Antimycobacterial activity of substituted isosteres of pyridine- and pyrazinecarboxylic acids

Article abstract of DOI:10.1021/jm9708745

Pyrazines and pyridines substituted with alkylated tetrazoles, esterified vinylogous carboxytic acids, and ketosulfides were synthesized as precursors of antimycobacterial agents which, after penetration of the mycobacterial cell wall, could be biotransfo

Full text of DOI:10.1021/jm9708745

2436
J . Med. Chem. 1998, 41, 2436-2438
An tim ycoba cter ia l Activity of Su bstitu ted Isoster es of P yr id in e- a n d
P yr a zin eca r boxylic Acid s
Gerald A. Wa¨chter, Matt C. Davis, Arnold R. Martin,* and Scott G. Franzblau†
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, and
GWL Hansen’s Disease Center, Baton Rouge, Louisiana 70894
Received December 31, 1997
Pyrazines and pyridines substituted with alkylated tetrazoles, esterified vinylogous carboxylic
acids, and ketosulfides were synthesized as precursors of antimycobacterial agents which, after
penetration of the mycobacterial cell wall, could be biotransformed by esterases or peroxidase-
catalases. The expected products are tetrazoles, a vinylogous carboxylic acid, and CH-acidic
ketosulfoxides, isosteres of pyrazinoic and nicotinic acids, which should inhibit mycobacterial
growth when released inside the bacterial cell. The growth inhibitory activity of the synthesized
compounds against the H37Rv strain of Mycobacterium tuberculosis was determined to assess
the viability of this concept. It was shown that all of the compounds designed as lipophilic
precursors were more active than the unmodified polar isosteres of pyrazinoic and nicotinic
acids.
In tr od u ction
resulting â-ketosulfoxide would be acidic enough to
imitate an acid function. These sulfides could, however,
not overcome drug resistance which is due to the lack
of a catalase-peroxidase. We here describe the synthe-
ses of these compounds and their activity against the
H37Rv strain of M. tuberculosis.
In 1985 the incidence of tuberculosis in the United
States increased for the first time since national record-
ing began in 1953.1 This fact and frequently observed
resistance to drugs used in the treatment of tuberculosis
make the development of new antitubercular drugs an
important task. In analogy to the antitubercular drugs
isoniazid (INH) and pyrazinamide (PZA), we assumed
that precursors of isosteres of isonicotinic and pyrazinoic
Ch em istr y
The pivaloyloxymethyl (POM) esters of the known
tetrazoles 1-310,11 (Table 1) were prepared as mixtures
of N(1)- and N(2)-alkylated isomers which were sepa-
rated by column chromatography on silica gel. Com-
parison of compounds 4, 6, and 8 with their respective
isomers 5, 7, and 9 showed that the former were the
major products, their N-CH2 protons showed reso-
nances at lower field, and their melting points were
lower. These characteristics are known to indicate
substitution at N(2).12 The reduction of the known
sulfoxides 10 and 1113 with a large excess of sodium
metabisulfite yielded the sulfides 13 and 14, while the
sulfoxide 1214 could not be reduced to its corresponding
sulfide 15 with this method.15 Instead, 15 was prepared
from bromoacetylpyrazine and sodium methyl sulfide.16
The 4-hydroxy group of the known 4-hydroxy-7-azacou-
marin17 was acetylated with acetyl chloride, benzoyl
chloride, and ethyl chloroformate to give compounds
17-19.
acids could possess antimycobacterial activity. Esters
of pyrazinoic acid have been shown to be more active
than the parent acid.2-5,6 These esters are presumably
activated by an esterase which we expected to also
activate esters of isosteres of pyrazinoic and isonicotinic
acids in a comparable way. We chose tetrazole ana-
logues of these acids and 4-hydroxy-7-azacoumarin and
attached groups which could be cleaved by esterases
within the mycobacterium. These compounds could also
overcome the observed drug resistance of certain strains
of Mycobacterium tuberculosis against PZA and INH
which has been attributed to a deficiency of nicotina-
midase or catalase-peroxidase necessary for their acti-
vation.7,8 Indeed, esters of pyrazinoic acid have been
shown to possess activity against pyrazinamide-resis-
tant isolates.2-5 In addition, we chose to synthesize
sulfides which could possibly be transformed to sulfox-
ides by the catalase-peroxidase enzyme system of M.
