Synthesis and hydrolytic stability studies of albendazole carrier prodrugs

Article abstract of DOI:10.1016/S0960-894X(01)00214-1

Three N-acyl (2, 3, and 4), two N-alkoxycarbonyl (5 and 6), and one N-acyloxymethyl (7) derivatives of albendazole (1) have been prepared and assessed as potential prodrugs. The determination of the aqueous solubility and partition coefficient, as well as the conversion of these derivatives to 1 in buffer solution, human plasma, and pig liver esterase were determined.

Full text of DOI:10.1016/S0960-894X(01)00214-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1359–1362
Synthesis and Hydrolytic Stability Studies of
Albendazole Carrier Prodrugs
Francisco Hernandez-Luis,a,* Alicia Hernandez-Campos,a
Lilian Yepez-Mulia,b Roberto Cedillob and Rafael Castilloa
a
´
Departamento de Farmacia, Facultad de Quımica, UNAM, C.U. Mexico D.F. 04510, Mexico
´ ´
Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias, IMSS, Mexico D.F., Mexico
b
Received 9 August 2000; accepted 21 March 2001
Abstract—Three N-acyl (2, 3, and 4), two N-alkoxycarbonyl (5 and 6), and one N-acyloxymethyl (7) derivatives of albendazole (1)
have been prepared and assessed as potential prodrugs. The determination of the aqueous solubility and partition coefficient, as
well as the conversion of these derivatives to 1 in buffer solution, human plasma, and pigliver esterase were determined. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
ethyl chloroformate for 12 h, led to a mixture of 5 and
6. After workup and recrystallization of the residue
from isopropyl ether, 5 could be obtained. Compound 6
was isolated by evaporatingthe mother liquor and
washingthe residue with ethyl acetate–petroleum ether.
For the synthesis of 7, commercial 8 was treated with di-
tert-butyldicarbonate to give 9, which could be con-
verted to 10 accordingto the procedure of Binderup et
al.6 A solution of 10 in DMF was then added to a pre-
vious 2 h reaction mixture of 1 and NaH in DMF. After
12 h of reaction, water was added, and the suspension
was filtered to give 1-[4-(tert-butoxy-carbonylamino-
methyl)benzoyloxymethyl]-albendazole, as a mixture of
two isomers. Compound 11 was obtained by recrys-
tallization of the residue from benzene; a second portion
remained in the mother liquor together with the other
Albendazole (1) is a potent, broad spectrum anthelmin-
tic agent used mainly in the treatment of intestinal hel-
minthiosis.1 Also, it has been demonstrated that 1 is
effective against some tissue-dwelling infestations, such
as trichinellosis, hydatid disease and neurocysti-
cercosis.2 However, high doses and long treatment are
required in the latter cases, due mainly to the poor
solubility and absorption of 1.2,3 In previous studies
various N-acyl, N-alkoxycarbonyl, and N-acyloxy-
methyl derivatives of other benzimidazole carbamates
were evaluated as possible prodrugform with enhanced
solubility properties.4,5 In order to increase the bio-
availability of 1 by the prodrugapproach, we have syn-
thesized three N-acyl (2, 3, and 4), two N-
alkoxycarbonyl (5 and 6), and one N-acyloxymethyl (7)
derivatives of this compound (Table 1). In this work,
aqueous solubility, partition coefficients and stability in
buffer solution, human plasma, and pigliver esterase of
derivatives synthesized were measured.
