Antimycobacterial activity of 5-arylidene aromatic derivatives of hydantoin

Article abstract of DOI:10.1016/S0014-827X(01)01194-6

Various 5-(chlorobenzylidene)-2-isoniazido and 5-(chlorobenzylidene)-2-amino substituted derivatives of imidazoline-4-one were synthesized and evaluated in the primary assay for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Eight of them exhibited >90% inhibition in the primary screening at 12.5 μg/ml. For these primarily selected compounds the actual MIC and IC₅₀ values were determined. Two of the isoniazid derivatives, for which MIC≤3.13 μg/ml and SI>10, were selected for further screening and investigated for efficacy in vitro in a TB-infected macrophage model. The most promising compound, 5-(3-chlorobenzylidene)-2-(isonicotinoylhydrazino)-imidazoline-4-one, with activity in vitro comparable with rifampin (MIC=0.8 μg/ml, SI>78) was tested in vivo in the animal tuberculosis model but exhibited insignificant activity. For several compounds the primary screening of antimycobacterial activity against Mycobacterium avium (ATCC 25291) was conducted as well, but none of them demonstrated satisfactory activity.

Full text of DOI:10.1016/S0014-827X(01)01194-6

Il Farmaco 57 (2002) 355–362
www.elsevier.com/locate/farmac
Antimycobacterial activity of 5-arylidene aromatic derivatives of
hydantoin
Ewa Szyman´ska, Katarzyna Kiec´-Kononowicz *
Jagiellonian Uni6ersity Medical College, Faculty of Pharmacy, Department of Chemical Technology of Drugs, Pl 30-688 Krako´w,
Medyczna 9, Poland
Received 18 December 2000; accepted 13 December 2001
Abstract
Various 5-(chlorobenzylidene)-2-isoniazido and 5-(chlorobenzylidene)-2-amino substituted derivatives of imidazoline-4-one were
synthesized and evaluated in the primary assay for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv.
Eight of them exhibited \90% inhibition in the primary screening at 12.5 mg/ml. For these primarily selected compounds the
actual MIC and IC50 values were determined. Two of the isoniazid derivatives, for which MIC53.13 mg/ml and SI\10, were
selected for further screening and investigated for efficacy in vitro in a TB-infected macrophage model. The most promising
compound, 5-(3-chlorobenzylidene)-2-(isonicotinoylhydrazino)-imidazoline-4-one, with activity in vitro comparable with rifampin
(MIC=0.8 mg/ml, SI\78) was tested in vivo in the animal tuberculosis model but exhibited insignificant activity. For several
compounds the primary screening of antimycobacterial activity against Mycobacterium a6ium (ATCC 25291) was conducted as
´
well, but none of them demonstrated satisfactory activity. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights
reserved.
Keywords: 5-Z-Arylideno hydantoins; 5-Chlorobenzylidene-2-aminoimidazoline-4-ones; Antimicrobial activity; Antimycobacterial activity
1. Introduction
mechanism conferring INH resistance, are not com-
pletely explained yet [6,8–10]. Isoniazid has bacterio-
static and bactericidal activity against M. tuberculosis
and also against strains resistant to other antimy-
cobacterial drugs. For these reasons the antimycobac-
terial pharmacophore moiety of INH is willingly
introduced in different molecules to improve their ac-
tivity against drug-sensitive strains and to fight against
multi-drug resistant Mycobacteria.
The group of various 5-chlorobenzylidene deriva-
tives of imidazoline-4-one with structure I was previ-
ously synthesized and evaluated in the primary assay
for their antimycobacterial activity [11–14]. Four of
those compounds exhibited \90% inhibition of M.
tuberculosis growth and were selected for further tests.
Now we present the synthesis and in vitro screening of
new imidazoline-4-one derivatives, possessing amine or
isoniazid residue (structures I and II). For active
compounds from this and previous group [13]
results of further assays against M. tuberculosis are
described.
Research programs into the discovery of new an-
timycobacterial drugs and improving their evaluation
criteria are under way in many laboratories [1–6].
Increasing resistance of Mycobacterium tuberculosis to
currently available therapy and the large number of
epidemic infections due to Mycobacterium a6ium com-
plex have become an important health problem in
several countries [7]. The knowledge of specific con-
stituents of a mycobacterial cell and their biochemical
roles as well as the knowledge of the mechanism of
the action of available drugs have advanced consider-
ably in recent years and may permit a more rational
design of new drugs acting on specific targets.
