In vitro antimycobacterial activity of 5-chloropyrazinamide.
ABSTRACT 5-Chloropyrazinamide and 5-chloropyrazinoic acid were evaluated for in vitro activity against Mycobacterium tuberculosis, Mycobacterium bovis, and several nontuberculous mycobacteria by a broth dilution method. 5-Chloropyrazinamide was more active than pyrazinamide against all organisms tested. It is likely that this agent has a different mechanism of action than pyrazinamide.
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ABSTRACT: A series of pyrazinamide derivatives with alkylamino substitution was designed, synthesized and tested for their ability to inhibit the growth of selected mycobacterial, bacterial and fungal strains. The target structures were prepared from the corresponding 5-chloro (1) or 6-chloropyrazine-2-carboxamide (2) by nucleophilic substitution of chlorine by various non-aromatic amines (alkylamines). To determine the influence of alkyl substitution, corresponding amino derivatives (1a, 2a) and compounds with phenylalkylamino substitution were prepared. Some of the compounds exerted antimycobacterial activity against Mycobacterium tuberculosis H37Rv significantly better than standard pyrazinamide and corresponding starting compounds (1 and 2). Basic structure-activity relationships are presented. Only weak antibacterial and no antifungal activity was detected.Bioorganic & medicinal chemistry letters 01/2013; · 2.65 Impact Factor
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ABSTRACT: To develop new potential antimycobacterial drugs, a series of pyrazinamide derivatives was designed, synthesized and tested for their ability to inhibit the growth of selected mycobacterial strains (Mycobacterium tuberculosis H37Rv, Mycobacterium kansasii and two strains of Mycobacterium avium). This Letter is focused on binuclear pyrazinamide analogues containing the -CONH-CH2- bridge, namely on N-benzyl-5-chloropyrazine-2-carboxamides with various substituents on the phenyl ring and their comparison with some analogously substituted 5-chloro-N-phenylpyrazine-2-carboxamides. Compounds from the N-benzyl series exerted lower antimycobacterial activity against M. tuberculosis H37Rv then corresponding anilides, however comparable with pyrazinamide (12.5-25μg/mL). Remarkably, 5-chloro-N-(4-methylbenzyl)pyrazine-2-carboxamide (8, MIC=3.13μg/mL) and 5-chloro-N-(2-chlorobenzyl)pyrazine-2-carboxamide (1, MIC=6.25μg/mL) were active against M. kansasii, which is naturally unsusceptible to PZA. Basic structure-activity relationships are presented.Bioorganic & medicinal chemistry letters 04/2013; · 2.65 Impact Factor
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ABSTRACT: Analogs of pyrazinamide (=pyrazine-2-carboxamide; PZA), an essential component of short-course antituberculous chemotherapy, such as 5-chloropyrazinamide (5-Cl-PZA) act as competitive inhibitors of NADPH binding to purified mycobacterial fatty acid synthase I (FAS I) as shown by Saturation Transfer Difference (STD) NMR studies. In addition, pyrazinoic acid esters (POE) and 5-Cl-POE reversibly bind to FAS I with the relatively greater affinity of longer-chain esters for FAS I, clear from the STD amplification factors. The competitive binding of PZA and 5-Cl-PZA clearly illustrates that both agents bind FAS. In contrast to PZA, at low NADPH concentrations 5-Cl-PZA is a cooperative inhibitor of NADPH binding.Chemistry & Biodiversity 11/2012; 9(11):2582-96. · 1.81 Impact Factor
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
Copyright © 1998, American Society for Microbiology
Feb. 1998, p. 462–463 Vol. 42, No. 2
In Vitro Antimycobacterial Activity of 5-Chloropyrazinamide
MICHAEL H. CYNAMON,1* ROBERT J. SPEIRS,1AND JOHN T. WELCH2
Veterans Affairs Medical Center, Syracuse, New York 13210,1and Department of Chemistry,
SUNY at Albany, Albany, New York 122222
Received 21 July 1997/Returned for modification 2 October 1997/Accepted 1 December 1997
5-Chloropyrazinamide and 5-chloropyrazinoic acid were evaluated for in vitro activity against Mycobacterium
tuberculosis, Mycobacterium bovis, and several nontuberculous mycobacteria by a broth dilution method. 5-Chlo-
ropyrazinamide was more active than pyrazinamide against all organisms tested. It is likely that this agent has
a different mechanism of action than pyrazinamide.
