Syntheses and antibacterial activities of tizoxanide, an /V-(Nitrothiazolyl)salicylamide, and its O-aryl glucuronidef

Article abstract of DOI:10.1039/a806676k

Mild hydrolysis of the broad-spectrum anaerobic antibacterial and antiparasitic agent nitazoxanide 1 affords tizoxanide 2, which is a major metabolite of 1 retaining most of its activity; further metabolism of 2 leads to the 0-aryl glucuronide 3, efficien

Full text of DOI:10.1039/a806676k

View Article Online / Journal Homepage / Table of Contents for this issue
44 J. CHEM. RESEARCH (S), 1999
J. Chem. Research (S),
1999, 44±45$
Syntheses and Antibacterial Activities of Tizoxanide,
an N-(Nitrothiazolyl)salicylamide, and its O-Aryl
Glucuronide$
Jean-Francois Rossignola and Andrew V. Stachulskib
aThe Romark Institute for Medical Research, 6200 Courtney Campbell Causeway, Suite 870,
Tampa, Florida, USA
bSalford Ultrafine Chemicals and Research Ltd., Synergy House, Guildhall Close, Manchester
Science Park, Manchester M15 6SY, UK
Mild hydrolysis of the broad-spectrum anaerobic antibacterial and antiparasitic agent nitazoxanide 1 affords tizoxanide 2,
which is a major metabolite of 1 retaining most of its activity; further metabolism of 2 leads to the O-aryl glucuronide 3,
efficiently synthesised in four steps from benzyl salicylate and showing slight antibacterial activity.
The thiazole derivative nitazoxanide 1, ®rst described by
Rossignol,1 is a broad-spectrum antibacterial and anti-
parasitic agent, particularly ecacious against anaerobic
bacteria2 and as an anthelmintic and antiprotozoal agent.3,4
The desacetyl metabolite of 1, tizoxanide 2, is itself a potent
antibacterial and antiparasitic agent.2 Further metabolism of
2 leads to the glucuronide 3 and sulfate 4 conjugates. In this
paper, we describe the conversion of 1 to 2 and a convenient
synthesis of 3, both to test for any remaining bioactivity
and as an analytical standard.
Scheme 1 Reagents and conditions: i, Ag2O, isoquinoline,
0±20 8C; ii, cyclohexene, Pd±C, PriOH, heat;
‡
.
.
iii, EtN C N(CH2)3NH Me2Cl , 1-hydroxybenzotriazole,
4-dimethylaminopyridine, 2-amino-5-nitrothiazole, DMF;
iv, NaOH, MeOH aq., 0±20 8C then pH 6
Tizoxanide 2 was readily obtained from nitazoxanide 1 in
excellent yield by hydrolysis with aqueous HCl at 50 8C
as previously given in the patent literature.5 However, 2
could not be coupled to the bromosugar 5 using either the
Koenigs±Knorr or lithium phenolate6 methods, and acid-
catalysed reactions with the tetraacetate 67,8 or imidate 79
were also unavailing, the low organic solubility of 2 being a
major problem.
A conjugate was obtained when 2 was reacted with the
1-hydroxysugar 810 under Mitsunobu conditions.11 The 1H
NMR spectrum [in particular dH 1.8 (s) and 5.9 (d)] was
consistent with the orthoester 911 rather than the desired
glucuronide.
Acid-catalysed condensation of benzyl salicylate 10 with 6
or 7 also proved quite ine€ective: by contrast a 2,6-
dimethylphenol has been successfully glucuronidated12 using
the classical Helferich procedure (6 ‡ tosic acid). Coupling
of the lithium phenolate of 10 with 5 in methanol gave a
low yield (24%) of a more polar product which proved
to be the partially deprotected ester 11. Rather than try to
progress 11, whose unprotected OH groups were likely to
cause problems, a literature procedure for the glucuronida-
tion of methyl salicylate13 was very satisfactorily adapted
(Scheme 1).
Koenigs±Knorr reaction of 5 with 10 gave the conjugate
12 in 61% yield after chromatography. The chemical shift
and coupling constant (J ˆ ca. 8) of the anomeric proton
in 12 were consistent with a b-glucuronide. Debenzylation
of 12 using catalytic transfer hydrogenation gave acid 13
in 80% yield: the condensation of 13 with 2-amino-
5-nitrothiazole was performed using the water-soluble
carbodiimide method shown.14 Chromatography a€orded
product 14 in excellent purity and 67% yield. The esters
were cleaved using aq. NaOH, and after acidi®cation to
pH 6 the sodium salt of glucuronide 3 precipitated in
80% yield. By high-performance liquid chromatographic
analysis this material appeared identical with the authentic
metabolite.
