1,4-Diarylpiperazines and analogs as anti-tubercular agents: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2011.12.035

Despite progress in modern chemotherapy to combat tuberculosis, the causative pathogen Mycobacterium tuberculosis (M.tb.) is far from eradicated. Bacillary resistance to anti-mycobacterial agents, bacillary persistence and human immunodeficiency virus (HIV) co-infection hamper current drug treatment to completely cure the infection, generating a constant demand for novel drug candidates to tackle these problems. A small library of novel heterocyclic compounds was screened in a rapid luminometric in vitro assay against the laboratory M.tb. strain H37Rv. A group of amidines was found to have the highest potency and was further evaluated for acute toxicity against C3A hepatocytes. Next, the most promising compounds were evaluated for activity against a multi-drug resistant clinical isolate. The group of amidines was also tested for their ability to kill intracellular M.tb. residing in mouse J774A.1 macrophages. Finally, we report on a correlation between the structural differences of the compounds and their anti-mycobacterial activity.

Full text of DOI:10.1016/j.ejmech.2011.12.035

European Journal of Medicinal Chemistry 49 (2012) 95e101
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1,4eDiarylpiperazines and analogs as anti-tubercular agents: Synthesis
and biological evaluation
,
,
D. Forge a 1, D. Cappoen b 1, J. Laurent a, D. Stanicki a, A. Mayence c, T.L. Huang c, L. Verschaeve d,
K. Huygen b , J.J. Vanden Eynde
,
a,
**
*
a University of Mons-UMONS, Laboratory of Organic Chemistry, 20 place du Parc, B-7000 Mons, Belgium
b Scientific Institute of Public Health WIV-ISP, Service Immunology, Engelandstraat 642, B-1180 Ukkel, Belgium
c Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125, USA
d Scientific Institute of Public Health WIV-ISP, Operational Directorate of Public Health and Surveillance, J. Wytsmanstraat 14, B-1050 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Despite progress in modern chemotherapy to combat tuberculosis, the causative pathogen Mycobacte-
rium tuberculosis (M.tb.) is far from eradicated. Bacillary resistance to anti-mycobacterial agents, bacillary
persistence and human immunodeficiency virus (HIV) co-infection hamper current drug treatment to
completely cure the infection, generating a constant demand for novel drug candidates to tackle these
problems.
Received 22 November 2011
Received in revised form
19 December 2011
Accepted 22 December 2011
Available online 21 January 2012
A small library of novel heterocyclic compounds was screened in a rapid luminometric in vitro assay
against the laboratory M.tb. strain H37Rv. A group of amidines was found to have the highest potency and
was further evaluated for acute toxicity against C3A hepatocytes. Next, the most promising compounds
were evaluated for activity against a multi-drug resistant clinical isolate. The group of amidines was also
tested for their ability to kill intracellular M.tb. residing in mouse J774A.1 macrophages. Finally, we report
on a correlation between the structural differences of the compounds and their anti-mycobacterial
activity.
Keywords:
Mycobacterium tuberculosis
1,4-Diarylpiperazines
Anti-mycobacterial activity
Cytotoxicity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
(MDR) M.tb., predominantly caused by non-compliance to the
current drug regimen, has rendered the actual treatment ineffec-
Since the introduction of streptomycin in the early 20th
century, modern chemotherapy has played a significant role in
mankind’s fight against tuberculosis (TB) [1,2]. The actual standard
first line treatment regimen is comprised of a combination of four
drugs, i.e. isoniazid, rifampicin, ethambutol and pyrazinamide.
Despite its efficacy to kill actively replicating Mycobacterium
tuberculosis (M.tb.), the pathogen has not been eradicated [3].
Although some interesting new drug candidates are under devel-
opment, the current drug regimen is more than 40 years old.
