-ani-
lino substituent of flufenamic acid. The nature of the
amino acid pro-moiety also affects the rate of hydrolysis
at pH 7.4, but to a lesser extent than the ester moiety. For
example, the phenylalanine piperidine 3a and diethyla-
mide 3b hydrolyze at the same rate as their glycine coun-
terpart 3e. In contrast, the proline derivative 3c is hydro-
lyzed ca. three times more slowly than the sarcosine
derivative 3d, a result that can be accounted in terms of
the steric crowding exerted by the bulky pyrrolidine
ring.
boxylic acid. For example, the glycine derivative of flufe-
namic acid 3e is hydrolyzed about 18 times faster than
the more sterically hindered phenylalanine counterpart
3b. Similarly, compound 3g, the glycine derivative of
diclofenac, is hydrolyzed approximately seven times fas-
ter than the phenylalanine counterpart 3h. The diclofe-
nac derivatives hydrolyze four to ten times faster than
their flufenamic acid analogues, reflecting the sterical
hindrance exerted by the ortho-anilino substituent on the
latter compounds.
Relative bioavailability studies
The plasma concentration-time curves of diclofenac after
oral administration of the free drug and of the prodrugs
3g, 3h, and 2a are shown in Fig. 3. Detectable concentra-
tions of diclofenac were observed for 4 h both when free
drug and prodrugs were given orally to rats. This result
indicates that compounds 3 are hydrolyzed in vivo to the
parent diclofenac and thus confirm the prodrug nature
of these compounds. The peak plasma concentration Cmax
for the free drug was 3.374 lg/mL within 1 h (Tmax),
whereas for the prodrug 2a it was 2.330 lg/mL after a per-
iod of 30 min. There was a reduction in the extent of
drug absorption between the free drug and the prodrugs
2a, 3g, and 3h as revealed by the area under the plasma
concentration curve (AUC) (Table 2). Equimolar doses of
2a and 3h resulted in 68% and 61% level of plasma diclo-
Hydrolysis in human plasma
Compounds 3 are quantitatively hydrolyzed to the corre-
sponding parent carboxylic acid in human plasma, with
first-order kinetics for at least four half-lives. As with the
hydrolysis in pH 7.4 buffer, no traceable amounts of the
rearrangement product were detected for any com-
pound, which contrasts with the formation of ca. 20% of
rearrangement product for the human plasma hydroly-
sis of the closely related secondary N-acyloxyalkoxycarbo-
nyl derivatives of aniline 9 (R = Ph) but compares with
that of the benzylamine counterpart 9 (R = CH2Ph)
(Fig. 2), which hydrolyzed in human plasma almost
exclusively to the parent amine [27].
Human plasma enzymes accelerated the rate of hydro-
lysis of aminocarbonyloxymethyl esters 3, as revealed by
the half-lives for hydrolysis in 80% human plasma ran-
ging from 4 min to 7 h (Table 1). Reactivity in human
plasma appears to be dependent on the nature of substi-
tuents in both the amino acid carrier and the parent car-
Figure 2. Molecular structure of secondary carbamates 8 and
N-acyloxyalkoxycarbonyl derivatives of aniline (R = Ph) and ben-
zylamine (R = CH2Ph) 9.
Figure 3. Mean plasma concentration-time profiles of diclofenac
after oral administration of diclofenac sodium and prodrugs 2a,
3g, and 3h in rats.
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Prodrugs of Diclofenac and Flufenamic Acid
37
dichloromethane (50% w/v) was added at room temperature
(4.5 mL for 1 mmol of the protected compound). After stirring
for 30 min the solvent was evaporated and the residue dissolved
in a solution of NaOH. The mixture was extracted with ethyl
acetate, and the organic phase dried (anhydrous Na2SO4) and eva-
porated to afford the deprotected compound 6. To a dichloro-
methane solution of the deprotected compound 6 was added
chloromethyl chloroformate (1.1 mol eq., in dichloromethane).
