Antimycobacterial agents. Novel diarylpyrrole derivatives of BM212 endowed with high activity toward Mycobacterium tuberculosis and low cytotoxicity

Article abstract of DOI:10.1021/jm0602662

On the basis of suggestions derived either from a pharmacophoric model for antitubercular agents or from a structure-activity relationship analysis of many pyrroles previously described by us, we report here the design and synthesis of new analogues of 1,

Full text of DOI:10.1021/jm0602662

4946
J. Med. Chem. 2006, 49, 4946-4952
Antimycobacterial Agents. Novel Diarylpyrrole Derivatives of BM212 Endowed with High
Activity toward Mycobacterium tuberculosis and Low Cytotoxicity
Mariangela Biava,*,† Giulio Cesare Porretta,† Giovanna Poce,† Sibilla Supino,† Delia Deidda,‡ Raffaello Pompei,‡
Paola Molicotti,§ Fabrizio Manetti,*,# and Maurizio Botta#
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente AttiVe, UniVersita` La Sapienza, P. le A. Moro 5,
00185 Rome, Italy, Cattedra di Microbiologia Applicata, Facolta` di Farmacia, UniVersita` degli Studi di Cagliari, Via Porcell 4,
09124 Cagliari, Italy, Dipartimento di Scienze Biomediche, UniVersita` di Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy, and
Dipartimento Farmaco Chimico Tecnologico, UniVersita` degli Studi di Siena, Via Alcide de Gasperi 2, 53100 Siena, Italy
ReceiVed March 9, 2006
On the basis of suggestions derived either from a pharmacophoric model for antitubercular agents or from
a structure-activity relationship analysis of many pyrroles previously described by us, we report here the
design and synthesis of new analogues of 1,5-(4-chlorophenyl)-2-methyl-3-(4-methylpiperazin-1-yl)methyl-
1H-pyrrole (BM212). Various substituents with different substitution patterns were added to both positions
1 and 5 of the pyrrole nucleus to evaluate their influence on the activity toward Mycobacterium tuberculosis
(MTB) and atypical mycobacteria. Biological data showed that, although some nontuberculosis mycobacterial
strains were found to be sensitive, MIC values were higher than those found toward MTB. The best compound
(1-(4-fluorophenyl)-2-methyl-3-(thiomorpholin-4-yl)methyl-5-(4-methylphenyl)-1H-pyrrole, 5) possessed a
MIC of 0.4 µg/mL (better than BM212 and streptomycin) and a very high protection index (160), better
than BM212, isoniazid, and streptomycin (6, 128, and 128, respectively). Finally, molecular modeling studies
were performed to rationalize the activity of the new compounds in terms of both superposition onto a
pharmacophoric model for antitubercular compounds and their hydrophobic character.
Introduction
for effective TB control. In view of this situation, in 1993, the
WHO declared TB a global emergency.8
Humankind’s battle with tuberculosis (TB) dates back to
antiquity. TB, which is caused by Mycobacterium tuberculosis
(MTB), was a much more prevalent disease in the past than it
is today, and it was responsible for the deaths of about 1 billion
people during the last two centuries.1 Improved sanitation and
living conditions significantly reduced the incidence of the
disease even before the advent of chemotherapy. However,
despite the widespread use of the bovine Calmette-Guerin
vaccine, which had a great impact on further reduction in TB
incidence, TB still remains a leading infectious disease world-
wide, especially in third world countries. In fact, MTB is the
cause of death of almost 3 million people each year, and it is
positioned as the leading bacterial infectious agent.2-4
One of the major obstacles to global control of TB is
represented by the reactivation of the disease in patients who
carry a latent infection, in which the bacteria are thought to be
in a slow growing or nongrowing state9,10 and are recalcitrant
to treatment by conventional anti-TB drugs.11,12
An additional difficulty derives from atypical mycobacteria,
which include the Mycobacterium aVium complex (MAC), an
opportunistic pathogen in AIDS patients.13 Clinical management
of MAC infections, especially among AIDS patients, is difficult,
partly because of the severely depressed state of the host defense
mechanisms in such patients14 and partly because of the
intrinsically low susceptibility of MAC isolates to many
antimycobacterial drugs.14,15
The increase of TB during recent years was largely due to
HIV-1 infection, immigration, increased trade, and globaliza-
tion.5 Moreover, also the increasing emergence of a drug-
resistant TB, especially multidrug-resistant TB (MDR-TB) is
particularly alarming. MDR-TB has already caused several fatal
outbreaks6,7 and poses a significant threat to the treatment and
control of the disease in some parts of the world, where the
incidence of MDR-TB can be as high as 14%.5 In addition, the
TB situation may become even worse with the spread of HIV-1
worldwide, a virus that weakens the host immune system, allows
latent TB to reactivate, and makes the person more susceptible
to reinfection with either drug-susceptible or drug-resistant
strains. The lethal combination of drug-resistant TB and HIV-1
infection is a growing problem that presents serious challenges
The current recommended standard TB chemotherapy, called
DOTS (directly observed treatment, short-course) has a cure
rate of up to 95% and is recommended by the WHO for treating
every TB patient. Although DOTS can cure TB, the length of
the therapy makes patient compliance difficult, and noncompli-
ance is a frequent source of drug-resistant strains.7,16 In fact,
MDR strains, resistant to many first-line agents, as well as some
of the second-line drugs, are increasingly being found.7,17
As a consequence, without more effective treatments, the
number of infections caused by MDR-TB will probably in-
crease out of control. Therefore, the development of new
antimicrobial drugs with potent anti-TB and/or anti-MAC
activity, and new protocols for chemotherapy of the patients
with refractory tuberculosis and MAC infections, are urgently
needed.18
* Corresponding authors. (M.B.) Phone: +39 (0)6 4991 3812; fax: +39
(0)6 4991 3133; e-mail: mariangela.biava@uniroma1.it. (F.M.) Phone: +39
(0)577 234330; fax: +39 (0)577 234333; e-mail: manettif@unisi.it.