Development of novel drug candidates is a slow process and M.tb.
is a fearsome opponent [1,3,4]. With the unique structure of its
thick, lipid-rich cell wall, it is hardly permeable for chemothera-
peutic compounds [5]. The emergence of multi-drug resistant
tive in many cases [5,6]. This is well documented through the data
presented by the World Health Organization (WHO) [7]. Drug
regimens with various second-line agents are needed for the
treatment of MDR M.tb. On the flip side, expansion of the various
drug regimens has resulted in a heavier burden for patients
because of the increased toxicity of the treatment. At the same
time, healthcare organizations have faced an increase in adminis-
tration and production costs. Additional resistance to fluo-
roquinolones (FQ) and (second-line) injectable drugs has led to
emergence of extensively-drug resistant (XDR) M.tb., which caused
new outbreaks of the disease at the start of the 21st century [5e7].
Another trait of M.tb. that makes it a difficult target for
chemotherapy is that the bacillus is capable to become metaboli-
cally inactive and to reside in a dormant stage within the host
phagocytic cells. These ‘persister’ bacteria are estimated to survive
in one third of the world population as a latent infection, but so far
no active drugs against these dormant bacilli are available for
therapy [8e10]. Moreover, the AIDS pandemic has exacerbated the
situation, as persons co-infected with HIV present with an annual
10% risk to reactivate a latent TB infection, as compared to non-HIV
* Corresponding author. Tel.: þ3223733370; fax: þ3223733367.
** Corresponding author. Tel.: þ3265373337.
E-mail addresses: kris.huygen@wiv-isp.be (K. Huygen), Jean-Jacques.Vandeneynde@
umons.ac.be (J.J. Vanden Eynde).
1
Both authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.035

96
D. Forge et al. / European Journal of Medicinal Chemistry 49 (2012) 95e101
infected subjects in whom the risk is estimated to be 10% on a life-
time basis. Drugedrug interactions between antiretroviral drugs
and anti-tuberculosis drugs frequently occur and limit the treat-
ment possibilities [11e13]. Although anti-tuberculosis drug devel-
opment is a far from trivial matter, there is an ongoing supply of
novel anti-tuberculosis drug candidates [14].
M.tb. and the ability of the library to inhibit intracellular growth of
M.tb. residing in macrophages. Determination of cytotoxicity (NI50
of the selected compounds was also included in our screening
procedure.
)
2. Results and discussion
The piperazine structure constitutes an important research area
in organic synthesis due to its pronounced biological activities.
Previously, we have reported that the 1,4-diarylpiperazines show
activity against Pneumocystis carinii, trypanosoma and leishmania
[15e17]. Furthermore, piperazine derivatives have shown activity
against the laboratory strain M.tb. H37Rv, with piperazine
1,4-disubstituted by two N-alkyl benzamidine groups identified as
the most active compound against tuberculosis [18]. Interestingly,
the piperazine core is also present in rifampicin, one of compounds
used in the first line treatment of TB.
So far, the structureeactivity relationship of these piperazine
derivatives has not been evaluated. Therefore, we synthesized an
expanded series of heterocyclic structures to better understand the
influence of structure on microbial activity and cell cytotoxicity.
The importance of parameters such as the lipophilicity, the
substitution pattern on the benzene ring as well as the presence of
electron-donating or electron-withdrawing groups (both halogen)
has been reported in earlier structureeactivity analyses of other
anti-mycobacterial compounds [19e21]. In this study, we examined
the potency of 1,4-diarylpiperazine derivatives against M.tb., MDR
The synthesis of the different heterocyclic compounds was
previously described. Briefly, as illustrated in Fig. 1, compounds
1e12 were obtained by a nucleophilic displacement of fluorine in 4-
fluoro derivatives by the nitrogen atoms of piperazine or diethylene
amine [22,23]. Conversion of the nitriles into amidines (compounds
13e29) was realized through the Pinner Reaction. Treatment with
dichloromethane, methanol and gaseous hydrochloric acid gave the
intermediate bisbenzenecarboximidic esters which were subse-
quently reacted with ammonia or the appropriate primary amine
(Fig. 1).
