Synthesis and in vitro anti-Mycobacterium activity of N-alkyl-1,2- dihydro-2-thioxo-3-pyridinecarbothioamides. Preliminary toxicity and pharmacokinetic evaluation

Article abstract of DOI:10.1021/jm991044p

Disseminated infections with Mycobacterium tuberculosis (MT) and Mycobacterium avium complex (MAC) are increasingly opportunistic diseases in patients with advanced acquired human immunodeficiency syndrome (AIDS). A series of N-alkyl-1,2-dihydro-2-thioxo-3-pyridinecarbothioamides has been synthesized, and MICs for MT and MAC strains, either standard or isolated from infected patients, have been determined. Preliminary tests show a good activity and a very low toxicity for some derivatives. Pharmacokinetic studies in the rat show a very rapid elimination from the body after intravenous administration and a poor absorption after oral administration.

Full text of DOI:10.1021/jm991044p

J . Med. Chem. 2000, 43, 199-204
199
Syn th esis a n d in Vitr o An ti-Mycoba cter iu m Activity of
N-Alk yl-1,2-d ih yd r o-2-th ioxo-3-p yr id in eca r both ioa m id es. P r elim in a r y Toxicity
a n d P h a r m a cok in etic Eva lu a tion
Giuseppe Pagani,† Massimo Pregnolato,† Daniela Ubiali,† Marco Terreni,*,† Claudio Piersimoni,‡
Francesco Scaglione,§ Franco Fraschini,§ Alicia Rodr´ıguez Gasco´n,| and J ose´ Luis Pedraz Mun˜oz|
Dipartimento di Chimica Farmaceutica, Universita` degli Studi, I-27100 Pavia, Italy, Dipartimento di Microbiologia Clinica,
Policlinico “Umberto I - Torrette”, I-60020 Ancona, Italy, Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica,
Universita` degli Studi, I-20129 Milano, Italy, and Laboratory of Pharmacy and Pharmaceutical Technology,
Faculty of Pharmacy, University of the Basque Country, Vitoria, Spain
Received March 23, 1999
Disseminated infections with Mycobacterium tuberculosis (MT) and Mycobacterium avium
complex (MAC) are increasingly opportunistic diseases in patients with advanced acquired
human immunodeficiency syndrome (AIDS). A series of N-alkyl-1,2-dihydro-2-thioxo-3-pyridi-
necarbothioamides has been synthesized, and MICs for MT and MAC strains, either standard
or isolated from infected patients, have been determined. Preliminary tests show a good activity
and a very low toxicity for some derivatives. Pharmacokinetic studies in the rat show a very
rapid elimination from the body after intravenous administration and a poor absorption after
oral administration.
In tr od u ction
Among the several pathogens afflicting AIDS-infected
patients, two groups of mycobacteria pose a significant
problem to the clinical management: the Mycobacte-
rium tuberculosis (MT) and the Mycobacterium avium
complex (MAC).1-3 Disseminated MAC (dMAC) infection
is the most frequent complication4,5 and occurs late in
F igu r e 1. Structures of the considered compounds.
the course of AIDS when patients are severely immu-
nocompromised.6,7 Although several reports suggest that
losis becomes drug resistant through random, spontane-
ous genetic mutation, and administration of a single
chemotherapy of dMAC infections may reduce symp-
toms and prolong survival, attempts to eradicate the
drug often leads to the development of a bacterial
infections have been unsuccessful.3,8 Conventional an-
population resistant to that drug. Consequently, effec-
timycobacterial agents such as isoniazid, rifampicin, and
tive regimens for the treatment of MT also must contain
pyrazinamide are inactive against almost all strains of
multiple drugs. Generally, a four-drug regimen with
MAC,9 and single drugs such as rifabutin, ethambutol,
isoniazid, rifampin, pyrazinamide, and ethambutol is
preferred for the initial, empiric treatment of MT. This
and ciprofloxacin are inadequate for therapy.10
The most valuable group of antimicrobial agents
availaible for the treatment of dMAC disease is repre-
sented by the new macrolides such as azithromycin,
clarithromycin, and roxithromycin.3 However, the selec-
tion of resistant mutants occurs at such a rate with
macrolide therapy that at least two agents have to be
used. Combinations of agents for dMAC treatment are
highly variable in content, but all associations should
contain one of the advanced generation macrolides.
The control of MT infections is still considered of
relevance, not only for the survival of AIDS patients.