tuberculosis, a reaction that has been observed with
horseradish peroxidase.9 The methylene group of the
Resu lts a n d Discu ssion
The MIC values in BACTEC 6A media18 (pH ) 6) of
the tetrazoles 4-9 ranged from 13 to 105 µg/mL, the
MICs of the sulfides 13, 14, and 15 were 105, 105, and
210 µg/mL, and the MIC of the benzoyl-substituted
† GWL Hansen’s Disease Center.
S0022-2623(97)00874-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/23/1998

Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2437
Ta ble 1. Substituted Pyridines and Pyrazines 1-15 and
Azacoumarins 16-19
which makes ionization impossible and may facilitate
penetration of the cell wall, leads to compounds of
interesting activity. The tetrazoles 4-9 show up to 4
times higher in vitro activity than PZA which is cur-
rently used in the therapy of tuberculosis. Different
rates of hydrolysis may contribute to the observed
differences in activity of the alkyated tetrazoles 4-9.
These results show that the modification to less polar
precursors can increase the activity of isosteres of
pyrazinoic and pyridinecarboxylic acids. Although we
have not tested the described compounds against pyrazi-
namide-resistant strains of M. tuberculosis, it is reason-
able to expect that they should be effective against
certain strains whose resistance is due to the lack of
pyrazinamidase.
compd
X
R
mp (°C)
formulaa
1c,10 CH 3-(5-tetrazolyl)
251-253 C6H5N5
259-261 C6bH5N5
203-205 C5H4N6
2c,10 CH 4-(5-tetrazolyl)
3c,11
N
2-(5-tetrazolyl)
4
5
6
7
8
9
CH 3-[2-POM-(5-tetrazolyl)] 81-82
C12H15N5O2
CH 3-[1-POM-(5-tetrazolyl)] 100-101 C12H15N5O2
CH 4-[2-POM-(5-tetrazolyl)] 80-81
CH 4-[1-POM-(5-tetrazolyl)] 90-91
C12H15N5O2
C12H15N5O2
C11H14N6O2
N
N
2-[2-POM-(5-tetrazolyl)] 86-87
2-[1-POM-(5-tetrazolyl)] 109-110 C11H14N6O2
10c,13 CH 3-COCH2SOCH3
11c,13 CH 4-COCH2SOCH3
70-82
84-87
C8H9NO2S
C8H9NO2S
119-120 C7H8N2O2S
12c,14
13
14
N
2-COCH2SOCH3
CH 3-COCH2SCH3
CH 4-COCH2SCH3
liquid
liquid
59-61
C8H9NOS‚HCl
C8H9NOS‚HCl
C7H8N2OS
15
N
2-COCH2SCH3
H
COCH3
COPh
16c,17
17
235-237 C8H5NO3‚H2O
153-154 C10H7NO4
124-126 C15H9NO4
Exp er im en ta l Section
18
Ch em istr y. Melting points were determined on an Elec-
trothermal capillary melting point apparatus and are uncor-
rected. 1H NMR spectra were recorded on a Varian Gemini
spectrometer at 200 MHz in deuterated solvents with TMS
as internal standard. APCIMS were measured with a Finne-
gan TSQ 7000 and EIMS with a Hewlett-Packard 5988A at
200 °C, at 75 eV. Elemental analyses were performed at
Desert Analytics, Tucson, AZ, and are within 0.4% of the
calculated values except where stated otherwise (Table 1). For
column chromatography silica gel 60, 63-200 µm, Macherey
& Nagel, Du¨ren, Germany, was used.