Anti-mycobacterial activity of the 1,4-diarylpiperazines was
tested in a luminometric assay. In this assay a luminescent H37Rv
M.tb. strain is used, which has been transformed with the pSMT1
plasmid encoding the luxAB genes from V. harveyi driven by the
constitutive hsp60 promoter [24]. This tool enables rapid assess-
ment of bacterial replication and of the influence of anti-
mycobacterial agents [25].
The first screen was done for all the derivatives at concentra-
tions of 10
mM, 1 mM and 0.1 mM. All the derivatives showed
Fig. 1. Synthesis of compounds 1e29.

D. Forge et al. / European Journal of Medicinal Chemistry 49 (2012) 95e101
97
antimicrobial activity against M.tb. H37Rv as reported in Table 1. For
further screening, only compounds that induced a 90% growth
reduction at 1 mM after 7 days of culture were selected. Compounds
19e22 met this requirement with inhibitions ranging between 91%
and 97%. Furthermore, the results revealed that the nature of the
substitution on the phenyl ring had a considerable impact on the
anti-mycobacterial activity.
Secondly, a symmetric structure did not improve the inhibition
of bacterial growth (compound 1 versus 4, 2 versus 5, 3 versus
6 and 13 versus 26). Data reported by Chabria and al showed
that the substitution at the para-position of the phenyl ring
influenced the anti-mycobacterial activity [19]. In the case of the
1,4ediarylpiperazines, the efficacy was not influenced by the
position (compounds 7e12).
Firstly, the presence of nitro-, nitrile-, amine- or amidine group
did not appear to influence the activity (compounds 1e3 and 13).
Importantly, the biological activity seemed to correlate with the
length of the carbon chain on the amidine nitrogen. A remarkable
Table 1
Anti-mycobacterial activity (% inhibition of Mycobacterium tuberculosis) and calculated LogP (www.molinspiration.com) of heterocyclic derivatives (1e29).
% of inhibition with regard to the positive control at day 7
LogP
[ ] ¼ 10
mM
[ ] ¼ 1
mM
[ ] ¼ 0.1
mM
R1
N
N
R2
1
2
3
4
5
6
R1 ¼ H R2 ¼ NO2
R1 ¼ H R2 ¼ NH2
R1 ¼ H R2 ¼ CN
26
30
23
13
27
22
<10
<10
<10
10
10
<10
<10
<10
<10
<10
<10
<10
3.59
2.71
3.38
3.55
1.78
3.14
R1 ¼ NO2 R2 ¼ NO2
R1 ¼ NH2 R2 ¼ NH2
R1 ¼ CN R2 ¼ CN
R
R
NH HN
7
8
9
10
11
12
R1 ¼ NO2 R2 ¼ NO2 (para substitution)
16
<10
13
10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
3.20
3.1
3.15
2.79
2.7
R1 ¼ NO2 R2 ¼ NO2 (ortho substitution)
R1 ¼ NO2 R2 ¼ NO2 (meta substitution)
R1 ¼ CN R2 ¼ CN (para substitution)
R1 ¼ CN R2 ¼ CN (ortho substitution)
R1 ¼ CN R2 ¼ CN (meta substitution)
2.74
NH.HCl
NHR
N
N
13
14
15
16
17
18
19
20
21
22
R ¼ H
14
16
10
33
66
95
99
100
100
100
<10
<10
<10
<10
<10
39
91
94
94
97
<10
<10
<10
<10
<10
<10
49
47
61
68
2.01
2.38
2.76
3.26
3.82
4.33
4.83
5.35
5.85
7.87
R ¼ CH3
R ¼ C2H5
R ¼ C3H7
R ¼ C4H9
R ¼ C5H11
R ¼ C6H13
R ¼ C7H15
R ¼ C8H17
R ¼ C12H23
23
67
19
<10
4.13
R =
24
25
R ¼ C2H5eC6H5
97
97
56
39
<10
<10
4.19
4.36
R ¼ C2H5eC6H4eF
ClH.HN
H2N
NH.HCl
NH2
N
N
26
17
<10
<10
0.40
NH.HCl
NHR
NH HN
27
28
R ¼ H
<100
<10
<10
1.66
3.78
4.49
63
19
10
R =
29
INH
R ¼ C6H13
93
100
48
100
23
98

98
D. Forge et al. / European Journal of Medicinal Chemistry 49 (2012) 95e101
activity was found for the compounds 20e22, with carbon chains
concentrations ranging from 0.264 mM to 0.052 mM. Compounds
of 7, 8 and 12 carbon atoms respectively. After 7 days of treatment
with 1 mM of compound 22, only 3% of bacteria were surviving. The
19e22 showed a lower MIC50 than the first line drug isoniazid,
which was used as a positive control in the assay.
