Synthesis, antimycobacterial activities and cytotoxicity on V79 of 3-[4′-Y-(1,1′-biphenyl)-4-yl]-N,N-dimethyl-3-(4-X-phenyl)-2- propen-1-amine derivatives

Article abstract of DOI:10.1016/S0223-5234(01)01263-6

The derivatives of 3-(4′-bromo-[1,1′-biphenyl]-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2- propen-1-amine (5a-m) were synthesised through a Friedel-Crafts acylation followed by Wittig reaction. The effects of the compounds on standard strains of Mycobacterium sp. (ATCC) and M. tuberculosis isolated from clinical specimens were evaluated. Also the toxicity was determined on V79 cells line using neutral red uptake (NRU), nucleic acid content (NAC) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to measure the cellular viability.

Full text of DOI:10.1016/S0223-5234(01)01263-6

Eur. J. Med. Chem. 36 (2001) 843–850
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01263-6/FLA
Laboratory note
Synthesis, antimycobacterial activities and cytotoxicity on V79 of
3-[4%-Y-(1,1%-biphenyl)-4-yl]-N,N-dimethyl-3-(4-X-phenyl)-2-propen-1-amine
derivatives
Ana O. de Souzaa, Rubens R. Santos Ju´niorb, Joa˜o F. Ferreira-Ju´lioa, Jaime A. Rodriguezc,
Patr´ıcia S. Melod, Marcela Haund, Daisy N. Satob, Nelson Dura´na,e*
aInstituto de Qu´ımica, Biological Chemistry Laboratory, Universidade Estadual de Campinas, C.P. 6154,
CEP 13083-970 Campinas, S.P., Brazil
bInstituto Adolfo Lutz, Ribeira˜o Preto, S.P., Brazil
cUniversidad de Talca, Talca, Chile
dCell Culture Laboratory, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, S.P., Brazil
eNCA, Universidade de Mogi das Cruzes, Mogi das Cruzes, S.P., Brazil
Received 6 March 2001; revised 23 July 2001; accepted 31 July 2001
Abstract – The derivatives of 3-(4%-bromo-[1,1%-biphenyl]-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine (5a–m) were synthe-
sised through a Friedel–Crafts acylation followed by Wittig reaction. The effects of the compounds on standard strains of
Mycobacterium sp. (ATCC) and M. tuberculosis isolated from clinical specimens were evaluated. Also the toxicity was determined
on V79 cells line using neutral red uptake (NRU), nucleic acid content (NAC) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
´
zolium bromide (MTT) reduction to measure the cellular viability. © 2001 Editions scientifiques et me´dicales Elsevier SAS
synthesis / cytotoxicity / V79 cells / antimycobacterial activity / 2-propen-1-amine
1. Introduction
Moreover, these conventional antitubercular agents
are also known to exhibit only restricted activity
against nontuberculosis mycobacteria. AIDS is
greatly accompanied by the development of oppor-
tunistic infections, particularly those involving the
Mycobacterium avium complex. Also, it is very
difficult to treat the infections caused by the M. avium
complex because of its diversified drug resistance pat-
terns to the majority of the antimycobacterial agents
employed in clinics [3].
In a preliminary communication, we showed the
antimycobacterial activity of the derivatives 5a and 5c
in figure 1 [4]. In this study, new derivatives 5i–m
were synthesized and the whole series (5a–m) (figure
1) were evaluated against several strains of mycobac-
teria. The antimycobacterial activities were evaluated
through a microplate Alamar Blue assay [5] and the
toxicity of the compounds 5j–m was determined on
V79 cells line [6].
Tuberculosis caused by members of the Mycobac-
terium tuberculosis complex is an infectious disease
that has damaged humanity since the beginning of the
century. An extra complication was detected in the
presence of multidrug-resistant or synergism with the
human acquired immune deficiency syndrome (AIDS)
epidemic. At this moment, there is no new drug
generation able to eliminate the bacillus, and the
World Health Organization has reported the increase
in the number of infections and mortality [1]. In fact,
mycobateria are known to easily acquire resistance to
streptomycin, isoniazid, ethambuthol, pirazinamide
and rifampicin, the most effective agents used in
tuberculosis chemotherapy [2].
* Correspondence and reprints
E-mail address: duran@iqm.unicamp.br (N. Dura´n).