19
CO2Et
73-76
C11H9NO5
a
Analyses were within 0.4% of the calculated values except
where noted (footnote b). C: calcd, 49.0; found, 48.4. c Reference
b
for known compound.
Ta ble 2. MIC Values of Compounds 1-19
MIC,a (µg/mL)/
(µM)
MIC,a (µg/mL)/
(µM)
compd
cmpd
pyrazinamideb
52/400
isoniazidb
<0.02/0.15
>210/1100
>210/1100
>210/1100
210/1000
210/1000
105/500
>210/1200
>210/1000
210/800
1
2
3
4
5
6
7
8
9
>210/1400
>210/1400
>210/1400
13/50
105/400
26/100
52/200
26/100
13/50
10
11
12
13
14
15
16
17
18
19
Ca u tion : The preparation of tetrazoles 1-3 according to
the cited literature involves the formation of potentially
explosive hydrazoic acid!
Gen er a l P r oced u r e for th e P r ep a r a tion of th e (P iv-
a loyloxym eth yl)tetr a zoles 4-9. A 10.0-mmol portion of the
respective tetrazole 1, 2,10 or 311 and 10.0 mmol of chloromethyl
pivalate were heated in 50 mL of dry DMF at 95 °C for 3 h.
The mixture was poured on water and extracted three times
with EtOAc. The combined extracts were washed with water,
dried with anhydrous Na2SO4, and evaporated in vacuo. The
residue was crystallized from petroleum ether at -20 °C. The
mother liquors were concentrated to give a second crop. The
combined materials were chromatographed on silica gel with
EtOAc or EtOAc-petroleum ether mixtures to obtain the pure
isomers which were crystallized from petroleum ether (PE) or
petroleum ether/ethyl ether mixtures (PE/DEE). 4: yield 73%
>210/900
a
Determined in BACTEC 6A; see the Experimental Section for
b
description of MIC determination. For comparison.
coumarin 19 was 210 µg/mL (Table 2). The MIC values
of all other compounds were greater than 210 µg/mL,
limited solubility prohibiting an accurate determination.
All values were determined using 2-fold dilutions and
vary within one dilution. For compounds whose MICs
were above 210 µg/mL, the percentage of inhibition of
M. tuberculosis at 1600 µMs4 times the MIC of pyrazin-
amideswas determined to allow for comparison of their
activities. The unsubstituted tetrazoles 1-3, as already
reported by others,11,19 exhibited only minimal activity
(33-50% inhibition of growth indices relative to drug-
free controls). The sulfoxides 10-12 were devoid of any
activity. In the azacoumarin series the acetate 17 and
the ethyl carbonate 18 (82% and 83%) showed activity
only slightly above that of the weakly active unsubsti-
tuted 4-hydroxy-7-azacoumarin (16) (39%). Pyrazinoic
acid showed 71% inhibition under these conditions.
As expected, the tetrazoles 1-3, the sulfoxides 10-
12, and the 4-hydroxy-7-azacoumarin show only insig-
nificant or no inhibition of M. tuberculosis at all, which,
we believe, is due to their structural relationship to the
polar heteroaromatic acids isonicotinic and pyrazinoic
acids which impedes penetration of the mycobacterial
cell wall. The results also show that modification of
these compounds to more lipophilic precursors can
indeed increase their activity. However, only the trans-
formation of 1-3 to the N-substituted tetrazoles 4-9,
1
(PE); H NMR (CDCl3) δ 1.24 (s, 9H), 6.52 (s, 2H), 7.43-7.50
(m, 1H), 8.46 (dt, 1H, J ) 2.0/7.8 Hz), 8.75 (dd, 1H, J ) 1.5/
1
4.8 Hz), 9.42 (d, 1H, J ) 2.1 Hz). 5: yield 7% (PE/DEE); H
NMR (CDCl3) δ 1.22 (s, 9H), 6.35 (s, 2H), 7.57 (dd, 1H, J )
4.8/8.0 Hz), 8.20 (dd, 1H, J ) 2.0/8.0 Hz), 8.88 (dd, 1H, J )
1
1.6/4.8 Hz), 9.06 (d, 1H, J ) 2.2 Hz). 6: yield 71% (PE); H
NMR (CDCl3) δ 1.24 (s, 9H), 6.56 (s, 2H), 8.06 and 8.81
(AA′BB′, 4H, J ) 6.0 Hz). 7: yield 7% (PE/DEE); 1H NMR
(CDCl3) δ 1.21 (s, 9H), 6.38 (s, 2H), 7.75 and 8.90 (AA′BB′,
1
4H, J ) 5.8 Hz). 8: yield 41% (PE/DEE); H NMR (CDCl3) δ
1.13 (s, 9H), 6.82 (s, 2H), 8.71 (dd, 1H, J ) 1.5/2.5 Hz), 8.82
(m, 1H), 9.63 (d, 1H, J ) 1.5 Hz). 9: yield 29% (PE/DEE); 1H
NMR (CDCl3) δ 1.23 (s, 9H), 6.60 (s, 2H), 8.74-8.80 (m, 2H),
9.51 (d, 1H, J ) 1.5 Hz).