The recent emergence of drug-resistant MT, in fact, also
has become a serious concern. Mycobacterium tubercu-
four-drug regimen is highly effective even for isoniazid-
resistant organisms.11
Because of the concern of the resistance to most of
the commonly used drugs displayed by the considered
mycobacteria, our studies have been focused on the
development of new potential therapeutic agents. The
1,2-benzisothiazol-3(2H)-ones I (BITs) (Figure 1) are
known to possess several biological activities, and the
antimicrobial properties have been largely described.12-14
Some N-hydroxyalkyl derivatives of BIT have been
claimed to possess in vitro antibacterial activity against
MT and other mycobacteria.15 Recently, our interest in
1,2-benzisothiazol-3(2H)-ones and their corresponding
thiono derivatives II has increased,16 and the reported
antibacterial activities have enhanced the development
of new derivatives. A series of N-(2-hydroxyethyl)-1,2-
benzisothiazol-3(2H)-one and -thione carbamic esters
have been recently synthesized and tested against
Mycobacterium avium strains.17
* Author for correspondence: Marco Terreni, Dipartimento di
Chimica Farmaceutica, Universita` degli Studi, Viale Taramelli 12,
I-27100 Pavia, Italy. Phone: +39-0382-507265. Fax: +39-0382-507589.
E-mail: markt@chifar1.unipv.it.
† Dipartimento di Chimica Farmaceutica, Universita` degli Studi,
I-27100 Pavia, Italy.
‡ Policlinico “Umberto I-Torrette”.
In a preliminary approach, our attention has been
directed to the synthesis of bioisosteric derivatives of
§ Dipartimento di Farmacologia, Universita` degli Studi.
| University of the Basque Country.
10.1021/jm991044p CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/07/2000

200 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Pagani et al.
Sch em e 1
Ta ble 1. In Vitro Anti-M. Avium and Anti-M. Tuberculosis
Activities of Compounds 1-6 (MIC µg/mL)
BIT in which the benzene ring is replaced by a hetero-
cycle such as pyridine. Thus, N-alkylisothiazole[5,4-b]-
pyridin-3(2H)-ones III and thiones IV, reported in
Figure 1, have been synthesized. Compounds III and
IV and their intermediates of synthesis (V, VI) have
been evaluated in vitro against MAC (ATCC 15769) and
MT, either standard (H37-Rv ATCC) or isolated from
blood of infected patients (MT-1, MT-2). For the most
active structure VI, the study has been enlarged to
several N-alkyl derivatives in order to evaluate the
effect of the N-alkyl side chain on anti-MAC and anti-
MT activities toward both standard and wild strains.
The most interesting substances have been preliminar-
ily evaluated for the in vivo toxicity and pharmaco-
kinetic studies in the rat.
MAC MT
compd
ATCC 15769a H37-Rv ATCCa MT-1b MT-2b
1
8
1
8
16
1
4
8
1
8
8
2
8
2a
3a ,bc
4
2
4
8
8
5
6
8
8
16
e2
8
8
e2
16
8
e2
16
ethambutol
NDd
b
aStandard strains. Wild-type strains (isolated from infected
patients). c The biological test has been conducted on the isomeric
mixture because of the rapid equilibrium of the single isomers in
d
polar solvents.17,18 ND: not determined.
Resu lts a n d Discu ssion
The first study on the bioisosteric isothiazole[5,4-b]-
pyridin-3(2H)-ones and related compounds (Figure 1:
III-VI) has been conducted choosing the n-hexyl group
for the N-substitution in order to prepare model deriva-
tives for anti-mycobacteria tests. The synthesis has been
performed by reacting the 3H-1,2-dithiolo[3,4-b]pyri-
dine-3-thione 1 and the 3H-1,2-dithiole[3,4-b]pyridin-
3-one 4 with n-hexylamine. The obtained N-hexyl-1,2-
dihydro-2-thioxo-3-pyridinecarbothioamide 2a and
N-hexyl-1,2-dihydro-2-thioxo-3-pyridinecarbamide 5 have
been cyclized by iodine treatment to give the N-hexyl-
isothiazolo[5,4-b]pyridine-3(2H)-thione 3a in equilibri-
um with its isomer N-hexyl-3-imino-3H-1,2-dithiolo[3,4-
b]pyridine 3b 18,19 and the N-hexylisothiazolo[5,4-b]-
pyridine-3(2H)-one 6, respectively (Scheme 1).
F igu r e 2. N-Alkyl-1,2-dihydro-2-thioxo-3-pyridinecarbothio-
amides 2a -2p .
increases the lypophilicity and affects a modification on
the electronic structure, suggesting a possible relation-
ship with the improved biological activity. Comparing
the MICs for compounds 5 and 6 with 2a and 3a -b, it
is evident that the thioxo-analogues are generally more
active against MAC and MT strains, in accordance with
results previously reported.18
The in vitro activity of all synthesized compounds 1-6
on mycobacteria strains has been determined by the
radiometric broth dilution method, and results are
reported in Table 1. Although it is not difficult to isolate
the two isomeric forms 3a and 3b, an equilibrium
between the two structures easily occurs upon being
dissolved in polar solvent (DMSO, DMF, acetone, wa-
ter).18,19 As a consequence, the biological tests for 3a and
3b have been performed on the isomeric mixture.
