Piperazine derivatives: Synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase and SAR studies

Article abstract of DOI:10.1016/j.ejmech.2014.11.034

The Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) catalyzes hydride transfer to long-chain enoyl thioester substrates. MtInhA is a member of the mycobacterial type II dissociated fatty acid biosynthesis system, and is the bona fide target for isoniazid, the most prescribed drug for tuberculosis treatment. Here, a series of piperazine derivatives was synthesized and screened as MtInhA inhibitors, which resulted in the identification of compounds with IC₅₀ values in the submicromolar range. A structure-activity relationship (SAR) evaluation indicated the importance of the chemical environment surrounding the carbonyl group for inhibition. In addition, the structure of one selected compound was supported by crystallographic studies, and experimental geometrical values were compared with semi-empirical quantum chemical calculations. Furthermore, the mode of inhibition and inhibitory dissociation constants were determined for the nine most active compounds. These findings suggest that these 9H-fluoren-9-yl-piperazine-containing compounds interact with MtInhA at the enoyl thioester (2-trans-dodecenoyl-CoA) substrate binding site.

Full text of DOI:10.1016/j.ejmech.2014.11.034

Accepted Manuscript
Piperazine derivatives: Synthesis, inhibition of the Mycobacterium tuberculosis enoyl-
acyl carrier protein reductase and SAR studies
Mariane Rotta , Kenia Pissinate , Anne Drumond Villela , Davi Fernando Back ,
Luis Fernando Saraiva Macedo Timmers , José Fernando Ruggiero Bachega ,
Osmar Norberto de Souza , Diógenes Santiago Santos , Luiz Augusto Basso , Pablo
Machado
PII:
S0223-5234(14)01066-6
10.1016/j.ejmech.2014.11.034
EJMECH 7527
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 July 2014
Revised Date: 13 November 2014
Accepted Date: 18 November 2014
Please cite this article as: M. Rotta, K. Pissinate, A.D. Villela, D.F. Back, L.F.S. Macedo Timmers, J.F.R.
Bachega, O.N. de Souza, D.S. Santos, L.A. Basso, P. Machado, Piperazine derivatives: Synthesis,
inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase and SAR studies,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.11.034.
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ACCEPTED MANUSCRIPT
Piperazine derivatives: synthesis, inhibition of the Mycobacterium tuberculosis
enoyl-acyl carrier protein reductase and SAR studies
1
,2
1,2
1,3
4
Mariane Rotta , Kenia Pissinate , Anne Drumond Villela , Davi Fernando Back ,
1
,2,5
1,5
Luis Fernando Saraiva Macedo Timmers
, José Fernando Ruggiero Bachega
,
,
1
,2,5
1,2*
1-3
Osmar Norberto de Souza
, Diógenes Santiago Santos
, Luiz Augusto Basso
1
,2*
Pablo Machado
1
Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em
Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do
Sul, 90619-900, Porto Alegre, Rio Grande do Sul, Brazil
2
Programa de Pós-graduação em Biologia Celular e Molecular, Pontifícia Universidade
Católica do Rio Grande do Sul, 90619-900, Porto Alegre, Rio Grande do Sul, Brazil
3
Programa de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia
Universidade Católica do Rio Grande do Sul, 90619-900, Porto Alegre, Rio Grande do
Sul, Brazil
4
Departamento de Química, Laboratório de Materiais Inorgânicos, Universidade Federal
de Santa Maria, 97105-900, Santa Maria, Rio Grande do Sul, Brazil
5
Laboratorio de Bioinformática, Modelagem e Simulação de Biossistemas - LABIO,
Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900, Porto Alegre, Rio
Grande do Sul, Brazil
*
Corresponding authors. Phone/Fax: +55 51 3320 3629
E-mail addresses: diogenes@pucrs.br (D.S. Santos); pablo.machado@pucrs.br (P.
Machado)

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Abstract
The Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein
reductase (MtInhA) catalyzes hydride transfer to long-chain enoyl thioester substrates.
MtInhA is a member of the mycobacterial type II dissociated fatty acid biosynthesis
system, and is the bona fide target for isoniazid, the most prescribed drug for
tuberculosis treatment. Here, a series of piperazine derivatives was synthesized and
screened as MtInhA inhibitors, which resulted in the identification of compounds with
IC50 values in the submicromolar range. A structure-activity relationship (SAR)
evaluation indicated the importance of the chemical environment surrounding the
carbonyl group for inhibition. In addition, the structure of one selected compound was
supported by crystallographic studies, and experimental geometrical values were
compared with semi-empirical quantum chemical calculations. Furthermore, the mode
of inhibition and inhibitory dissociation constants were determined for the nine most
active compounds. These findings suggest that these 9H-fluoren-9-yl-piperazine-
containing compounds interact with MtInhA at the enoyl thioester (2-trans-dodecenoyl-
CoA) substrate binding site.
Keywords: Mycobacterium tuberculosis, tuberculosis, enoyl-ACP reductase, InhA
inhibition, piperazines
Abbreviations: TB, tuberculosis; M. tuberculosis, Mycobacterium tuberculosis; MDR-
TB, multidrug-resistant tuberculosis; HIV, human immunodeficiency virus; FAS, fatty
acid synthase; ACP, acyl carrier protein; MtInhA, enoyl-ACP reductase from
Mycobacterium tuberculosis; NADH, nicotinamide adenine dinucleotide; INH,
isonicotinic acid hydrazide; KatG, catalase-peroxidase; SAR, structure activity
2