lipophilicity of a drug, characterized by its LogP value, is well
known to play an important role in the penetration of a compound
into bacterial cells [26,27]. LogP values (the log of the octanol/water
partition coefficient) were calculated for the 29 compounds using
www.molinspiration.com software, which determines the hydro-
phobic parameters of the substituents (Table 1). Molecules with
high LogP values have high hydrophobicity [28]. As reported in the
literature, an increase in the lipophilic nature of the derivatives
improved the anti-mycobacterial activity. Indeed, in our study, the
inactive compounds presented a hydrophilic character (LogP < 4).
With more than 4 carbon atoms on the side chain (starting with
compound 18), the compounds become hydrophobic but we could
The compounds were also screened for acute cytotoxicity in
a neutral red (NR) dye uptake assay. The assay was performed on
C3A hepatocytes because toxicity of novel drug candidates often
results in liver necrosis. Hepatoxicity is also one of the problems
encountered when treating patients with some of the current
second-line TB drugs [3]. NR uptake is based on the ability of viable
cells to endocytose and bind the dye neutral red (3-amino-7-
dimethylamino-2-methyl-phenazine). The NI50 is the concentra-
tion at which there is a reduction of 50% in NR uptake as a result of
the inability of dead or damaged cells to endocytose NR [29]. The
compounds were tested for acute toxicity at concentrations ranging
from 10 mM to 0.1 mM.
only detect an activity at 10
carbon atoms on the amidine group resulted in killing of 90% of
M.tb at a 1 M concentration. Compound 22 with 12 carbon atoms
had the highest LogP value (7.87) and displayed the strongest anti-
mycobacterial activity.
m
M. Presence of a chain of at least 7
The results showed that toxicity increased and NI50 lowered
progressively with the elongation of the carbon chain. Thus for
compound 19, the NI50 was more than 10 fold lower than for the
parent molecule 13. Further elongation of the amidine group by
extra carbon residues did not increase the toxicity much further
m
Moreover, the linearity of the carbon chain plays an important
role for its activity. For the same number of carbon atoms,
a compound with a linear carbon chain was more active than its
analog bearing a branched chain (compound 18 versus 23). Simi-
and we found a plateau value between 5 mM and 6 mM for
compounds 19e22. As a measure of the compound selectivity, the
selectivity index (SI) was calculated for each compound by dividing
the NI50 by the MIC50. As shown in Table 2, when we compared
compound 22 with the parent molecule 13, the SI was enhanced
almost 50 fold. The results are graphically summarized in Fig. 2,
showing the NI50 and MIC50 values of the compounds and the
resulting SI as plotted against the LogP values.
We also studied the susceptibility of a multi-drug resistant M.tb.
strain for 5 of the most potent derivatives, using the BACTEC 460-TB
system which is regarded as the golden standard in M.tb. suscep-
tibility testing [30]. A clinical MDR isolate of M.tb. was spoligotyped
and classified as a LAM-1 strain. The isolate was resistant to isoni-
azid, rifampicin, rifabutin and prothionamid. The MIC99 value of the
various compounds i.e. the concentration at which 99% of the
bacterial growth was inhibited is shown in Table 3. Compounds
larly,
a linear carbon chain was more active than a cycle
(compound 19 versus 24). Introducing a fluorine atom onto the
phenyl ring did not improve the anti-mycobacterial activity in our
analysis, in contrast to what has been reported in an earlier study
[21]. A comparison between compounds 19 and 29 showed the
importance of maintaining the piperazine core into the structure
of the compounds. Replacing the piperazine ring by an ethyl-
enediamine moiety decreased the anti-mycobacterial activity at
a concentration of 1 mM. Similarly, the activity of compound 23
was also enhanced in comparison to the analog 28.