† Universita` La Sapienza.
However, in the last 40 years, only a few drugs have been
approved by the Food and Drug Administration (FDA) to treat
TB, reflecting the inherent difficulties in discovery and clinical
testing of new agents and the lack of pharmaceutical industry
research in the area.19 In particular, in addition to the current
‡ Universita` degli Studi di Cagliari.
§ Universita` di Sassari.
# Universita` degli Studi di Siena.
10.1021/jm0602662 CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/07/2006

Antimycobacterial Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4947
drugs approved by the FDA for the treatment of TB and to the
drugs that commonly are recommended for the treatment of TB
but are not FDA approved,20 a variety of other compounds or
classes of compounds is under investigation as potential
antimycobacterial drugs. Among them, pyrrole derivatives were
first reported by us in 199821 as antimycobacterial agents. The
most potent derivative (BM212) showed minimum inhibitory
concentration (MIC) values ranging from 0.7 to 1.5 µg/mL
toward several strains of MTB. Activity toward drug-resistant
strains of MTB was similar to that found toward sensitive strains.
Finally, it was also active toward nontuberculosis mycobacteria,
with MICs higher that that against MTB.
Design of New Pyrrole Derivatives. In an effort to search
novel derivatives of BM212 endowed with a better biological
profile, we have previously reported the synthesis and both
antimycobacterial and antifungal activities of many pyrrole
derivatives,22-24 finding that most of such compounds showed
interesting antimycobacterial properties. Moreover, modifica-
tions of BM212 (based on the suggestion that a thiomorpholin-
4-yl substituent onto a five-membered heterocyclic ring is a
crucial key for antitubercular activity)25 allowed the identifica-
tion of several very active antitubercular compounds and showed
the importance of the (thiomorpholin-4-yl)methyl moiety at the
pyrrole C3 and a p-halophenyl substituent at both N1 and
C5.26-29
Figure 1. (A) Graphical representation of the superposition pathway
of compound 5 onto the pharmacophoric model for antitubercular
compounds. The phenyl ring at the nitrogen of the pyrrole ring is
accommodated within the aromatic ring feature RA2, the sulfur atom
of the thiomorpholine ring at C3 is the hydrogen-bond acceptor group
HBA, and the p-methylphenyl substituent at position 5 is able to fill
both the hydrophobic feature HY (with the methyl group) and the
additional aromatic ring RA1 (with its phenyl portion). The methyl
substituent at C2 lies in an empty region of space where no pharma-
cophoric feature is located. (B) Superposition of 9 into the pharma-
cophoric model showing an alternative binding mode due to the reversed
location of substitutents at N1 and C5 of the pyrrole ring. Portions of
the pharmacophoric model are color-coded: green for hydrogen-bond
acceptor groups (HBA), orange for aromatic rings (RA), and blue for
hydrophobics (HY).
Moreover, in the absence of any knowledge regarding both
the drug target and the biologically active conformation of the
previous antitubercular agents, a ligand-based drug design
approach has been applied to better understand the relationships
between the structure of compounds and their activity and also
to aid in the design of more potent inhibitors. Accordingly, the
application of a pharmacophoric generation technique led us to
build26 and optimize30 a pharmacophoric model for antimyco-
bacterial compounds, consisting of four chemical features
represented by a hydrophobic region (HY), a hydrogen-bond
acceptor group (HBA), and two aromatic rings (RA1 and RA2)
(Figure 1).
Analysis of the superposition mode of previously published
antitubercular compounds suggested that a phenyl ring bearing
a lipophilic group (such as a methyl, ethyl, or isopropyl
substituent) at the para position could have profitable interactions
with the HY feature of the pharmacophoric model, with a
consequent enhancement in activity.28 On the basis of such a
hypothesis, in an attempt to increase the fitting to the pharma-
cophoric model and, consequently, to optimize the interactions
with the hypothetical receptor, we pursued our studies by
synthesizing new pyrrole derivatives maintaining the alternative
presence of a (thiomorpholin-4-yl)methyl or a (4-methylpiper-
azin-1-yl)methyl moiety (as in BM212) at the C3 position of
the pyrrole ring and introducing a methyl group into one of the
phenyl rings at N1 or C5, to allow for a better superposition
with the corresponding pharmacophoric feature HY.
Scheme 1a
a Reagents: (a) 1: 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bro-
mide, NEt3, 75-78 °C, 5 h and 2: 2 N HCl. (b) Amine, p-toluensulfonic
acid, 100 °C, 5 h. (c) 1: Thiomorpholine or N-methylpiperazine, CH3CN,
HCHO, CH3COOH, rt, 8 h and 2: NaOH 20% w/v.
In this paper, we report the synthesis of the new derivatives
1-28, characterized by (i) a p-fluorophenyl ring at position 1
or 5, found to be important for antitubercular activity; (ii) an
additional phenyl group (at position 5 or 1, respectively) bearing
F, Cl, and Me substituents with different substitution patterns,
to test the hypothesis that a more lipophilic group (such as the
methyl one) could improve activity, probably on the basis of
the fact that the mycobacterial cell wall structure is very waxy,
hydrophobic, and characterized by a high lipid content, and thus,
it requires a hydrophobic compound to be penetrated; (iii) a
(thiomorpholin-4-yl)methyl or a (4-methylpiperazin-1-yl)methyl
moiety at position 3 of the pyrrole nucleus, to support previous
findings suggesting that thiomorpholine31 derivatives were in
principle more active than the corresponding N-methylpiperazine
analogues.