Table 1. Synthesized derivatives of albendazole (1–7)
Chemistry
Compd
R1
R2
R3
Compounds 2–4 were prepared by known procedures.4
Treatment of a suspension of 1 (Smith & Kline, Mexico)
and Na2CO3 in dichloromethane, with an equivalent of
1
2
3
4
5
6
7
H
COCH3
COCH2CH3
S(CH2)2CH3
S(CH2)2CH3
S(CH2)2CH3
S(CH2)2CH3
S(CH2)2CH3
H
H
H
H
H
COCH2CH2CH3
COOCH2CH3
COOCH2CH3
CH2OOCC6H4NH2 2HCl
H
S(CH2)2CH3
H
*Correspondingauthor. Tel.: +52-5-622-5287; fax: +52-5-622-5329;
e-mail: franher@servidor.unam.mx
S(CH2)2CH3
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00214-1

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F. Hernandez-Luis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1359–1362
1360
isomer, but their separation was not successful. Com-
pound 11 was dissolved in ethyl acetate and a 3 N solu-
tion of hydrochloric acid in ethanol was added. The
mixture was stirred for 3 h at room temperature. The
precipitate was collected and recrystallized from metha-
nol–ether to give the dihydrochloride of 7 (Scheme 1).
The structures of 2–7 were established by IR, 1H NMR,
13C NMR, and MS data.7ꢀ12
Hydrolysis in aqueous solutions
All rate studies were performed in aqueous buffer solu-
tion at 37ꢂ0.2 ꢁC. The buffers used were hydrochloric
acid, acetate, phosphate, and borate buffers. The reac-
tions were initiated by adding100 mL of a stock solution
of the prodrugs in methanol to 10 mL of preheated
buffer solution in screw-capped test tubes; the final
concentration beingabout 4 ꢃ10ꢀ4 M (1% methanol).14
At appropriate intervals, 10 mL of samples were taken
and chromatographed immediately. Appearance of 1
was followed by HPLC as previously described by
Hurtado et al.15
Determination of apparent partition coefficients and
aqueous solubility
The apparent partition coefficients of 1–7 were deter-
mined usingHPLC retention times, as previously
described by Thomas et al.13 In this case, the reference
compounds (benzaldehyde, benzene, toluene, chloro-
benzene, xylene, fluorene and anthracene) were detected
at 255 nm, whereas 1–7 were detected at 310 nm. The
logof the capacity factor ( K) in each mobile phase
composition was calculated usingthe equation:
Hydrolysis in biological media
Compounds 1–7 were incubated at 37 ꢁC in 80% human
plasma; at an initial concentration of 4ꢃ10ꢀ4 M (1%
methanol). At various time points, samples of plasma
were withdrawn and extracted with a Sep-Pak C18
cartridge. Appearance of 1 was followed by HPLC as
previously described.15 Enzymatic hydrolysis was con-
ducted similarly with pigliver esterase (Simg a Co.)
instead of plasma.
log K ¼ log½ðtr À t0Þ=t0ꢀ
where t0 refers to the column ‘dead volume’ (elution
time of methanol, a nonretained compound). The logof
the predicted capacity factor at 0% of methanol (log K0)
was determined for each standard by linear regression
analysis of log K versus percent methanol. Calculation
of log K0 values of 1–7 was then done allowingpre-
dicted P0/w values to be calculated usingthe regression
equation obtained for the standards of known P0/w.
Results and Discussion
Prodrugs 2–6 were prepared by one-step reaction and
obtained in low yields (30%) due to difficulties that
appeared duringthe separation of each isomer. In the
case of 2–4, it was not possible to obtain the two iso-
mers due to instability of one of them.
1
All solubility studies were performed in 0.02 M phos-
phate buffer of pH 6.0 at 25 ꢁC by addingan excess
amount of the compounds to the buffer in screw-capped
test tubes.5 It was ensured that saturation equilibrium
was established with exception of compounds 2, 3, and
4, which were evaluated after 15 min due to stability
problems (>25% degradation). The pH values of the
solution were recorded prior to their filtration through
0.45 mm pore size Millipore filters. The solutions were
analyzed for compound content usinga UV spectro-
photometric assay.