The results show that the pyridinecarbothioamide 2a
displays the best activity (MICs: 1-2 µg/mL). Com-
pounds 1 and 4 (MICs: 8-16 µg/mL and 2-8 µg/mL,
respectively), the oxo-analogue pyridinecarbamide 5
(MICs: 8-16 µg/mL), the cyclic compounds 3a -3b
(MICs: 4-8 µg/mL), and 6 (MICs: 8-16 µg/mL) are, in
fact, less active.
The preliminary microbiological data indicate that
the N-alkyl-1,2-dihydro-2-thioxo-3-pyridinecarbothio-
amide structure is the most active. Thus, the research
has been focused on the study of the carbothioamidic
moiety, and the effect of the N-alkyl substitution has
been evaluated. Through the same procedure employed
for the synthesis of the N-hexyl-2-mercapto-3-pyridin-
ecarbothioamide 2a , a series of primary alkylamines
have been reacted with 3H-1,2-dithiole[3,4-b]pyridin-
3-thione 1. The reaction gave the 1,2-dihydro-2-thioxo-
3-pyridinecarbothioamides 2a -2p in 75-83% yields
1
(Figure 2). H NMR spectrum of compound 2a shows
As reported in a previous study,17 the substitution of
sulfur for oxygen in the ketobenzisothiazole system
the typical pyridinic signals at δ 6.95 (q, 1H, 5-H), 7.67
(q, 1H, 4-H), 9.42 (q, 1H, 6-H) and the multiplet of the

Syntheses of Antimycobacterial Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 201
Ta ble 2. In Vitro Anti-M. Avium and Anti-M. Tuberculosis Activities of Compounds 2a -2p (MIC µg/mL)
MAC ATCC
15769a
MAC
MAC
AN1b
MAC
AN2b
MAC
AN3b
MT H37-Rv
ATCCa
compd
R
cLogP
ISS 486b
MT-1b
MT-2b
2a
2b
2c
2d
2e
2f
2g
2h
2i
2l
2m
2n
2o
2p
C6H13
CH3
C2H5
(CH2)2-OH
C3H7
C4H9
C5H11
cyclohexyl
C7H15
C8H17
3.66
1.58
1.92
ND
2.41
2.83
3.24
ND
4.08
4.50
4.91
5.33
7.83
8.67
ND
1
4
4
8
2
1
1
4
2
4
8
16
8
0.5
4
4
8
1
2
1
4
4
8
8
32
8
32
2
1
8
4
4
2
4
1
4
4
8
8
32
8
32
1
1
4
8
8
4
4
2
4
4
4
8
32
8
32
0.5
1
4
8
4
2
4
1
4
4
4
8
32
8
32
2
1
2
0.5
2
2
4
1
1
1
4
4
4
4
4
2
2
1
1
8
4
4
4
1
2
2
0.5
0.5
1
4
4
C9H19
4
16
64
16
64
e2
16
64
32
64
e2
C
C
C
10H21
16H33
18H37
16
16
32
e2
64
ND
ethambutol
a
b
Standard strains. Wild-type strains (isolated from infected patients). ND: not determined.
CH2-NH group at δ 3,85 (J NH-CH ) 5.0 Hz) as well as
2
in all prepared compounds.18
In vitro activities of the synthesized compounds
versus M. tuberculosis (H37-Rv ATCC, MT-1, 2) and M.
avium (MAC ATCC 15769, MAC ISS 486, MAC AN1,
AN2, AN3) expressed as MIC (µg/mL) have been re-
ported in Table 2. The biological results show a remark-
able activity especially for compounds 2c and 2i against
M. tuberculosis H37-Rv ATCC (MIC: 0.5 µg/mL); these
substances result, however, as less active on the MAC
strains (MICs: 4-8 µg/mL for 2c and 2-4 µg/mL for
2i). Compounds 2a and 2b exhibit a positive activity
against M. tuberculosis (MICs: 1-2 µg/mL). Besides,
compound 2a has a good activity versus MAC wild
strains (MICs: 0.5-2 µg/mL), even higher than that of
ethambutol for MAC ISS 486 and MAC AN3. It is of
relevance that 2a also displays a comparable activity
with the reference drug against all MT strains, result-
ing, indeed, in the most active compound both against
MAC and MT strains.
F igu r e 3. Plot of clogP vs -log MMIC for compounds 2a -
2n .
