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

Article abstract of DOI:10.1016/j.bmcl.2008.03.033

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

Full text of DOI:10.1016/j.bmcl.2008.03.033

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2649–2651
N-D-Aldopentofuranosyl-N0-[p-(isoamyloxy)phenyl]-thiourea
derivatives: Potential anti-TB therapeutic agents
Avraham Liav,* Shiva K. Angala, Patrick J. Brennan and Mary Jackson
Department of Microbiology, Immunology and Pathology, Colorado State University, 1682 Campus Delivery,
Fort Collins, CO 80523, USA
Received 7 January 2008; revised 6 March 2008; accepted 7 March 2008
Available online 14 March 2008
Abstract—Thiocarlide (THC; N,N0-bis[p-(isoamyloxy)phenyl]-thiourea; also known as isoxyl) has been used in the past as anti-
tuberculosis agent. In an effort to improve the therapeutic value of THC several N-pentofuranosyl-N0-[p-(isoamyloxy)phenyl]-thio-
urea derivatives were synthesized by coupling of an aniline derivative and pentofuranosyl isothiocyanates. The MIC values of the
new products against M.tb indicate that this new approach to the synthesis of potential anti-TB therapeutic agents was successful.
Ó 2008 Elsevier Ltd. All rights reserved.
O
The human pathogen Mycobacterium tuberculosis
(M. tb) causes tuberculosis and is responsible for the
deaths of millions of people, the most by any single
infectious agent.1
O
S
N
N
H
H
1
Fig. 1. The structure of THC.
One of the therapeutic agents that were used in the clin-
ical treatment of tuberculosis in the 1960s was a deriva-
tive of thiourea known as thiocarlide (THC; N,N0-bis[p-
(isoamyloxy)phenyl]-thiourea; 1, Fig. 1; also known as
IsoxylÒ). This powerful compound was first chemically
synthesized in 1951.2 The minimum inhibitory concen-
tration (MIC) value of THC against most clinical iso-
lates of M. tb, including multi-drug resistant ones, was
determined to be 2 lg/ml.3 Nevertheless, the clinical
use of THC was discontinued, apparently because of
the poor bioavailability of the highly non-polar
product.4
tives. These compounds were obtained by the coupling
of protected pentofuranosyl isothiocyanate with p-iso-
amyloxy aniline (Scheme 1), as previously described by
us5 and others.6–8 However, we have used acetates as
the protecting groups instead of the TBDMS groups
used previously5 as resynthesis of the arabino product
showed an increase in the yield of the final product be-
cause of the milder deprotecting conditions employed.
The isothiocyanates were synthesized from the corre-
sponding tetraacetates.9 All of the fully acetylated D-ald-
opentofuranoses are already described.9 The general
scheme consists of conversion of the pentose into the cor-
responding methyl pentofuranoside, and subsequent
acetylation.10 We then subjected these intermediates to
acetolysis, using a mixture of glacial acetic acid, acetic
anhydride, and a 5% solution of sulfuric acid in acetic
acid. Under these conditions the tetraacetates were ob-
tained in high yields (90–95%) and no purification of
the products was necessary. The 1H NMR data of the tet-
raacetates are in agreement with the reported values,9
thus confirming that the products exist in the furanose
form. Conversion of the acetates into the bromides was
carried out by treatment with trimethylsilyl bromide in
In an effort to improve the therapeutic value of THC, we
have synthesized several N-glycosyl-N0-[p-(isoamyl-
oxy)phenyl]-thiourea derivatives and determined their
MIC values against M.tb.5 The arabinfuranosyl product
(2, Fig. 2), the only pentosyl derivative in the previous
work,5 turned out to be the most promising one, exhib-
iting an MIC value of 2.5 lg/ml. This encouraging result
prompted us to focus our attention on the synthesis and
testing of the remaining D-aldopentofuranosyl deriva-
Keywords: Thiocarlide; Thiourea; Isoxyl; Mycobacterium tuberculosis.