In the H NMR spectrum for 2, the H-7 absorption is
shifted down-field from that in 1 by about 1.0 ppm in
DMSO, because of the orientation of the acetyl car-
1
bonyl group. Since the H NMR spectra of 3, 4, and 5
exhibit similar shifts of their H-7 absorption, they must
also be 1/5 disubstituted. In 6, H-7 appears as a doublet
at 7.75 (J=2 Hz). Differentiation between 5 and 6 could
be made from the couplingconstant of the signal for H-
7, the proton deshielded by N1-COOC2H5 group (Table
2).16 Compounds 2–4, with –COR group, and 5 and 6
with –COOC2H5 group, would be expected to exist in
Scheme 1. (a) O[CO2C(CH3)3]2, tert-BuOH; (b) ClCH2OSO2Cl, CH2Cl2, H2O, [CH3(CH2)3]4N(HSO4), NaHCO3; (c) 1, DMF; (d) HCl/EtOH.

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F. Hernandez-Luis et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1359–1362
1361
Table 2. Chemical shifts in H-7 and H-4 for compounds 1–6
Table 4. kobs for the hydrolysis of prodrugs 5–7
kobs (10ꢀ3 minꢀ1 a
Compd
H-7 (ppm) (Hz)
H-4 (ppm) (Hz)
)
1
2
3
4
5
6
7.33 (J=10)
7.97 (J=10)
8.01 (J=9)
7.99 (J=9)
7.70 (J=10)
7.75 (J=2)
7.42 (J=2)
7.41 (J=2)
7.43 (J=2)
7.42 (J=2)
7.53 (J=2)
7.51 (J=10)
Compd
pH 5.0
pH 7.4
Plasma
Esterase
5
6
7
2.19ꢂ0.59
4.21ꢂ0.93
3.01ꢂ1.15
5.73ꢂ1.08
5.78ꢂ0.95
5.62ꢂ2.08
83.29ꢂ10.24
96.33ꢂ23.12
12.43ꢂ5.68
100.14ꢂ29.35
99.14ꢂ34.56
15.29ꢂ3.34
aMeanꢂstandard deviation.
Table 3. Meltingpoints, log P, and solubility for compounds 1–7
Compd
mp (ꢁC)
log Pa
Sb (mM)a [lmax (nm)]
Table 5. t1=2 for the hydrolysis of prodrugs 5–7
t1=2 (min)
1
2
3
4
5
6
7c
205
128
145
138
75
3.07ꢂ0.36
2.82ꢂ0.28
3.17ꢂ0.26
3.92ꢂ0.31
3.92ꢂ0.31
3.19ꢂ0.57
2.91ꢂ0.42
16.2ꢂ5.36 (295)
58.2ꢂ2.33 (332)
42.9ꢂ5.22 (328)
17.4ꢂ1.27 (334)
85.3ꢂ4.32 (317)
83.6ꢂ5.43 (312)
114ꢂ8.34 (322)
Compd
pH 5.0
pH 7.4
Plasma
Esterase
5
6
7
316.43
164.60
230.23
120.94
119.89
123.30
8.32
7.19
55.75
6.92
6.99
45.32
89
ndd
aMeanꢂstandard deviation.
bS, solubility pH 6.
cDihydrochloride salt.
dnd, not determined.
Table 6. Influence of pH on the hydrolysis rate for compound 7
pH
kobs (minꢀ1 a
)
pH
kobs (minꢀ1 a
)
the endo conformation which relieves the dipole–dipole
interaction between this group and the NHCOOCH3 in
the 2-position.17 The N1-substitution position in 7 was
1
2
3
4
5
0.3203ꢂ0.1305
0.2826ꢂ0.0680
0.0323ꢂ0.0057
0.0087ꢂ0.0010
0.0030ꢂ0.0011
6.
7.4
8
0.0036ꢂ0.0015
0.0056ꢂ0.0020
1.8066ꢂ0.6651
8.0010ꢂ1.1861
1
assigned by H NOESY spectroscopy.18
9
The meltingpoints, log P and solubility of 1–7 are
shown in Table 3.
aMeanꢂstandard deviation.