Chemistry. Synthesis of the target compounds is shown in
Scheme 1. Briefly, a reaction of a suitable benzaldehyde 29
with methyl vinyl ketone 30, according to the Stetter condi-
tions,28 was very versatile in the preparation of the 1,4-diketones
31a-h. Following the usual Paal-Knoor (thermal) condensa-
tion, compounds 31a-h, after prolonged reflux in the presence

4948 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
BiaVa et al.
Table 1. Structure, In Vitro Antimycobacterial Activity against M.
tuberculosis 103471, Cytotoxicity, and Protection Index (PI) of the New
Pyrrole Derivatives 1-28 and Compounds Used as References (BM212,
Isoniazid, Streptomycin, and Rifampicin)
of the appropriate amine, cyclized to yield the expected 1,5-
diarylpyrroles 32a-n in satisfactory yield. Construction of the
side chain at C3 was achieved in good yield by reaction of
compounds 32a-n with formaldehyde and thiomorpholine or
N-methylpiperazine, according to the Mannich reaction condi-
tions.
Biology. Compounds were preliminarly assayed in a rapid
screening test for their activity toward two clinical strains (i.e.,
Mycobacterium fortuitum CA10 and M. tuberculosis B814) at
the single dose of 100 µg/mL. Those substances that resulted
as active in this first screening against at least one of the two
mycobacteria were analyzed for their minimum inhibitory
concentration (MIC). Only compounds showing MIC values of
16 µg/mL or lower were then studied for their inhibitory activity
toward M. tuberculosis CIP 103471 and a panel of atypical
mycobacteria, such as Mycobacterium marinum CIP 6423, M.
aVium CIP 103317, and Mycobacterium smegmatis CIP 10359.
MIC values, expressed as µg/mL, were determined for each
compound on each of the test mycobacteria.
Cytotoxic activity assays was performed in Vero cells to
determine the maximum nontoxic dose (MNTD50 expressed as
µg/mL), defined as the drug concentration that decreased cell
multiplication less than 50% of the control.
In vitro activity of compounds 1-28 toward MTB 103471,
as well as the cytotoxicity and protection index (PI), were
reported in Table 1. Moreover, in vitro activity toward atypical
mycobacteria was also reported (Table 2). Isoniazid, strepto-
mycin, rifampicin, and BM212 were used as reference com-
pounds.
MIC
MNTD50
compd
Ra
R1
R2
(µg/mL)b (µg/mL)c PId logPe
1f
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
B
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
2-Cl-Ph
2-Cl-Ph
2-F-Ph
2-F-Ph
4-CH3-Ph 4-F-Ph
4-CH3-Ph 4-F-Ph
3-CH3-Ph 4-F-Ph
3-CH3-Ph 4-F-Ph
2-CH3-Ph 4-F-Ph
2-CH3-Ph 4-F-Ph
2,4-Cl2-Ph 4-F-Ph
2,4-Cl2-Ph 4-F-Ph
2,4-F2-Ph 4-F-Ph
2,4-F2-Ph 4-F-Ph
2-Cl-Ph
2-Cl-Ph
2-F-Ph
2-F-Ph
4
8
0.5
>16
0.4
4
2
16
4
>16
2
8
0.5
4
2
4
0.5
8
1
16
4
16
8
4
2
16
4
64
8
4
8
8
2
64
16
16
8
8
8
8
8
32
8
8
16
16
2
4
2
16
16
4
32
>64
64
1
6.04
2f
0.25 5.56
3f
32
5.58
5.10
5.86
5.38
5.86
5.38
5.86
5.38
6.70
6.23
5.78
5.31
6.04
5.56
5.58
5.10
5.86
5.38
5.86
5.38
5.86
5.38
6.70
6.23
5.78
5.31
6.02
4f
5f
4-CH3-Ph
4-CH3-Ph
3-CH3-Ph
3-CH3-Ph
2-CH3-Ph
2-CH3-Ph
2,4-Cl2-Ph
2,4-Cl2-Ph
2,4-F2-Ph
2,4-F2-Ph
4-F-Ph
160
2
2
0.5
2
6f
7
8
9
10
11f
12f
13f
14f
15f
16f
17f
18f
19f
20f
21
22
23
24
25f
26f
27f
28f
32
2
32
2
4
2
16
1
32
0.5
2
4-F-Ph
4-F-Ph
4-F-Ph
1
2
>16
1
4
2
16
0.7
0.25
0.50
0.30
Compounds 5 and 17 were also evaluated for their activity
toward intracellular MTB (Table 3), as well as toward a panel
of 11 resistant and multi-drug-resistant (MDR) clinical isolate
strains of MTB (Table 4).
4
0.5
8
1
BM212
Isoniazid
Streptomycin
Rifampicin
4-Cl-Ph
4-Cl-Ph
5.6
128
>128
213
Results and Discussion
Biological data reported in Table 1 showed a general trend
that confirmed previous suggestions on the importance of the
(thiomorpholin-4-yl)methyl moiety as a substituent of the pyrrole
ring.27-29 In fact, thiomorpholine derivatives showed very good
in vitro activity toward MTB and poor toxicity, with a biological
profile better than that of the corresponding N-methylpiperazine
analogues. It is also important to note that compounds 3, 5,
and 17, in addition to showing a high in vitro activity toward
MTB (MIC of 0.5, 0.4, and 0.5 µg/mL, respectively, comp-
arable to that found for isoniazid, 0.25 µg/mL, streptomycin,
0.5 µg/mL, and rifampicin, 0.3 µg/mL), were also characterized
by a very low toxicity. In particular, 5 showed a high protec-
tion index (160), better than that of BM212 (6), isoniazid
(128), and streptomycin (128) and slightly lower than that of
rifampicin (213).
