Antimycobacterial Agents. 1. Thio Analogues of Purine

Article abstract of DOI:10.1021/jm030389b

Thio analogues of purine, pyridine, and pyrimidine were prepared based on the initial activity screening of several analogues of these heterocycles against Mycobacterium tuberculosis (Mtb). Certain 6-thio-substituted purine analogues described herein showed moderate to good inhibitory activity. In particular, two purine analogues 9-(ethylcarboxymethyl)-6-(decylthio)-9H-purine (20) and 9-(ethylcarboxymethyl)-6-(dodecylthio)-9H-purine (21) exhibited MIC values of 1.56 and 0.78 μg/mL respectively against the Mtb H₃₇Rv strain. N⁹-Substitution apparently enhances the antimycobacterial activity in the purine series described herein.

Full text of DOI:10.1021/jm030389b

J . Med. Chem. 2004, 47, 273-276
273
Brief Articles
An tim ycoba cter ia l Agen ts. 1. Th io An a logu es of P u r in e†
Ashish K. Pathak, Vibha Pathak, Lainne E. Seitz, William J . Suling, and Robert C. Reynolds*
Drug Discovery Division, Southern Research Institute, P.O. Box 55305, Birmingham, Alabama 35255-5305
Received August 12, 2003
Thio analogues of purine, pyridine, and pyrimidine were prepared based on the initial activity
screening of several analogues of these heterocycles against Mycobacterium tuberculosis (Mtb).
Certain 6-thio-substituted purine analogues described herein showed moderate to good
inhibitory activity. In particular, two purine analogues 9-(ethylcarboxymethyl)-6-(decylthio)-
9H-purine (20) and 9-(ethylcarboxymethyl)-6-(dodecylthio)-9H-purine (21) exhibited MIC values
of 1.56 and 0.78 µg/mL respectively against the Mtb H37Rv strain. N9-Substitution apparently
enhances the antimycobacterial activity in the purine series described herein.
In tr od u ction
On the basis of reasonable activity shown by several
purine analogues (data not shown) in initial screening
by the TAACF, we prepared a small 6-thiopurine library
for antimycobacterial screening. This library can be
further subdivided into 2-H, 2-Cl, and 2-OH analogues
of the primary 6-thiopurine grouping. On the basis of
the activity screening for our initial library by the
TAACF, we further prepared certain 6-thio-N9-substi-
tuted purine analogues. Promising activity suggested
the importance of N9-substitution. Additionally, a dis-
connection approach recommended preparation of small
sets of 2-thio-substituted pyrimidine/pyridine and 4-thio-
substituted pyridine analogues as portions of the active
6-thiopurine nucleus.
Ch em istr y. We carried out the synthesis of several
diverse 6-thioaryl/alkyl purines, 2-thioaryl/alkyl pyri-
midine and 2- and 4-thioaryl/alkyl pyridine analogues
for screening against Mtb and Mycobacterium avium
complex (MAC) strains. A total of 28 analogues of
6-thiopurine, 26 analogues of 2-chloro-6-thiopurine, 32
analogues of 2-hydroxy-6-thiopurine, 16 analogues of
2-thiopyrimidine, 16 analogues of 4-thiopyridine, and
7 analogues of 2-thiopyridine were prepared in the
initial phase to determine antibacterial inhibitor activity
and only purine analogues have shown promising activ-
ity (see Supporting Information). A total of 19 purine
analogues have shown g50% inhibitory activity against
Mtb H37Rv (Chart 1) and are discussed herein.