After stirring at –108C during 25 min and 75 min at room tem-
perature, the reaction mixture was filtered, the filtrate was
washed with water, dried, and evaporated to obtain the corre-
sponding chloromethylcarbamate 7. A solution of the sodium
salt of the appropriate carboxylic acid (1.1 mol eq.) and tetrabu-
tylammonium bromide (1.1 mol equivalent) in tetrahydrofuran
(5 mL) was added to a solution of the intermediate 7 in tetrahy-
drofuran (5 mL) and triethylamine (1.1 mol equivalent). After
stirring for 24 h at room temperature, the solvent was evapo-
rated and the resultant compound purified by column chroma-
tography using a mixture of diethyl ether and light petroleum
(40:60) as eluent.
fenac of that from an equimolar dose of diclofenac
sodium (100%). These results are comparable with pre-
vious studies with nitrosothiolesters of diclofenac, which
also display a reduced oral bioavailability compared to
the parent drug [28]. For prodrugs 3, this effect might be
ascribed to the high lipophilicity, as indicated by the log
Papp values around 4 (Table 1). In conclusion, the novel
aminocarbonyloxymethyl prodrugs 3 of flufenamic acid
and diclofenac, containing amino acid amide pro-moi-
eties, display an unusually high stability in pH 7.4 buffers
combined with a good susceptibility to plasma-mediated
regeneration to the parent drug. Moreover, the pre-
viously reported O fi N intramolecular acyl transfer
from the carbamate conjugate base observed with the
ester counterparts 2 was completely suppressed in pro-
drugs 3. The quantitative delivery of the parent drug in
buffers and in human plasma suggests that the oral bioa-
vailability can be further improved by increasing aque-
ous solubility using amino acid carriers with ionizable
groups.
Synthesis of 3a
N-[N-(tert-Butoxycarbonyl)phenylalanyl]piperidine 5a
Prepared from BocPheOSuc, 4a (905.9 mg, 2.5 mmol) and piperi-
This project was supported by Fundaꢀ¼o para a CiÞncia e Tec-
nologia (POCTI) and by the Academy of Finland.
1
dine (212.9 mg, 2.5 mmol). Yield: 86% (714.7 mg, 2.1 mmol). H-
NMR: 1.01 (1H, brs, CH pip), 1.41–1.79 (14H, m, (CH3)3 and CH
pip), 2.95–3.05 (3H, m, NCH pip and CH2Ph), 3.25 (1H, ddd, J =
18.0, 7.7, 3.4, NCH pip), 3.44-3.50 (2H, m, NCH pip), 4.85 (1H, q, J =
7.3, a-CH), 5.45 (1H, brs, J = 7.9, NH), 7.18–7.28 (5H, m, ArH).
Experimental
N-(Phenylalanyl)piperidineamide 6a
Materials and chemicals
Yield: 95% (473.9 mg, 2.0 mmol). 1H-NMR: 1.16–1.28 (1H, brs, CH
pip), 1.41–1.61 (5H, m, CH2 pip), 2.73 (2H, brs, NH2), 2.86 (1H, dd,
J = 13.2, 8.5, CHPh), 2.96 (1H, 26dd, J = 13.2, 6.1, CHPh), 3.11–
3.14 (1H, m, NCH pip), 3.31–3.36 (1H, m, NCH pip), 3.48–3.60
(2H, m, NCH2 pip), 4.10 (1H, dd, J = 8.0, 7.0, a-CH), 7.20–7.32 (5H,
m, ArH).
Melting points were determined using a Kofler camera Bock
Monoscop M (C. Reichert, Vienna, Austria) and are uncorrected.
Elemental analyses were performed either by Instituto Tecnolꢀ-
gico Nuclear, Sacavꢁm, Portugal or by Perkin-Elmer Analyzer
2400, Department of Pharmaceutical Chemistry, University of
Kuopio. FTIR spectra were recorded using a Nicolet Impact 400
spectrophotometer (Nicolet, Madison, WI, USA). The high-per-
formance liquid chromatography (HPLC) system consisted of a
Merck Hitachi L-7110 pump with a L-7400 UV detector, a manual
sample injection module equipped with a 20 lL loop, a Merck
LiChrospherm 100 RP8 125 cm64.6 mm (5 lm) column equipped
with a Merck LiChrocart pre-column (Merck, Germany). 1H- and
13C-NMR spectra were recorded in CDCl3 solutions and were per-
formed by Bruker AM 400 WB (Bruker). Coupling constants J are
expressed in Hz. The carboxylic acids were purchased from
Sigma. All other chemicals and solvents were of reagent grade,
except buffers substances and HPLC solvents which were analyti-
cal grade and LiChrosolvm grade, respectively. Column chromato-
graphy was performed using silica gel 60 mesh 70–230 (Merck).