Regarding activity toward atypical mycobacteria, the new
compounds were in principle found to be more active against
MTB than toward M. aVium (Table 2). The only exception to
this trend was represented by 12, found 4-fold more active
toward M. aVium (MIC of 2 vs 8 µg/mL), as well as 6 and 26
that showed an identical in vitro activity toward both myco-
bacteria (4 µg/mL). Moreover, the corresponding thiomorpholine
compounds 11 and 25 also showed an interesting inhibitory
activity toward M. aVium (4 and 2 µg/mL, respectively). These
results were in good agreement with the high activity showed
by BM212 (in which a chloro substituent was present in both
the phenyl rings at N1 and C5) against M. aVium. All the
remaining compounds revealed themselves to be completely
inactive against atypical mycobacteria, thus showing a good
a A ) thiomorpholin-4-yl and B ) 4-methylpiperazin-1-yl. b MIC )
minimum inhibitory concentration toward M. tuberculosis. c MNTD50
)
maximal nontoxic dose toward Vero cells. d PI ) protection index, as the
ratio between cytotoxicity and in vitro activity toward M. tuberculosis.
e Calculated by means of the AlogP98 method (see ref 36). f Compound
submitted to an international patent application (see ref 37).
selectivity versus MTB. The sole compound 18 was found to
have a very relevant inhibitory activity toward M. smegmatis
(0.3 µg/mL).
Compounds 5 and 17 were also evaluated for their activity
toward intracellular MTB. It is important to note that while the
inhibitory activity toward extracellular MTB accounts for the
ability of tested compounds to treat active tuberculosis, assays
on intracellular MTB assess the ability of tested compounds to
inhibit mycobacteria during the latent phase of tuberculosis,
before the latent tuberculosis infection itself progresses to active
disease. Biological results reported in Table 3 show that both
compounds exert bactericidal activity on intracellular myco-
bacteria at a 3 µg/mL concentration, comparable to that of
rifampicin, used as the reference compound. This result is very
important because mycobacteria can reside for years inside
lymphoid cells and macrophages (during the latent phase of
tuberculosis), and many traditional drugs are unable to get throw
it. Moreover, combating latent tuberculosis infection is one of
the major challenges mainly for reducing the high rate of
progression to active disease in immunocompromised individu-
als (in fact, progression is higher in persons with concomitant
HIV infection).

Antimycobacterial Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4949
Table 2. In Vitro Activity (Expressed as MIC Values in µg/mL) of the
New Pyrrole Derivatives 1-28 and Reference Compounds (BM212,
Isoniazid, Streptomycin, and Rifampicin) toward Atypical Mycobacteria
(M. smegmatis, M. marinum, and M. aVium)
of clinical isolates of MTB, with the exception of the 326/04
strain (32 µg/mL). Similarly, 17 was characterized by an activity
of 0.5 µg/mL toward 43/05 and 158/97, while the remaining
clinical isolates were inhibited at a 2 µg/mL concentration. Also,
compound 17 was inactive toward the isolate 326/04, known
to be sensitive to rifampicin, while resistant to isoniazid,
streptomycin, and ethambutol. These experimental evidences
make these compounds extremely interesting when compared
to the compounds now used in therapy, which tend to be less
active against drug-resistant mycobacteria.
MIC (µg/mL)
compd
M. aVium
M. marinum
M. smegmatis
1
2
3
4
16
16
16
>16
8
>16
>16
>16
>16
8
>16
>16
>16
>16
16
5
6
7
8
9
4
8
16
8
>16
4
2
16
16
>16
>16
8
16
8
16
16
>16
16
>16
2
>16
>16
>16
>16
>16
>16
>16
16
>16
>16
>16
>16
>16
8
8
Computational Investigations. With the aim of further
investigating the relationships between the structure of the new
compounds and their in vitro activity toward MTB, we resorted
to a ligand-based drug design approach based on a pharma-
cophoric model previously reported by us for antitubercular
agents.30 It is important to note that the pharmacophore should
be considered as a qualitative model mainly intended to identify
common chemical features shared by our antimycobacterial
compounds. On this basis, the model proved to rationalize the
major structure-activity relationships of the studied compounds.
In fact, it is able to account for the influence that substituents
and substitution patterns at both positions 1 and 5 exert in
defining the activity of 1,5-diarylpyrrole derivatives toward
MTB. However, the pharmacophoric model is not fully able to
explain differences in activity (among the two classes of
compounds) when they probably derive from a different
substituent at position 3 (namely, the N-methylpiperazinomethyl
and thiomorpholinomethyl moiety).