Despite the availability of highly active antitubercular
agents, tuberculosis (TB) remains one of the primary
causes of human death and suffering worldwide.1 Of
even greater concern is the development of highly drug-
resistant forms of the disease, particularly multiple
drug-resistant tuberculosis (MDR-TB) that is both dif-
ficult and expensive to treat.2 TB treatment is also
complicated in AIDS patients undergoing chronic, com-
bined therapies for HIV and attendant opportunistic
diseases.3 The sequencing of the Mtb genome has
greatly facilitated the identification of potential new and
unique targets within this organism.4
Another approach to identification of new antituber-
cular agents is based on broad screening of chemical
libraries against the tuberculosis bacillus. An extensive
library of heterocycles, developed over the last five
decades at Southern Research Institute (SRI), was
screened through the Tuberculosis Antimicrobial Ac-
quisition and Coordination Facility (TAACF).5 Due to
the paucity of biochemical data concerning these het-
erocycles in bacteria and the interesting preliminary
activity shown against mycobacteria, a modest number
of analogues in these series were pursued. It was also
deemed appropriate due to the fact that both purines
and pyrimidines are not represented in the current
clinical antitubercular regimens, suggesting that active
compounds in these series may target new biochemical
mechanisms, potentially allowing treatment of MDR-
TB. The purine ring system is a key structural element
of substrates and ligands of many biosynthetic, regula-
tory, and signal transduction proteins including cellular
kinases, G proteins, and polymerases.6,7 Very little work
has been done to explore the potential of purine ana-
logues as antitubercular agents. Recently, syntheses of
purine analogues possessing antitubercular activity
have been published.8
The purine analogues 1 and 2 were prepared by the
reactions of 6-mercaptopurine with the corresponding
alkyl/benzyl halides in the presence of K2CO3 in DMAc
at room temperature. The compounds 3-11 were ob-
tained by reactions of 6-chloropurine or 2,6-dichloro-
purine with the corresponding alkyl/aryl thiols in 2-pro-
panol using (CH3)3COK while heating at 50 °C. The
2-hydroxy purine analogues 12-19 were prepared by
the treatment of 6-thioxanthine with alkyl/benzyl ha-
lides in 0.1 N NaOH.
To evaluate the effect of N9-substitution on inhibitory
activity, N9-alkylation of a small number of active
6-thio-substituted purines was performed. The alkyla-
* Corresponding author. Phone: (205)581-2454, Fax: 205-581-2877,
e-mail: reynolds@sri.org.
† Part of this paper was presented as a poster at the 221st American
Chemical Society National Meeting at San Diego, CA, April 1-5, 2001.
10.1021/jm030389b CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/27/2003

274 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Brief Articles
Ta ble 1. Antimycobacterial Activity against Mtb of
6-Thio-Substituted Purine Analogues
Ch a r t 1
compd
no.
% inhib
Mtb H37Rv 6.25 µg/mL
MIC90
Mtb H37Rv
MICa
MtbH37Ra
1
52b
98
69b
69b
78b
99b
65b
72
56
86
53
94
59
62
55
50
52
61
50
-
>12.5
3.13
>12.8
>12.8
>12.8
>12.8c
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
>12.8
16
2d
3
>12.5
>12.5
>12.5
>12.5
>12.5
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
-
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
EMB
16
>12.8c
8
>12.8c
16
16
2-4
Sch em e 1
a
b
MIC are given in µg/mL. Determined at 12.5 µg/mL. c Indi-
cated partial inhibition of growth representing an estimated 50%
or more. IC50 against VERO cells >62.5, selectivity index (SI) >
d
20 (SI ) IC50/MIC) and activity against Mtb Erdman in monolay-
ers of mouse bone marrow macrophages EC90 ) 2.54 µg/mL. EMB
) ethambutol.
Ta ble 2. Antimycobacterial Activity of N9-Substituted Purines
against Mtb
MICa
IC50
a
% inhib
MIC90
compd. Mtb H37Rv
no.
against
against
VERO selectivity
6.25 µg/mL Mtb H37Rv Mtb H37Ra cells index (SI)b
20
21
22
23
24
25
26
27
28
29
30
31
32
33
100
98
99
0
99
100
25
17
0
22
0
0
99
0
1.56
0.78
>12.8c
8
>62.5
>10
n.d
>40
>12.8
n.d
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
3.13
32
>12.8c
>12.8
>12.8c
4
n.d
n.d
n.d.
n.d.
n.d
n.d
n.d
n.d
n.d
n.d
n.d.
n.d.
n.d
n.d
n.d
n.d
n.d
n.d
4
16
4
>12.8
>12.8
16
>10
n.d
>3.19
n.d
>6.25
32
a
b
MIC are given in µg/mL. SI ) IC50/MIC. c Indicated partial
inhibition of growth representing an estimated 50% or more.
Positive control, ethambutol, MIC 2-4 µg/mL against Mtb H37Ra.
“n.d.” indicates not determined.
tion was carried out with ethyl bromoacetate in the
presence of K2CO3 using DMAc as solvent and resulted
in 6,9-disubstituted purine analogues 20-29 (Scheme
1A). Upon evaluation of the antimycobacterial activity
of 20 that has shown promising antimycobacterial
activity, four N9-alkylated analogues of 2 were also
synthesized (30-33) as shown in Scheme 1B. All the
new analogues were characterized using NMR, FABMS/
MS-ESI spectroscopic, and CHN analysis before evalu-
ating for antimycobacterial activity. Compound 29 was
found to contain 7% of the N7-substituted analogue (by
NMR analysis) even after several column chromato-
graphic purifications and was used as such for antimy-
cobacterial screening.