Biological Data
The antibacterial activities of compounds 1, 2 and 3 were
compared. Minimum inhibitory concentrations (MICs) in
the range 1±10 mg cm were observed for all three com-
pounds against Helicobacter pylori, 3 being about tenfold
$This is a Short Paper as de®ned in the Instructions for Authors,
Section 5.0 [see J. Chem. Research (S), 1999, Issue 1]; there is there-
fore no corresponding material in J. Chem. Research (M).
3

View Article Online
J. CHEM. RESEARCH (S), 1999 45
‡
t, ArH) and 8.11 (1 H, d, ArH); m/z (CI, NH3) 472 (MNH4
100%).
,
less e€ective than 1 or 2. Against Sarcocystis neurona, 1 and
3
2 showed MICs of 2 mg cm while the MIC of 3 was
Methyl 1-[2-N-(5-Nitrothiazol-2-yl)carboxamido]phenyl-2,3,4-tri-
O-acetyl-ꢂ-D-glucopyranuronate 14.Ð1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (0.81 g, 4.25 mmol) was added to a
stirred suspension of acid 13 (1.75 g, 3.85 mmol), 4-N,N-dimethyl-
aminopyridine (0.5 g, 4.10 mmol), 1-hydroxybenzotriazole mono-
hydrate (0.65 g, 4.25 mmol) and 2-amino-5-nitrothiazole (0.615 g,
4.24 mmol) in DMF (25 cm3) at 0 8C. After 2 h at 20 8C, then 16 h
at 0 8C the solution was concentrated to near dryness, then
extracted with CH2Cl2 (2Â 25 cm3) and worked up for a neutral
40 mg cm 3. Against strains of the aerobic Gram-positive
and Gram-negative bacteria Staphylococcus aureus, Entero-
coccus faecalis, Morganella morganii, Escherichia coli and
Pseudomonas aeruginosa all three compounds were inactive
3
at up to 512 mg cm
Further biological results, with a discussion of the mode
.
of action of these compounds, will be published separately.
product. Evaporation gave
a brown solid (2.63 g) which was
chromatographed on silica. Appropriate fractions were pooled and
evaporated to a sticky solid which on trituration with diethyl ether
deposited the product 14 as a ¯aky yellow solid (1.51 g, 67%), mp
262±264 8C (Ko¯er block, from CH2Cl2±methanol±diethyl ether)
(Found: C, 47.5; H, 4.15; N, 7.15. C23H23N3O13S requires C, 47.5;
Experimental
For general directions, see an earlier paper from these
laboratories.15 Mass spectra were recorded on a Varian-Saturn
GC-ITD instrument in the electron-impact (EI) mode for compound
2, on a Kratos MS 25 instrument for chemical ionisation (CI)
spectra and on a Kratos Concept 1S instrument for the fast atom
bombardment (FAB) mode. Antibacterial screening was performed
using either an agar dilution technique in a Wilkens Chalgren
medium containing 10% blood at an inoculum of 109 colony form-
ing units (CFU) cm 3, for the anaerobic bacteria, or in a Mueller
1
H, 4.0; N, 7.2%); ꢀmax (Nujol)/cm 3350 (sharp), 1750, 1665, 1625
(w), 1605 (m), 1530 and 1350; ꢁ [(CD3)2SO] 1.94, 1.98, 2.05 (9 H,
3 s, 3x CH3CO), 3.68 (3 H, s, CH3O), 4.80 (1 H, d, 5-H), 5.05
(2 H, t) and 5.48 (3 H, m, 2-H ‡ 3-H ‡ 4-H), 5.67 (1 H, d, 1-H),
7.20±7.30 (2 H, m, ArH), 7.55±7.70 (2 H, m, ArH) 8.71 (1 H, s,
‡
40-H) and 13.39 (1 H, br s, NH); m/z (CI, NH3) 582 (MH , 100%).
Hinton agar medium at an inoculum of 106 CFU cm in Mueller
Hinton broth for the aerobic bacteria.