Because differences in lipophilicity showed the strongest
influence on the anti-mycobacterial activity, this parameter was
further examined in a more detailed study of parent compound 13
and its derivatives 14e22. The minimal inhibitory concentration
18e22 showed MIC99 values that ranged between 0.95
mM and
3.85 M. The order of potency was the same as the one observed for
m
at which 50% of the bacterial growth is inhibited (MIC50
)
M.tb. H37Rv. The lowest potency/highest MIC99 was found for
compound 18, and the highest potency/lowest MIC99 for compound
22. These low MIC99 concentrations suggest that the compounds
have another mechanism of action than the three TB drugs against
which the LAM-1 strain was found to be resistant.
Because M.tb. is mainly an intracellular pathogen, it was inter-
esting to investigate whether the amidine derivatives 13e22
showed antimicrobial activity against M.tb. H37Rv residing inside
macrophages. In this assay we infected a murine macrophage cell
line J774A.1. Cells were then treated with the compounds for five
was determined, as well as the acute toxicity of the analogs. As
shown in Table 2, the previous findings were confirmed. Elonga-
tion of the carbon chain of the substituent from 1 to 6 carbon
residues, and from a LogP of 2.38 to a LogP of 4.83 lowered the
MIC50 considerably (14e19). For compounds bearing a chain of 6, 7,
8 or 12 carbon atoms, the MIC50 was lowered to sub-micromolar
Table 2
MIC50, NI50 and the calculated selectivity index and LogP value of the 10 selected
amidine derivatives on the drug-sensitive M. tuberculosis strain H37Rv.
days at a concentration of 2 mM, 1 mM and 0.5 mM. The intracellular
a
b
Compound
MIC50
NI50
SIc
LogPd
anti-mycobacterial activity increased progressively for compounds
13e20 (Table 4, Fig. 3). The compound with the highest observed
intracellular activity was compound 19, which showed 88.32%
13
14
15
16
17
18
19
20
21
22
INH
29.612
15.947
11.950
7.010
2.162
1.198
0.264
0.091
0.072
0.052
0.465
66.93
70.98
41.69
27.51
17.36
11.24
6.71
5.21
5.18
5.61
2.26
4.45
3.49
3.92
8.03
9.39
25.38
57.20
72.19
107.52
ND
2.01
2.38
2.76
3.26
3.82
4.33
4.83
5.35
5.85
7.87
ND
growth inhibition at a 2
mM concentration as compared to an
untreated, negative control. For compounds 21 and 22, activity
against intracellular M.tb. was lower than for compounds 19 and 20,
despite increased LogP values and derivative 22, the most prom-
ising candidate in the extracellular assays, only inhibited 44.9% of
the bacterial growth at a concentration of 2
mM. Compounds 19e21
ND
showed a higher antimicrobial effect against intracellular bacteria
than the first line drug isoniazid at 2 mM concentration.
a
MIC50, is the minimal concentration (in mM) at which 50 percent of the bacterial
growth is inhibited.
b
NI50 the minimal concentration at which the uptake of neutral red is reduced by
3. Conclusions
50%.
c
SI, the selectivity index, is the NI50 divided by the MIC50 and is a measure of the
selectivity.
A series of heterocyclic compounds were evaluated as poten-
tial drug candidates for the chemotherapy of TB. Some of the
d
LogP, calculated on www.molinspiration.com.