844
A.O. de Souza et al. / European Journal of Medicinal Chemistry 36 (2001) 843–850
4. Results and discussion
2. Chemistry
4.1. Chemistry
2-Propen-1-amine derivatives 5 were prepared by
Friedel–Crafts reaction between 4-bromo-biphenyl
and the corresponding benzoyl chloride to give the
ketones 3a–m and by subsequent Wittig reaction of
the latter with b-(N,N-dimethylamino)ethyltriphenyl-
phosphonium bromide (figure 1) [7].
4.1.1. Synthesis
The compound 1i was prepared in a 50 mL flask
containing 7.2 mmol of 4-acetoxybenzoic acid; 27 mmol
of thionyl chloride was added and the mixture was
refluxed under stirring for 6 h. The excess thionyl chlo-
ride was removed by distillation under vacuum to re-
cover the 4-acetoxybenzoic acid chloride (1i) [8]. The
ketone 3i was synthesized by a classical Friedel–Crafts
reaction between the corresponding acid chloride (1i)
and 4-bromobiphenyl (2) (figure 1). Briefly, the acid
chloride 1a–i (2 mmol) was added dropwise under agita-
tion at room temperature to a solution containing 4-
bromobiphenyl (1.72 mmol), AlCl3 (3 mmol) and CS2 (4
mL). The mixture was refluxed for 7 h and cooled to
room temperature by addition of crushed ice. The sol-
vent was distilled and the reaction product was ex-
tracted with CH2Cl2 (100 mL) and treated with
saturated solution of Na2CO3 for 3i. The organic layer
was separated, dried over MgSO4, filtered and evapo-
rated under reduced pressure to give the solid crystalline
product 3i. The purification was performed through
re-crystallisation with CH2Cl2–hexane. Compound 3m
was prepared using 1.36 mmol of 3g and refluxed with
5.27 mmol of AlCl3 and 7 mL of benzene for 4 h under
stirring [9]. The reaction was stopped with water addi-
tion and acidified with HCl. The product 3m was ex-
tracted with CH2Cl2 and purified by re-crystallisation
with CH2Cl2–hexane. Compound 5i was prepared in a
flask containing b-dimethylamino ethyl triphenyl phos-
3. Biology
The mycobacteria type strains and the M. tubercu-
losis isolated from clinical specimens were subcultured
on Lowenstein–Jensen medium, and subcultured in
Middlebrook 7H9 broth medium, until a bacterial
density corresponding to a 1.0 McFarland turbidity
standard. The suspensions were further diluted 1:25 in
Middlebrook 7H9 broth medium before the inocula-
tion (4×105 mycobacteria mL−1) [4].
The cytotoxicity effect expressed as cell viability
was assayed on a permanent lung fibroblast cell line
derived from Chinese hamsters (V79). V79 fibroblasts
were grown as monolayers in Dulbecco’s modification
of Eagle’s medium (DMEM), supplemented with
heat-inactivated foetal calf serum, penicillin and
streptomycin in a humidified incubator with CO2 in
air. At the end of incubation, three independent end-
points for cytotoxicity were evaluated: 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), nucleic acid content (NAC) and neutral red
uptake (NRU) [6].
Figure 1. General procedure for the synthesis of compounds 5a–m. (A) Friedel–Crafts reaction with reflux during 7 h; and (B)
Wittig reaction overnight.

A.O. de Souza et al. / European Journal of Medicinal Chemistry 36 (2001) 843–850
845
Table I. Qualitative analysis of bromine from the compound
3m by EDXRF.
For the compounds 3i and 5i, there is a characteristic
peak at l 2.35 and 2.43 due to the hydrogens of their
acetyl group and the compound 5l showed a broad band
at l 3.81 due to the hydrogens of the amine group. For
3m, the NMR showed the absence of the hydrogen of
the methoxyl group (ꢀOCH3) (present in 3g) and in the
IR spectra, the band at 1256.4 cm−1 attributed to the
ꢀOCH3 group of 3g was absent and band at 3322.9
cm−1 was observed due to hydroxyl group of 3m. Al-
though the compound 3m has been characterized by
1H-NMR and MS, a further technique, EDXRF, was
used in order to confirm the results. The MS showed the
absence of the isotopes of bromine, which was present in
3g (table I).