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
0.3
16
8
>16
>16
>16
>16
>16
16
>16
>16
>16
25
>16
>16
>16
>16
8
8
16
16
100
16
32
0.6
4
16
16
0.4
32
8
BM212
Isoniazid
Streptomycin
Rifampicin
64
8
32
Analysis of the orientation of the most active compound (5)
into the pharmacophoric model showed that such a compound
was able to perfectly fulfill all the features (Figure 1A). In detail,
the side chain at position 5 was embedded into the HY-RA1
system, the methyl group corresponding to the hydrophobic
substituent HY and the phenyl ring to one of the aromatic ring
features (RA1) of the model. Moreover, the remaining aromatic
ring feature (RA2) was matched by the phenyl ring at position
1, while, as expected, the sulfur atom of the thiomorpholine
ring was the hydrogen-bond acceptor group. Changing the
substitution pattern of the p-methyl group as in the m-methyl
and o-methyl derivatives (7 and 9, respectively), the binding
mode onto the pharmacophore was found to be reversed (Figure
1B). In fact, while the side chain at position 3 remained in the
region of the HBA feature, the substituent at position 1 partially
filled HY and RA1 with its p-fluoro and phenyl group,
respectively. Consequently, RA2 was matched by the phenyl
ring at position 5. However, the m-methyl and, particularly, the
o-methyl substituents exerted a conformational constraint toward
the phenyl ring at C5, leading to a reorientation of its plan. As
a consequence, both the m-methylphenyl and the o-methylphenyl
substituents were unable to fully satisfy the directionality prop-
erty of the RA2 feature (the fit toward this feature decreased,
possibly leading to less profitable interactions with the aromatic
counterpart of the putative receptor). This result, in addition to
the partial fit of the p-fluorophenyl group onto the HY-RA1
system, could account for the decrease in the activity of these
derivatives. In a similar way, the o-chloro derivative 1, showing
the same orientation of both 7 and 9, possessed an activity lower
than that of the corresponding fluoro analogue 3. This was
accounted by the pharmacophoric model on the basis of the
fact that the conformational rearrangement found for 7 and 9
with respect to 5 was also shown by 1 in comparison to 3. As
a consequence, concerning the latter compound, the fluoro being
smaller than the chloro substituent, the phenyl ring at position
5 was able to fulfill RA2. Moreover, the remaining pharma-
0.3
Table 3. Activity of Compounds 5 and 17 toward Intracellular
(Intramacrophagic) M. tuberculosis
inhibition of intramacrophagic
compd
mycobacteria (MIC, µg/mL)
5
17
3
3
3
Rifampicin
Table 4. In Vitro Activity of Compounds 5 and 17 toward a Panel of
Clinical Isolates of M. tuberculosis Resistant to Different Antitubercular
Agents
sensitivity or resistancea
isolate streptomycin isoniazid rifampicin ethambutol
MIC (µg/mL)
clinical
17
5
149/03
421/96
586/98
43/05
158/97
134/02
520/98
326/04
296/04
482/98
275/05
S
S
S
S
S
R
R
R
S
S
R
r
r
r
r
s
s
s
r
s
r
r
s
s
s
s
s
r
s
s
r
2
2
2
0.5
0.5
2
0.5
0.5
0.5
0.5
0.5
0.5
0.5
s
s
s
s
r
s
r
s
s
s
2
>32
32
r
s
s
2
2
2
0.5
0.5
0.5
a s: sensitive and r: resistant. Streptomycin, isoniazid, rifampicin, and
ethambutol were tested at single doses of 1.0, 0.1, 1.0, and 5.0 µg/mL,
respectively.
Finally, compounds 5 and 17 were also evaluated for their
activity toward a panel of 11 resistant and multi-drug-resistant
clinical isolate strains (MDR-TB) of MTB. Table 4 shows that
all of the tested strains were inhibited by such compounds at
concentrations ranging from 0.5 to 2 µg/mL. The sole exception
was represented by the 326/04 strain, sensitive to such com-
pounds at concentrations higher than 32 µg/mL. In detail, 5
showed a very good activity (0.5 µg/mL) toward the whole panel

4950 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
BiaVa et al.
Reversing the substitution pattern at positions 1 and 5 of 1-14
led to compounds 15-28. Among them, similar to that found
for compounds 1-14, thiomorpholine derivatives were more
active than the corresponding N-methylpiperazine analogues.
Moreover, an activity of 17 (0.5 µg/mL), identical to that found
for 3, was rationalized through the analysis of its superposition
mode onto the pharmacophoric model. In fact, 17 showed an
orientation reversed in comparison to that of 3, with the
substituent at position 5 (a p-fluorophenyl moiety) matching
the HY-RA1 pharmacophoric portion, while the N1 phenyl
ring was embedded into the RA2 feature. Despite the 180˚
rotation around the pyrrole ring, the substituent at position 3
was located in such a way that the thiomorpholine sulfur atom
corresponded to the hydrogen-bond acceptor group. A similar
orientation was found for 15, characterized by an activity 4-fold
lower than that of 17 (2 vs 0.5 µg/mL), in agreement with what
was previously reported for 3 and 1, respectively. Compound
19, able to fulfill the whole pharmacophoric model as 5 did,
showed an activity of 1 µg/mL, comparable to that of 5 (0.4
µg/mL). Moreover, compound 25, bearing a 2,4-dichloro phenyl
group at N1, was characterized by an activity very similar to
that of the corresponding monochloro analogue 15 (1 vs 2
µg/mL), following the same trend found for 11 and 1.
However, the activity of 27, expected to be similar to that of
17, was found to be 4-fold lower (2 vs 0.5 µg/mL), and it was
impossible to be rationalized on the basis of suggestions only
derived from the superposition to the pharmacophoric features.
Finally, the methyl group at position 2 of the pyrrole ring,
located in empty regions of space with no contact involving
the features of the pharmacophore, could be considered as a
nonpharmacophoric portion of the antitubercular compounds
reported, but it possibly contributed to define the mutual
orientation (conformations) of substituents at both positions 1
and 3.
