N-glycosyl-N′-[p-(isoamyloxy)phenyl]-thiourea derivatives: Potential anti-TB therapeutic agents

Article abstract of DOI:10.1080/00397910701865777

Thiocarlide (THC; N,N′-bis[p-(isoamyloxy)phenyl]-thiourea; also known as Isoxyl) has been used in the past as an anti-tuberculosis agent. In an effort to improve the therapeutic value of THC, several N-glycosyl-N′-[p- (isoamyloxy)phenyl]-thiourea derivati

Full text of DOI:10.1080/00397910701865777

This article was downloaded by: [University of Virginia, Charlottesville]
On: 24 September 2012, At: 16:41
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office:
Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Synthetic Communications: An International
Journal for Rapid Communication of
Synthetic Organic Chemistry
Publication details, including instructions for authors and subscription
information:
http://www.tandfonline.com/loi/lsyc20
N‐Glycosyl‐N′‐[p‐(isoamyloxy)phenyl]‐thiourea
Derivatives: Potential Anti‐TB Therapeutic
Agents
Avraham Liav a , Shiva K. Angala a & Patrick J. Brennan a
a Department of Microbiology, Immunology, and Pathology, Colorado State
University, Fort Collins, Colorado, USA
Version of record first published: 17 Apr 2008.
To cite this article: Avraham Liav, Shiva K. Angala & Patrick J. Brennan (2008):
N‐Glycosyl‐N′‐[p‐(isoamyloxy)phenyl]‐thiourea Derivatives: Potential Anti‐TB Therapeutic Agents, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry, 38:8,
1176-1183
To link to this article: http://dx.doi.org/10.1080/00397910701865777
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial
or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or
distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the
contents will be complete or accurate or up to date. The accuracy of any instructions, formulae,
and drug doses should be independently verified with primary sources. The publisher shall not
be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or
howsoever caused arising directly or indirectly in connection with or arising out of the use of this
material.

Synthetic Communicationsw, 38: 1176–1183, 2008
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910701865777
N-Glycosyl-N0-[p-(isoamyloxy)phenyl]-
thiourea Derivatives: Potential Anti-TB
Therapeutic Agents
Avraham Liav, Shiva K. Angala, and Patrick J. Brennan
Department of Microbiology, Immunology, and Pathology, Colorado
State University, Fort Collins, Colorado, USA
Abstract: Thiocarlide (THC; N,N0-bis[p-(isoamyloxy)phenyl]-thiourea; also known as
Isoxylw) has been used in the past as an anti-tuberculosis agent. In an effort to improve
the therapeutic value of THC, several N-glycosyl-N0-[p-(isoamyloxy)phenyl]-thiourea
derivatives were synthesized by coupling an aniline derivative and glycosyl isothiocya-
nates. The minimum inhibitory concentration (MIC) values of the new products against
M. tuberculosis were determined.
Keywords: Isoxyl, Mycobacterium tuberculosis, thiocarlide, thiourea
INTRODUCTION
The human pathogen Mycobacterium tuberculosis (M.tb) causes tuberculosis
and is responsible for the most deaths of people by any single infectious
agent.[1] Nearly 33% of the world’s population is infected with M.tb, and
10% are predicted to develop the disease during their lifetimes.[2] One of
the therapeutic agents used in the clinical treatment of tuberculosis in the
1960s was a derivative of thiourea, known as thiocarlide (THC; N,N0-bis[p-
(isoamyloxy)phenyl]-thiourea, 1, Fig. 1, also known as Isoxylw). This
powerful compound was first chemically synthesized in 1951.[3] The
minimum inhibitory concentration (MIC) of THC against most clinical
isolates of M.tb, including multidrug-resistant ones, was determined to be
Received in the USA October 30, 2007
Address correspondence to Avraham Liav, Department of Microbiology, Immu-
nology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
E-mail: avraham.liav@colostate.edu
1176