N-[2-(Chloromethyloxycarbonylamino)-3-
phenylpropanoyl]piperidine 7a
To a solution of 6a (2.0 mmol, in 5 mL CH2Cl2) was added chloro-
methyl chloroformate (2.0 mmol, 263.0 mg in 5 mL CH2Cl2)
yielding an oil (292.1 mg, 0.9 mmol). Yield: 46%. 1H-NMR: 1.05–
1.90 (6H, m, CH2 pip), 3.00–3.04 (2H, m, NCH2 pip), 3.12–3.27
(2H, m, CH2Ph), 3.46-3.52 (2H, m, CH2 pip), 4.90 (1H, q, J = 7.5, a-
CH), 5.69 (1H, d, J = 6.2, OCHCl), 5.73 (1H, d, J = 6.2, OCHCl), 6.13
(1H, broad d, J = 8.0, NH), 7.16–7.30 (5H, m, ArH).
Compound 3a
Prepared from 7a (292.1 mg, 0.9 mmol) and flufenamic acid
(264.3 mg, 0.9 mmol). Yield: 39% (210.8 mg, 0.4 mmol). M. p. 58–
608C. mmax/cm–1: 3315, 1732, 1710, 1629. 1H-NMR: 1.36–1.56 (6H,
m, CH2CH2CH2), 2.95–3.04 (3H, m, NCH2 and CHPh), 3.21–3.26
(1H, m, CHPh), 3.44–3.51 (2H, m, NCH2), 4.89 and 4.90 (1H, ddd, J
= 8.8, 8.7, 5.4, a-CH), 5.93 (1H, d, J = 8.4, NH), 5.96 (2H, s, OCH2O),
6.80–6.82 (1H, m, ArH), 7.14–7.50 (11H, m, ArH), 8.04–8.06 (1H,
m, ArH), 9.46 (1H, s, NH). EIMS (%): 570.1 (100) [M+]. Calculated for
C30H30N3O5F3 : C, 63.3; H, 5.3; N, 7.4%. Found: C, 63.2; H, 5.4; N,
7.4%.
General procedures for the synthesis of prodrugs
3a–3h
Typically, the amine (2.5 mmol) was added dropwise to a stirred
solution of the N-t-Boc-amino acid-N-hydroxysuccinimidyl ester 4
(2.5 mmol) in dichloromethane (5 mL) (Scheme 2). After 48 h at
room temperature the solvent was evaporated and the residue
purified by column chromatography (eluant: diethyl ether). To
the protected compound 5, a solution of trifluoroacetic acid in
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Arch. Pharm. Chem. Life Sci. 2007, 340, 32–40
Synthesis of 3b
N-(Chloromethyloxycarbonyl)proline diethylamide 7c
To a solution of 6c (275.8 mg in 5 mL CH2Cl2) was added chloro-
methyl chloroformate (208.9 mg in 5 mL CH2Cl2) yielding an oil
N-[N-(tert-Butoxycarbonyl)phenylalanyl]-N,N-
diethylamine 5b
1
(170.8 mg, 0.7 mmol). Yield: 40%. H-NMR: 1.12 (3H, t, J = 7.0,
CH3CH2), 1.23 (3H, t, J = 7.0, CH3CH2), 1.71–1.78 (1H, m, CH Pro),
1.85–2.05 (2H, m, CH2 Pro), 2.25–2.32 (1H, m, CH Pro), 3.27–3.44
(6H, m, CH3CH2 and NCH2 Pro), 4.32 (1H, brs, a-CH), 5.95, 5.98 and
6.06 (2H, 36overlapping doublets, J = 5.6, OCH2Cl).
Prepared from BocPheOSuc 4a (906.0, mg, 2.5 mmol) and dieth-
ylamine (182.8 mg, 2.5 mmol). Yield: 78% (624.9 mg, 1.9 mmol).