Additional calculations were performed in an attempt to
rationalize the lower MIC values usually found for thiomor-
pholine derivatives in comparison to the corresponding N-
methylpiperazine compounds. For this purpose, log P values
of the new pyrrole derivatives were calculated with Cerius2
software32 (Table 1) with the aim of evaluating if any correlation
between antitubercular activity and lipophilicity of such com-
pounds occurred. Results showed that log P values of the
thiomorpholine derivatives were in all cases higher than those
found for the corresponding N-methylpiperazine counterparts,
supporting the hypothesis that a more hydrophobic character is
preferred for the antitubercular potency of a compound (at least
in vitro).33 Accordingly, the analysis of log P values showed
that compounds with MIC values in the range between 0.4 and
1 µg/mL showed high hydrophobicity (5.58-6.70). On the
contrary, compounds with the lowest activity (namely, 4, 8, 10,
20, 22, 24, and 28) were characterized by the lowest hydro-
phobicity values, their log P values ranging from 5.10 to 5.38.
Figure 2. Superposition of compound 6 into the pharmacophoric
model. Both the substituted phenyl rings follow a superposition pathway
similar to that found for compound 5. Moreover, the N-methylpiperazine
ring adopts a conformation with its methylene moiety at position 1 in
an axial conformation. However, such a disfavored conformer is the
solely able to allow for the match of N4 into the HBA feature of the
pharmacophore.
cophoric features were well-satisfied by the N1 p-fluorophenyl
group (HY and RA1) and by the side chain at position 3, similar
to that found for 5, accounting for the comparable MIC values
determined for 3 and 5 (0.5 and 0.4 µg/mL, respectively). As
expected, the activity of 13 (0.5 µg/mL) was higher than that
of the corresponding dichloro derivative 11 (2 µg/mL) and
comparable to that of the monofluoro derivative 3 (0.5 µg/mL).
Finally, the dichloro compound 11 showed an activity value
slightly lower than that of the corresponding monochloro
analogue 1 (4 µg/mL).
As already evidenced in our previous work, derivatives
bearing a (4-methylpiperazin-1-yl)methyl moiety at position 3
of the pyrrole ring showed an activity toward M. tuberculosis
lower than that of the corresponding (thiomorpholin-4-yl)methyl
analogues. In particular, the o-chloro derivatives 1 and 15
showed better activity (a 2-fold difference) than 2 and 16,
respectively. A 16-32-fold difference was also found for the
corresponding o-fluoro derivatives (compare 17 vs 18 and 3 vs
4, respectively). In an attempt to rationalize these results, an
analysis of the fitting mode of the (thiomorpholin-4-yl)methyl
and (4-methylpiperazin-1-yl)methyl moieties allowed us to find
the N4 of the piperazine as the hydrogen-bond acceptor feature,
similar to the sulfur atom of the thiomorpholine ring. However,
such a conformer was disfavored because it suffered from the
fact that the methylene substituent at N1 was in an axial
conformation (Figure 2). This result could, at least in part,
account for the better activity usually found for thiomorpholine
derivatives in comparison to the corresponding N-methylpip-
erazine analogues. However, as a consequence of the inability
of the pharmacophoric model to fully explain the differences
in activity usually found between the two classes of compounds,
we are aware of the fact that a further refinement of the model
is required to better account for the relationships between the
structure of compounds and their antimycobacterial activity.
Moreover, it is important to note that, by definition, the Catalyst
software is unable to codify into a pharmacophoric model all
the molecular determinants contributing to activity. As an
example, the software does not take into account electronic
effects possibly involved in defining the activity. On this basis,
we cannot exclude that the pharmacophoric model is unable to
fully explain the difference in activity of the two classes of
compounds and that different variables, in addition to the fit to
the pharmacophoric model, could account for the lower activity
of N-methylpiperazine derivatives with respect to thiomorpholine
compounds.
Conclusion
We report here a series of novel 1,5-diarylpyrroles designed
on the basis of both structure-activity relationships collected
from analogue compounds previously reported and suggestions
derived from the analysis of a pharmacophoric model for
antitubercular compounds. Several of the new derivatives were
found to have an in vitro activity toward M. tuberculosis better
than that of the hit compound BM212, and comparable to that
of isoniazid, streptomycin, and rifampicin, used as the reference
compounds. In addition, they were also characterized by low
cytotoxicity with a protection index up to 160, which was better

Antimycobacterial Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4951
General Procedure for the Preparation of Compounds 1-28.
These compounds were prepared by means of the Mannich reaction
as shown in Scheme 1, starting from a suitable pyrrole and
thiomorpholine or N-methylpiperazine in acetonitrile and acetic acid,
in the presence of formaldehyde. In detail, to a stirred solution of
an appropriate pyrrole 32a-n (5.6 mmol), in acetonitrile (20 mL),
a mixture of thiomorpholine (0.57 g, 5.6 mmol) or N-methylpip-
erazine (0.56 g, 5.6 mmol), formaldehyde (0.18 g, 5.6 mmol) (40%
in water), and 5 mL of acetic acid, was added dropwise. After the
addition was complete, the mixture was stirred at room temperature
for 8 h. The mixture was then treated with a solution of sodium
hydroxide (20%, w/v) and extracted with ethyl acetate. The organic
extracts were combined, washed with water, and dried. After the
removal of solvent, the residue was purified by column chroma-
tography, using aluminum oxide and ethyl acetate for (thiomor-
pholin-4-yl)methyl derivatives and chloroform for (4-methylpiperazin-
1-yl)methyl derivatives. The eluates were combined after TLC
control, and the solvent was removed to give 1-28 as solids in
satisfactory yield. Recrystallization from diethyl ether gave the
required products.