Potential Anti-TB Therapeutic Agents
1177
Figure 1. Structure of THC.
2 mg/ml.[4] In spite of this promising result, the clinical use of THC was dis-
continued, apparently because of the poor bioavailability of the highly
nonpolar product.[5]
In an effort to synthesize THC analogs with better hydrophilic character,
we have examined a novel approach, consisting of the substitution of one of
the isoamyloxyphenyl moieties in 1 by a carbohydrate. The synthesis of
di-N,N0-glycosyl derivatives of thiourea (both symmetrical and nonsymmetrical)
has already been described.[6–8] Syntheses of mono-N-glycosyl-N0-alkyl and
even N-glycosyl-N0-aryl-thiourea derivatives have also been reported.[7,9–11]
However, the synthesis of N-glycosyl-N0-aryl thiourea derivatives in
which the aryl moiety is p-isoamyloxyphenyl (as in 1) has not been described
yet. In this article, we report the synthesis of this new type of thiourea derivatives
(Fig. 2).
The products described in this article were obtained by the coupling of a
suitably protected glycosyl isothiocyanate with p-isoamyloxyphenyl aniline
(4) following the general schemes described in the literature,[9,10,12] but in
several cases more efficient reagents were utilized.
The synthesis of the key intermediate 4 was accomplished by alkylation of
p-nitrophenol (2) with p-isoamyloxy bromide in the presence of a crown ether,
potassium carbonate in acetone to give 3,[13] and subsequent reduction of 3.
The reduction of the nitro function could be achieved by using tin chloride in
ethanol,[14] but a product with a higher degree of purity (95%, as judged by
1H NMR and thin-layer chromatography, TLC) was obtained when Pd/C 10%
catalyst and ammonium formate in ethanol were employed (Scheme 1).[15]
The yields in this two-step synthesis were high (87% and 85% respectively).
Figure 2. Structures of the N-glycosyl-N0-(isoamyloxyphenyl)-thiourea derivatives.

1178
A. Liav, S. K. Angala, and P. J. Brennan
Scheme 1. Synthesis of p-isoamyloxy-aniline: (a) isoamyloxy bromide, anhydrous
potassium carbonate, 18-crown-6 ether, boiling acetone, 15 h; (b) Pd/C 10%,
ammonium formate, ethyl alcohol, 3 h, rt.
This approach has been utilized in the past,[16] but the reduction step was signifi-
cantly inconvenient (a mixture of sodium sulfide and sulfur in refluxing methanol
for 15 h). Alternatively, product 4 has been obtained in the past by treatment of
p-isoamyloxy acetamidophenol with 50% aqueous sodium hydroxide solution
1
under reflux.[17] The structure of 4 was confirmed by H NMR: a new signal,
corresponding to the amino group protons appeared (d 3.459), and the signals
of the aromatic protons shifted to a higher field, as expected (from d 8.161
and 6.938 to d 6.810 and 6.678).
Conversion of 2-acetamido-2-deoxy-b-D-glucopyranose tetraacetate (5)
into the a-bromide 6 and subsequent treatment with potassium thiocyanate
(Scheme 2) gave the corresponding isothiocyanate (7), which was found to
have the b-configuration. In support of the structure of 7, there was a significant
shift of the H-1 signal in its 1H NMR spectrum: from d 6.291 (in 6) to 5.305.
Finally, coupling of the isothiocyanate 7 with the amine 4 in pyridine[10] and
subsequent O-deacetylation yielded the N-glucosyl thiourea derivative 8.
The analytical data for 8 are in agreement with the structure and with the
b-D-configuration. In a similar manner, L-rhamnose was converted into the
a-L-N-rhamnosyl thiourea derivative 10 (via the corresponding isothiocyanate
9, not shown). The synthesis of the analogous D-arabino product (12)
proceeded by the same scheme, using 2,3,5-tri-O-tert butyldimethylsilyl
(TBDMS)-a-D-arabinofuranosyl bromide[18] as the starting material. The use
of TBDMS groups for protection in the case of the arabino product is
necessary to ensure that the product will exist in the furanose configuration.
Scheme 2. Synthesis of the N-acetylglucoseamine-isothiocyanate derivative: (a) 30%
HBr/AcOH, DCM, 2 h, rt; (b1) KSCN, tetrabutylammonium hydrogen sulfate, 4A
molecular sieves, acetonitrile, 2 h, rt; (b2) mixture of the thiocyanate and 6, 2 h, 65 8C.