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Su lfid es
13 a n d 14. A 6.0-g (32.8 mmol) portion of the respective
sulfoxide 10 or 1113 was dissolved in 150 mL of water and
heated to 70 °C; 47 g of Na2S2O5 (250 mmol) was added with
vigorous stirring. After 30 min the reaction mixture was
cooled to ambient temperature, diluted with 300 mL of water,
and extracted three times with 100-mL portions of CH2Cl2.
The combined extracts were dried with MgSO4. The solvent
was evaporated and the liquid residue purified by distillation
at reduced pressure. Hydrochlorides were prepared by addi-
tion of HCl to a solution of the free bases in dry diethyl ether.
1
13: yield 37%; H NMR (CDCl3) δ 2.13 (s, 3H), 3.77 (s, 2H),
7,45 (t, 1H, J ) 5.8 Hz), 8.29 (d, 1H, J ) 5.9 Hz), 8.80 (d, 1H,

2438 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Notes
1
(2) Cynamon, M. H.; Klemens, S. P.; Chou, T.-S.; Gimi, R. H.; Welch,
J . T. Antimycobacterial Activity of a Series of Pyrazinoic Acid
Esters. J . Med. Chem. 1992, 35, 1212-5.
J ) 5.6 Hz), 9.21 (s, 1H). 14: yield 44%; H NMR (CDCl3) δ
2.10 (s, 3H), 3.69 (s, 2H), 7.75 and 8.82 (AA′BB′, 4H, J ) 5.7
Hz).
(3) Cynamon, M. H.; Gimi, R. H.; Gyenes, F.; Sharpe, C. A.;
Bergmann, K. E.; J an, H.-J .; Gregor, L. B.; Rapolu, R.; Luciano,
G.; Welch, J . T. Pyrazinoic Acid Esters with Broad Spectrum in
vitro Antimycobacterial Activity. J . Med. Chem. 1995, 38, 3902-
7.
(4) Speirs, R. J .; Welch, J . T.; Cynamon, M. H. Activity of n-Propyl
Pyrazinoate against Pyrazinamide Resistant Mycobacterium
tuberculosis: Investigations into Mechanism of Action of and
Mechanism of Resistance to Pyrazinamide. Antimicrob. Agents
Chemother. 1995, 39, 1269-71.
(5) Yamamoto, S.; Toida, I.; Watanabe, N.; Ura, T. In Vitro Anti-
mycobacterial Activities of Pyrazinamide Analogues. Antimicrob.
Agents Chemother. 1995, 39, 2088-91.
(6) Heifets, L. B.; Flory, M. A.; Lindholm-Levy, P. J . Does Pyrazinoic
Acid as an Active Moiety of Pyrazinamide Have Specific Activity
against Mycobacterium tuberculosis? Antimicrob. Agents Chemoth-
er. 1989, 33, 1252-4.