Compound 7 was the most soluble of all compounds (8-
fold higher solubility than 1). In relation to 5 and 6,
their higher solubility could be ascribed to a decreased
crystal lattice energy achieved by removing the NH-
proton in 1, and manifested in the pronounced melting
point decrease (>100 ꢁC).4 Compounds 2–4 did not
show significant stability in aqueous solution, for this
reason they were not used in further studies. The differ-
ence in the logP values for the derivatives 2–6 is as
expected on the basis of the p substituents values.19
The logarithms of kobs were plotted against pH. The
pH-rate profile was U-shaped indicatingthe occurrence
of apparent specific acid and base catalysis as well as
a spontaneous or water-catalyzed reaction according
to the followingrate expression: kobs=k0+kHaH+
kOHaOH, where aH and aOH refer to hydrogen ion and
hydroxide ion activity, respectively.5 The latter was cal-
culated from the measured pH at 37 ꢁC accordingto the
followingequation: log aH=pH ꢀ13.62.20 Values of the
second-order rate constants for the apparent specific
acid (kH) and specific base (kOH) catalyzed decomposi-
tion were determined from the straight line portion of
the pH–rate profile at low and high pH values, respec-
tively. The value of the apparent first-order rate con-
stant for spontaneous decomposition (k0) was obtained
from the plateau region of the pH-rate profile. The
The hydrolysis of 5, 6, and 7 at 37 ꢁC in the two buffer
solutions tested, pigliver esterase, and human plasma
displayed first-order kinetics to give 1. Order rate con-
stants observed (kobs) were calculated from slopes of
linear plots of the logarithm% formed 1 versus time.
The kobs and half-lives (t1=2=0.693/kobs) for the hydro-
lysis are listed in Tables 4 and 5.
values of the rate constants were kH=2.11 Mꢀ1 minꢀ1
,
k0=3.65ꢃ10ꢀ3 minꢀ1, kOH=37.45 Mꢀ1 minꢀ1. Com-
pound 7 is most stable at weakly acidic pH. Studies are
in progress to determine the permeability of 5–7 into
Caco-2 cells.
As appears from data, plasma and esterase markedly
increase the rate of hydrolysis in relation to buffer
solutions. This indicates that 5, 6, and 7 may be expec-
ted to be readily cleaved in vivo. It is of interest to note
that the rates of biological hydrolysis observed for 5 and
6 are considerably higher than that of 7. The position of
the ethoxycarbonyl group, in 5 and 6, has only a minor
influence in the hydrolysis in plasma, esterase, and buf-
fer solution at pH 7.4.
Acknowledgements
This study was sponsored by project CONACYT
G34851-M and DGPA-PAPIIT IN204998. We are
very thankful to: Rosa Isela del Villar, Georgina
Chavez, and Maricela Gutierrez from the School of
Chemistry, UNAM, for the determination of all
spectra.
The influence of pH on the hydrolysis rate was studied
for the compound 7 at 37 ꢁC. The kobs obtained are
shown in Table 6.

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1362
References and Notes
13C NMR (DMSO-d6) d 13.05, 13.72, 21.90, 35.19, 52.72,
64.47, 114.54, 118.88, 125.06, 129.30, 131.83, 141.05, 145.05,
149.69, 152.89 ppm; M(EI) m/z 337 (M+, 100%). Anal. calcd
C15H19N3O4S: C, 53.40; H, 5.68; N, 12.46; found: C, 53.38; H,
5.61; N, 12.41.
1. Singh, S.; Sharma, S. Med. Res. Rev. 1991, 11, 581.
2. Sharma, S. Adv. Drug Res. 1994, 25, 104.
3. Jung, H. J. Clin. Pharmacol. 1992, 32, 28.
4. Nielsen, L. S.; Slok, F.; Bundgaard, H. Int. J. Pharm. 1994,
102, 231.
11. Methyl 1-ethoxycarbonyl-6-(propylthio)-2-benzimidazole-
carbamate (6). IR (KBr) n 3314, 2960, 1764, 1736, 1536, 1374,
1
5. Nielsen, L. S.; Bundgaard, H.; Falch, E. Acta Pharm. Nord.
1992, 4, 43.