D. Forge et al. / European Journal of Medicinal Chemistry 49 (2012) 95e101
99
Fig. 2. MIC50, NI50 and selectivity index (SI) in function of the calculated lipophilicity of compounds 13e22.
synthesized compounds showed significant activity against a drug-
sensitive M.tb. H37Rv strain. Linearity of the side chain and the
presence of a piperazine core in the structure are necessary for an
efficient inhibition of bacterial growth. Analogs 13e22 were further
investigated in detail and revealed a wide MIC50 range varying
derivatives might decrease, which could partially prevent the
compounds from reaching the unknown intracellular target.
Another possibility would be that longer carbon chains could
promote cyclization within the cell and this could significantly
alter the properties of the compound.
between 29.61
m
M and 0.05
m
M. Elongation of the carbon chain in
The results obtained with the BACTEC 460-TB showed that
a multi-drug resistant LAM-1 strain was susceptible to this class of
compounds. Because this strain is resistant to isoniazid, rifampicin,
rifabutin and prothionamid, the 1,4ediarylpiperazines probably
have a different mechanism of action than these classical antibi-
otics. Additional studies are needed to determine whether this class
of piperazine derivatives is suitable for further development as
candidate drugs against MDR TB. Although compounds with
increased LogP showed enhanced potency, increased LogP might
also result in lowered bioavailability and lowered tissue distribu-
tion [31]. To address this question, in vivo studies combined with
pharmacokinetics and LogD determination, taking into account the
pKa of the compounds are needed. Future efforts for a structural
optimization of this class of compounds such as the synthesis of
piperazine salts are ongoing.
the amidine side chain altered the lipophilicity and resulted in
enhanced anti-mycobacterial activity. Compound 22 with a LogP
of 7.87, exhibited a MIC50 value 570 fold lower than the parental
compound 13 which had a LogP of 2.01. However, with increasing
anti-mycobacterial activity, compounds also showed increasing
acute toxicity, as detected in a C3A hepatocyte based neutral red
uptake assay. Thus derivative 22 was also 12 fold more toxic than
the parent compound 13, but overall selectivity index of compound
22 was still 50 fold higher than the SI of compound 13.
The piperazine derivatives were also active against intracellular
M.tb. H37Rv and the best compounds in this assay (19e21)
inhibited the multiplication in murine macrophages more effi-
ciently that the standard drug isoniazid. It remains to be deter-
mined to what extent the results of the macrophage infection
assay will be confirmed in an in vivo mouse infection model.
Compounds with high LogP were more active in the macrophage
infection assay, but beyond a LogP of 5.35 (compound 20) the
potency diminished progressively. This could possibly be attrib-
uted to an increased difficulty for compounds with higher
lipophilicity to cross the cell membrane lipid bilayer of the
macrophage and the lack of a specific transporter. In light of these
findings, the results of the acute toxicity test on C3A hepatocytes
could be interpreted differently. We observed that further
increasing the lipophilicity of compounds 19 to 22 did not further
increase the cytotoxicity. A possible explanation would be that
beyond a certain threshold the permeability of the cells to the
4. Experimental section
4.1. Testing in vitro mycobacterial growth inhibition
Identification of the candidates and selection of the most active
derivatives from the library was done by a rapid luminometric
Table 4
Activity of the amidine derivatives 13e22 against intracellular M.tb. H37Rv.
Inhibition (%)
Compounds
2
m
M
1
mM
0.5 mM
Table 3
13
14
15
16
17
18
19
20
21
22
INH
1.83 ꢀ 8.23
0.41 ꢀ 8.85
6.43 ꢀ 6.01
ꢁ0.47 ꢀ 10.45a
ꢁ0.8 ꢀ 2.78a
3.93 ꢀ 7.15
In vitro activity of compounds 18e22 against a multi-
drug-resistant clinical isolate of M. tuberculosis.