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relationship; HTS, high-throughput screening; K , Inhibitory dissociation constant for
ii
the ESI complex; K , Inhibitory dissociation constant for the EI complex; MIC,
is
minimal inhibitory concentration; TEA, triethylamine; HBTU, 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; DMF, N,N-dimethylformamide;
DD-CoA, 2-trans-dodecenoyl-CoA; DMSO, dimethyl sulfoxide; K , Michaelis
m
constant;
DMAP,
4-(N,N-dimethylamino)pyridine;
PIPES,
1,4-
piperazinediethanesulfonic acid
3

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1
. Introduction
Tuberculosis (TB) is an infectious disease primarily caused by Mycobacterium
tuberculosis (M. tuberculosis), and it remains a major global health concern. According
to the World Health Organization, an estimated 8.6 million new TB cases with
approximately 1.3 million deaths were reported in 2012 [1]. The emergence of M.
tuberculosis strains resistant to first- and second-line TB drugs has highlighted the need
for novel and effective treatments [2]. Additional major concerns include TB-HIV
coinfection and latent TB. In 2012, approximately 1.1 million of the estimated TB cases
and 0.3 million deaths were described to occur among people who were HIV-positive
[
1]. In addition, one-third of the worldwide population has been reported to be at risk
for reactivation from latent TB, which presents challenges in the eradication of this
disease [3]. Within this context, worldwide efforts have been directed at the
development of new anti-TB drugs [4]. Following more than 40 years, a new TB drug
named bedaquiline was approved for clinical use [5]. However, the restrict indications
of this drug, possible side effects [6], and the rapid appearance of new drug-resistant TB
strains strengthen the need to continuing efforts towards the development of novel
antimycobacterial compounds.
The mycobacterial type II dissociated fatty acid biosynthesis system (FAS-II) has
emerged as an attractive, validated target for the development of novel anti-TB
molecules [7]. The FAS-II system elongates acyl fatty acid precursors yielding the long
carbon chain of the meromycolate branch of mycolic acids, the hallmark of
mycobacteria [8,9]. Mycolic acids are high-molecular-weight α-alkyl, β-hydroxy fatty
acids, which appear mostly as bound esters in tetramycolylpentaarabinosyl cluster in the
mycobacterial cell wall [10,11]. These mycolic acids have been associated with M.
tuberculosis virulence [9], the ability of M. tuberculosis to survive and replicate inside
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macrophages and with the inability of many antimycobacterial compounds to penetrate
into the M. tuberculosis cytosol [8,10]. The fatty acid precursors required for M.
tuberculosis mycolic acid biosynthesis are synthesized by successive rounds of
elongation and reduction reactions via the type I and type II fatty acid synthase (FAS-I
and FAS-II) systems to yield, respectively, the α-branch and the meromycolate chain
[
10,11]. Encoded by the inhA gene, M. tuberculosis enoyl-ACP reductase (MtInhA)
catalyzes the final essential enzymatic step in fatty acid elongation in the FAS-II
pathway, converting 2-trans-enoyl-ACP to acyl-ACP via a hydride transfer from the 4S
hydrogen of NADH to the C3 position of the 2-trans-enoyl-CoA(ACP) substrate.
MtInhA has been identified as the primary target of isoniazid (INH), which is the
frontline drug for TB chemotherapy, thereby validating this target for anti-TB drug
discovery [12-14]. As a prodrug, INH requires oxidation by mycobacterial katG-
encoded catalase-peroxidase (KatG) [15,16], which leads to the formation of the final
covalent INH-NAD adduct that has been shown to be a slow, high-affinity competitive
inhibitor of MtInhA [13,17]. Notably, mutations in the katG gene have been linked to
clinical resistance in 25-50% of newly diagnosed cases of INH-resistant TB [18,19].
Therefore, compounds able to directly inhibit MtInhA, without KatG-assisted
activation, could in theory target INH-resistant M. tuberculosis strains harboring katG
gene mutations. We have thus been investigating compounds bound to transition metal
complexes as self-activating species in the whole mycobacterial cell context with some
encouraging results [20,21]. Moreover, virtual screening and pharmacophore-based
approaches have resulted in the discovery of new MtInhA inhibitors in the micromolar
range [22]. In contrast to the recently reported methyl thiazoles that interact with
MtInhA in a “Tyr158-out” binding mode [23], direct inhibitors such as pyrrolidine
carboxamides [24] and piperazine-indole derivatives [25] have explored polar
5