One of the most active drugs, isoniazid (INH), con-
tinues to be well established for the treatment of tu-
berculosis. The mechanism of its action, as well as the
* Corresponding author.
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec´-Kononowicz).
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(01)01194-6

356
E. Szyman´ska, K. Kiec´-Kononowicz / Il Farmaco 57 (2002) 355–362
2. Chemistry
The synthesis of compounds 1–6 is presented in
Scheme 1. The starting 5-(2-/, 3-/, 4-chlorobenzylidene)-,
5-(2,4-/, 2,6-dichlorobenzylidene)-2-thiohydantoins
were prepared in the Knovenagel condensation of the
appropriate benzaldehydes with 2-thiohydantoin ac-
cording to the described procedure [15].
In the reaction of thiohydantoin derivatives with
methyl iodide the intermediate methylthio-products
were prepared and reacted with benzylamine or isoni-
azid to give the designed compounds. The reactions
were carried out in toluene. Other cited compounds
(7–10) were already described elsewhere [11–13].
Scheme 1.

E. Szyman´ska, K. Kiec´-Kononowicz / Il Farmaco 57 (2002) 355–362
357
3. Experimental
isolated by suction and recrystallized from acetic acid
(1–5) or methanol (6).
3.1. Chemistry
3.1.2.1. 5-(2-Chlorobenzylidene)-2-(isonicotinoylhydra-
zino)-imidazoline-4-one (1). Yield 35%, m.p. 270–273
°C, Rf=0.29, H NMR l (ppm) 6.56 (s, 1H, ArꢀCHꢁ),
7.28–7.48 (m, 2H, Har-4, Har-5), 7.49 (def.d, 1H, J=
7.1 Hz, Har-3), 7.78 (br.s, 2H, Har-3%, Har-5%), 8.30 (br.s,
1H, Har-6), 8.73 (def.d, 2H, J=5.8 Hz, Har-2%, Har-6%),
10.81 (br.s, 1H, NH), 11.47 (br.s, 2H, N3H, NHCO);
IR w (cm−1): 3348 (ꢀNH), 1724, 1691 (CꢁO), 1645
(CꢁN), 1628 (ArꢀCHꢁ). Anal. (C16H12O2N5Cl×1.5
CH3COOH): C, H, N.
3.1.1. Material and methods
1
The chemical structures of the obtained compounds
were confirmed by elemental and spectral analyses (IR,
1H NMR). IR spectra were recorded with FT/IR-410
Spectrophotometer (Jasco Corp., Japan) using KBr
1
discs. H NMR spectra were determined with Varian
Mercury 300 MHz spectrometer, in DMSO-d6 solution
with TMS as an internal standard. All chemical shifts
are quoted in l values. Elemental analyses (C, H, N)
were within 90.3 from the theoretical values.
The purity of the compounds was checked by thin-
layer chromatography performed with Merck silica gel
GF254 aluminum sheets, using the developing system:
2-propanol:chloroform:aqueous ammonia, 11:9:2. Spots
were detected by their absorption under UV light. The
melting points (uncorrected) were determined on Mel-
Temp melting point apparatus (Laboratory Devices
Inc., USA).
Starting materials: 5-(2-chlorobenzylidene)-, 5-(3-
chlorobenzylidene)-, 5-(4-chlorobenzylidene)-, 5-(2,6-
dichlorobenzylidene)- and 5-(2,4-dichlorobenzylidene)-
2-thiohydantoin were prepared according to the litera-
ture procedure [15]. The methods of synthesis of
the compounds: 5-(2,4-dichlorobenzylidene)-2-(2,4-
dichloroanilino)-imidazoline-4-one (7), 5-(2,4-dichloro-
benzylidene)-2-(4-chloroanilino)-imidazoline-4-one (8),
5-(2-chlorobenzylidene)-2-(3-chloroanilino)-imidazo-
3.1.2.2. 5-(3-Chlorobenzylidene)-2-(isonicotinoylhydra-
zino)-imidazoline-4-one (2). Yield 23.5%, m.p. 303–305
1
°C, Rf=0.31, H NMR l (ppm) 6.32 (s, 1H, ArꢀCHꢁ),
7.28–7.52 (m, 2H, Har-4, Har-5), 7.57–7.78 (m, 1H,
Har-6), 7.79 (def.d, 2H, J=5.8 Hz, Har-3%, Har-5%), 8.06
(br.s, 1H, Har-2), 8.74 (def.d, 2H, J=5.5 Hz, Har-2%,
Har-6%), 10.83 (br.s, 1H, NH), 11.19 (br.s, 1H, N3H),
11.69 (br.s, 1H, NHCO); IR w (cm−1): 3420 (ꢀNH),
1756, 1692 (CꢁO), 1636 (CꢁN), 1628 (ArꢀCHꢁ). Anal.