1H-NMR: 0.99 (3H, t, J = 7.0, CH3CH2), 1.10 (3H, t, J = 7.0, CH3CH2),
1.40 (9H, s, (CH3)3), 2.92–3.14 (5H, m, NCH2 and CH2Ph), 3.5 (1H,
dq, J = 14.0, 6.3, NCH) 4.73 (1H, apparent q, J = 7.5, a-CH), 5.37 (1H,
d, J = 7.5, NH), 7.16–7.24 (5H, m, ArH).
Compound 3c
Prepared from 7c (170.8 mg, 0.7 mmol) and flufenamic acid
(sodium salt) (182.8 mg, 0.7 mmol). Yield: 37% (122.0 mg,
N-(Phenylalanyl)-N,N-diethylamine 6b
1
0.2 mmol). Oil. mmax/cm–1: 3325, 1717, 1687, 1644. H-NMR: 1.01,
1
Yield: 95% (407.6 mg,1.9 mmol). H-NMR: 1.10 (3H, t, J = 7.0,
1.13, 1.21 and 1.28 (6H, 46t, J = 7.0, CH3CH2), 1.85–1.92 (2H, m,
CH2 Pro), 2.05–2.24 (2H, m, CH2 Pro), 3.11–3.74 (6H, m, CH3CH2
and NCH2 Pro), 4.55 and 4.66 (1H, 26dd, J = 8.2, 3.6, a-CH), 5.92,
5.93, 5.98, and 6.08 (2H, 46d, J = 5.8, OCH2O), 6.79–6.84 (1H, m,
ArH), 7.25–7.49 (6H, m, ArH), 8.03-8.06 (1H, m, ArH), 9.42, 9.46
(1H, 26s, NH). EIMS (%): 508.1 (91) [M+]. Calculated for
C25H28N3O5F3 : C, 59.2, H, 5.7, N, 8.3%. Found: C, 59.0, H, 5.7, N,
8.3%.
CH3CH2), 1.15 (3H, t, J = 7.0, CH3CH2), 2.60 (2H, s, NH2), 2.97 (2H,
m, CH3CH2), 3.12 (1H, m, CHHPh), 3.26 (2H, m, CH3CH2), 3.52 (1H,
m, CHHPh), 4.10 (1H, dd, J = 8.7, 7.0, a-CH), 7.20–7.40 (5H, m,
ArH).
N-[2-(Chloromethyloxycarbonylamino)-3-
phenylpropanoyl]-N,N-diethylamine 7b
To a solution of 6b (407.6 mg in 5 mL CH2Cl2) was added chloro-
methyl chloroformate (1.9 mmol, 238.5 mg in 5 mL CH2Cl2)
yielding an oil (309.7 mg, 1.0 mmol). Yield: 54%. H-NMR: 1.01
Synthesis of 3d
1
(3H, t, J = 7.0, CH3CH2), 1.05 (3H, t, J = 7.0, CH3CH2), 2.90–3.15 (5H,
m, NCHH and CH2Ph), 3.50–3.58 (1H, dq, J = 12.5, 8.0, NCHH),
4.78–4.81 (1H, m, a-CH), 5.67 (1H, d, J = 5.8, OCHCl), 5.75 (1H, d, J
= 5.8, OCHCl), 6.09 (1H, d broad, J = 8.0, NH), 7.17-7.29 (5H, m,
ArH).
N-(tert-Butoxycarbonyl)sarcosine diethylamide 5d
Prepared from BocSarOSuc 4c (715.7 mg, 2.5 mmol) and diethy-
lamine (182.8 mg, 2.5 mmol). Yield: 79% (482.6 mg, 2.0 mmol).
1H-NMR: 1.13 (3H, t, J = 7.0, CH3CH2), 1.19 (3H, t, J = 7.0, CH3CH2),
1.43 and 1.47 (9H, 26s, (CH3)3), 2.93 and 2.95 (3H, 26s, NCH3),
3.28-3.32 (2H, m, NCH2CH3), 3.35–3.42 (2H, m, NCH2CH3), 3.94
and 4.05 (2H, 26s, NCH2CO).