(7) Butler, W. R.; Kilburn, J . O. Susceptibility of Mycobacterium
tuberculosis to Pyrazinamide and its Relationship to Pyrazina-
midase Activity. Antimicrob. Agents Chemother. 1983, 24, 600-
1.
(8) Heym, B.; Zhang, Y.; Poulet, S.; Young, D.; Cole, S. T. Charac-
terization of the KatG Gene Encoding a Catalase-Peroxidase
Required for the Isoniazid Susceptibility of Mycobacterium
tuberculosis. J . Bacteriol. 1993, 175, 4255-9.
(9) Kobayashi, S.; Nakano, M.; Goto, T.; Kimura, T.; Schaap, A. P.
An Evidence of the Peroxidase Dependent Oxygen Transfer from
Hydrogen Peroxide to Sulfides. Biochem. Biophys. Res. Commun.
1986, 135, 166-71.
(10) McManus, J . M.; Herbst, R. M. Tetrazole Analogues of Pyridi-
necarboxylic Acids. J . Org. Chem. 1959, 24, 1462-3.
(11) Kushner, S.; Dalalian, H.; Sanjurjo, J . L.; Bach, F. L., J r.; Safir,
S. R.; Smith, V. K., J r.; Williams, J . H. Experimental Chemo-
therapy of Tuberculosis. II. The Synthesis of Pyrazinamides and
Related Compounds. J . Am. Chem. Soc. 1952, 74, 3617-21.
(12) Butler, R. N. Tetrazoles. In Comprehensive Heterocyclic Chem-
istry; Katriztky, A. R., Rees, C. W., Potts, K. T., Eds.; Pergamon
Press Inc.: New York, 1984; Part 4A, Vol. 5, pp 791-838.
(13) Freedman, H. H.; Fox, A. E.; Shavel, J ., J r.; Morrison, G. C.
Oxisuran: A Differential Inhibitor of Cell Mediated Hypersen-
sitivity. Proc. Soc. Exp. Biol. Med. 1972, 139, 909-12.
(14) Alvarez-Ibarra, C.; Cuervo-Rodrigues, R.; Fernandes-Monreal,
M. C.; Ruiz, M. P. Synthesis, Configurational Assignment, and
Conformational Analysis of â-Hydroxy Sulfoxides, Bioisosteres
of Oxisuran Metabolites, and Their O-Methyl Derivatives. J .
Org. Chem. 1994, 59, 7284-91.
2-(Meth ylth ioa cetyl)p yr a zin e, 15. R-Bromoacetylpyra-
zine was prepared from acetylpyrazine by bromination in
glacial acetic acid at 50 °C. The crude product was chromato-
graphed on silica gel with a CH2Cl2/EtOAc mixture; 1.18 g (5.9
mmol) of this material was dissolved in 4 mL of MeOH, and a
solution of 413 mg (5.9 mmol) of sodium methylthiolate in 4
mL of MeOH was added at room temperature. After 1 h 10
mL of water was added, and the product was extracted with
CH2Cl2. The crude product was chromatographed on silica gel
with a CH2Cl2/MeOH mixture. Recrystallization from hexanes
gave the product as pale-yellow crystals: yield 25%; 1H NMR
(CDCl3) δ 2.15 (s, 3H), 3.90 (s, 2H), 8.65 (s, 1H), 8.75 (s, 1H),
9.32 (s, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of th e 4-Su b-
stitu ted 7-Aza cou m a r in s 17-19. 4-Hydroxy-7-azacoumarin
was obtained according to Dejardin and Lapiere;18 3.3 mmol
of triethylamine was added to a suspension of 3.0 mmol of 16
in 15 mL of CH2Cl2. After the starting material was dissolved
completely, 10 mL of petroleum ether was added, and the
mixture was cooled on an ice bath; 3.6 mmol of acetyl chloride
or benzoyl chloride in 3 mL of cold petroleum ether was added
slowly. The resulting slurry was stirred for 30 min, then the
ice bath was removed, and stirring was continued for another
30 min. The solvents were removed in vacuo, and the residue
was stirred with 50 mL of ice water. The crude product was
obtained by filtering. It was dried over CaCl2 and recrystal-
lized from EtOAc or EtOAc/petroleum ether. For the synthesis
of 19: The reaction with ethyl chloroformate was carried out
at - 20 °C; 100 mL of cold diethyl ether was added to the
mixture which was then slowly brought to room temperature.