Element
Concentration (%)
Error
Al
P
Cl
Ca
Fe
Cu
Zn
Br
1.149
0.124
0.038
0.016
0.001
0.003
0.001
0.641
98.028 (difference)
0.0680
0.0103
0.0026
0.0005
0.0001
0.0001
0.0001
0.0028
–
OCHN
phonium bromide (2.15 mmol) and THF (25 mL) and a
solution of n-butyllithium in 2.5 mmol hexane (2.21
mmol) 0 °C with stirring under argon atmosphere. After
30 min, the ketone 3i was added to the mixture and the
reaction left overnight at room temperature with stir-
ring. The products 5i was extracted with CH2Cl2 and
purified by percolation in a silica gel column and by
TLC using ethyl acetate–CH3OH 5% as eluent. Com-
pound 5j was prepared with 0.4 mmol of 5i, and
refluxed for 4–5 h with 5 mL of potassium methoxide
(Scheme 3). Compound 5j was obtained through an
extraction with CH2Cl2 in acid solution. The solvent was
removed by distillation under vacuum and 5j was
purified by TLC using ethyl acetate–CH3OH 5%–tri-
ethylamine 0.4% as eluent. Compound 5l was prepared
by reaction of 0.11 mmol of 5e, 6 mmol of iron (Fe0)
and 25 mL of an alcoholic solution EtOH–HCl (2
mol L−1), and refluxed for 6 h [10]. The solution was
alkalinised to pH 8.3 with an alcoholic solution of KOH
15% and 38 mL of an alcoholic solution of H2SO4 (3
mol L−1) was added. After filtration and distillation of
the EtOH the organic product 5l was extracted with
CH2Cl2. The product was purified first by percolation in
a silica gel column and by TLC in silica gel using ethyl
acetate–CH3OH 5% as eluent. Two other tentative ap-
proaches to synthesize 5l were carried out by reduction
of 5e with NaBH2S3 [11] and by high-pressure hydro-
genation in ethanolic solution for 30 min [12] with Pd/C
10%, but in both cases, the reactions were ineffective.
The structural data to the new compounds 3i and
5i–m were similar to those of 3a–h and 5a–h, respec-
EDXRF showed that the bromine concentration in
the sample of 3m was insignificant as shown in table I.
The elements OCHN represented 98.028% (by differ-
ence) of the composition of the sample (3m−C19H14O2).
If bromine is present in the molecule (C19H14BrO2) it
would be 22.6% and not 0.641 as detected. Probably,
this insignificant percentage is related to a residue
present in the sample.
The geometric isomers E and Z of the compounds 5a
and 5c were prepared with a good yield as previously
reported and were now evaluated on mycobacteria
strains [14] (table III).
4.2. Biology
The susceptibility patterns of the M. tuberculosis iso-
lated from clinical specimens are listed in table II. From
the 12 tested strains only one resistant strain to INH
(ALI 258), one resistant to INH and RFP (ALI 3082)
and one resistant to INH, EMB, RFP and SM (3415)
were found. The others strains were totally susceptible
to the classical drugs used in tuberculosis chemotherapy.
One week after inoculation, the results from visually
determined MICs were obtained using Alamar Blue dye
which is a general indicator of cellular growth and/or
viability; the blue, nonfluorescent, oxidized form, be-
comes pink and fluorescent upon reduction.
The antimycobacterial activities of the all compounds
are summarised in table III. Compounds 5a and 5c with
their respective geometric isomers showed good antimy-
cobacterial activity against all the tested strains, as
compared EMB, table IV), mainly on the isolates of
clinical specimens. For the clinical specimens strains (ALI
strains) studied, the compounds 5a–e and 5j–m showed
an MIC between 4 and 49 mmol L−1. Since the com-
pound 5f exhibited very low antimycobacterial activities
1
tively. In the H-NMR spectra, there are peaks at the
aromatic region associated with the hydrogen of the
phenyl and biphenyl rings. The groups ꢀCH and ꢀCH2
are responsible for the two triplets and two doublets due
to the presence of the geometric isomers E and Z [7].