An tim ycoba cter ia l Activity. All synthesized pu-
rine, pyrimidine, and pyridine analogues were screened
for their antimycobacterial activity against two strains
of Mtb H37Rv and H37Ra, and three strains of MAC
(NJ 211, NJ 168, and NJ 3404). Pyrimidine and pyridine
analogues were found inactive. The 6-thio-substituted
purine analogues, having more than 50% inhibition
against H37Rv, are presented in Table 1, and activity
results of 6,9-substituted purine analogues are pre-
sented in Table 2.
Act ivit y a ga in st Mt b H37R a a n d MAC St r a in s.
The compounds were tested against Mtb H37Ra and
MAC strains using a colorimetric microdilution broth
assay at two concentration 1.28 and 12.8 µg/mL.9 Most
of the analogues (1-19) were found to be inactive
against H37Ra, and none of the compounds were active
(MIC >12.8 µg/mL) against MAC. However, N9-alky-
lated 2-hydroxy-6-thio-substituted purines analogues
26, 27, and 29 showed improved MICs (4 µg/mL) against
Mtb H37Ra.
Activity a ga in st Mtb H37Rv Str a in . All compounds
were initially screened for their antimycobacterial activ-
ity at 6.25 µg/mL (few at 12.5 µg/mL) against H37Rv
strain by the TAACF. Compounds exhibiting g90%

Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 275
equiv) in the presence of dry K2CO3 (1.2 equiv) under argon
atmosphere. Deionized water was added, and the reaction was
neutralized by addition of acetic acid. The solid obtained was
filtered, washed with deionized water followed by diethyl ether,
and dried overnight in vacuo over P2O5 at 25 °C.
inhibition in the initial screen were retested at and
below 6.25 mg/mL using 2-fold dilution to determine the
MIC90. In particular, 6-(decylthio)purine (2) had the
most promising activity with a MIC90 of 3.13 µg/mL. To
evaluate the effect of N9-alkylation on the antimyco-
bacterial activity, compound 20 was synthesized from
compound 2 and tested. The alkylated analogue 20
possessed a MIC90 of 1.56 µg/mL. On the basis of this
observation, we synthesized other N9-alkylated purine
analogues (21-29) of a few active samples for activity
screening (Table 2). Compound 21 showed excellent
activity with an MIC90 of 0.78 µg/mL, making it the most
active analogue against Mtb H37Rv in the present series.
Meanwhile, to investigate the effect of different N9-
alkylating substitutions on antimycobacterial activity,
compound 2 was also derivatized to compounds 30-33,
but none of these purine analogues showed enhanced
activity as compared to compound 20. The N9-ethylcar-
boxymethyl substitution in compound 2 to give com-
pound 20 showed a 2-fold increase in inhibitory activity,
clearly indicating the value of N9-alkylation for en-
hanced antimycobacterial activity in purines. A more
lipophilic analogue, 32, possessed a MIC90 of 3.13 µg/
mL, which is 2-fold less active compared to compound
20 that contained an N9-ethyl ester. The results de-
scribed herein show that 6-thio-substituted purines
exhibit good antimycobacterial activity, in particular,
more hydrophobic analogues at both the 6- and 9
positions.
Gen er a l P r oced u r e for Syn th esis of 6-Th ioa r yl P u -
r in es 3-7 a n d 2-Ch lor o-6- th ioa lk yl/a r yl P u r in es 9-11.
The starting chloro compound (6-chloropurine or 2,6-dichlo-
ropurine) in dry i-PrOH was heated under reflux for 3-5 h
with mercaptoaryl/alkyl (1.2 equiv) in the presence of (CH3)3-
COK (1.2 equiv) under argon atmosphere. Deionized water was
added, and the resulting solid was filtered, washed with
deionized water followed by diethyl ether, and dried overnight
in vacuo over P2O5 at 25 °C.
Gen er a l P r oced u r e for Syn th esis of 2-Hyd r oxy-6-
t h iob en zyl/a lk yl P u r in es 12-19. 2-Hydroxy-6-mercapto-
purine in 0.1 N NaOH was reacted with the appropriate
benzyl/alkyl halide (1.2 equiv) at room temperature for 4-6
h. Deionized water was added followed by neutralization with
acetic acid. The solid obtained was filtered, washed with
deionized water followed by diethyl ether, and dried overnight
in vacuo over P2O5 at 25 °C.