Compound 3b
Prepared from 7b (309.67 mg, 0.99 mmol) and flufenamic acid
(sodium salt) (278.4 mg, 1.0 mmol). Yield: 39% (217.5 mg,
0.4 mmol). Oil. mmax/cm–1: 3323, 3275, 1736, 1699, 1633. 1H-NMR:
0.99 (3H, t, J = 7.0, CH3CH2) and 1.04 (3H, t, J = 7.0, CH3CH2), 2.90–
3.11 (5H, m, NCH2 and CH2Ph), 3.49–3.57 (1H, m, NCH), 4.78 and
4.80 (1H, ddd, J = 8.53, 8.35, 6.18, a-CH), 5.83 (1H, d, J = 8.7, NH),
5.94 (1H, d, J = 5.81, OCHO), 5.97 (1H, d, J = 5.81, OCHO), 6.80–
6.83 (1H, m, ArH), 7.17–7.50 (11H, m, ArH), 8.01–8.04 (1H, m,
ArH), 9.46 (1H, s, NH). EIMS (%): 558 (100) [M+]. Calculated for
C29H30N3O5F3 : C, 62.5, H, 5.4, N, 7.5%. Found: C, 62.4; H, 5.4; N,
7.5%.
Sarcosine diethylamide 6d
Yield: 90% (257.0 mg, 1.8 mmol). Oil 1H-NMR: 1.10 (3H, t, J = 7.0,
CH3CH2), 1.20 (3H, t, J = 7.0, CH3CH2), 2.54 (3H, s, NCH3), 3.26 (2H,
q, J = 7.0, CH3CH2), 3.39 (2H, q, J = 7.0, CH3CH2), 3.58 (2H, s, NCH2),
3.86 (1H, brs, NH).
N-(Chloromethyloxycarbonylsarcosine diethylamide 7d
To a solution of 6d (257.0 mg in 5 mL CH2Cl2) was added chloro-
methyl chloroformate (1.8 mmol, 229.5 mg in 5 mL CH2Cl2)
yielding an oil (421.3 mg, 1.8 mmol). Yield: 100% (syn/anti ratio
= 0.5) [29, 30]. 1H-NMR: 1.13 (3H, t, J = 7.0, CH3CH2), 1.22 (3H, t, J =
7.0, CH3CH2), 3.04 (3H, s, NCH3), 3.29 (2H, q, J = 7.2, NCH2), 3.39
(2H, q, J = 7.0, NCH2), 4.05 and 4.11 (2H, 26s, COCH2N), 5.76 and
5.79 (2H, 26s, OCH2Cl).
Synthesis of 3c
N-(tert-Butoxycarbonyl)proline diethylamide 5c
Prepared from BocProOSuc 4b (780.8 mg, 2.5 mmol) and diethy-
lamine (182.8 mg, 2.5 mmol). Yield: 68% (459.6 mg, 1.7 mmol).
1H-NMR: 1.10 and 1.14 (3H, 26t, J = 7.0, CH3CH2), 1.23 and 1.25
(3H, 26t, J = 7.0, CH3CH2), 1.41 and 1.45 (9H, 26s, (CH3)3), 1.64–
2.22 (4H, m, 26CH2 pro), 3.20–3.66 (6H, m, NCH2 and CH2 Pro),
4.44 and 4.60 (1H, 26dd, J = 4.0, 8.3, a-CH).
Compound 3d
Prepared from 7d (421.3 mg) and flufenamic acid (sodium salt)
(500.5 mg, 0.7 mmol). Yield: 37% (317.8 mg, 0.7 mmol). Oil. mmax
/
cm–1: 3335, 1727, 1697, 1663. 1H-NMR: 1.13, 1.17, 1.21, 1.26 (6H,
46t, J = 7.1, CH3CH2), 3.03 and 3.04 (3H, 26s, NCH3), 3.21, 3.28,
3.29, 3.38 (4H, 46q, J = 7.0, CH3CH2), 4.02 and 4.10 (2H, 26s,
NCH2), 5.98 and 6.03 (2H, 26s, OCH2O), 6.79–6.84 (1H, m, ArH),
7.25–7.49 (6H, m, ArH), 8.04–8.06 (1H, m, ArH), 9.41 and 9.45
(1H, 26s, NH). EIMS (%): 482.1 (100) [M+]. Calculated for
C23H26N3O5F3 : C, 57.4, H, 5.4, N, 8.7%. Found: C, 56.7; H, 5.6; N,
8.7%.