P r elim in a r y Toxicity a n d P h a r m a cok in etic
Eva lu a tion
Among the more active compounds (2a -l), two de-
rivatives have been selected for a preliminary toxicity
and pharmacokinetic study. To evaluate the influence
of the hydrophobicity properties on the pharmacokinetic
profile, N-ethyl and N-heptyl-1,2-dihydro-2-thioxo-3-
pyridinecarbothioamides (2c and 2i respectively) have
been considered. These compounds have, in fact, differ-
ent cLogP values (namely 1.92 for 2c and 4.08 for 2i)
but similar and positive biological activity.
Toxicity. A single dose of compounds 2c and 2i was
orally administered to rats and mice (5 males and 5
females/species/group/dose) at a dose ranging from 200
to 2000 mg/kg. Intraperitoneal administration was only
performed at the dose of 200 mg/kg, being that a higher
dose would not be applicable because of the low solubil-
ity of the compounds. Observations of mortality and
anomalous clinical signs were made during 14 days but
no clinical effects and mortality were observed.
According to these experimental conditions, LD50 after
intraperitoneal administration of compounds 2c and 2i
is higher than 200 mg/kg, for both mice and rats. No
toxic effects were in fact observed at the dose of 200 mg/
kg i.p. The lack of toxicity of the compounds following
oral administration is probably due to their poor bio-
availability by this route (see Pharmacokinetic Studies).
P h a r m a cok in etic Stu d ies. The pharmacokinetic
evaluation of 2c and 2i has been performed after
intravenous and oral administration to rats (20 mg/kg).
The analysis of 2c and 2i was performed from plasma
samples, by gas chromatography-mass spectrometry.
Further procedure details are reported in the Experi-
To verify the existence of a correlation between the
lipophilicity apported to the moiety by the different
N-alkyl side chains and the biological activity, the
relationship between hydrophobicity of the derivatives
and anti-Mycobacterium activity has been evaluated. An
attempt to find a relationship between the calculated
LogP20 (Table 2) and the -log MMIC (expressed in
mmol/mL) of compounds 2a -2n (chain length: C1-C10)
for all strains considered resulted in a quadratic fit
equation with r values higher than 0.92. The best
correlation has been found for MAC ATCC 15769. The
equation is reported below, and the curve fitting is
depicted in Figure 3.
-log MMIC ) -0.2224(LogP)2 + 1.4341 LogP - 0.03439
(r ) 0.992; S ) 0.113; n ) 10)
The curves obtained present a maximum of antitu-
bercular activity for clogP values between 2.4 and 4.1
(MICs: 0.5-4 µg/mL). Molecules having higher clogP
values (chain length C8-C18) have lower activities,
probably because of the interference of long alkyl chains
with cellular lipophilic materials.

202 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Pagani et al.
Ta ble 3. Pharmacokinetic Values of 2c and 2i after i.v. Administration to Rats
AUC0-∞
compd
t
1/2λ (min)
Cl (L/min)
Vss (L)
MRT (min)
(mg min/L)
2c
2i
37.18 ( 19.71
28.0 ( 15.6
0.02 ( 0.007
0.05 ( 0.03
0.93 ( 0.34
1.28 ( 0.51
47.65 ( 14.52
28.9 ( 14.4
1110 ( 384
494.8 ( 193.7
mental Section, while the pharmacokinetic parameters
are reported in Table 3. After intravenous administra-
tion of 2c, the elimination half-life (t1/2λ) was 37.18 min
and the clearance (Cl) and steady-state volume of
distribution (Vss) were 0.02 L/min and 0.93 L/kg, re-
spectively. The area under the plasma concentration-
time curve from zero to infinity (AUC0-∞) was 1110 mg
min/L, and the mean residence time (MRT) was 47.65
min. Concerning 2i intravenous administration, the
elimination half-life (t1/2λ) was 28.0 min and the clear-
ance (Cl) and steady-state volume of distribution (Vss)
were 0.05 L/min and 1.28 L/kg, respectively. The area
under the plasma concentration-time curve from zero
to infinity (AUC0-λ) was 494.8 mg min/L, and the mean
residence time (MRT) was 28.9 min. These data show
that both compounds are rapidly eliminated from the
body; however, the results obtained indicate that the
less lipophilic compound 2c shows a higher bioavail-
ability, compared with the N-heptyl analogue 2i, after
intravenous administration.
hypothesized that the compounds are well orally ab-
sorbed, but undergo significant first-pass metabolism,
leading to poor or undetectable oral bioavailability.
Oxidative metabolism of both sulfur substituents (as in
thiobarbiturates and ethionamide) or oxidative N-dealkyl-
ation is highly likely. The higher bioavailability after
intravenous administration of the less lipophilic N-ethyl
derivative 2c, in comparison with the N-heptyl analogue
2i, indicates that an increased water solubility (or a
decreased lipophilicity) is a likely desirable property in
order to improve the pharmacokinetic profile. Further
studies are in progress and will be the matter of
forthcoming papers.