14.92 ꢀ 6.69
22.46 ꢀ 10.48
26.07 ꢀ 8.33
50.56 ꢀ 10.40
64.70 ꢀ 10.10
88.07 ꢀ 5.49
83.32 ꢀ 7.04
71.64 ꢀ 6.19
44.89 ꢀ 8.72
51.39 ꢀ 6.32
13.37 ꢀ 10.52
12.93 ꢀ 6.92
31.56 ꢀ 6.11
53.65 ꢀ 8.80
79.23 ꢀ 5.50
64.19 ꢀ 8.89
31.14 ꢀ 9.96
23.38 ꢀ 7.98
29.38 ꢀ 8.68
5.62 ꢀ 9.61
a
Compound
MIC99
(
m
M)
25.61 ꢀ 10.05
44.72 ꢀ 9.92
67.13 ꢀ 5.83
61.10 ꢀ 6.00
15.24 ꢀ 9.75
10.72 ꢀ 10.21
9.77 ꢀ 8.84
18
19
20
21
22
3.85
2.11
1.48
1.17
0.95
a
MIC99 refers to the minimal inhibitory concentration
at which 99% of the bacterial growth is inhibited.
a
Negative values reflect higher growth in treated than in untreated cultures.

100
D. Forge et al. / European Journal of Medicinal Chemistry 49 (2012) 95e101
4.3. Macrophage infection assay
Compounds were tested on a murine J774.A1 macrophage cell
line infected with luminescent M.tb. H37Rv transformed with
the pSMT1 luciferase reporter plasmid. The J774 macrophages
were grown at 37 ꢂC, 5% CO2 in complete DMEM medium
[DMEM (Gibco) þ10% FCS (Gibco) þ 1% Non essential amino acids
(Gibco) þ 1% Sodium Pyruvate (Gibco) þ 1% Glutamax (Gibco) þ
0.1% 2-mercaptoethanol (Gibco) þ 1% gentamycin (Gibco)] until
a semi confluent layer was formed. The macrophages were washed
and seeded onto a flat-bottomed 96 microwell plate at a cell density
of 40 000 cells per well. The cells were left to recover overnight and
were washed three times. M.tb. H37Rv was grown at 37 ꢂC in
7H9 þ 10% FCS þ 0.2% hygromycin (Roche) to an O.D between 0.6
and 1.0. The fully grown bacterial suspension was measured and
brought into complete DMEMPen/Fung [DMEM medium þ 0.1%
penicillin (Roche) þ 0.8% Fungizone (Gibco) without gentamycin].
Compounds were solubilized in DMSO at stock concentrations of
1 mM. Serial dilutions of each compound were made in DMEMPen/
Fung in which 2 times the final concentration of each compound
Fig. 3. Activity of the amidine derivatives 13e22 at 2
H37Rv versus their LogP.
mM against intracellular M.tb.
in vitro assay. In this assay a luminescent H37Rv M.tb. strain is used,
which has been transformed with the pSMT1 plasmid encoding the
luxAB genes from V. harveyi driven by the constitutive hsp60
promoter [24]. A glycerol stock of M.tb. kept at ꢁ80ꢂ was thawed,
diluted in synthetic Sauton mediumþ 10% Fetal calf serum (FCS)
was prepared. A volume of 100
DMEMPen/Fung containing 4000 RLU of bacteria (multiplicity of
infection of 0.1.) and 100 L of the serial compound dilutions were
mL of the bacterial suspension in
m
added to the macrophage cultures. To measure the effects of the
compounds on intracellular growth of M.tb, the infected macro-
phages were washed three times on day 5 to remove all extracel-
and seeded at 1000 relative light units (RLU) in a 180 mL volume per
well of a flat-bottomed 96 microwell plate. Stock solutions of the
compounds were made at 1 mM in DMSO (Sigma). Further dilu-
tions were made in Sauton medium þ10% FCS and added to the
lular bacteria, incubated 1 h with 1% gentamicine, lysed with 200
1% Triton X-100 (Sigma) and the wells washed four times with
200 L PBS. The lysate was transferred in a 2.5 mL Eppendorf tube
together with the 4 PBS washings.100 L of 1% n-decanal in ethanol
mL
wells to reach a 10, 1 and 0.1
mM final concentration of the
m
compounds in the wells. Cultures were incubated at 37 ꢂC in
a humidified 5% CO2 incubator and after 7 days of culture the
number of luminescent bacteria in each well were enumerated.