Proline diethylamide 6c
Yield: 95% (275.8 mg, 1.6 mmol). 1H-NMR: 1.12 (3H, t, J = 7.0,
CH3CH2), 1.23 (3H, t, J = 7.0, CH3CH2), 1.71–1.78 (1H, m, CH Pro),
1.85–2.05 (2H, m, CH2 Pro), 2.25–2.32 (1H, m, CH Pro), 3.15 (1H,
brs, NH), 3.27–3.44 (6H, m, CH3CH2 and NCH2 Pro), 4.32 (1H, brs,
a-CH).
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Arch. Pharm. Chem. Life Sci. 2007, 340, 32–40
Prodrugs of Diclofenac and Flufenamic Acid
39
= 6.1, OCHCl), 6.01 (1H, d broad, J = 7.8, NH), 7.17-7.32 (5H, m,
ArH).
Synthesis of 3e
N-(tert-Butoxycarbonyl)glycine diethylamide 6e
Prepared from BocGlyOSuc 4d (2.4 g, 8.9 mmol) and diethyla-
mine (650.9 mg, 8.9 mmol). Yield: 90% (1.9 g, 8.0 mmol). 1H-
NMR: 1.10 (3H, t, J = 7.0, CH3CH2), 1.30 (3H, t, J = 7.0, CH3CH2), 1.47
(9H, s, (CH3)3), 3.17–3.67 (4H, m, 26CH3CH2), 4.00 (2H, d, J = 4.0,
NHCH2CO), 5.57 (1H, brs, NHCH2CO).
Compound 3f
Prepared from 7f (300.6 mg, 0.9 mmol) and flufenamic acid
(sodium salt) (258.7 mg, 0.9 mmol ). Yield: 27% (142.9 mg,
0.3 mmol). Oil mmax/cm–1: 3325, 1737, 1698, 1639. 1H-NMR: 2.89–
2.93 (3H, m, CH2NCH morphol), 2.97 (1H, dd, J = 13.3, 9.2, CHPh),
3.07 (1H, dd, J = 13.3, 5.3, CHPh), 3.23–3.29 (1H, m, NCH), 3.40-
3.60 (4H, m, CH2OCH2), 4.85 (1H, ddd, J = 8.8, 8.7, 5.4, a-CH), 5.90
(1H, d, J = 8.5, NH), 5.97 (2H, s, OCH2O), 6.80–6.83 (1H, m, ArH),
7.17–7.50 (11H, m, ArH), 8.03 (1H, d, J = 7.9, ArH), 9.45 (1H, s,
NH). EIMS (%): 572.0 (100) [MH+]. Calculated for C29H28N3O6F3 : C,
60.9; H, 4.9; N, 7.4%. Found: C, 60.8; H, 5.0; N, 7.4%.
Glycine diethylamide 6e
Yield: 66% (689.9 mg, 5.3 mmol). 1H-NMR: 1.07 (3H, t, J = 7.0,
CH3CH2), 1.23 (3H, t, J = 7.0, CH3CH2), 3.10–3.93 (8H, m, CH3CH2,
CH2CO and NH2).
N-(Chloromethyloxycarbonylglycine diethylamide 7e
To a solution of 6e (5.3 mmol, 689.9 mg in 10 mL CH2Cl2) was
added chloromethyl chloroformate (5.3 mmol, 683.4 mg in
10 mL CH2Cl2) yielding an oil ( 532.2 mg, 2.4 mmol). Yield: 45%.
1H-NMR: 1.13 (3H, t, J = 7.0, CH3CH2), 1.30 (3H, t, J = 7.0, CH3CH2),
3.30 (2H, q, J = 7.0, CH3CH2), 3.51 (2H, q, J = 7.0, CH3CH2), 4.13 (2H,
d, J = 4.0, NHCH2CO), 5.87 (2H, s, OCH2Cl), 6.23 (1H, brs,
CONHCH2).
Synthesis of compound 3g
Prepared from 7e (302.3 mg, 1.4 mmol) and diclofenac (sodium
salt) (432.6 mg, 1.4 mmol). Yield: 13% (83.3 mg, 0.2 mmol). M. p.