Exp er im en ta l Section
Ch em istr y. All reagents are commercial reagent grade and
were purchased from their suppliers and used without further
purification. Melting points were measured using a Kofler hot-
1
stage apparatus and are uncorrected. H NMR were recorded
at 300 MHz using a Bruker ACE-300 spectrometer in CDCl3
1
or DMSO-d6. H chemical shift (δ) were reported with Me4Si
After oral administration of 2c and 2i to the rats, drug
levels in the plasma samples were under detection level.
In some samples, the presence of the compounds was
detectable under quantification levels.
(δ ) 0.00 ppm) as internal standard. The following abbrevia-
tions are used: br ) broad, s ) singlet, d ) doublet, dd )
double doublet, t ) triplet, and m ) multiplet. Mass spectra
were obtained on a Finnigan MAT 8222 spectrometer via the
direct inlet. Electron ionization was performed at 70 eV and
0.5 mA with a source temperature of 250 °C. Elemental
analyses were within (0.4% of the theoretical values and were
performed on a Carlo Erba 1106 elemental analyzer. All
reactions were monitored by thin-layer chromatography car-
ried out on 0.25 mm Merck silica gel (60 F254) and visualized
by UV light (λ ) 264 or 365 nm); flash chromatography was
performed using silica gel 60 (60-200 µm, Merck). Compound
3a ,b was prepared as previously reported.18
Con clu sion s
All synthesized compounds generally display a posi-
tive activity against Mycobacterium tuberculosis and
Mycobacterium avium complex strains, both standard
(MAC ATCC 15769, MT H37-Rv ATCC) and isolated
from infected patients (MAC ISS 486, MAC AN1, 2, 3;
MT1, 2). The significant anti-mycobacterial activity
observed indicate that these substances could be con-
sidered as important leads for new potential anti-
Mycobacterium drugs development.
Gen er a l P r oced u r e for 1,2-Dih yd r o-2-th ioxo-3-p yr i-
d in eca r both ioa m id e Der iva tives, 2a -2p . A mixture of 3H-
1,2-dithiolo[3,4-b]pyridine-3-thione 1 (16 mmol) and the ap-
propriate amine (19 mmol) was refluxed in 150 mL of ethanol
for 30-60 min, the optimum reaction time being determined
by TLC monitoring (eluent: hexane/ethyl acetate). After
cooling, the solvent was removed under reduced pressure and
the residue was recrystallized from the appropriate solvent.
Analytical data of compounds 2a ,e,f are consistent with those
reported in the previous paper.18
N-Met h yl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in eca r b ot h io-
a m id e, 2b. This compound was obtained as yellow crystals
(ethyl acetate/hexane), mp 130-133 °C: 1H NMR (CDCl3) δ
3.40 (d, 3H, CH3, J ) 5.0 Hz), 6.99 (dd, 1H, 5-H, J 4,5 ) 6.0 Hz,
J 6,5 ) 7.5 Hz), 7.72 (dd, 1H, 4-H, J 4,5 ) 6.0 Hz, J 6,4 ) 1.5 Hz),
9.41 (dd, 1H, 6-H, J 6,5 ) 7.5 Hz, J 6,4 ) 1.5 Hz), 10.6 and 12.5
(br s, 2H, NH and SH); MS m/z (% ra) 184 (14), 182 (100), 118
(62), 103 (7), 91 (14), 78 (15). Anal. (C7H8N2S2) C, H, N.
N -E t h yl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in e ca r b ot h io-
a m id e, 2c. This compound was obtained as yellow crystals
(ethyl acetate/petroleum ether), mp 150-152 °C: 1H NMR
(CDCl3) δ 1.45 (t, 3H, CH3, J ) 7.5 Hz), 3.90 (m, 2H, N-CH2,
J ) 5.0 Hz), 6.98 (dd, 1H, 5-H, J 6,5 ) 7.5 Hz, J 4,5 ) 6.0 Hz),
7.70 (dd, 1H, H-4, J 4,5 ) 6.0 Hz, J 6,4 ) 1.5 Hz), 9.39 (dd, 1H,
6-H, J 6,5 ) 7.5 Hz, J 6,4 ) 1.5 Hz), 12.4 (br s, 2H, NH and SH);
MS m/z (% ra) 198 (38), 196 (100), 181 (31), 168 (46), 155 (23),
137 (22), 104 (70), 77 (12). Anal. (C8H10N2S2) C, H, N.