*
Corresponding author. Tel.: +1 970 491 5537; e-mail: avraham.liav@
colostate.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.033

2650
A. Liav et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2649–2651
O
S
R
N
H
N
H
O
HO
O
HO
O
HO
O
HO
OH
HO
R=
OH
HO
OH
OH
OH
OH
5
2
4
3
Fig. 2. The structures of the N-D-pentofuranosyl-N0-p-(isoamyloxy) phenyl-thiourea products.
RO
RO
O
O
RO
O
a
b
OMe
OR
HO
N
C
S
OR
RO
OR
RO
OR
RO
R=Ac
8
7
6
O
1. c
O
S
O
9
H2N
N
N
OH HO
H
H
2. d
5
Scheme 1. Synthesis of N-D-lyxofuranosyl-N0-[p-(isoamyloxy)phenyl]-thiourea. Reagents and conditions: (a) AcOH, Ac2OH, 5% H2SO4; rt, 2.5H.
(b1) BrTMS, CH2Cl2; rt, 1 h. (b2) KSCN, tetrabutylammonium hydrogen sulfate, 4A molecular sieves, acetonitrile; rt, 2 h. (b3) The products from
(b1) and (b2), acetonitrile, 65°, 2 h; (c) isoamyloxy aniline, pyridine; rt, 70 min. (d) M NaOCH3, MeOH.
methylene chloride.11 The isothiocyanates were prepared
from the corresponding bromides by treatment with
potassium thiocyanate and tetrabutyl ammonium hydro-
gen sulfate. The pentofuranosyl isothiocyantes were
found to be unstable, and their purification by column
chromatography on silica gel could not be achieved. After
extraction of the crude isothiocyanates with a petroleum
ether–ethyl acetate 3:1 mixture, they were coupled to p-
(isoamyloxy) aniline (9, Scheme 1) as described before.5
Using this procedure (Scheme 1) we first prepared the
D-xylo and D-lyxo products (4 and 5, respectively). The
analogous D-ribo product (3) could be obtained by the
same procedure, but a cleaner product was obtained when
the commercially available 2,3,5-tri-O-benzoyl-D-ribofu-
ranose was used as the starting material. The synthesis of
the arabinose product (2) has already been described by
us.5 However, in that synthesis TBDMS groups were
used, and the removal of these groups (by treatment with
ammonium fluoride in methanolic ammonium hydroxide
at 65°) in the final step was not as efficient as the O-deacet-
ylation described in this report. Accordingly, we repeated
the synthesis of 2, using tetra-O-acetyl-D-arabinofura-
nose as a starting material.
arated by chromatography. The signals of the anomeric
protons appeared at low field, as expected (d 5.88–5.45).
In all cases, these signals were broad and the determina-
tion of the coupling constants was not feasible. The ring
carbohydrate protons usually appeared as complex mul-
tiplets.The signals of the isoamyloxyphenyl moiety ap-
peared as a pair of doublets in the aromatic region
(J = 9.0 Hz; except the spectrum of the D-lyxo product
which showed two pairs of doublets, one for each of
the two anomers), a two proton-triplet at around d
4.0 ppm, a one-proton multiplet at d 1.96–1.82 ppm, a
two proton-doublet of doublets at d 1.7 ppm, and a pair
of three protons—singlets at d 1.03 ppm and d 1.01 ppm.
The new products were tested as growth inhibitors against
M. tuberculosis H37Rv, using the microplate alamar blue
dye assay.12 Re-testing of the arabino analog (2) showed it
to be more potent than THC itself (which was tested
under the same conditions) while the ribo analog (3)
displayed an MIC value in the same range as THC
(Table 1).13 The D-xylo analog (4) was somewhat less
active and the D-lyxo product (5) showed activity only
at a concentration of 50 lg/mL. The sensitivity of the
products to the stereochemical configuration suggests that
the carbohydrate group not only adds needed hydrophi-
licity to the molecule but also adds specificity.