846
A.O. de Souza et al. / European Journal of Medicinal Chemistry 36 (2001) 843–850
(around 68–79 mmol L−1) and 5h (around 34–68
mmol L−1) on standard mycobacteria strains no mea-
surements with the ALI strain were done. The values for
5g and 5i, which are similar to 5f, are not presented in
table III. It is interesting to note that the isomers 5aE,
5aZ, 5cE and 5cZ showed activities similar to that of
the 5a and 5c showing that there is no advantage in the
use of the isolated isomers, at least for the in vitro
assays. M. avium, M. malmoense, M. celatum and M.
intracellulare were less susceptible to the compounds
which exhibited higher MIC values than the clinical
specimen ones. This result was expected since nontuber-
Table II. Susceptibility patterns of clinical isolates of M. tuberculosis (n=12).
Strains
Drugs and tested concentrations (mg mL−1
)
PZA 200
INH 0.2
EMB 2
RFP 40
SM 4
ALI 3415
ALI 258
ALI 3082
ALI 3236
ALI 282
ALI 536
ALI 909
ALI 544
ALI 452
ALI 3342
ALI 552
ALI 329
S
S
S
S
S
S
S
S
S
S
S
S
R
R
R
S
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
S
S
S
R
S
R
S
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
S
S
S
ALI, Adolfo Lutz Institute; S, fully susceptible, R, resistant; PZA, pirazinamid; SM, streptomycin; EMB, ethambutol; INH,
isoniazid; RFP, rifampicin.
Table III. MIC (mmol L−1) of compounds 5a–m against M. tuberculosis H37Ra (H37Ra), M. tuberculosis H37Rv (H37Rv), M.
kansasii (Mk), M. a6ium (Ma), M. malmoense (Mm), M. cellatum (Mc), M. intracellulare (Mi) and strains of clinical specimens of
M. tuberculosis (identified by numbers).
Strains
Compound
5a 5aE
20.0
5aZ
10.0
5b
5cE
34.0
8.5
34.0
34.0
34.0
34.0
68.0
17.0
5cZ
17.0
5c
34.0
5d
5e
5f
5h
5j
5l
19.6
9.8
19.6
19.6
78.0
78.0
78.0
ND
5m
H37Ra
H37Rv
Mk
Ma
Mm
Mc
Mi
ALI 3415
ALI 282
ALI 536
ALI 909
ALI 544
ALI 452
ALI 3342
ALI 552
ALI 258
ALI 3082
ALI 329
ALI 3236
10.0
10.0
40.0
40.0
80.0
40.0
80.0
40.0
55.0
55.0
20.0
20.0
55.0
10.0
20.0
55.0
20.0
ND
5.0
5.0
9.5 36.5 79.0 68.0 39.0
19.0 36.5 79.0 34.0 5.0
38.0 73.0 79.0 68.0 39.0
19.0 73.0 79.0 68.0 39.0
9.5 73.0 79.0 68.0 39.0
38.0 36.5 79.0 68.0
19.0 73.0 79.0 68.0 78.0
49.0
6.0
10.0
20.0
40.0
80.0
40.0
40.0
40.0
55.0
55.0
10.0
10.0
55.0
ND
10.0
20.0
40.0
40.0
20.0
40.0
40.0
55.0
55.0
20.0
20.0
55.0
10.0
20.0
55.0
40.0
ND
4.0
34.0
34.0
34.0
34.0
68.0
34.0
54.0
54.0
17.0
17.0
54.0
8.5
8.5
34.0
34.0
17.0
17.0
68.0
34.0
54.0
54.0
17.0
17.0
54.0
8.5
14.0
37.5
37.5
5.0
18.7
ND
55.0 54.0
55.0 54.0
ND
ND
55.0 54.0
ND
ND
55.0
ND
ND
98.0
98.0
98.0
48.6
98.0
49.0
49.0
ND
ND
ND
ND
ND
ND
ND
ND
ND
49.0
5.0
ND
36.5 ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
39.0
54.0 18.5 ND
54.0 18.5 ND
ND
ND
54.0 ND
ND
ND
ND
55.0
39.0
ND
ND
ND
39.0
19.5
ND
55.0
19.5
17.0
34.0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8.5
17.0
8.5
17.0
ND
ND
ND
ND
10.0
55.0
20.0
ND
17.0
8.5
17.0
ND
17.0
54.0
17.0
ND
54.0 ND
ND
ND
ND
ND
ND
ND
20.0
ND
ND
ND
ND
34.0
ND, not determined; ALI, Adolfo Lutz Institute.