Examples. 1-(4-Fluorophenyl)-2-methyl-3-(thiomorpholin-4-
yl)methyl-5-(2-chlorophenyl)-1H-pyrrole (1). Mp 95 °C (yield
83%); 1H NMR (CDCl3) 7.27-7.25 (m, 2 H), 7.10-7.04 (m, 4H),
7.03-6.92 (m, 2H), 6.28 (s, 1H), 3.49 (s, 2H), 2.80 (m, 4H), 2.72
(m, 4H), 2.08 (s, 3H). Anal. (C22H22ClFN2S) C, H, N, S, Cl, F.
1-(2-Methylphenyl)-2-methyl-3-(thiomorpholin-4-yl)methyl-
5-(4-fluorophenyl)-1H-pyrrole (23). Oil (yield 50%); 1H
NMR (CDCl3) 7.26-7.23 (m, 4H), 7.00-6.98 (m, 2H), 6.80-6.75
(m, 2H), 6.31 (s, 1H), 3.51-3.44 (m, 2H), 2.76 (m, 4H), 2.70 (m,
4H), 1.96 (s, 3H), 1.90-1.77 (m, 3H). Anal. (C23H25FN2S) C, H,
N, S, F.
than that found for both isoniazid and streptomycin and
comparable to that of rifampicin. Although the new compounds
cannot yet be considered as potential clinical candidates,
nevertheless, they are of great interest as hit structures in the
search of new antitubercular agents.
Experimental Procedures
Chemistry. All chemicals used were of reagent grade. Yields
refer to purified products and are not optimized. Melting points
were determined in open capillaries on a Gallenkamp apparatus
and are uncorrected. Microanalyses were carried out by means of
a Perkin-Elmer 240C or a Perkin-Elmer Series II CHNS/O Analyzer
2400. Merck silica gel 60 (230-400 mesh) was used for column
chromatography. Merck TLC plates, silica gel 60 F254 were used
for TLC. Fluka aluminum oxide (activity II-III, according to
Brockmann) was used for chromatographic purifications. Fluka
Stratocrom aluminum oxide plates with fluorescent indicators were
used for thin-layer chromatography (TLC) to check the purity of
the compounds.
1H NMR spectra were recorded with a Bruker AC 400 spec-
trometer in the indicated solvent (TMS as internal standard): the
values of the chemical shifts are expressed in ppm and the coupling
constants (J) in Hz. Mass spectra were recorded on either a Varian
Saturn 3 spectrometer or a ThermoFinnigan LCQ-deca.
General Procedure for the Preparation of Pentane-1,4-diones
31a-h. These compounds were prepared according to the Stetter
reaction as shown in Scheme 1, by reaction of suitable benzalde-
hyde, triethylamine, methyl vinyl ketone, and 3-ethyl-5-(2-hydroxy-
ethyl)-4-methylthiazolium bromide. Briefly, a mixture of the
appropriate benzaldehyde 29a-h (0.09 mol), triethylamine (19.5
mL, 0.14 mol), methyl vinyl ketone 30 (5.8 mL, 0.07 mol), and
3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide (3.53 g,
0.014 mol) was heated at 75-80 °C for 5 h under nitrogen and
then cooled. The residue was treated with 2 N HCl (10 mL). After
extraction with dichloromethane, the organic layer was washed with
aqueous sodium hydrogen carbonate and water. The organic
fractions were dried over Na2SO4, filtered, and concentrated to give
a crude orange liquid. After chromatography on aluminum oxide
(activity II-III, according to Brockmann) (hexane/ethyl acetate,
7:3 v/v), the desired compounds 31 were isolated as light yellow
solids that, after recrystallization from cyclohexane, gave an
analytical sample as white needles.
Examples. 1-(2,4-Difluorophenyl)pentane-1,4-dione (31g). Mp
41 °C (yield 45%), NMR (CDCl3) 7.95 (m, 1H), 6.90 (m, 1H),
6.83 (m, 1H), 3.23 (m, 2H), 2.88 (t, 2H), 2.23 (s, 3H). Anal.
(C11H10F2O2) C, H, F.
1-(2,4-Dichlorophenyl)pentane-1,4-dione (31f). Mp 37 °C
(yield 40%), NMR (CDCl3) 7.73 (m, 1H), 7.45 (m, 1H), 7.15 (m,
1H), 3.20 (m, 2H), 2.62 (t, 2H), 2.23 (s, 3H). Anal. (C11H10Cl2O2)
C, H, Cl.
General Procedure for the Preparation of 1,5-Diarylpyrroles
32a-n. These compounds were prepared by means of the Paal-
Knorr reaction as shown in Scheme 1, by condensing a 1,4-diketone
with the appropriate amine. Briefly, a mixture of the proper diketone
31a-h (2.28 mmol) and the suitable amine (2.28 mmol) in the
presence of p-toluensulfonic acid (30 mg, 0.17 mmol) was heated
at 100 °C for 5 h. The reaction mixture was cooled, filtered, and
concentrated. The crude material was purified by chromatography
with cyclohexane as the eluent to give, in satisfactory yield, the
expected 1,5-diarylpyrrole as a solid. Recrystallization from cy-
clohexane gave the required product.
Microbiology. Compounds. Compounds 1-28 and reference
drugs were dissolved in DMSO at a concentration of 10 mg/mL
and stored cold until used.