105–1060C. mmax/cm–1. 3393, 3335, 3090, 2982, 1746, 1644; H-
1
NMR: 1.07 (3H, t, J = 7.2, CH3CH2), 1.13 (3H, t, J = 7.2, CH3CH2), 3.18
(2H, q, J = 7.2, CH3CH2), 3.34 (2H, q, J = 7.2, CH3CH2), 3.80 (2H, s,
ArCH2CO), 3.94 (2H, d, J = 4.0, NHCH2CO), 5.74 (2H, s, OCH2O),
5.93 (1H, broad t, CONHCH2), 6.49 (1H, d, J = 8.0, ArH), 6.69 (1H,
brs, ArNHAr), 7.16–7.28 (6H, m, ArH). EIMS: 482 (69) [MH+], 505
(80) [MNa+], 988 (78) [M2Na+]. Calculated for C22H25N3O5Cl2, C,
54.78; H, 4.22; N, 8.71%. Found: C, 54.62; H, 4.43; N, 8.07%.
Compound 3e
Prepared from 7e (243 mg, 1.1 mmol) and flufenamic acid
(sodium salt) (309.3 mg, 1.1 mmol). Yield: 65% (332.1 mg,
0.7 mmol). M. p. 119–1200C. mmax/cm–1: 3333, 3279, 1739, 1677,
1631. 1H-NMR: 1.14 (3H, t, J = 7.2, CH3CH2), 1.21 (3H, t, J = 7.2,
CH3CH2), 3.26 (2H, q, J = 7.2, CH3CH2), 3.41 (2H, q, J = 7.2, CH3CH2),
4.04 (2H, d, J = 4.0, NCH2CO), 6.02 (2H, s, OCH2O), 6.08 (1H, broad
t, CONHCH2), 6.80–6.84 (1H, m, ArH), 7.27–7.55 (6H, m, ArH),
8.06 (1H, dd, J = 8.2, 1.4, ArH), 9.45 (1H, s, NH). EIMS: 468.9 (45)
[MH+], 935.9 (35) [M2H+], 957.9 (100) [M2Na+]. Calculated for
C22H24N3O5F3; C, 56.5; H, 5.2; N, 9.0%. Found: C, 56.4, H, 5.4, N,
8.92%.
Synthesis of compound 3h
Prepared from 7a (824.8 mg, 2.6 mmol) and diclofenac (sodium
salt) (839.8 mg, 2.6 mmol). Yield: 23% (343.0 mg, 0.6 mmol). M. p.
33–340C. mmax/cm–1: 3325, 3266, 3060, 2973, 1737, 1629. 1H-
NMR: 0.91 (3H, t, J = 7.2, CH3CH2), 0.97 (3H, t, J = 7.2, CH3CH2),
2.80–3.01 (6H, m, CH3CH2, CH3CH2, PhCH2), 3.79 (2H, s, ArCH2CO),
4.70 (1H, ddd, J = 8.8, 8.4, 6.4, NHCH), 5.68 (1H, d, J = 6.0, OCH2O),
5.71 (1H, d, J = 6.0, OCH2O), 5.80 (1H, d, J = 6.0, CONHCH), 6.49
(1H, broad d, J = 8.8, ArH), 6.68 (1H, s, ArNHAr), 6.87–7.28 (11H,
m, ArH); 13C-NMR: 12.75 (CH3CH2), 14.12 (CH3CH2), 38.29 (CH3CH2),
40.25 (CH3CH2), 40.55 (CH2Ph), 41.61 (NHCH2CO), 51.97
(HNCH(CH2Ph)CO, 80.52 (OCH2O), 118.54–153.71 (CHAr), 170.03
(CH2CO), 171.25 (NHCO). Calculated for C29H31N3O5Cl2: C, 60.84;
H, 5.46; N, 7.34%. Found: C, 60.18; H, 5.64; N, 6.99%.
Synthesis of 3f
N-[(tert-Butoxycarbonyl)phenylalanyl]morpholine 5f
Prepared from BocPheOSuc 4a (905.9 mg, 2.5 mmol) and mor-
pholine (217.8 mg, 2.5 mmol). Yield: 87% (729.0 mg, 2.2 mmol).