A.O. de Souza et al. / European Journal of Medicinal Chemistry 36 (2001) 843–850
847
Table IV. MIC (mmol L−1) of classical drugs used in the
explain using the same argument as before. Also, it is
interesting to notice that 5c (p-Br) and 5d (p-I) are the
most effective on the different mycobacteria strains and
acted efficiently in almost all the tested strains, standard
or from clinical specimens (H37Ra,, Ma, Mm, Mi,
ALI-282, ALI-536l, ALI-452, ALI-3342 and ALI-258).
Other comparison can be made between compounds 5j
and 5m. Compound 5j seems to be more effective with
lower MIC than 5m, which does not contain a bromine
atom at the 4%-position of the biphenyl ring. Probably,
the bromine gives a lipophilic character to the molecule
increasing the ability of the drug to pass through the
membrane and consequently there is an increase of the
antimycobacterial activity.
In our study the reference drugs INH, RFP, SM and
EMB were used on standard strains and the respective
MIC are given in table IV. The MIC of 5a–m are
indicative of a moderated antimycobacterial activity if
the data are compared with that of reference drugs,
since the MIC doses are higher than those of INH,
RFP, SM and EMB with some exceptions as discussed
before.
The toxicity to mammalian cells V79 was determined
for 5b, 5d, 5f, 5g, and 5j–m. The results are expressed as
MTT, NRU and NAC assays. In table V it is possible to
observe the IC50 values of all the compounds (except
compound 5i which was not studied since it exhibited a
low antimycobacterial activity) of the series obtained by
extrapolation of the data. Compound 5j was the most
toxic of the series with lower IC50 values. However,
compounds 5d, 5h and 5l are less toxic to V79 cells with
higher IC50 values than the others. Probably in this case,
a combination of inductive (sI) and MR effects [19] are
involved in the toxicity. In the reference drugs INH, SM
and BEM did not show any cytotoxicity in V79 cells up
to 1000 mmol L−1, but RFP exhibited IC50 values of 200,
470 and 360 mmol L−1 for NAC, NRU and MTT,
respectively (table V).
chemotherapy of tuberculosis.
Strains
MIC (mmol L−1
)
INH RFP
SM
EMB
14.4
M. tuberculosis
H37Rv
50.22 50.036 50.086
50.22 50.036 50.086
M. tuberculosis
H37Ra
M. a6ium
M. kansasii
M. malmoense
M. cellatum
3.6
]3.65
0.3
0.69
51.37
51.37
0.69
]14.4
\14.4
]14.4
7.2
]3.65 ]0.6
\3.65 ]0.6
\3.65 ]0.6
INH, isoniazid; RFP, rifampicin; SM, streptomycin; EMB,
ethambutol.
Table V. Cytotoxicity of compounds 5a–m on V79 cells.
Compound
IC50 (mmol L−1
)
NAC
NRU
4.98 a
6.32
11.0 a
35.45
5.8 a
MTT
10.0 a
12.0
5.7 a
\40
7.5 a
\25
5a
5b
5c
5d
5e
5f
8.6 a
10.0 a
10.8 a
24.39 a
9.2 a/8.2 b
\25
\20
5g
5h
5j
7.0 a
5.02
20.0 a
7.55
8.30
40.0 a
7.19
48.0 a
6.66
5l
25.51
9.57
200.0
28.97
16.84
470.0
28.03
18.02
360.0
5m
RFP c
a De Conti et al. (1998) [6].
b De Conti et al. (1996b) [13].
c SM, INH and EMB did not exhibit any cytotoxicity up to
1000 mmol L−1 concentration.
culosis mycobacteria are less susceptible to the
chemotherapy of tuberculosis.
In the case of H37Ra and H37Rv strains, the halogen
at the p-position in the phenyl moiety of the compounds
5b–d exerts a significant effect on their activity at lower
IC50 values than the MIC values. Presumably, in this
strain the amino group (5l) also exerts an efficient
volume effect due to its molar refractivity (MR) value
similar to the halogens [20]. Similar explanation for the
p-OH group (5j) is proposed, since the MR value is
slightly lower than the others mentioned before. In the
cases of ALI-282, ALI-536, ALI-452 and 258 INH in
which are involved the compounds 5a–d, it is hard to
5. Conclusions
The derivatives of 3-(4%-bromo-[1,1%-biphenyl]-4-yl)-
3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine (5a–
m) were synthesised and showed a moderated
antimycobacterial activity. Due to the wide range of
activity in all the mycobacteria strains, compound 5c
was the best choice for the preparations of new for-
mulations (free form, inclusion complex with b-cy-
clodextrin and liposomes) in order to improved its

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A.O. de Souza et al. / European Journal of Medicinal Chemistry 36 (2001) 843–850
6.2.1. Compound 3i
effectiveness on intracellular mycobacteria (macro-
phage) or in infected animal. Also, improvements are
required to obtain new derivatives of this compounds
which able to achieve more effective antimycobacte-
rial activity with lower toxicity to the mammalian
cells.