Potential Anti-TB Therapeutic Agents
1179
Removal of the TBDMS groups was achieved by treatment with ammonium
fluoride as described before.[18] In this case, the final product was isolated as
a mixture of the a and b anomers (as indicated by 1H NMR).
The final products, 8, 10, and 12, were fully characterized by 1H NMR, mass
spectroscopy, elemental analysis, and optical rotation. The 1H NMR data for the
gluco (7) and the rhamno (9) intermediates appear in the Experimental section.
Attempts to obtain an analytical sample of the arabino-isothiocyanate intermedi-
ate (11, not shown) failed because of the instability of the product.
The new products were tested as growth inhibitors against M.tb H37Rv,
using the microplate alamar blue dye assay.[19] Encouragingly, one product
(12) was found to have an MIC value of 2.5 mg/mL, which is almost as
good as that of the parent THC product (Table 1). Further testing of this
product will be conducted.
EXPERIMENTAL
p-Isoamyloxy Aniline (4)
A mixture of p-nitrophenol (270 mg, 1.94 mmol), isoamyl bromide (0.6 mL,
4.76 mmol), anhydrous potassium carbonate (290 mg, 2.10 mmol), and
18-crown-6 ether (74 mg) in acetone (6 mL) was stirred at 64 8C under
nitrogen for 16 h. The mixture was cooled and diluted with acetone, and the
insoluble solid material was filtered off and washed with acetone. The filtrate
was dried, and the residue was chromatographed on silica gel (Sigma, 70–
230 mesh). Elution with ethyl acetate–petroleum ether 1:1 gave the chromato-
graphically pure product (3; 355 mg, 87% yield). The homogeneity of the
1
product was established by H NMR as well. Part of 3 (200 mg, 0.95 mmol)
was treated with Pd/C 10% catalyst (120 mg) and ammonium formate
(500 mg, 7.93 mmole) in ethanol (5 mL) at rt for 3 h. The mixture was filtered
through Celite,w and the filtrate was dried to give the product 4, which was
found by 1H NMR and TLC to be 95% pure (145 mg, 85% yield).
Table 1. MIC values of products 8, 10,
and 12 against M. tuberculosis H37Rv
Product
MIC values (mg/mL)
8
10
12
50–100
8
2.5
Note: MIC values were determined by
the microplate alamar blue dye assay.
INH (isoniazid) was used as a standard
at a concentration of 0.35 mg/mL.

1180
A. Liav, S. K. Angala, and P. J. Brennan
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-glucopyranosyl
isothiocyanate (7)
A solution of hydrogen bromide in acetic acid (30%; 0.4 mL, 1.48 mmol) was
added to a cold (ice bath) suspension of 2-acetamido-2-deoxy-b-D-glucopyra-
nose-1, 3,4,6-tetraacetate (220 mg, 0.56 mmol) in methylene chloride
(2.5 mL). The mixture was then stirred at room temperature for 2 h. The
excess of the hydrogen bromide was removed by evaporation under nitrogen,
and the residue was diluted with ethyl acetate. The organic solution was
washed with a saturated sodium bicarbonate solution and dried to give the cor-
responding bromide. In a separate flask, potassium thiocyanate (133 mg,
1 mmol) was treated with tetrabutylammonium hydrogen sulfate (160 mg,
0.47 mmol) and molecular sieves (4A, 750 mg) in acetonitrile (10 mL) at
room temperature for 3 h. This mixture was then added to the crude bromide,
and the new mixture was stirred at 65 8C for 1.5 h. The mixture was cooled,
and the solid material was filtered off and washed with acetone. The filtrate
was dried, and the residue was chromatographed on silica gel. Elution with
ethyl acetate gave the chromatographically homogeneous product (116 mg;
52%). The homogeneity of the product was established by 1H NMR as well.
N-(2-Acetamido-2-deoxy-b-D-glucopyranosyl]-N0-
(p-isoamyloxyphenyl)thiourea (9)
p-Isoamyloxyaniline (4, 62 mg, 0.34 mmole) was dissolved in pyridine
(1 mL), and the solution was added to the isothiocyanate intermediate 7.
The mixture was stirred at room temperature for 2 h and dried. The residue
was chromatographed on silica gel, and the chromatographically homogenous
product was eluted with methylene chloride–methanol 9:1. Yield 159 mg
(94%). Part of the product (28 mg) was dissolved in methanol (1 mL), and
M sodium methoxide solution in methanol (0.2 mL) was added. The
mixture was stirred at room temperature for 2 h and neutralized with
Dowex 50 (Hþ). The resin was filtered off, and the filtrate was dried. The
residue was chromatographed on silica gel. Elution with methylene
chloride–methanol 5:1 gave the product (16 mg, 76%). The homogeneity of
1
the product was established by H NMR, mass spectrometry, and elemental
analysis (CHN). The product was also characterized by optical rotation.
ANALYTICAL DATA
Compound 3
1H NMR (300 MHz, CDCl3): d 8.161 (d, J ¼ 9.0 Hz, 2H, aromatic), 6.938
(d, J ¼ 9.0 Hz, 2H, aromatic), 4.084 (t, J ¼ 6.6 Hz, 2H, CH2), 1.920–1.784