The structures of the new products were confirmed by
mass spectrometry and H NMR spectroscopy. In the
1
positive electro spray mode the products exhibited the
393 ion which corresponds to M+Na. The H NMR
spectra showed that the D-xylo and D-ribo products
were anomerically pure while the other products existed
as a mixture of the two anomers that could not be sep-
1
Further testing of the best product (2) in mice, including
the bioavailability assay, is now in progress. The bio-
availability assay estimates the drug levels in mice at
specific time points post oral dosing, using the growth

A. Liav et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2649–2651
2651
Table 1. MIC values of THC and products 2–5 against M. tuberculosis
H37
The mixture was stirred at room temperature for 2 h and
dried. In a separate flask a mixture of potassium thiocy-
anate (250 mg), tetrabutylammonium hydrogen sulfate
(205 mg) and molecular sieves (4A, 1 g) in acetonitrile
(10 mL) was stirred at room temperature for 2 h and then
added to the crude bromide. The reaction mixture was
stirred at 65° for 2 h. It was cooled and filtered through
celite. The filtrate was dried and petroleum ether–ethyl
acetate 3:1 was added to the residue. The organic extract
was filtered and the filtrate was dried to give the crude
isothiocyanate that was not purified. The crude isothiocy-
anate was then treated with p-isoamyloxy aniline5
(100 mg, 0.6 mmol) in pyridine (2 mL) at room tempera-
ture for 70 min. The mixture was dried and the residue was
chromatographed on silica gel (Sigma, mesh 70–230).
Elution with ethyl acetate–petroleum ether 1:1 removed an
un-identified by-product. Continued elution with the same
solvent system gave the coupled product. After re-chro-
matography of mixed fractions a combined sample of
165 mg (54%) was obtained. Part of the product (115 mg)
was treated with M sodium methoxide solution (0.15 mL)
in methanol (1.5 mL) at room temperature for 50 min. The
mixture was neutralized with Dowex 50 (H+) and the resin
was filtered off and washed with methanol. The filtrate was
dried and the residue was chromatographed on silica gel.
Elution with methylene chloride–methanol 9:1 removed
fast moving impurities. Continued elution with methylene
chloride–methanol 7:1 gave the product (52 mg, 60%).
Compound 2: 1H NMR (CD3OD, 300 MHz) d 7.26 (d,
2H, J = 9.0 Hz), 6.97 (d, 2H, J = 9.0 Hz), 5.88 (bs, 0.4H,
H-1 a), 5.58 (bd, 0.6H, H-1 b), 4.05 (t, 2H, J = 6.6 Hz),
3.96-3.90 (m, 2H), 3.82–3.58 (m, 2H), 3.64 (dd, 1H,
J = 3.4 Hz, J = 12.1 Hz), 1.96–1.82 (m, 1H), 1.71 (dd, 2H,
J = 6,6 Hz, J = 13.2 Hz), 1.03 (s, 3H), 1.01(s, 3H); MS
ESI+ m/z 393.141 (M+Na)+.
Product
MIC values (lg/mL)
THC
2.5–5.0
2
3
4
5
1.56–3.125
3.125–6.25
6.25–12.5
50
The MIC values were determined by the microplate alamar blue dye
assay. INH (isoniazid) was used as a standard at a concentration of
0.35 lg/mL.
of M. tuberculosis H37Rv as an indicator for drug activ-
ity.14 Also, the cLogP (log of the octanol/water parti-
tion coefficient) value of the new products, 2 and 3, is
1.56, which is significantly lower than that of THC
(6.22), and this decrease in cLogP may result in an in-
crease in bioavailability.
Acknowledgment
The work was supported by Grant #RO1 AI063054
from the National Institute of Allergy and Infectious
Diseases, NIH.
References and notes
1. (a) Rouhi, A. M. Chem. Eng. News 1999, 77, 52; (b) Dolin,
J. P.; Raviglione, M. D.; Kochi, A. Bull. World Health
Org. 1994, 72, 213.
2. Anonymous. Antibiot. Chemother. 1970, 16, 187.
3. Phetsuksiri, B.; Baulard, A. R.; Cooper, A.; Minnikin, D.
E.; Douglas, J. D.; Besra, G. S.; Brennan, P. J. Antimicrob.
Agents Chemother. 1999, 43, 1042.
4. Lambelin, G. Antibiot. Chemother. 1970, 16, 84.
5. Liav, A.; Angala, S. K.; Brennan, P. J. Synth. Commun., in
press.