Pyrazinamide (PZA) is a first-line agent for the treatment of
tuberculosis (1, 4) and an essential element of experimental
preventive therapy regimens (6, 9). PZA appears to function as
a prodrug of pyrazinoic acid (PA) and is converted to PA
intracellularly. The biochemical basis for the antituberculosis
activity of PA has not been established (7).
It is known that the majority of Mycobacterium tuberculosis
isolates resistant to PZA in vitro have low levels of pyrazin-
amidase activity, as do Mycobacterium bovis isolates (8, 10–12).
PZA-susceptible and -resistant isolates are generally suscepti-
ble to PA in vitro, but PA is not active in vivo (5). A series of
esters of PA and 5-substituted PA have been found to have
enhanced in vitro activity against both PZA-susceptible and
-resistant M. tuberculosis as well as against PZA-resistant M.
bovis, Mycobacterium kansasii, and Mycobacterium avium iso-
lates (2, 3). The aim of this study was to evaluate the in vitro
activity of 5-chloro-PZA (5-Cl PZA) and 5-Cl PA against var-
ious mycobacterial isolates, including PZA-resistant M. tuber-
PZA was obtained from Sigma Chemical Company, St.
Louis, Mo. PA was obtained from Aldrich Chemical Company,
Milwaukee, Wis. 5-Cl PZA and 5-Cl PA were synthesized from
5-chloropyrazinoyl chloride. 5-Cl PZA was obtained as follows:
to 30 ml of NH4OH, 3.55 g (20 mmol) of 5-Cl-pyrazinoyl
chloride in 25 ml of dry tetrahydrofuran was added at 0°C over
a 30-min period. After the addition was complete, the reaction
mixture was stirred for another 30 min. The reaction mixture
was diluted with 30 ml of ether, and the formed precipitate was
filtered. The filtercake was washed with 30 ml of ether, and the
filtrate was separated. The aqueous layer was extracted twice
with 20 ml of ether each time, and the combined organic layer
was dried over MgSO4. After filtration and evaporation of the
solvent, the crude product was recrystallized from EtOH. The
yield was 78.6%. The melting point was 206 to 210°C, infrared
3,400, 3,436, 1,700 cm?1,1H NMR (CDCl3? 9.16 [J ? 1.6 Hz,
d,1H], 8.53 [J ? 1.6 Hz, d], 7.5 [br, 1 H], 5.82 [br, 2H]). 5-Cl
PZA and 5-Cl PA were ?95% pure.
Stock solutions were prepared by dissolving each compound
in modified 7H10 broth (7H10 agar formulation with agar and
malachite green omitted), pH 5.8, with 10% oleic acid-albu-
min-dextrose-catalase (OADC) enrichment (Difco Laborato-
ries, Detroit, Mich.) at a concentration of 2,048 ?g/ml. Stock
solutions were sterilized by passage through a 0.22-?m-pore-
size membrane filter. Stock solutions of PA and 5-Cl PA were
adjusted to pH 5.8 with 1 N KOH prior to sterilization. Serial
twofold dilutions of each compound were made in modified
7H10 broth (concentrations ranged from 2,048 to 0.5 ?g/ml).
Strains of M. tuberculosis (ATCC 27294, ATCC 35801, and
ATCC 35828), M. bovis (ATCC 35720 and ATCC 27289),
Mycobacterium smegmatis (ATCC 19420), and Mycobacterium
fortuitum (ATCC 49403) were obtained from the American
Type Culture Collection, Rockville, Md. Isolates of PZA-re-
sistant M. tuberculosis were kindly provided by Salman Siddiqi
(Becton Dickinson Diagnostic Instrument Systems, Sparks,
Md.). M. avium strain 101 (serotype 1) was provided by Lowell
Young (Kuzell Institute for Arthritis and Infectious Diseases,
California Pacific Medical Center Research Institute, San
Francisco, Calif.). M. avium ATCC 49601 (serotype 1) is a
clinical isolate from a patient with AIDS at State University of
New York Health Science Center, Syracuse, N.Y. M. kansasii
strain S was a clinical isolate from a patient at the Veterans
Affairs Medical Center, Syracuse, N.Y.