After filtration the residue was treated with water and
extracted with diethyl ether. The combined ethereal solutions
were dried, concentrated to 40 mL, and left in the freezer for
15 h. The solution was filtered; evaporation of the filtrate gave
the slightly yellow product. 17: yield 74% (EtOAc); 1H NMR
(CDCl3) δ 2.48 (s, 3H), 6.80 (s, 1H), 7.52 (d, 1H, J ) 5.1 Hz),
8.57 (d, 1H, J ) 5.1 Hz), 8.78 (s, 1H). 18: yield 86% (EtOAc/
PE); 1H NMR (CDCl3) δ 6.90 (s, 1H), 7.57-7.65 (m, 4H), 7.73-
7.77 (m, 1H), 8.20-8.26 (m, 1H), 8.59 (d, 1H, J ) 5.2 Hz), 8.83
1
(s, 1H). 19: yield 38%; H NMR (CDCl3) δ 1.46 (t, 3H, J )
7.2 Hz), 4.44 (q, 2H, J ) 7.2 Hz), 6.94 (s, 1H), 7.63 (d, 1H, J )
5.2 Hz), 8.58 (d, 1H, J ) 5.2 Hz), 8.78 (s, 1H).
Deter m in a tion of Biologica l Activity. The compounds
were tested for inhibition of M. tuberculosis H37Rv ATCC 27294
using the BACTEC 460 system as previously described.18
Percent inhibition was calculated as 1 - (growth index of test
sample/growth index of control) × 100. The minimum inhibi-
tory concentration is defined as the lowest concentration which
inhibited 99% of the inoculum.
(15) Russel, G. A.; Sabourin, E. T. â-Keto-Sulfoxides. IV. Conversion
into â-Keto Sulfides, Vinyl Ethers, and Enol Acetates. J . Org.
Chem. 1969, 34, 2336.
(16) Garc´ıa-Ruano, J . L.; Pedregal, C.; Rodriguez, J . H. Synthesis
and Conformational Analysis of some Oxisuran Metabolites and
their O-Methylderivatives. Tetrahedron 1987, 43, 4407-16.
(17) Dejardin, J .-V.; Lapiere, C.-L. Synthesis in the Domain of the
Azacoumarins III. Hydroxy-4-aza-5-coumarin and hydroxy-4-
aza-7-coumarin. Bull. Soc. Chim. Fr. 1979, 279-98.
(18) Collins, L. A.; Franzblau, S. G. Microplate Alamar Blue Assay
versus BACTEC 460 System for High Throughput Screening of
Compounds Against Mycobacterium tuberculosis and Mycobac-
terium avium. Antimicrob. Agents Chemother. 1997, 41, 1004-
9.
Ack n ow led gm en t. This work was funded by the
Arizona Disease Control Research Commission, Phoe-
nix, AZ, Contract 9617, and Intraagency Agreement
Y-02-AI-30123 with the National Institutes of Allergy
and Infectious Disease, National Institutes of Health.
The authors thank Anita Biswas for her reliable techni-
cal assistance in the determination of the bioactivities.
(19) Browner-Van Stratten, B.; Solinger, D.; Van de Westeringh, C.;
Veldstra, H. Tetrazole Analogues of Physiologically or Pharma-
cologically Active Carboxylic Acids. Recl. Trav. Chim. Pays Bas
1958, 77, 1129-34.
Refer en ces
(1) CDC. Tuberculosis Morbidity-United States, 1994. MMWR -
Morbidity and Mortality Weekly Report 1995, 44, 387-9 and 395.
J M9708745