(C16H12O2N5Cl×CH3COOH): C, H, N.
3.1.2.3. 5-(4-Chlorobenzylidene)-2-(isonicotinoylhydra-
zino)-imidazoline-4-one (3). Yield 80%, m.p. 301–303
1
°C, Rf=0,41, H NMR l (ppm) 6.33 (s, 1H, ArꢀCHꢁ),
7.41 (def.d, 2H, J=7.7 Hz, Har-3, Har-5), 7.78 (def.d,
2H, J=4.9 Hz, Har-3%, Har-5%), 7.80 (br.s, 2H, Har-2,
Har-6), 8.74 (def.d, 2H, J=4.8 Hz, Har-2%, Har-6%), 10.90
(br.s, 1H, NH), 11.38 (br.s, 2H, N3H, NHCO); IR w
(cm−1): 3428 (ꢀNH), 1730, 1690 (CꢁO), 1664 (CꢁN),
1628 (ArꢀCHꢁ). Anal. (C16H12O2N5Cl×CH3COOH):
C, H, N.
line-4-one
(9)
and
5-(2-chlorobenzylidene)-2-(4-
chloroanilino)-imidazoline-4-one (10) are described in
separate publications [11,13].
3.1.2. General procedure for synthesis of: 5-(2-chloro-
benzylidene)-2-(isonicotinoylhydrazino)-imidazoline-
4-one (1), 5-(3-chlorobenzylidene)-2-(isonicotinoyl-
hydrazino)-imidazoline-4-one (2), 5-(4-chlorobenzyl-
idene)-2-(isonicotinoylhydrazino)-imidazoline-4-one (3),
5-(2,4-dichlorobenzylidene)-2-(isonicotinoylhydrazino)-
imidazoline-4-one (4), 5-(2,6-dichlorobenzylidene)-2-
(isonicotinoylhydrazino)-imidazoline-4-one (5), 5-(4-
chlorobenzylidene)-2-(benzylamino)-imidazoline-4-one
(6)
3.1.2.4. 5-(2,4-Dichlorobenzylidene)-2-(isonicotinoylhy-
drazino)-imidazoline-4-one (4). Yield 66%, m.p. 268–
270 °C, Rf=0.36, 1H NMR l (ppm) 6.88 (s, 1H,
ArꢀCHꢁ), 7.49 (d.d, 2H, J1=8.5 Hz, J2=2.0 Hz,
Har-3%, Har-5%), 7.69–7.70 (m, 3H, Har-3, Har-5, Har-6),
8.85 (def.d, 2H, J=8.5 Hz, Har-2%, Har-6%), 12.02 (br.s,
1H, NHCO); IR w (cm−1): 3461 (ꢀNH), 1762, 1703
(CꢁO), 1630 (ArꢀCHꢁ). Anal. (C16H11O2N5Cl2): C, H,
N.
To the stirred solution of sodium (0.04 mol) in 200
ml of ethanol arylidene-2-tiohydantoin (0.04 mol) and
methyl iodide (0.04 mol) were added. After stirring at
room temperature for 30 min the product was filtered
off, washed with water and dried. Methylthio derivative
was shown to be analytically pure.
A mixture containing 5 mmol of methylthio deriva-
tive of arylidene-2-thiohydantoin and 5.5 mmol of iso-
niazid or benzylamine in 30 ml of toluene was refluxed
for 9 h, and then allowed to cool. The solid was
3.1.2.5. 5-(2,6-Dichlorobenzylidene)-2-(isonicotinoylhy-
drazino)-imidazoline-4-one (5). Yield 86%, m.p. 275–
277 °C, Rf=0.27, 1H NMR l (ppm) 6.07 (s, 1H,
ArꢀCHꢁ), 7.24–7.43 (m, 3H, Har-3, Har-4, Har-5), 7.72
(def.d, 1H, J=5.8 Hz, Har-3%), 7.78 (def.d, 1H, J=5.8
Hz, Har-5%), 8.71 (def.d, 2H, J=4.7 Hz, Har-2%, Har-6%),
10.08 (br.s, 1H, N3H), 10.58 (d, 1H, NH), 11.77 (br.s,
1H, NHCO); IR w (cm−1): 3430 (ꢀNH), 1753, 1687
(CꢁO), 1639 (ArꢀCHꢁ). Anal. (C16H11O2N5Cl2): C, H,
N.