3
1-[2-N-(5-Nitrothiazol-2-yl)carboxamido]phenyl-ꢂ-D-glucopyranosid-
3
uronic Acid 3.ÐA 2.5 mol dm NaOH solution (5 cm3) was added
2-Hydroxy-N-(5-nitrothiazol-2-yl)benzamide (Tizoxanide) 2.ÐA
suspension of 2-acetoxy-N-(5-nitrothiazol-2-yl)benzamide (nitazox-
anide, 1, 100 g, 0.326 mol) in 37% w/v HCl (500 cm3) was stirred
and heated at 50 8C for 24 h.5 The resulting slurry was cooled and
®ltered, then the ®ltrate was well washed with deionized water
until the washings were neutral and dried at 50 8C to give tizoxanide
in one portion to a stirred suspension of the ester 14 (1.45 g,
2.50 mmol) in methanol (17.5 cm3) at 0 8C. On warming to 20 8C
over 1 h, a yellow solution resulted which was acidi®ed to pH 6.9
with acetic acid, followed by evaporation to dryness. The residue
was triturated with aq. ethanol, 1:4 (20 cm3) then the yellow solid
was ®ltered to give the sodium salt of the product 3 (1.03 g, 89%),
mp >200 8C (decomp.) from aq. ethanol (Found: m/z, 464.0367.
1
2 (85 g, 98%), mp 254 8C; ꢀmax/cm (Nujol) 1670; ꢁ [220 MHz,
‡
1
(CD3)2SO] 7.00±7.15 (2 H, m, ArH), 7.60 (1 H, t, ArH), 8.00 (1 H,
d, 6-H) and 8.75 (1 H, s, 4'-H); m/z (Me3Si derivative, EI) 338
[MSi(CH3)3 ], 193 (100%, cleavage of thiazole fragment).
C16H15N3O10SNa requires MH , 464.0376); ꢀmax (Nujol)/cm
3700±2500 (br), 3540, 3260, 3100 (w), 1645 (sh), 1620, 1600, 1535
and 1350; ꢁ(D2O) 3.53 (2 H, m) and 3.67 (1 H, t, 2-H ‡ 3-H ‡
4-H), 3.83 (1 H, d, 5-H), 5.14 (1 H, d, J 8, 1-H), 7.16 (1 H, t, ArH),
7.29 (1 H, d, ArH), 7.54 (1 H, dt, ArH), 7.74 (1 H, dd, ArH)
‡
Methyl 1-[2-(Benzyloxycarbonyl)phenyl]-ꢂ-D-glucopyranuronate 11.
ÐThe bromosugar 5 (0.60 g, 1.5 mmol) was added in one portion
‡
to
a solution of benzyl salicylate 10 (0.34 g, 1.5 mmol) and
and 8.38 (1 H, s, 40-H); m/z (FAB ‡ve ion, glycerol) 442 (MH ,
LiOHÁH2O (0.063 g, 1.5 mmol) in methanol (1.5 cm3) which was
stirred at 0 8C. After 1 h, the temperature having risen to 20 8C, the
solution was diluted with water containing a few drops of acetic
acid, then extracted with CH2Cl2 (3Â5 cm3). Evaporation gave
crude product (0.60 g) which was chromatographed to a€ord the
product 11 as a solid (0.15 g, 24%) which on trituration with
diethyl ether and recrystallisation (methanol±diethyl ether) had mp
164±166 8C (Found: C, 60.1; H, 5.3. C21H22O9 requires C, 60.3; H,
5.3%); ꢁ [(CD3)2SO], inter alia, 3.68 (3 H, s, CH3O), 4.12 (1 H, d,
5-H), 5.23 (1 H, d, 1-H), 5.34 (2 H, m, ArCH2O) and 7.10±7.70
‡
‡
free acid), 464 (MH ) and 486 (MNa ). High-performance liquid
chromatographic analysis of the product (C18 ꢃ-Bondapak reverse-
phase column, aq. acetonitrile eluent) showed an area purity of
99.25%.
Received, 25th August 1998; Accepted, 25th September 1998
Paper E/8/06676K
References
‡
(9 H, m, ArH); m/z (CI, NH3) 436 (MNH4 , 12%).
Methyl
1 J.-F. Rossignol and H. Maisonneuve, Am. J. Trop. Med. Hyg.,
1984, 33, 511.
1-[2-(Benzyloxycarbonyl)phenyl]-2,3,4-tri-O-acetyl-ꢂ-D-
glucopyranuronate 12.ÐSilver(I) oxide (2.12 g, 0.91 mmol) was
added in portions to a stirred mixture of bromosugar 5 (3.30 g,
8.31 mmol) and benzyl salicylate 10 (3.78 g, 16.6 mmol) in isoquino-
line (4.6 g) at 0 8C, giving a thick slurry. On warming to 20 8C over
1 h no remaining 5 was seen (TLC in 1:1 EtOAc±hexane), so the
mixture was diluted with diethyl ether and ®ltered through Celite,
then the ®ltrate was worked up for a neutral product, followed by
evaporation to an orange oil which was washed with hexane (2Â),
decanting the mother liquors, to remove the bulk of the unreacted
2 L. Dubreuil, I. Houcke, Y. Mouton and J.-F. Rossignol,
Antimicrob. Agents Chemother., 1996, 40, 2266.