1
Compound 3: H NMR (CD3OD, 300 MHz) d 7.27 (2H,
J = 9.0 Hz), 6.98(d, 2H, J = 9.0 Hz), 5.84 (bs, 1H), 4.05 (t,
2H, J = 6.6 Hz), 3.94 (bs, 1H), 3.78 (t, 1H, J = 3.0 Hz),
3.76–3.58 (m, 2H), 3.52 (dd, 1H, J = 3.6 Hz, J = 12.8 Hz),
1.96–1.82 (m, 1H), 1.71 (dd, 2H, J = 6,6 Hz, J = 13.2 Hz),
1.03 (s, 3H), 1.01 (s, 3H); MS ESI+ m/z 393.136 (M+Na)+.
1
Compound 4: H NMR (CD3OD, 300 MHz) d 7.28 (2H,
6. Takahashi, H.; Nimura, N.; Ogura, H. Chem. Pharm. Bull.
1979, 27, 1147.
7. Saitz-Barria, C.; Torres-Pinedo, A.; Santoyo-Gonzales, F.
Synlett 1999, 12, 1898.
8. Prata, C.; Mora, N.; Lacombe, J.-M.; Maurizis, J.-C.;
Pucci, B. Carbohydr. Res. 1999, 321, 4.
J = 9.0 Hz), 6.95 (d, 2H, J = 9.0 Hz), 5.45 (bs, 1H), 4.05 (t,
2H, J = 6.6 Hz), 4.02 (t, 1H, partially obscured by the 4.05
signal, J = 6.6 Hz), 3.95 (t, 1H, J = 6.6 Hz), 1.96–1.92 (m,
1H), 1.76 (dd, 2H, J = 6,6 Hz, J = 13.2 Hz), 1.03 (s, 3H),
1.01 (s, 3H); MS ESI+ m/z 393.145 (M+Na)+.
1
Compound 5: H NMR (CD3OD, 300 MHz) d 7.29, 7.26
9. Bernard, L. K.; Barascat, J.-L.; Imbach, J.-L. Carbohydr.
Res. 1979, 69, 135.
(2d, 2H, J = 9.0 Hz), 6.98, 6.96 (2d, 2H, J = 9.0 Hz), 5.82
(bs, 0.5H, H-1 a), 5.72 (bd, 0.5H, H-1 b), 4.06–4.02 (m,
1H, obscured by the 4.04 signal), 4.04 (t, 2H, J = 6.6 Hz),
3.97 (dd, 1H, J = 2.1 Hz, J = 4.8 Hz), 3.86 (dd, 1H,
J = 2.1 Hz, J = 11.5 Hz), 3.80–3.65 (m, 2H), 1.98–1.84
(m, 1H), 1.71 (dd, 2H, J = 6,6 Hz, J = 13.2 Hz), 1.03 (s,
3H), 1.01 (s, 3H); MS ESI+ m/z 393.139 (M+Na)+.
11. Liav, A.; Brennan, P. J. Tetrahedron Lett. 2005, 46, 2937.
12. Collins, L. A.; Franzblau, S. G. Antimicrob. Agents
Chemother. 1997, 41, 1004.
13. A provisional patent application covering these products
was filed.
14. Gruppo, V.; Johnson, C. M.; Marietta, K. S.; Scherman, H.;
Zinc, E. E.; Crick, D. C.; Adams, L. B.; Orme, I. M.;
Lenaerts, A. J. Antimicrob. Agents Chemother 2006, 50, 1245.
10. General procedure for the synthesis of N-D-aldopentofur-
anosyl-N0-[p-(isoamyloxy)phenyl]-thiourea derivatives: To
a cold (ice bath) solution of the methyl 2,3,5-tri-O-acetyl-
pentofuranoside (610 mg) in acetic acid (1.6 mL) and
acetic anhydride (1.6 mL) was added a 5% solution of
sulfuric acid in acetic acid (0.8 mL). The ice bath was
removed and the mixture was stirred at room temperature
for 3 h. It was neutralized with solid sodium bicarbonate
and the product was extracted with ethyl acetate. The
organic solution was washed with water and dried to give
656 mg (98%) of the tetraacetate. A part of the product
(290 mg, 0.92 mmol) was dissolved in methylene chloride
(0.6 mL) and trimethylsilyl bromide (0.6 mL) was added.