N-H yd r oxyet h yl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in eca r -
both ioa m id e, 2d . This compound was obtained as yellow
crystals (2-propanol/ethanol/water), mp 151-153 °C: 1H NMR
(DMSO-d6) δ 3.70 (m, 2H, N-CH2, J ) 5.0 Hz), 3.80 (m, 2H,
In vitro antibacterial activity of compounds 2a and
2g against MAC strains appears to be promising: MICs
are considerably low, and these compounds deserve
further attention. Activity against MT of compounds 2c
and 2i is interesting. It is, indeed, very similar to the
activity shown by ethambutol. However, in vitro drug
susceptibility testing against first-line drug-resistant
strains is mandatory to confirm good activity shown on
drug-susceptible ones. Compound 2a exhibits a broad
anti-mycobacterial activity covering both MAC and MT.
Furthermore, the preliminary studies of toxicity after
intraperitoneal administration carried out for two de-
rivatives, the N-ethyl and the N-heptyl compounds (2c
and 2i), are very favorable. However, the pharmacoki-
netic data obtained following intravenous administra-
tion of the two analogues to rats indicate that they are
rapidly cleared (either hepatically or renally or both),
while the data obtained after oral administration,
appear to be not significative because of the very low
levels detected. This means that neither the ethyl nor
the heptyl derivatives (2c and 2i, respectively) have
suitable properties for oral or parenteral administration.
Since no urinary or fecal analyses or analyses for
potential metabolites have been performed, it could be

Syntheses of Antimycobacterial Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2 203
CH2OH, J ) 5.0 Hz), 4.85 (t, 1H, OH), 7.00 (m, 1H, 5-H, J 6,5
) 7.5 Hz, J 4,5 ) 6.0 Hz), 7.90 (dd, 1H, H-4, J 4,5 ) 6.0 Hz, J 6,4
) 1.5 Hz), 8.55 (dd, 1H, 6-H, J 6,5 ) 7.5 Hz, J 6,4 ) 1.5 Hz), 12.01
(s, 1H, NH), 13.97 (s, 1H, SH); MS m/z (% ra) 214 (26), 196
(21), 181 (100), 168 (55), 152 (44), 137 (59), 104 (75), 92 (17),
77 (16). Anal. (C8H10N2S2) C, H, N.
N-P en t yl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in eca r b ot h io-
a m id e, 2g. This compound was obtained as yellow crystals
(ethyl acetate/hexane), mp 123-126 °C: 1H NMR (CDCl3) δ
3.90 (m, 2H, N-CH2, J ) 5.0 Hz), 6.95 (dd, 1H, 5-H, J 6,5 ) 7.5
Hz, J 4,5 ) 6.0 Hz), 7.67 (dd, 1H, 4H, J 4,5 ) 6.0 Hz, J 6,4 ) 1.5
Hz), 9.42 (dd, 1H, 6-H, J 6,5 ) 7.5 Hz, J 6,4 ) 1.5 Hz), 12.3 and
12.45 (br s, 2H, NH and SH); MS m/z (% ra) 240 (30), 205 (70),
181 (24), 168 (100), 155 (20), 137 (34), 104 (48), 77 (14), 55
(16). Anal. (C11H16N2S2) C, H, N.
N-Cycloh exyl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in eca r b o-
th ioa m id e, 2h . This compound was obtained as yellow
crystals (water), mp 151-155 °C: 1H NMR (DMSO-d6) δ 1.20-
2.10 (m, 11H, cyclohexyl), 4.40 (m, 2H, N-CH2, J ) 5.0 Hz),
6.95 (dd, 1H, 5-H, J 6,5 ) 7.5 Hz, J 4,5 ) 6.0 Hz), 7.85 (dd, 1H,
4H, J 4,5 ) 6.0 Hz, J 6,4 ) 1.5 Hz), 8.50 (dd, 1H, 6-H, J 6,5 ) 7.5
Hz, J 6,4 ) 1.5 Hz), 12.10 (s, 1H, NH), 13.92 (s, 1H, SH); MS
m/z (% ra) 250 (22), 168 (100), 137 (7), 104 (15), 83 (26), 77
(7), 55 (50), 41 (29). Anal. (C12H16N2S2) C, H, N.
N-H ep t yl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in eca r b ot h io-
a m id e, 2i. This compound was obtained as yellow crystals
(ethanol/water), mp 106-108 °C: 1H NMR (CDCl3) δ 3.82 (m,
2H, N-CH2, J ) 5.0 Hz), 6.92 (br t, 1H, 5-H), 7.82 (br s, 1H,
4-H), 9.30 (d, 1H, 6-H), 9.50 and 12.38 (br s, 2H, NH and SH);
MS m/z (% ra) 268 (23), 233 (100), 203 (38), 181 (47), 168 (80),
137 (36), 104 (50), 57 (75). Anal. (C13H20N2S2) C, H, N.