3 R. Romerio Cabello, L. Roberto Guerroro, M. R. Munoz
Garcia and A. Geyne Cruz, Trans. R. Soc. Trop. Med. Hyg.,
1997, 91, 701.
4 O. Doumbo, J.-F. Rossignol, E. Pichard, H. Traore,
M. Dembele, M. Diakite, F. Traore and D. A. Diallo, Am. J.
Trop. Med. Hyg., 1997, 56, 637.
5 J.-F. Rossignol and R. Cavier, Chem. Abstr., 1975, 83, 28216.
6 B. Berrang, C. E. Twine, G. L. Hennessee and F. I. Carroll,
Synth. Commun., 1975, 5, 231.
7 G. N. Bellenback, J. W. Long, D. G. Benjamin and J. A.
Lindquist, J. Am. Chem. Soc., 1955, 77, 3310.
10. Chromatography a€orded the product 12 as a foam (2.75 g,
61%) (Found: m/z, 562.1933. C27H32NO12 requires MNH4
‡
,
1
562.1924); ꢀmax (CHCl3)/cm 1750 (vs), 1610, 1590 (sh) and 1490;
ꢁ (CDCl3) 2.09 (9 H, s, 3Â CH3CO), 3.77 (3 H, s, CH3O), 4.21 (1 H,
d, 5-H), 5.20 (1 H, m, 1-H), 5.30±5.40 (3 H, m, 2-H ‡ 3-H ‡ 4-H),
8 K. Honma, K. Nakazima, T. Uematsu and A. Hamada, Chem.
Pharm. Bull., 1976, 24, 394.
9 B. Fischer, A. Nudelman, M. Ruse, J. Herzig, H. E. Gottlieb
and E. Keinan, J. Org. Chem., 1984, 49, 4988.
5.37 (2 H, s, PhCH2O), 7.10±7.25 (2 H, m, ArH) 7.35±7.55 (6 H,
‡
m, ArH) and 7.81 (1 H, dd, ArH); m/z (CI, NH3) 562 (MNH4
65%).
,
Methyl
1-(2-Carboxyphenyl)-2,3,4,-tri-O-acetyl-ꢂ-D-glucopyran-
10 A. Nudelman, J. Herzig, H. E. Gottlieb, E. Keinan and
J. Sterling, Carbohydr. Res., 1987, 162, 145; N. Pravdic and
D. Keglevic, J. Chem. Soc., 1964, 4633.
11 G. T. Badman, D. V. S. Green and M. Voyle, J. Organomet.
Chem., 1990, 388, 117.
12 T. Yoshioka, Y. Aizawa, T. Fujita, K. Nakamura, K. Sasahara,
H. Kuwano, T. Kinoshita and H. Horikoshi, Chem. Pharm.
Bull., 1991, 39, 2124.
uronate 13.ÐA solution of ester 12 (2.71 g, 4.98 mmol) in propan-2-
ol (75 cm3) and cyclohexene (5 cm3) was stirred and heated at gentle
re¯ux for 0.5 h with Pd-C (0.3 g). The catalyst was ®ltered o€, then
the ®ltrate was evaporated to a foam which was dissolved in 4%.
aq. NaHCO3 (25 cm3) and washed with diethyl ether (2Â). Cautious
acidi®cation of the aq. phase then extraction with Et2O gave on
evaporation the acid 13 as a colourless foam (1.84 g, 80%) (Found:
C, 52.4; H, 4.9; m/z, 472.1465.
H, 4.8%; MNH4 , 472.1455); ꢀmax (Nujol)/cm
1760 (br, s), 1610 (m) and 1495; ꢁ (220 MHz, CDCl3) 2.00±2.10
(9 H, 3 s, 3 ÂCH3CO), 3.73 (3 H, s, CH3O), 4.34 (1 H, d, 5-H),
5.35±5.45 (4 H, m, 1-H to 4-H), 7.28 (2 H, m ArH), 7.62 (1 H,
C
20H22O12 requires C, 52.85;
3700±2500(br),
13 C. D. Lunsford and R. S. Murphey, J. Org. Chem., 1956, 21, 580.
14 J. C. Sheehan, P. A. Cruickshank and G. L. Boshart, J. Org.
Chem., 1961, 26, 2525.
15 A. V. Stachulski, D. E. Nichols and F. Scheinmann, J. Chem.
Res. (S), 1996, 30.
‡
1