Mycobacteria were grown in modified 7H10 broth, pH 6.6,
with 10% OADC enrichment and 0.05% Tween 80 (13). Cell
suspensions were diluted in modified 7H10 broth, pH 5.8, to
yield 1 Klett unit of M. tuberculosis, M. bovis, and M. smegmatis
per ml and 0.1 Klett unit of M. avium, M. kansasii, and M.
fortuitum per ml (Klett-Summerson colorimeter; Klett Manu-
facturing, Brooklyn, N.Y.) or approximately 5 ? 105CFU/ml.
A 0.1-ml volume of culture suspension was added to each tube
containing drug in 1.9 ml of modified 7H10 broth, pH 5.8,
yielding a final inoculum of approximately 2.5 ? 104CFU/ml.
Susceptibility testing was performed with modified 7H10 broth,
pH 5.8, because some isolates of M. tuberculosis grow poorly at
pH 5.6, the standard pH used for susceptibility testing in agar.
Inoculum size was determined by titration and counting from
duplicate 7H10 agar plates (BBL Microbiology Systems, Cock-
eysville, Md.). A tube without drug was included for each
isolate as a positive control. Tubes were incubated on a rotary
shaker (190 rpm) at 37°C for 24 h to 2 weeks. The MIC was
defined as the lowest concentration of drug that yielded no
The broth dilution MICs of PZA, 5-Cl PZA, PA, and 5-Cl
PA for the M. tuberculosis isolates (n ? 7) are shown in Table
1. The MIC ranges of PZA and 5-Cl PZA were from 32 to
?2,048 ?g/ml and from 8 to 32 ?g/ml, respectively. The MIC
ranges of PA and 5-Cl PA were from 16 to 64 ?g/ml and from
64 to 256 ?g/ml, respectively. The MICs of 5-Cl PZA and PA
for M. tuberculosis are more favorable than are those of PZA
and 5-Cl PA. PZA-resistant isolates retain susceptibility in
vitro to 5-Cl PZA, PA, and 5-Cl PA, suggesting that 5-Cl PZA
can circumvent the requirement for activation by mycobacte-
* Corresponding author. Mailing address: Department of Medicine,
Veterans Affairs Medical Center, 800 Irving Ave., Syracuse, NY 13210.
Phone: (315) 477-4597. Fax: (315) 424-6233. E-mail: CYNAMON
rial amidase. The MICs of 5-Cl PZA for nontuberculous my-
cobacteria are lower than those of 5-Cl PA, PZA, or PA. The
activity against M. avium is noteworthy, particularly in light of
the poor activity of 5-Cl PA.
The presumption that PZA is a prodrug for PA is supported
by previous studies (3, 8). The lower MICs of PA relative to
those of PZA for M. tuberculosis are consistent with this hy-
pothesis. While the mechanism of action of PA remains to be
defined, assumptions based upon the effect of PA increasing
the intracellular pH are confounded by the observation that
5-Cl PA is significantly less effective than PA against M. tuber-
culosis. The largest difference, an eightfold increase in the MIC
of 5-Cl PA relative to that of PA, is found with organisms such
as ATCC 35828, which are resistant to PZA and deficient in
When the activity of PZA relative to 5-Cl PZA is considered,
these organisms are more susceptible to the substituted com-
pound. If PZA is activated by hydrolysis to PA, inhibition is not
likely to be based upon acidification by PA acting as a proton
donor. According to the Hammet relationship, 5-Cl PA should
be a stronger acid and therefore a more potent inhibitor than
PA. It is unclear whether 5-Cl PZA has a different mechanism
of action than PZA or whether it functions as a prodrug with
an alternative method of activation. The hypothesis that 5-Cl
PZA has an alternative activation pathway is not consistent
with the observation that 5-Cl PA is less effective than PA
against the same organisms.
This study was supported in part by the NCDDG-0I program, co-
operative agreement U19-AI40972 with NIAID.
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TABLE 1. MICs of pyrazinamide analogs for various mycobacteria
MIC (?g/ml) of:
PZA 5-Cl PZA PA5-Cl PA
M. tuberculosis strain
M. bovis strain
M. kansasii S
M. smegmatis 19420
M. fortuitum 49403
M. avium 49601
VOL. 42, 1998NOTES 463