1334, 1288, 1220, 1186 cmꢀ1; H NMR (DMSO-d6) d 0.95 (t,
3H, CH3CH2CH2S–), 1.37 (t, 3H, OCH2CH3), 1.57 (m, 2H,
CH3CH2CH2S–), 2.92 (t, 2H, CH3CH2CH2S–), 3.71 (s, 3H,
OCH3), 4.45 (q, 2H, –OCH2CH3), 7.29 (dd, J=2 Hz, J=9 Hz,
1H, H-5), 7.51 (d, J=9 Hz, 1H, H-4), 7.75 (d, J=2 Hz, 1H, H-
7), 10.32 (s, 1H, exchangeable, NHCO–); 13C NMR (DMSO-
d6) d 12.98, 13.66, 21.90, 35.59, 52.69, 64.47, 114.57, 119.03,
125.89, 131.21, 131.49, 138.87, 144.43, 149.57, 153.113 ppm;
M(EI) m/z 337 (M+, 100%). Anal. calcd C15H19N3O4S: C,
53.40; H, 5.68; N, 12.46; found: C, 53.41; H, 5.63; N, 12.60.
12. Methyl 1-[(4-aminomethyl)benzoyloxy-5-(propylthio)-2-
benzimidazolecarbamate dihydrochloride (7). IR (KBr) n
3432, 2958, 2606, 1768, 1730, 1614, 1454, 1252, 1228, 1076
6. Binderup, E.; Hansen, E. T. Synth. Commun. 1984, 14, 857.
7. Methyl 1-acetyl-5-(propylthio)-2-benzimidazole-carbamate
(2). IR (KBr) n 3312, 2962, 1718, 1646, 1612, 1320, 1284, 1248,
;
1188, 1116 cmꢀ1 1H NMR (DMSO-d6) d 0.9 (t, 3H,
CH3CH2CH2S–), 1.59 (m, 2H, CH3CH2CH2S), 2.78 (s, 3H,
CH3CO–), 2.9 (t, 2H, CH3CH2CH2S–), 3.7 (s, 3H, –OCH3),
7.15 (dd, J=2 Hz, J=10 Hz, 1H, H-6), 7.4 (d, J=2 Hz, 1H,
H-4), 7.99 (d, J=10 Hz, 1H, H-7), 12.0 (s, 1H, exchangeable,
–NHCO–) ppm; M(EI) m/z 307 (M+, 20%), 265 (100%).
Anal. calcd C14H17N3O3S: C, 54.66; H, 5.53; N, 13.66; found:
C, 54.66; H, 5.53; N, 13.66.
8. Methyl 1-propyl-5-(propylthio)-2-benzimidazole-carbamate
(3). IR (KBr) n 3312, 2964, 1722, 1648, 1602, 1310, 1276, 1152,
cmꢀ1
;
1H NMR (CDCl3/MeOD)
d
1.038 (t, 3H,
CH3CH2CH2S–), 1.68 (m, 2H, CH3CH2CH2S–), 2.97 (t,
2H, CH3CH2CH2S–), 4.03 (s, 3H, –OCH3), 4.17 (s, 2H,
–CH2NH2HCl), 6.62 (s, 2H, –CH2OOC–), 7.48 (dd, J=2 Hz,
J=10 Hz, 1H, H-6), 7.62 (d, J=10 Hz, 2H, H-20, H-60), 7.67
(d, J=2 Hz, 1H, H-4), 7.77 (d, J=10 Hz, 1H, H-7), 8.09 (d,
J=10 Hz, 2H, H-30, H-60); 13C NMR (CDCl3/MeOD) d 13.38,
22.60, 36.41, 43.11, 54.91, 66.04, 112.44, 114.02, 127.11,
127.54, 128.79, 129.55, 129.75, 131.15, 136.94, 139.57, 145.17,
153.36, 165.45 ppm. Anal. calcd C21H26N4O4SCl2: C, 50.39;
H, 5.24; N, 11.20; found: C, 50.23; H, 5.16; N, 11.04.
13. Thomas, B. F.; Compton, D. R.; Martin, R. J. Pharmacol.
Exp. Ther. 1990, 255, 624.