IR (KBr, cm−1): 1744.1 (w(CꢁO), OCOCH3), 1642.5
1
(w(CꢁO), ArCOAr). H-NMR (300 MHz, CDCl3/TMS,
l): 8.0–7.1 (m, 12H, Ar), 2.35 (s, 3H, ArOCOCH3).
13C-NMR (75 MHz, CDCl3/TMS, l): 195.07 (CꢁO),
168.77 (CꢁO), 144.02 (C), 138.80 (C), 136.45 (C), 135.21
(C), 132.12 (CH), 132.00 (CH), 131.92 (CH), 131.71
(CH), 130.74 (CH), 128.85 (CH), 128.77 (CH), 127.92
(CH), 126.84 (CH), 126.68 (C), 126.41 (C), 125.40 (CH),
6. Experimental protocols
122.64 (C), 122.14 (CH), 122.03 (CH), 121.75 (CH),
21.17 (CH3). MS (m/z): 396/394 ([M ], 22), 354/352
(73), 261/259 (20), 152 (53), 121 (100), 93 (8), 65 (8).
M.p. 185–195 °C; UV (nm, CH2Cl2) u=254. Yield
80%.
+
’
6.1. Materials and methods
The chemicals used in this study were purchased from
Aldrich–Sigma or Merck without further purification,
except the solvent THF, which was treated with anhy-
drous calcium hydride, refluxed with metallic sodium
and distilled.
The drugs pyrazinoic acid amide (PZA), isonicotinic
acid hydrazide (INH), ethambuthol dihydrochloride
(EMB), streptomycin sulfate (SM) were obtained from
Sigma–Aldrich and Rifampicin (RFP) from Merck.
6.2.2. Compound 3m
IR (KBr, cm−1): 3322.9 (w(ꢀOH)), 1644.1 (w(CꢁO)
1
ArCOAr). H-NMR (300 MHz, CDCl3/TMS, l): 8.35–
+
’
7.31 (m, 13 H, Ar). MS (m/z) 274 ([M ], 100), 181 (79),
152 (44), 121 (94). M.p. 161 °C. Yield ꢀ100%.
6.2.3. Compound 5i
IR (film, cm−1) 1752 (w(CꢁO), ArOCOCH3). 1H-
NMR (300 MHz, CDCl3/TMS, l): 7.8–6.8 (m, 24H Ar,
E/Z), 6.33 (1t, 1H, ꢀCH, E/Z, J=6.96 Hz), 6.30 (1t,
1H, ꢀCH, E/Z, J=6.96 Hz), 6.28 (1t, 1H, ꢀCH, E/Z,
J=6.96 Hz), 3.45 (1d, 2H, ꢀCH2, E/Z, J=7.32 Hz),
3.32 (1d, 2H, ꢀCH2, E/Z, J=6.96 Hz), 2.43 (s, 6H, CH3
6.2. Chemistry
Reactions were monitored and compound purities
were determined by TLC on UV-254 plates. Mass spec-
tra (MS) were taken on a VG Autospec spectrometer
under electron impact at an ionisation irradiation energy
of 70 eV. The ratios m/z and the relative intensities
corresponded to the calculated molecular weights of the
compounds prepared. Nuclear magnetic resonance spec-
tra of hydrogen and carbon (1H-NMR and 13C-NMR),
spectra were recorded in a Gemini (Varian, USA) or on
a Bruker apparatus at a working frequency of 300 or
ꢀOCOCH3, E/Z), 2.30 (s, 12H, ꢀN(CH3)2, E/Z). MS
+
’
(m/z): 451/449 ([M ], 27), 436 (8), 406 (5), 363 (13), 363
(13), 329 (41), 314 (92), 58 (100). Yield 40%.