N -Oct yl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in e ca r b ot h io-
a m id e, 2l. This compound was obtained as yellow crystals
(ethanol/water), mp 101-103 °C: 1H NMR (CDCl3) δ 3.85 (m,
2H, N-CH2, J ) 5.0 Hz), 6.99 (br t, 1H, 5-H), 7.70 (br s, 1H,
4-H), 9.38 (d, 1H, 6-H), 12.28 (br s, 1H, SH); MS m/z (% ra)
282 (4), 280 (16), 247 (100), 203 (26), 168 (91), 154 (33), 104
(24), 57 (35). Anal. (C14H22N2S2) C, H, N.
N -Non yl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in e ca r b ot h io-
a m id e, 2m . This compound was obtained as yellow crystals
(ethanol/water), mp 105-108 °C: 1H NMR (DMSO-d6) δ 3.90
(m, 2H, N-CH2, J ) 5.0 Hz), 7.00 (t, 1H, 5-H), 7.90 (d, 1H,
4-H), 9.40 (d, 1H, 6-H), 8.70 and 12.50 (br s, 2H, NH and SH);
MS m/z (% ra) 296 (4), 261 (100), 203 (36), 168 (83), 154 (9),
137 (16), 104 (24), 57 (36). Anal. (C15H24N2S2) C, H, N.
N -De cyl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in e ca r b ot h io-
a m id e, 2n . This compound was obtained as yellow crystals
(ethanol/water), mp 111-114 °C: 1H NMR (CDCl3) δ 3.86 (m,
2H, N-CH2, J ) 5.0 Hz), 6.98 (dd, 1H, 5-H, J 4,5 ) 6.0 Hz, J 6,5
) 7.5 Hz), 7.67 (dd, 1H, 4-H, J 4,5 ) 6.0 Hz, J 6,4 ) 1.5 Hz), 9.40
(d, 1H, 6-H), 12.19 and 12.41 (br s, 2H, NH and SH); MS m/z
(% ra) 310 (51), 275 (100), 203 (23), 168 (81), 137 (78), 104
(22), 57 (58). Anal. (C16H26N2S2) C, H, N.
N-H exa d ecyl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in eca r b o-
th ioa m id e, 2o. This compound was obtained as yellow
crystals (2-propanol), mp 94-97 °C: 1H NMR (DMSO-d6) δ
3.65 (m, 2H, N-CH2, J ) 5.0 Hz), 6.97 (t, 1H, 5-H), 7.88 (d,
1H, 4-H), 8.45 (d, 1H, 6-H), 11.96 and 13.93 (br s, 2H, NH
and SH); MS m/z (% ra) 394 (17), 359 (100), 240 (13), 203 (30),
160 (40), 137 (18), 104 (8), 57 (23). Anal. (C22H38N2S2) C, H,
N.
N -Oct a d e cyl-1,2-d ih yd r o-2-t h ioxo-3-p yr id in e ca r b o-
th ioa m id e, 2p . This compound was obtained as yellow
crystals (ethanol/water), mp 103-105 °C: 1H NMR (DMSO-
d6) δ 3.65 (m, 2H, N-CH2, J ) 5.0 Hz), 6.94 (dd, 1H, 5-H),
7.87 (dd, 1H, 4-H), 8.45 (dd, 1H, 6-H), 12.80 (br s, 1H, SH);
MS m/z (% ra) 422 (2), 387 (100), 240 (13), 203 (30), 160 (40),
137 (18), 104 (8), 57 (23). Anal. (C24H42N2S2) C, H, N.
3H-1,2-P yr id in d ith iol-3-on e, 4. A suspension of pyridin-
1,2-dithiol-3-thione (0.84 mol) in 300 mL of chloroform was
added, at room temperature, to a suspension of mercuric
acetate (0.18 mol) in 700 mL of glacial acetic acid. The reaction
was monitored by TLC (hexane/ethyl acetate 80:20) until
disappearance of the starting material. The raw material
was filtered through Celite and the clear solution obtained was
evaporated under vacuum to yield the product as a pure white
solid (yield: 98%), mp 94-96 °C: 1H NMR δ (CDCl3) 7.40 (dd,
1H, H-5, J 5,4 ) 7.5 Hz, J 5,6 ) 5.0 Hz), 8.23 (dd, 1H, H-4, J 4,5
7.5 Hz, J 4,6 ) 1.5 Hz), 8.85 (dd, 1H, H-6 J 6,5 ) 5.0 Hz, J 6,4
)
)
1.5 Hz); MS m/z (% ra) 169 (100) 141 (30), 105 (18), 77 (14).
Anal. (C6H3S2NO) C, H, N.