14. Jensen, E.; Bundgaard, H.; Falch, E. Int. J. Pharm. 1990,
58, 143.
15. Hurtado, M.; Sanchez, M.; Jung, H.; Medina, M. T.;
Sotelo, J. J. Chromatogr. 1989, 494, 403.
16. Perumal, S.; Vasuk, G.; Wilson, D. A. Mag. Res. Chem.
1990, 28, 257.
17. Haddadin, M. J.; Jarrar, A. A. Tetrahedron Lett. 1971, 20,
1651.
1114 cmꢀ1
;
1H NMR (DMSO-d6)
d
1.0 (t, 3H,
CH3CH2CH2S–), 1.15 (t, 3H, CH3CH2CO–), 1.6 (m, 2H,
CH3CH2CH2S–), 2.9 (t, 2H, CH3CH2CH2S–), 3.3 (q, 2H,
CH3CH2CO–), 3.65 (s, 3H, –OCH3), 7.15 (dd, J=2 Hz, J=9
Hz, 1H, H-6), 7.41 (d, J=2 Hz, 1H, H-4), 7.95 (d, J=9 Hz,
1H, H-7), 12.1 (br, 1H, –NHCO–) ppm; M(EI) m/z 321 (M+,
13%), 265 (100%). Anal. calcd C15H19N3O3S: C, 56.05; H,
5.96; N, 13.08; found: C, 56.66; H, 5.74; N, 13.46.
9. Methyl 1-butyl-5-(propylthio)-2-benzimidazole-carbamate
(4). IR (KBr) n 3334, 2958, 1716, 1632, 1588, 1270, 1098 cmꢀ1
;
1H NMR (DMSO-d6) d 1.0 (t, 3H, CH3CH2CH2S–), 1.2
(t, 3H, CH3CH2CH2CO–), 1.65 (m, 4H, CH3CH2CH2S–,
CH3CH2CH2CO–), 2.85 (t, 2H, CH3CH2CH2S–), 3.3 (t, 3H,
CH3CH2CH2CO–), 7.15 (dd, J=2 Hz, J=9 Hz, 1H, H-6),
7.41 (d, J=2 Hz, 1H, H-4), 8.01 (d, J=9 Hz, 1H, H-7), 12.1
(br, 1H, –NHCO–) ppm; M(EI) m/z 335 (M+, 11%), 265
(100%). Anal. calcd C16H21N3O3S: C, 57.29; H, 6.32; N,
12.54; found: C, 57.19; H, 5.94; N, 12.46.
10. Methyl
1-ethoxycarbonyl-5-(propylthio)-2-benzimida-
zolecarbamate (5). IR (KBr) n 3300, 2988, 1774, 1720, 1590,
18. Seela, F.; Burgeois, W.; Rosemeyer, H.; Wenzel, T. Helv.
Chim. Acta 1996, 79, 488.
1
1544, 1372, 1328, 1288, 1218, 1164 cmꢀ1; H NMR (DMSO-
d6) d 0.95 (t, 3H, CH3CH2CH2S–), 1.37 (t, 3H, –OCH2CH3),
1.55 (m, 2H, CH3CH2CH2S–), 2.92 (t, 2H, CH3CH2CH2S–),
3.71 (s, 3H, –OCH3), 4.45 (q, 2H, –OCH2CH3), 7.26 (dd,
J=1.8 Hz, J=9 Hz, 1H, H-6), 7.53 (d, J=1.8 Hz, 1H, H-4), 7.70
(d, J=9 Hz, 1H, H-7), 10.31 (s, 1H, exchangeable, NHCO–);
19. Leo A. J. In Comprehensive Medicinal Chemistry; Hansch,
C., Sammes, P. G., Taylor, J. B., Ramsden, C. A., Eds.; Per-
gamon: Oxford, 1990; Vol. 4, pp 295–300.
20. Bundgaard, H.; Nielsen, N. M. Acta Pharm. Suec. 1987,
24, 233.