6.2.4. Compound 5j
IR (film, CH2Cl2, cm−1) 3322 (w(ArOH)). H-NMR
1
(300 MHz, CDCl3/TMS, l): 7.54–6.64 (m, 24H Ar,
E/Z), 6.13 (1t, 1H, ꢀCH, E/Z, J=6.96 Hz) 6.00 (1t,
1H, ꢀCH, E/Z, J=6.96 Hz), 3.12 (1d, 2H, ꢀCH2, E/Z,
1
500 MHz for H and 75 or 125 MHz for 13C in CDCl3
J=6.96 Hz); 3.07 (1d, 2H, ꢀCH2, E/Z, J=6.96 Hz),
using tetramethylsilane (TMS) as the internal standard
(chemical shifts in l, ppm). Analytical TLC was carried
out on pre-coated plates (silica gel 60 F254) and spots
were visualised with UV light and in an I2 chamber. IR
spectra (KBr) were recorded on a Win-Bomem spec-
trophotometer (wmax in cm−1) and melting point in a
Quimis apparatus. All the compounds were character-
ised by the above-mentioned techniques and the data for
only the new compounds are listed since the data of
3a–h and 5a–h have been reported before [13]. Qualita-
tive analysis of bromine was performed for 3m by
energy dispersive X-ray fluorescence (EDXRF) in a
Tracor X-ray Model Spectrace 5000 (table I).
+
’
2.27 (s, 12H ꢀN(CH3)2, E/Z). MS (m/z): 409/407 ([M ],
100), 394/392 (34), 365/363 (41), 176 (47), 107 (29), 70
(46), 58 (63). Yield 40%.
6.2.5. Compound 5l
IR (film, CH2Cl2, cm−1): 3054 (w(NꢀH)), 1265
1
(w(CꢀN)). H-NMR (300 MHz, CDCl3/TMS, l): 7.60–
6.59 (m, 24H Ar, E/Z), 6.28 (1t, 1H, ꢀCH, E/Z, J=
6.96 Hz), 6.22 (1t, 1H, ꢀCH, E/Z, J=7.14 Hz), 3.81 (s,
4H ꢀNH2, E/Z), 3.43 (1d, 2H, ꢀCH2 E/Z, J=6.96 Hz),
3.39 (1d, 2H, ꢀCH2, E/Z, J=7.32 Hz), 2.49 (s, 12H
+
’
ꢀN(CH3)2, E/Z). MS (m/z) 408/406 ([M ] 100), 393/391
(50), 130 (34), 106 (55), 70 (30), 58 (55). Yield 30%.

A.O. de Souza et al. / European Journal of Medicinal Chemistry 36 (2001) 843–850
849
6.2.6. Compound 5m
method using Lowenstein–Jensen medium [16]. The crit-
ical concentration of the drugs were 200 mg mL−1 for
PZA; 0.2 mg mL−1 for INH; 2 mg mL−1 for EMB; 40
mg mL−1 for RFP and 4 mg mL−1 for SM. The stock
solutions were prepared in water 10 mg L−1 (except for
RFP which was prepared in methanol) and diluted in
Loweinstein–Jensen medium before solidification. The
isolates were considered resistant if there was a growth
on the medium containing drug more than 1% for INH,
RFP and EMB and 10% for PZA and SM compared
with the growth on the drug-free medium (table II).
The mycobacteria type strains and the M. tuberculosis
isolated from clinical specimens were subcultured in
Lowenstein–Jensen medium at 37 °C for 3 weeks and
subcultured in Middlebrook 7H9 broth medium at
37 °C for 10 days, until a bacterial density correspond-
ing to a 1.0 McFarland turbidity standard. The suspen-
sions were further diluted 1:25 in Middlebrook 7H9
broth medium before the inoculation (4×105
mycobacteria mL−1).