N-Hexyl-1,2-d ih yd r o-2-th ioxo-3-p yr id in eca r boa m id e,
5. A solution of 3H-1,2-dithiole[3,4-b]pyridin-3-one 4 (3.5
mmol) and n-hexylamine (4.2 mmol) in 20 mL of ethanol was
refluxed for 1 h, then the solvent was removed under reduced
pressure, and the residue was purified through flash chroma-
tography (CHCl3/acetone 80:20). This compound was obtained
as yellow crystals (ethanol/water), mp 108-112 °C: 1H NMR
(CDCl3) δ 3.48 (m, 2H, N-CH2, J ) 5.0 Hz), 6.91 (dd, 1H, 5-H,
J 6,5 ) 7.5 Hz, J 4,5 ) 6.0 Hz), 7.69 (dd, 1H, 4-H, J 4,5 ) 6.0 Hz,
J 6,4 ) 1.7 Hz), 8.82 (dd, 1H, 6-H, J 6,5 ) 7.5 Hz, J 6,4 ) 1.7 Hz),
10.78 (bs, 1H, NH o SH); MS m/z (% ra) 238 (68), 205 (30),
165 (14), 154 (12), 138 (45),111 (39), 100 (100), 67 (10). Anal.
(C12H18N2OS) C, H, N.
N-Hexylisoth ia zolo[5,4-b]p yr id in -3(2H)-on e, 6. An etha-
nolic solution of iodine (6%) was added dropwise to a stirred
mixture of N-hexyl-1,2-dihydro-2-thioxo-3-pyridinecarboamide
5 (6 mmol) and sodium hydrogen carbonate (10 mmol) in 100
mL of ethanol, until a persistent brown color is noted. A
precipitate was removed by filtration and washed with dichlo-
romethane. Collected organic phases were evaporated under
reduced pressure, and the residue was purified through flash
chromatography (hexane/ethyl acetate 70:30) to give pure
product (36% yield). Analytical data are in agreement with
those previously reported.18
Micr obiology. Stock solutions were made in dimethyl
sulfoxide (DMSO). Working solutions, whose concentrations
were 40-fold greater than the desired concentrations, were
prepared from stock solutions in sterile distilled water. It has
been verified that DMSO did not suppress or delay M. avium
or M. tuberculosis strains’ growth when added undiluted
(producing 5% concentration in the medium). Ethambutol was
employed as reference drug.
Ra d iom etr ic Meth od . The growth of bacteria was recorded
radiometrically by using the BACTEC 460-TB system (Becton
Dickinson, Sparks, USA). Growth in 7H12 liquid medium
(Becton Dickinson) containing 14C-labeled palmitic acid leads
to the consumption of this substrate, with subsequent release
of 14CO2 in the confined atmosphere above the medium.21 The
BACTEC instrument detects the amount of 14CO2 and records
it as a growth index (GI) on a scale from 0 to 999.
MIC Deter m in a tion . Determination of MIC for MAC
strains was performed as previously reported.17 Determination
of MIC for MT was performed according to the protocol
reported.22
Toxicity. Compounds 2c and 2i were suspended in car-
boximethylcellulose and orally administered (by gastric gav-
age) as a single dose to Sprague-Dawley rats and CD1 mice
(5 males and 5 females/species/group/dose) at a dose ranging
from 200 to 2000 mg/kg and by intraperitoneal administration
at the single dose of 200 mg/kg diluted in saline (higher doses
were not applicable because of the low solubility of the
compounds). The control group of mice and rats (5 males and
5 females/species/group) was treated with carboximethylcel-
lulose or saline by oral and i.p. routes, respectively. Observa-
tions of mortality and of anomalous clinical signs were made
15 min and 1, 2, and 4 h after treatment and every day during
the study (14 days).
P h a r m a cok in etic Stu d ies. Compounds (2c and 2i) were
dissolved in 1 mL of poly(ethylene glycol) 400. The animals
(rats Wistar, male, 250-300 g body weight) received a single
dose (20 mg/kg) of both compounds by intravenous or oral route
through a gastric tube. Intravenous drug administration and
blood samples were collected through a permanent cannula
inserted into the jugular vein. Plasma was obtained by
centrifugation and stored until analysis.
The analysis of 2c and 2i was performed from plasma
samples by gas chromatography-mass spectrometry. Col-

204 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 2
Pagani et al.
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Carrier gas: highly purified helium (0.5 mL/min). Column
temperatures: initial temperature, 100 °C for 3 min and then
10 °C/min to the final temperature of 300 °C. Injector tem-
perature: 220 °C. Detector temperature: 280 °C. Electron
impact: energy: 70 eV.
Compound levels in the plasma samples were determined
by monitoring the following ions: 233, 181, 168, 137, 104.
Pharmacokinetic parameters were determined according to a
noncompartmental analysis by using the WINNONLIN pro-
gram.23 The parameters were obtained from the usual rela-
tionship.24
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Controlled trial of 4 three-times-weekly regimens and a daily
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Ack n ow led gm en t. Financial support for this re-
search by the Istituto Superiore Sanita`-Roma (Progetto
Tubercolosi) is gratefully acknowledged. We thank Dr.
Pierangela Villani for her help in chemical synthesis.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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