Potential Anti-TB Therapeutic Agents
1181
(m, 1H, CH), 1.611 (dd, J ¼ 6.9, 13.5 Hz, 2H, CH2), 0.995, 0.973 (2s, 6H,
2CH3).
Compound 4
1H NMR (300 MHz, CDCl3): d 6.810 (d, J ¼ 9.0 Hz, 2H, aromatic), 6.678
(d, J ¼ 9.0 Hz, 2H, aromatic), 3.976 (t, J ¼ 6.6 Hz, 2H, CH2), 3.459 (s, 2H,
NH2), 1.980–1.740 (m, 1H, CH), 1.715 (dd, J ¼ 6.6, 13.2 Hz, 2H, CH2),
1.041, 1.019 (2s, 6H, 2CH3).
Compound 7
1H NMR (300 MHz, CDCl3): d 6.063 (d, J ¼ 8.7 Hz, 1H, NH), 5.366 (dd,
J ¼ 9.3, 10.5 Hz, 1H, H-3), 5.305 (d, J ¼ 9.3 Hz, H-1), 5.124 (t, J ¼ 9.6 Hz,
1H, H-4), 4.276 (dd, J ¼ 4.8, 12.6 Hz, 1H, H-6a), 4.179 (dd, J ¼ 2.4,
12.6 Hz, 1H, H-6b), 4.091–4.056 (m, 1H, H-2), 3.850–3.808 (m, 1H, H-5)
2.108, 2.079, 2.076, (3s, 9H), 1.975 (s, 3H), 10950–1.801 (m, 1H), 1.705 (dd,
J ¼ 6.6, 13.2 Hz, 2H), 1.015, 0.994 (2s, 6H).
Compound 8
1H NMR (300 MHz, methanol-d): d 7.198 (d, J ¼ 9.0 Hz, 2H), 6.97
(d, J ¼ 9.0 Hz, 2H), 5.558 (bd, J ¼ 8.1 Hz, 1H), 4.065 (t, J ¼ 6.6 Hz, 2H),
3.876 (m, 2H), 3.727 (dd, J ¼ 4.5, 11.7 Hz, 1H), 3.536 (dd, J ¼ 8.4, 9.9 Hz,
1H), 3.368 (m, 2H, partially obscured by the solvent signal), 2.03 (s, 3H),
1.898 (m, 1H), 1.719 (dd, J ¼ 6.6, 13.2 Hz, 2H), 1.029, 1.023 (2s, 6H);
mass spectrometry (electrospray positive ion): 442.4 (M þ 1); optical
rotation: [a]D þ 22.58 (c 1.1, methanol). Calc. for C20H31N3O6S: C, 54.40;
H, 7.07; N, 9.51. Found: C, 54.64; H, 7.31; N, 9.66.
Compound 9
1H NMR (300 MHz, CDCl3): d 5.376 (d, J ¼ 1.5, 1H, H-1), 5.164–5.102
(m, 2H), 4.925 (t, J ¼ 9.6 Hz, 1H, H-4), 3.922–3.823 (m, 1H, H-5), 2.036.
1.949, 1.873 (3s, 9H, COCH3), 1.133 (d, J ¼ 6.3 Hz, C-5-CH3).
Compound 10
1H NMR (300 MHz, methanol d): d7.329 (d, J ¼ 9.0 Hz, 2H), 6.946
(d, J ¼ 9.0 Hz, 2H), 5.880 (bs, 1H), 4.044 (t, J ¼ 6.6 Hz, 2H), 3.971 (dd,
J ¼ 2.4, 3.0 Hz, 1H), 3.623 (dd, J ¼ 3.0, 8.4 Hz, 1H), 3.585–3.465