358
E. Szyman´ska, K. Kiec´-Kononowicz / Il Farmaco 57 (2002) 355–362
3.1.2.6. 5-(4-Chlorobenzylidene)-2-(benzylamino)-imida-
zoline-4-one (6). Yield 48%, m.p. 237–238 °C, Rf=
0.86, H NMR l (ppm) 4.54 (br.s, 2H, ꢀCH2) 6.26 (s,
1H, ArꢀCHꢁ), 7.23–7.48 (m, 7H, Har-3, Har-5, Har-2%,
Har-3%, Har-4%, Har-5%, Har-6%), 8.06 (def.d, 2H, J=8.5
Hz, Har-2, Har-6), 10.92 (br.s, 1H, NH); IR w (cm−1):
3341 (ꢀNH), 1702, 1672 (CꢁO), 1608 (ArꢀCHꢁ). Anal.
(C17H14ON3Cl): C, H, N.
a one-time dose/animal (oral gavage) of the compound
at concentrations of 100, 300 or 1000 mg/kg. The
compounds were dissolved in an appropriate vehicle
(DMSO), administered in a solution if necessary. Three
animals per dose were observed post-administration for
a total of 1 week. The surviving mice were sacrificed
day 7 post-administration and the critical organs were
observed for evidence of drug toxicity.
1
Mice were exposed to an aerosol of M. tuberculosis
Erdman (ATCC 35801), which deposited approximately
50 bacilli into the lungs of the animal. Test compounds
were administered to groups of mice starting on day 20
post-inoculation. The dose of drug (25 mg/kg/day) was
given intraperitoneally once a day; an additional group
was given isoniazid as a positive control. The course of
the infection and the effect of the compound were
observed in the lungs and spleen on days 35 and 50 by
plating homogenates of harvested organs on nutrient
agar and determining bacterial numbers. The data is
expressed as the log 10 protection provided by a given
dose of the compound against the growth of the organ-
ism in the untreated control group.
3.2. Microbiology
3.2.1. In 6itro e6aluation of antimycobacterial acti6ity
[16–19]
Primary screening was conducted at 12.5 mg/ml
against M. tuberculosis H37Rv (ATCC 27294; Ameri-
can Type Culture Collection, Rockville, MD) in
BACTEC 12B medium using the BACTEC 460-radio-
metric system. Compounds demonstrating at least 90%
inhibition were re-tested against M. tuberculosis H37Rv
at lower concentration to determine the actual mini-
mum inhibitory concentration (MIC) in the Microplate
Alamar Blue Assay (MABA). The MIC was defined as
the lowest concentration inhibiting 99% of the inocu-
lum. Rifampin (Sigma Chemical Compound, St. Louis,
MO) or isoniazid were included as a positive drug
control.
Compounds were tested for cytotoxicity (IC50) in
VERO cells at concentrations less than or equal to 10
times the MIC. After 72 h exposure, viability was
assessed on the basis of cellular conversion of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) into a formazan product using the Promega
CellTiter 96 Non-radioactive Cell Proliferation Assay.
Compounds for which the selectivity index SI (=IC50/
MIC) \10 were tested for killing of M. tuberculosis
Erdman (ATCC 35801) in monolayers of mouse bone
marrow macrophages at 4-fold increasing concentra-
tions equivalent to 0.25, 1, 4 and 16 times the MIC.
The activity against singly-drug-resistant M. tubercu-
losis (strain resistant to isoniazid, rifampin, ethambutol,
capreomycin and ciprofloksacin) as well as drug-sensi-
tive M. tuberculosis (H37Rv and Erdman) for investi-
gated compounds was determined.
An INH control group, administered via oral gavage
at 25 mg/kg/day was included in each study.
3.2.3. E6aluation of anti-Mycobacterium a6ium acti6ity
Primary screening was conducted against Mycobac-
terium a6ium (ATCC 25291) in Middlebrook 7H9 broth
using the Microplate Alamar Blue Assay and in the
BACTEC 460-radiometric system. Clarithromycin was
included as a positive drug control.