Antimycobacterial Activity. Compounds were preliminarily
assayed against two freshly isolated clinical strains, M. fortuitum
CA10 and M. tuberculosis B814, according to the dilution method
in agar.34 Growth media were Mueller-Hinton (Difco) containing
10% OADC (oleic acid, albumine, and dextrose complex) for
M. fortuitum and Middlebrook 7H11 agar (Difco) with 10%
ADC (albumine and dextrose complex) for M. tuberculosis. The
substances were preliminarily screened at a single dose of 100
µg/mL. Compounds that resulted active in the preliminary test were
assayed for detecting the minumum inhibitory concentration (MIC).
Only compounds with MIC values of 16 µg/mL or less were then
further studied for inhibitory activity against a variety of myco-
bacteria in Middlebrok 7H9 broth using the NCCLS procedure.
The mycobacteria used for biological tests were M. tuberculosis
CIP 103471 and atypical mycobacteria, such as M. marinum CIP
6423, M. aVium CIP 103317, and M. smegmatis CIP 103599 (from
the Institute Pasteur collection, CIP). Results are reported in Tables
1 and 2. For each compound, MIC values (expressed in µg/mL)
were determined on each strain of mycobacteria.
The MIC was defined as the lowest concentration of drug that
yielded an absence of visual torbidity. Stock solutions of substances
were prepared by dissolving a known weight of compound in
DMSO. The stock solutions were sterilized by passage through a
0.2 µm nylon membrane filter. Serial 2-fold dilutions of the com-
pounds with water were prepared. The tubes were inoculated with
test mycobacteria at a concentration of 105 to 106 colony forming
units (CFU)/mL and incubated at 37 °C for 3-21 days. A control
tube without any drug was included in each experiment. Isoniazid,
streptomycin, rifampicin, and BM212 were used as controls.
Inhibitory Activity on Clinical Isolates of M. tuberculosis.
Compounds 5 and 17 were also assayed toward a panel of 11
clinical isolates of M. tuberculosis in Middlebrook 7119 broth
enriched with 10% ADC (Difco) using the macrodilution broth
method.
Examples. 1-(4-Fluorophenyl)-2-methyl-5-(2-chlorophenyl)-
1
1H-pyrrole (32a). Mp 127 °C (yield 60%); H NMR (CDCl3)
7.26 (m, 2H), 7.12-7.05 (m, 4H), 6.98-6.95 (m, 2H), 6.30 (m,
1H), 6.13-6.12 (m, 1H), 2.14 (s, 3H). Anal. (C17H13ClFN) C, H,
N, Cl, F.
1-(2-Methylphenyl)-2-methyl-5-(4-fluorophenyl)-1H-pyr-
1
role (32l). Mp 170 °C (yield 65%); H NMR (CDCl3) 7.27-7.24
(m, 4H), 7.04-7.00 (m, 2H), 6.82-6.78 (m, 2H), 6.33 (m, 1H),
6.09-6.08 (m, 1H), 1.99 (m, 3H), 1.82 (s, 3H). Anal. (C18H16FN)
C, H, N, F.
Cytotoxic Activity Assays. Vero cells were inoculated in 6-well
plates each containing 9 × 104 cells and incubated in Dulbecco’s
Minimum Essential Medium (DMEM) with 5% fetal calf serum

4952 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
BiaVa et al.
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(FS) for 24 h at 37 °C in a 5% CO2 incubator. After 24 h of culture,
the medium was changed, and a new medium containing decreasing
doses of the substances under study was added.
After 5 days, cells were trypsinized and counted in a Neubauer
chamber under a light microscope. All the tests were done in
triplicate. The maximal 50% nontoxic dose (MNTD50) was defined
as the drug concentration that decreased cell multiplication less than
50% with respect to the control.
Computational Methods. All calculations and graphic manipu-
lations were performed on a SGI Origin 300 server and SGI Octane2
workstations by means of the Catalyst 4.10 software package.35
All the compounds used in this study were built using the 2-D-
3-D sketcher of Catalyst. A representative family of conformations
was generated for each molecule using the poling algorithm and
the best quality conformational analysis method. Conformational
diversity was emphasized by selection of the conformers that fell
within a 20 kcal/mol range above the lowest energy conformation
found.
The Compare/Fit command within Catalyst was used to align
the studied compounds onto the pharmacophoric model. Particularly,
the Best Fit option was selected, which manipulated the conformers
of each compound to find, when possible, different mapping modes
of the ligand within the model.
The QSAR+ module of Cerius2 was used to calculate Alog P98
values of the studied compounds. Such a descriptor is an imple-
mentation of the atom type-based Alog P method using the latest
published set of parameters.36
Acknowledgment. Financial support from the Italian Min-
istero dell’Istruzione, dell’Universita` e della Ricerca (PRIN
2005037820) is gratefully acknowledged.
Supporting Information Available: Experimental details (de-
tails of synthesis and elemental analysis data of compounds). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(28) Biava, M.; Porretta, G. C.; Deidda, D.; Pompei, R.; Tafi, A.; Manetti,
F. Antimycobacterial compounds. New pyrrole derivatives of BM
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(31) In the text, we use the following notation. The terms thiomorpho-
line derivatives and N-methylpiperazine derivatives refer to com-
pounds bearing a (thiomorpholin-4-yl)methyl and a (4-methylpiper-
azin-1-yl)methyl moiety at position 3 of the pyrrole ring, respec-
tively.
(32) Cerius2, version 4.8.1 is distributed by Accelrys, Inc., Scranton Rd.,
San Diego, CA.
(33) Ragno, R.; Marshall, G. R.; Di Santo, R.; Costi, R.; Massa, S.;
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(35) Catalyst, version 4.10 is distributed by Accelrys, Inc., Scranton Rd.,
San Diego, CA.
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