1182
A. Liav, S. K. Angala, and P. J. Brennan
(m, 2H), 1.95–1.82 (m, 1H), 1.712 (dd, J ¼ 6.6, 13.2 Hz, 2H), 1.359
(d, J ¼ 6.0 Hz, 3H), 1.035, 1.014 (2s, 6H). Mass spectrometry (electrospray
positive ion): 385.1 (M þ 1); optical rotation: [a]D 2528 (c 1.3, methanol).
Calc. for C18H28N2O5S: C, 56.23; H, 7.34; N, 7.28. Found: C, 56.00; H,
7.20; N, 7.38.
Compound 12
1H NMR (400 MHz, methanol d): d 7.1949 (d, J ¼ 8.8 Hz, 2H), 6.904
(d, J ¼ 8.8 Hz, 2H), 4.05–3.85 (m, 3H), 3.78–3.45 (m, 3H), 3.28–3.27
(m, obscured by the solvent signal, 2H), 1.88–1.78 (m, 1H), 1.68–1.60
(m, 2H), 0.94 (d, J ¼ 6.8 Hz, 6H). Mass spectrometry (FAB): 371.2
(M þ 1); calc. for C17H26N2O5S: C, 55.12; H, 7.07; N, 7.56. Found: C,
54.85; H, 7.00; N, 7.38.
ACKNOWLEDGMENT
The work was supported by Grant RO1 AI063054 from the National Institute
of Allergy and Infectious Diseases, National Instiute of Health (NIH).
REFERENCES
1. (a) Rouhi, A. M. Tuberculosis: A tough adversary. Chem. Eng. News 1999, 77,
52–69; (b) Dolin, J. P.; Raviglione, M. D.; Kochi, A. Global tubeculosis
incident and mortality during 1990–2000. Bull. World Health Org. 1994, 72,
213–220.
2. Nunn, P.; Kochi, A. A deadly duo—TB and AIDS. World Health 1993, 46, 7–8.
3. Anonymous Discussion on isoxyl. Antibiot. Chemother. 1970, 16, 187–202.
4. Phetsuksiri, B.; Baulard, A. R.; Cooper, A.; Minnikin, D. E.; Douglas, J. D.;
Besra, G. S.; Brennan, P. J. Antimycobacterial activities of isoxyl and new deriva-
tives through the inhibition of mycolic acid synthesis. Antimicrob. Agents
Chemother. 1999, 43, 1042–1051.
5. Lambelin, G. Pharmacology and toxicology of Isoxyl. Antibiot. Chemother. 1970,
16, 84–95.
6. Camarasa, M. J.; Fernandez-Resa, P.; Garcia-Lopez, M. T.; De Las Heras, F. G.;
Mendez-Castrillon, P. P.; San Felix, A. A new procedure for the synthesis of
glycosyl isothiocynates. Synthesis 1984, 509–510.
7. Lopez, O.; Maya, I.; Fuentes, J.; Fernandez-Bolanos, J. G. Simple and efficient
synthesis of O-unprotected glycosyl thiourea and isourea derivatives from glyco-
sylamines. Tetrahedron 2004, 60, 61–72.
8. Babiano Caballero, R.; Fuentes Mota, J.; Galbis Perez, J. A. A new method for the
preparation of acylated glycosylamines and their transformations into glycosyl iso-
thiocyanates and N,N0-diglycosyl-thioureas. Carbohydr. Res. 1986, 154, 280–288.

Potential Anti-TB Therapeutic Agents
1183
9. Takahashi, H.; Nimura, N.; Ogura, H. A one-step synthesis of glycosylaminoi-
sothiaazolo [3,4] pyrimidines and glycosylaminoisothiazoles. Chem. Pharm.
Bull. 1979, 27, 1147–1152.
10. Saitz-Barria, C.; Torres-Pinedo, A.; Santoyo-Gonzales, F. Synthesis of bridged
thiourea calilx-sugar. Synlett 1999, 12, 1891–1894.
11. Gama, Y.; Shibuya, I.; Shimizu, M. Novel efficient synthesis of 4-dimethylamino-
2-glycosylaminoquinazolines by cyclodesulfurization of glycosylthioureas with
dimethylcyanamide. Chem. Pharm. Bull. 2002, 50, 1517–1519.
12. Prata, C.; Mora, N.; Lacombe, J.-M.; Maurizis, J.-C.; Pucci, B. Synthesis and
surface-active properties of glycosyl carbamates and thioureas. Carbohydr. Res.
1999, 321, 4–14.
13. Sivanandan, K.; Sandanaraj, B. S.; Thayumanavan, S. Sequences in dendrons and
dendrimers. J. Org. Chem. 2004, 69, 2937–2944.
14. Bellamy, F. D.; Ou, K. Selective reduction of aromatic nitro compounds with
stannous chloride in non acidic and non aqueous medium. Tetrahedron Lett.
1984, 25, 839–842.
15. Alaouui, A. E.; Schmidt, F.; Monneret, C.; Florent, J.-C. Protecting groups for glu-
curonic acid: Application to the synthesis of new paclitaxel derivatives. J. Org.
Chem. 2006, 71, 9628–9636.
16. Gertzke, J.; Wellner, E. Preparation of symmetrically disubstituted thioureas.
German (East) patent 19650712, 1966.
17. Vourinen, R.; Lampi, K. 4,4-Bis(isoamyloxy)thiocarbanilide. Finnish patent
36860 19680215, 1968.
18. Liav, A.; Brennan, P. J. Stereoselective synthesis of farnesylphosphoryl b-D-ara-
binofuranose. Tetrahedron Lett. 2005, 46, 2937–2939.
19. Collins, L. A.; Franzblau, S. G. Microplate alamar blue assay versus BACTAC 460
for high-throughput screening of compounds against Mycobacterium tuberculosis
and Mycobacterium avium. Antimicrob. Agents Chemother. 1997, 41, 1004–1009.