Gen er a l P r oced u r e for th e Syn th esis of N9-Alk yla ted
P u r in es 20-33. The 6-thio-substituted purine in DMAc and
the appropriate alkyl- or aryl-bromoacetyl derivative (1.5
equiv) were reacted in the presence of K2CO3 (1.2 equiv) at
room temperature. It was poured into deionized water and
neutralized with acetic acid. The crude product was extracted
with diethyl ether and dried over Na2SO4. The final compounds
were purified by column chromatography over silica gel G.
Activity a ga in st Mtb H37Ra Str a in , MAC. All compounds
were tested for their inhibitory activity against Mtb H37Ra
(ATCC 25177) and MAC NJ 211, NJ 168, and NJ 3404 strains.
The screening was performed at 1.28 and 12.8 µg/mL in
Middlebrook 7H9 broth supplemented with 0.2% glycerol and
ADC enrichment using a colorimetric (Alamar blue) microdi-
lution broth assay.9 The active compounds (e12.8 µg/mL) were
retested using 2-fold dilutions to obtain the actual MIC. In
this particular assay, the MIC was recorded as the lowest drug
concentration that inhibited the growth completely.
Activity a ga in st Mtb H37Rv Str a in a t th e TAACF .5 The
primary screen was conducted at either 6.25 or 12.5 µg/mL
against Mtb H37Rv (ATCC 27294) in BACTEC 12B medium
using the Microplate Alamar Blue Assay (MABA).11 Com-
pounds demonstrating at least 90% inhibition at 6.25 or e12.5
µg/mL were retested to determine the MIC90, defined as the
lowest concentrating inhibiting growth by 90% or higher.
Compounds 2, 20, 21, and 32 exhibited antimycobac-
terial activity in the preliminary screening against Mtb
H
37Rv and were further examined for toxicity (IC50) in
a mammalian cell line, VERO cells by the TAACF
(Tables 1 and 2). Compound 20 exhibited a better IC50
and selectivity index (SI) as compared to compound 21.
Compounds 2 and 20 were tested against Mtb Erdman
in monolayers of a mouse bone marrow macrophages
model,10 and the concentrations effecting 90% reduction
in the viable cell count after 7 days, compared to
untreated controls (EC90), were 2.53 µg/mL and 1.65 µg/
mL, respectively. These data demonstrated that certain
purine analogues exhibit high activity against Mtb
inside macrophages since the EC90:MIC90 (Mtb H37Rv)
ratios were calculated as 0.81 and 1.06, respectively. The
present study has demonstrated the future potential for
development of 6-thio-substituted purine analogues as
antimycobacterial agents.
Cytotoxicity a ga in st VERO cell Lin es a n d Selectivity
In d ex (SI).5 Concurrent with the determination of MIC90’s,
compounds 2, 20, 21, and 32 were tested for cytotoxicity (IC50
in VERO cells. The selectivity index is defined as the ratio of
the measured IC50 in VERO cells to the MIC90
)
.
Activity a ga in st Mtb Er d m a n in Ma cr op h a ges. Com-
pounds 2 and 20 were tested for killing of Mtb Erdman (ATCC
35801) in monolayers of mouse bone marrow macrophages.10
EC90 is defined as the concentrations effecting 90% reduction
in the viable cell counts after 7 days, compared to untreated
controls.
Exp er im en ta l Section
Anhydrous solvents and reagents from Aldrich were used
without further drying. Reactions were monitored by thin-layer
chromatography (TLC) on precoated E. Merck silica gel (60F254
)
plates (0.25 mm) and visualized using UV light (254 nm). Flash
chromatography was carried out on Fischer silica gel G 60
(230-400 mesh). Melting points, determined with a Mel-Temp
II capillary melting points apparatus, are uncorrected. 1H
NMR spectra were recorded on a Nicolet NT 300NB instru-
ment at 300 MHz. The coupling constants (J ) are reported in
hertz, and chemical shifts are reported in ppm (δ) relative to
residual solvent peak or internal standard. Microanalyses were
performed on a Perkin-Elmer 2400 CHN analyzer. FABMS
were recorded on a Varian/MAT 311A double-focusing mass
spectrometer either by adding NBA or LiCl and MS-ESI on a
BioTof-2 time-of-flight mass spectrometer.
Ack n ow led gm en t. Authors are thankful to TAACF
for screening against Mtb H37Rv. We are also thankful
to Dr. J . M. Riordan, Mr. M. D. Richardson, and Ms. J .
C. Bearden for spectral and analytical analyses. This
work was supported by NIH/NIAID grant R01AI45317.