1H-NMR: 1.38 (9H, s, (CH3)3), 2.87–2.99 (3H, m, NCH2 and CHPh),
3.02 (1H, dd, J = 12.2, 5.0, CHPh), 3.25–3.31 (1H, m, NCHH), 3.39-
3.61 (5H, m, NCHH and 26CH2O), 4.80 (1H, q, J = 8.0, a-CH), 5.45
(1H, d, J = 8.0, NH), 7.10–7.30 (5H, m, ArH).
Apparent partition coefficients
The apparent partition coefficients (log Papp) of prodrugs were
determined at room temperature using octanol-pH 7.4 phos-
phate buffer. Each phase was mutually saturated before the
experiment. The volumes of each phase were chosen so that
solute concentrations in the aqueous phase after distribution
were readily measurable. The compounds were dissolved in 1-
octanol and the octanol-phosphate buffer mixtures were shaken
for 30 min to reach an equilibrium distribution. Each phase was
analyzed separately by HPLC, but the organic phase was diluted
in acetonitrile (1:10) before HPLC analysis. Partition coefficients
Papp were calculated from the ratio of the peak area in octanol to
the peak area in buffer.
N-(Phenylalanyl)morpholine 6f
Yield: 90% (459.2 mg, 1.9 mmol). 1H-NMR: 2.91–3.10 (7H, m,
CH2Ph and CHHNCH2 and NH2), 3.35 (1H, brs, NCHH), 3.50 (2H,
brs, OCH2), 3.60 (2H, brs, OCH2), 4.10 (1H, t, J = 6.8, a-CH), 7.10–
7.30 (5H, m, ArH).
N-[(2-Chloromethyloxycarbonylamino)-3-
phenylpropanoyl]morpholine 7f
To a solution of 6f (459.2 mg in 10 mL CH2Cl2) was added chloro-
methyl chloroformate (1.9 mmol, 252.7 mg in 5 mL CH2Cl2)
yielding an oil (300.6 mg, 0.9 mmol). Yield: 47%. H-NMR: 2.62
(4H, m, CH2NCH2), 2.87 (2H, m, CH2Ph), 3.60 (4H, m, CH2OCH2),
4.83–4.88 (1H, m, a-CH), 5.71 (1H, d, J = 6.1, OCHCl), 5.74 (1H, d, J
Hydrolysis in buffer solution
1
Reaction mixtures were analysed using HPLC. Usually, a 10 lL
aliquot of a 10–2 M stock solution of substrate in acetonitrile was
added to 10 mL of the appropriate thermostatted buffer solu-
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40
L. Ribeiro et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 32–40
tion. At regular intervals, samples of the reaction mixture were
analysed by HPLC using the following conditions: mobile phase,
acetonitrile/water (the composition of which depended on the
compound); flow rate 1.0 mL/min; detector wavelength, 230 nm.
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Hydrolysis in human plasma
Human plasma was obtained from the pooled, heparinized
blood of healthy donors, and was frozen and stored at –708C
prior to use. For the hydrolysis experiments, the substrates were
incubated at 378C in human plasma that had been diluted to
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Animal model
Male Wistar rats (Harlan Iberica, Barcelona, Spain) weighing 150
to 200 g were randomized into four groups and each of these
groups subdivided into five groups containing five animals
each. Animals were administered, by oral gavage, either a fixed
dose (10 mg/kg) of diclofenac sodium or an equimolar dose of a
diclofenac derivative (2a, 3g, and 3h) in a suspension of propy-
lenglycol-400 (mg/mL). Blood samples were collected at 0 (pre-
dose), 30 min, 1, 2, and 4 h. (five rats per time point; five time
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mL (r2 A 0.998) range.
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Estimation of the pKa differences between the
carbamate group of prodrugs 2 and 3
To estimate the pKa differences between the secondary carba-
mate group of prodrugs 2 and 3, we have assumed that the equa-
tion established for secondary amides, RCONHR9, [21] applies:
pKa = 22–3.1 R*
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The difference between compounds 2 and 3 lies in the R9 sub-
stituent, which corresponds to the amino acid pro-moiety. For
the glycine derivatives, the Taft r* values for the methyl ester,
CH2CO2CH3, ethyl ester, CH2CO2CH2CH3, and amide, CH2CONH2
groups, are 1.06, 0.82 and 0.31, respectively; thus, the difference
in pKa ranges from 3.1(1.06–0.31) L 2.3 to 3.1 (0.82–0.31) L 1.6.
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