4. Results
In order to find a potential antimycobacterial activity
among 30 previously described [13] and 6 new amino-
and isoniazid derivatives of 5-arylidene hydantoin, the
compounds were tested according to the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility
(TAACF) screening program using the BACTEC 460
radiometric system. Eight of them (1–3, 6–10) showed
at least a 90% inhibition of growth of Mycobacterium
tuberculosis H37Rv at concentrations lower than 12.5
mg/ml, and were selected for further tests. The mini-
mum inhibitory concentrations for these compounds
were determined. The results are shown in Table 1.
Generally the monochloro substituted isoniazid
derivatives were active against M. tuberculosis at con-
centrations equal to or lower than 3.13 mg/ml. The
compound 2, 5-(3-chlorobenzylidene)-2-(isonicotinoyl-
hydrazino)-imidazoline-4-one (MIC=0.8 mg/ml), was
found to be almost as active as rifampin (MICRIF=0.5
mg/ml). The compounds 6–10 showed in vitro MIC
values versus M. tuberculosis higher than 12.5 mg/ml.
The minimum bactericidal concentration (MBC) was
determined for M. tuberculosis H37Rv, Erdman and for
the rifampin- and isoniazid-resistant strains by subcul-
turing onto drug-free solid media and enumerating
colony forming units following exposure in supple-
mented Middlebrook 7H9 media to drug concentra-
tions equivalent to and higher than the previously
determined MICs of the respective strains.
3.2.2. Tuberculosis animal model
Prior to animal screening, the maximally tolerated
dose (MTD) of an experimental compound was deter-
mined using C57BL/6 female mice by administration of

E. Szyman´ska, K. Kiec´-Kononowicz / Il Farmaco 57 (2002) 355–362
359
Table 1
Antibacterial activity against M. tuberculosis
Comp.
% Inhibition at 12.5 mg/ml of the compound
MIC vs. H37Rv (mg/ml)
IC50 (mg/ml)
SI (IC50/MIC)
1
2
3
4
5
6
7
8
9
99
99
99
0
17
97
96
100
91
99
3.13 a
0.8 b
48.56 a
\62.5 c
34.51 a
16 a
\78 c
11 a
3.13 a
\12.5 a
\12.5 a
\12.5 d
\12.5 d
\12.5 d
\12.5 d
\12.5 d
31 e
23 f
*
*
*
B2.5 e
B2 f
10
a MICRIF=0.25 mg/ml. IC50 for INH\1000; RIF=30; DMSO=0.0115.
b MIC RIF=0.5 mg/ml.
c MIC RIF=0. 25 mg/ml. IC50 for INH\1000; RIF=69; DMSO=0.0105.
d MIC RIF=0. 125 mg/ml.
e MIC RIF=0.125 mg/ml. IC50 for INH\1000; RIF=116; DMSO=0.0069.
f MIC Rifampin (RIF)=0.125 mg/ml. IC50 for isoniazid (INH)\1000; RIF=120; DMSO=0.0095.
* Insoluble in tissue culture medium, unable to test IC50
.
(MTD) is the highest one of all tested doses of 2 and it
amounts to 1000 mg/kg. In the aerosol standard mouse
model the compound 2 was considered inactive, as it
yielded only 0.08 log10 reduction in bacterial counts in
a lung tissue, as shown in Table 5. During the test the
yellow plagues on/within peritoneum-enlarged spleens
were observed.
All the compounds except 4 and 5 were screened to
assess cytotoxicity (IC50) to a VERO cell line. Unfortu-
nately, derivatives 8, 9 and 10 demonstrated very low
solubility in tissue culture medium, and the test could
not be carried out. For the remaining compounds the
selectivity index (SI) was calculated as it is presented in
Table 1.
Only compounds 6–10 were tested in the Microplate
Alamar Blue Assay (MABA) against Mycobacterium
a6ium as well, but none of them demonstrated satisfac-
tory activity as shown in Table 6. All the compounds
showed low inhibition at 12.5 mg/ml.
The isoniazid derivatives 1 and 2, for which SI\10,
were tested for efficacy in vitro in a TB-infected
macrophage model. The results are summarized in
Table 2. The columns labeled EC90 and EC99 list the
concentrations effecting 90 and 99% reduction in intra-
macrophage bacterial growth after 7 days relative to a
drug-free control. The ratio EC99/MIC compares the in
vitro activity against the bacillus to the activity against
the bacillus while it residues and flourishes within the
target host cell. This value should be less than 4 for
active agents. Under the experimental conditions the
most active compound 2 exhibited the ratio EC99/MIC
comparable with positive controls: rifampin and isoni-
azid (Table 2).