Su p p or tin g In for m a tion Ava ila ble: Detailed informa-
tion on synthetic methods, analytical, spectroscopic, and
antimycobacterial activity of purine, pyrimidine, and pyridine
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Syn t h esis of 6-(Ben zylt h io)p u r in e (1) a n d 6-(Decyl-
th io)p u r in e (2). 6-Mercaptopurine in DMAc was reacted at
room temperature for 2-6 h with benzyl/decyl chloride (1.2

276 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Brief Articles
Rev. Cell Dev. Biol. 1997, 13, 261-291. (c) Hengst, L.; Reed, S.
I. Inhibitors of the Cip/Kip family. Curr. Topics Microb. Immu-
nol. 1998, 227, 25-41.
Refer en ces
(1) (a) World Health Organization. Anti-tuberculosis drug resistance
in the world. The WHO/ IUATLD global project on anti-
tuberculosis drug resistance surveillance, 1997. (b) Bastian, I.;
Colebunders, R. Drugs 1999, 58, 633-661. (c) Butler D. New
fronts in an old war. Nature 2000, 406, 670-672.
(2) (a) Bodiang, C. K. Issue facing TB control (2.1) Tuberculosis
control in refugee populations: A focus on developing countries.
Scot. Med. J . 2000, 45, 25-28. (b) Van Scoy, R. E.; Wilkowske,
C. J . Antimycobacterial therapy. Mayo Clin. Proc. 1999, 74,
1038-1048. (c) Long, R. Drug-resistant tuberculosis. Can. Med.
Assoc. J . 2000, 163, 425-428.
(3) (a) Gazdic, A. Correlation between increased incidence of
pulmonary tuberculosis and AIDS. Med. Arch. 1998, 52, 207-
209. (b) Pozniak, A. HIV-associated tuberculosis in the era of
HAART. Int. J . Tuberc. Lung Dis. 2000, 4, 993-994.
(4) Cole, S. T.; Brosch, R.; Parkhill, J . et. al. Deciphering the biology
of Mycobacterium tuberculosis from the complete genome se-
quence. Nature 1998, 393, 537-544.
(5) Orme, I.; Secrist, J .; Ananthan, S. et. al. Search for new drugs
for treatment of tuberculosis. Antimicrob. Agents Chemother.
2001, 45, 1943-1946; http://taacf.org.
(6) J acobson, K. A.; Daly, J . W.; Manganiello, V. In Purines in
cellular signaling: Targets for new drugs; Springer: New York,
1990.
(7) (a) Norbury, C.; Nurse, P. Animal cell cycles and their control.
Annu. Rev. Biochem. 1992, 61, 441-470. (b) Morgan, D. Cyclin-
dependent kinases: engines, clocks, and microprocessors. Annu.
(8) (a) Bakkestuen, A. K.; Gundersen, L.-L.; Langli, G.; Liu, F.;
Nolsoe, J . M. J . 9-Benzylpurines with inhibitory activity against
Mycobacterium tuberculosis. Bioorg. Med. Chem. Lett. 2000, 10,
1207-1210. (b) Scozzafava, A.; Mastrolorenzo, A.; Supuran, C.
T. Antimycobacterial activity of 9-sulfonylated/selfenylated-6-
mercaptopurine derivatives. Bioorg. Med. Chem. Lett. 2001, 11,
1675-1678. (c) Gundersen, L.-L.; Nissen-Meyer, J . N.; Spilsberg,
B. Synthesis and antimycobacterial activity of 6-arylpurines:
The requirements for the N-9 substituent in active antimyco-
bacterial purines. J . Med. Chem. 2002, 45, 1383-1386.
(9) Suling, W. J .; Reynolds, R. C.; Barrow, E. L.; Wilson, L. N.; Piper,
J . R.; Barrow, W. W. Susceptibilities of Mycobacterium tuber-
culosis and Mycobacterium avium complex to lipophilic deazap-
teridine derivatives, inhibitors of dihydrofolate reductase. An-
timicrob. Agents Chemother. 1998, 42, 811-815.
(10) Skinner, P. S.; Furney, S. K.; J acobs, M. R.; Klopman, G.; Ellner,
J . J .; Orme, I. M. A bone marrow-derived murine macrophage
model for evaluating efficacy of antimycobacterial drugs under
relevant physiological conditions. Antimicrob. Agents Chemother.
1994, 38, 2557-2563.
(11) Collins, L.; Franzblau, S. G. Microplate alamar blue assay versus
BACTEC 460 system for high-throughput screening of com-
pounds against Mycobacterium tuberculosis and Mycobacterium
avium. Antimicrob. Agents Chemother. 1997, 41, 1004-1009.
J M030389B