IR (film, CH2Cl2, cm−1): 3322 (w(ArOH)). H-NMR
(500 MHz, CDCl3/TMS, l): 7.86 (s, 2H ꢀOH, E/Z),
7.53–6.7 (m, 26H, Ar, E/Z), 6.14 (1t, 1H, ꢀCH, E/Z,
J=6.96 Hz), 6.05 (1t, 1H, ꢀCH, E/Z, J=6.96 Hz), 3.28
(1d, 2H, ꢀCH2, E/Z, J=6.84 Hz), 3.25 (1d, 2H, ꢀCH2,
E/Z, J=6.84 Hz), 2.34 (s, 12H, ꢀN(CH3)2, E/Z). 13C-
NMR (500 MHz, CDCl3/TMS, l): 157.46 (C), 157.82
(C), 146.71 (C), 146.39 (C), 140.36 (C), 140.22 (C),
140.21 (C), 140.14 (C), 140.03 (C), 137.74 (C), 132.00
(CH), 130.64 (CH), 129.79 (CH), 128.56 (CH), 128.55
(CH), 128.49 (CH), 127.73 (CH), 127.20 (CH), 127.06
(CH), 126.74 (CH), 126.71 (CH), 126.66 (CH), 126.54
(CH), 120.08 (C), 118.33 (C), 115.53 (CH), 115.50 (CH),
1
57.23 (CH3), 56.95 (CH3), 43.48 (CH2), 43.43 (CH2). MS
+
’
(m/z): 329 ([M ], 100), 314 (41), 285 (50), 176 (29), 107
(30), 70 (26), 58 (45). Yield 80%.
6.2.7. Isolation of the isomers from compounds 5a and
5c
The geometric isomers E and Z of 5a were isolated
with preparative TLC on silica gel 2–25 mm (Aldrich)
using ethyl acetate–hexane–NH4OH (79:20:1) as eluent.
For the geometric isomers E and Z of 5c, a HPLC
separation was achieved by a Waters 600E Millipore
model with Hi-Chrom preparative HPLC column,
Spherisorb S5W 5m 25 cm×10 mm using ethyl acetate–
C6H14–TEA (79:20:1) as eluent [14].
6.4. Stock solutions
Stock solutions of compounds 5a–m were prepared in
DMSO at 1 g L−1 concentration and the dilutions were
performed in Middlebrook 7H9 broth medium to obtain
100 ml of dilution in each well. The drugs INH, RFP,
SM and EMB were used as references and the tests were
performed by the microplate Alamar Blue assay as
previous reported [5]. The visual MICs were defined as
the lowest drug concentration that prevented a colour
change from blue to pink.
6.3. Biological assays: antimicrobial susceptibility testing
The type strains of M. tuberculosis H37Rv ATCC
27294, M. tuberculosis H37Ra ATCC 25177, M. avium
ATCC 15769, M. kansasii ATCC 12478, M. intracellu-
lare ATCC 25169 and M. malmoense ATCC 29571 were
kindly provided by Dr Robert G. Cooksey of the Center
for Disease Control and Prevention—CDC (Atlanta,
USA) and the M. tuberculosis isolated from clinical
specimens were obtained from patients with tuberculosis
at Adolfo Lutz Institute and enumerated (ALI, Ribeira˜o
Preto, SP, Brazil). All isolates were grown on Lowein-
stein–Jensen medium and were examined for growth
rate, microscopy colony morphology and pigmentation.
For identification, they were submitted to biochemical
tests for niacin production, nitrate reduction, catalase,
arylsulphatase, pyrazinamidase, urease and lipase
(Tween 80 hydrolysis) [15].
6.5. Cytotoxicity to mammalian cells
The toxicities of the compounds were determined on
V79 cells (Chinese hamster lung fibroblast generously
supplied by Dr R. Meneghini from the Universidade de
Sa˜o Paulo, Brazil) by a multi-endpoints cytotoxicity
method [6]. The cytotoxicity effect expressed as cell
viability was assayed on a permanent lung fibroblast cell
line derived from Chinese hamsters (V79). V79 fibrob-
lasts were grown as monolayers in Dulbecco’s modifica-
tion of Eagle’s medium (DMEM), supplemented with
10% heat-inactivated foetal calf serum (FCS), 100
IU mL−1 penicillin and 100 mg mL−1 streptomycin in a
humidified incubator with 5% CO2 in air at 37 °C. Cells
were plated at density of 3×104 cell mL−1 in 24- or
96-well plates. The medium was removed 48 h after cell
seeding and replaced with medium containing the com-
The M. tuberculosis strains isolated from clinic speci-
mens were classified as sensitive or resistant to the drugs
PZA, INH, EMB, RFP and SM by the proportion

850
A.O. de Souza et al. / European Journal of Medicinal Chemistry 36 (2001) 843–850
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Acknowledgements
Support by Fapesp is acknowledged.
.