From each well the bacterial suspension was harvested and each
m
was added to the Eppendorf tube and emission of luminescence
was measured over a time span of 10 s with a Turner Modulus
Single Tube Luminometer from Biosystems. RLU values shown were
obtained from six replicate cultures.
well was washed four times with 200
ml of PBS (DIFCO). The
bacterial suspension and the washings were pooled in a 2.5 mL
Eppendorf tube in a total volume of 1 ml and luminescence was
4.4. Assesment of acute cytotoxicity
measured by adding 100
mL 1% n-decanal in ethanol (Sigma)
substrate to the mixture. The signal was measured over 10 s using
a Turner Modulus Single Tube Luminometer from Biosystems.
Percentage of growth inhibition was calculated by comparison
with RLU values detected in control M.tb. H37Rv cultures grown
without the test compounds. For the determination of MIC50
values, bacteria were grown for 6 days and concentration was
calculated as the minimal concentration in which 50% of the
bacterial growth (as measured in control, untreated cultures) was
inhibited.
The inhibitory concentration at which the uptake of neutral red
was reduced by 50% (NI50) was determined according to the
following protocol. C3A cells were grown in DMEM þ 10% FCS until
a semi confluent layer of cells was obtained. The cells were tryp-
sinized, washed and 40 000 cells were seeded per well in a flat-
bottomed 96 well plate and left for recovery at 37 ꢂC, 5% CO2. The
following days, the compounds were solubilized in DMSO to stock
concentrations of 1 mM. A serial dilution of each compound was
made in DMEM þ 10% FCS to achieve the final concentrations. The
C3A cells were washed and exposed to the derivatives by adding
the serial dilutions of the compounds to the wells. The plates were
left for incubation at 37 ꢂC, 5% CO2 for 24 h. After exposure, the cells
4.2. Testing anti-mycobacterial activity against multi-drug resistant
M.tb
were washed with 200
mL PBS and 200 mL neutral red working
The MDR strain used was a clinical isolate which had been
spolygotyped to be a LAM-1 strain and which showed resistance to
isoniazid, rifampicin, rifabutin and prothionamid. BACTEC 460-TB
was used to monitor the cultures. The compounds were solubi-
lized in DMSO (Sigma) at stock concentrations of 1 mM. Serial
dilutions of each compound were made in 7H9 þ 10% OADC, at 40
times the final concentrations. A bacterial pre-culture of M.tb LAM-
1 was made in a 4 mL BACTEC vial containing 7H9 based medium
until a Growth index (GI) of 300 was reached. When the pre-
solution (Sigma) were added per well. Subsequently the plates
were incubated for 3 h at 37 ꢂC, 5% CO2. The wells were washed
with 200 mL PBS and 200 mL of an ethanol/acetic acid (50%) mixture.
The plates were left on the shaker until the color became homo-
geneously purple and the optical density was measured at 530 nm
(NR max) and 620 nm (reference wavelength) with the Paradigm
detection platform.
Acknowledgments
culture was fully grown, 100
BACTEC vial together with 100
m
L were inoculated into a new 4 ml
L of the serial dilutions of the
m
The authors thank F.R.S.eFNRS for financial support (DS and
JJVE). Work by DC and KH was partially supported by FWO-
Vlaanderen (Grant G.0020.10N). We also want to thank Dr. M.
Fauville and Dr. V. Mathys (Scientific Institute of Public Health,
Brussels, Belgium) for the help on susceptibility testing of the MDR
compounds. The GI was measured each day. To determine the
MIC99, the cultures, treated with the compounds were compared
with an untreated culture that was diluted 100 times upon
inoculation.

D. Forge et al. / European Journal of Medicinal Chemistry 49 (2012) 95e101
101
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