For compounds 1–3 their in vitro activity (MIC)
against five singly-drug-resistant strains was determined
in the MABA assay. All the compounds showed high
levels of activity for RMP-R, EMB-R, KM-R, and
CIP-R strains, but were much less active against the
isoniazid-resistant one (Table 3).
5. Discussion
Continuing our studies on the biological activity of
imidazoline-4-one derivatives [14], and taking into con-
sideration strong antitubercular properties of INH, we
decided to synthesize a group of 5-arylidenehydantoins
containing isoniazid or an aromatic amine substituent
at 2 positions of the hydantoin ring, with a view to
Table 2
Macrophage assay
Comp.
EC90 (mg/ml)
EC99 (mg/ml)
EC99/MIC
As shown in Table 4, the derivatives 1 and 3 have
bacteriostatic rather than bactericidal profile of activity,
as their MBC values are greater than MICs (MBC/
MICꢀ1). Only 1 demonstrated bactericidal effect
against rifampin resistant strain (MBC/MIC:1).
Compound 2 was tested in vivo in the animal tuber-
culosis model. It showed no toxicity under the experi-
mental conditions. The maximally tolerated dose
2
1
0.38
16.71
0.85
\50
1.06 a
\15.97 b
EC90 and EC99, the lowest concentration effecting a 90 and 99%
reduction in colony forming units at 7 days compared to drug-free
controls.
a EC90 RIF=0.077 mg/ml; EC99 RIF=0.46 mg/ml.
b EC90 RIF=0.11 mg/ml; EC99 RIF=0.37 mg/ml; EC90 INH=
0.21 mg/ml; EC99 INH=0.4 mg/ml.

360
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E. Szyman´ska, K. Kiec´-Kononowicz / Il Farmaco 57 (2002) 355–362
361
Table 4
Minimum bactericidal concentration (MBC) assay
Comp. H37Rv MIC (mg/ml)
H37Rv MBC (mg/ml)
H37Rv MBC/MIC
RMP-R MBC (mg/ml)
INH-R MBC (mg/ml)
1
3
51.56
3.13
50
\100
\32
\32
1.56
25
\100
\100
Table 5
In vivo activity against M. tuberculosis
Compound MTD assay
Tested doses
(mg/kg)
Highest tolerated dose
(mg/kg)
Toxicity issues
(pathology)
Solubility issues Comments
2
100
IP injection with 26 gauge needle. No
ill effects apparent
300
1000
none
suspension at all
doses
1000
Murine TB model assay
Route
Dose (mg/kg)
Log protection
Lung
Control protection a
Spleen
0.75
Lung
1.13
Spleen
2.06
IP injection
25
0.08
a Isoniazid; the results are referred to day 50.
Table 6
improving the solubility and bioavailability of active in
vitro compounds are going to be conducted.
The effectiveness of compounds 1–5 against M.
a6ium complex is now being studied.
a
Antibacterial activity against M. a6ium
Comp.
MIC (mg/ml)
Inhibition%
6
7
8
9
10
\12.5
\12.5
\12.5
\12.5
\12.5
42
11
66
53
71
Acknowledgements
Antimycobacterial data were provided by the Tuber-
culosis Antimicrobial Acquisition and Coordinating
Facility (TAACF) through a research and development
contract with US National Institute of Allergy and
Infectious Diseases. This work was partly supported by
the Polish State Committee for Scientific Research;
project Wl/102/P/F.
a MIC Clarithromycin=2 mg/ml (98% inhibition).
obtaining antimycobacterial agents. During the primary
screening the activity of these compounds against M.
tuberculosis was confirmed. In further studies, com-
pounds 1–3 possessing in their structure isoniazid
residue showed high levels of antimycobacterial activity
in vitro. However, the most effective compound 2
(MIC=0.8 mg/ml), the activity of which was compara-
ble with rifampin in macrophage assay as well, was
shown to be inactive in vivo. It is supposed that be-
cause of the very low solubility of this group of hydan-
toin derivatives, the compound 2 administered by IP
injection (in the form of suspension) was not absorbed
and was observed on/within peritoneum-enlarged
spleens in the form of yellow plagues.
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