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interactions involving a ribose hydroxyl, the Tyr158 hydroxyl and a hydrogen bond
acceptor in the compounds. Consistent with our strategy [22], this substrate-protein-
ligand interaction has been observed as a pharmacophoric point in virtual screening
campaigns for novel MtInhA inhibitors. However, the structural and electronic
requirements for these hydrogen bond donor-acceptor pairs have not been extensively
examined.
Therefore, in this study, we evaluated the inhibition of MtInhA by piperazine-based
compounds combined with structure activity relationship (SAR) studies. These
compounds have been primarily obtained through high-throughput screening (HTS)
approaches and exhibit submicromolar inhibition of MtInhA enzyme activity (Figure 1)
[
25-27]. Herein, novel piperazine derivatives were synthesized, and the structure of one
compound was elucidated using X-ray diffraction. In addition, the mode of inhibition,
inhibitory dissociation constants (K and/or K ) were determined.
ii
is
2
. Results and discussion
2
.1. Chemistry
First, we sought to synthesize 1-(9H-fluoren-9-yl)-piperazine derivatives bearing
modifications around the carbonyl hydrogen bond acceptor. Using 1-(9H-fluoren-9-yl)-
piperazine (3), compounds 2 and 4-8 were synthesized in low to good yields using
classical methods (Scheme 1). The amides 2 and 4a-j were obtained through acylation
reactions of 3 using synthesized or commercially available benzoyl chlorides in the
presence of triethylamine (TEA) as the base and dichloromethane (CH Cl ) as the
2
2
solvent. The reaction mixture was maintained at 0 °C during the addition of the
reactants and then allowed to warm to 25 °C with subsequent stirring for 16 h. The 9H-
6

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fluoren-9-yl-piperazines 2 and 4a-j were isolated with 7-87% yield. Notably, one-pot
synthesis of benzoyl chlorides following acylation reactions resulted in lower product
yields in comparison with a direct acylation protocol. In addition, piperazine 4k was
synthesized from an active ester of pyridine-4-carboxylic acid and 1-(9H-fluoren-9-yl)-
piperazine (3). The ester was formed in the presence of 2-(1H-benzotriazole-1-yl)-
1
,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and TEA using N,N-
dimethylformamide (DMF) as solvent. The reaction mixture was stirred at 45 °C for 4 h
to afford 4k with 83% yield.
Direct sulfonylation was used for the synthesis of benzenesulfonyl derivatives 5a-b
to obtain the desired products in 61-82% yield. Additionally, to synthesize piperazine 6,
the cinnamic acid was previously prepared through condensation between benzaldehyde
and malonic acid as previously described [28]. Thereafter, the α,β-unsaturated acid was
converted to the respective chloride and reacted with 1-(9H-fluoren-9-yl)-piperazine (3)
in a one-pot synthesis to afford 6 with 26% yield. The stereochemistry of the double
bond was assigned based on the observed proton coupling constants (J) of 15 Hz.
Finally, two S 2 nucleophilic substitution reactions yielded compounds 7 and 8. The 2-
N
bromo-N-phenylacetamide precursor was synthesized as previously described [29] and
was reacted with amine 3 in DMF in the presence of potassium carbonate at room
temperature for 24 h. Piperazine 8 was synthesized via an identical protocol using
(bromomethyl)benzene as the alkylating agent. Products 7 and 8 were obtained with
5
1% and 55% yield, respectively.
Compounds 10-12 were synthesized (Scheme 2) to evaluate the effect of increasing the
degrees of freedom of phenyl-like groups inserted into the 1-(9H-fluoren-9-yl) ring on
MtInhA inhibition. This enhanced conformational flexibility may enable stronger
hydrophobic interactions with the enzyme or result in entropy penalties in molecular
7

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recognition [30]. Compound 10 was synthesized through acylation of 1-
(diphenylmethyl)-piperazine (9) with benzoyl chlorides. The benzoyl chlorides were
obtained from a commercial source or synthesized from the desired benzoic acids using
of thionyl chloride. Independent of the source of the acylating agent, the reaction with
amine 9 was performed in dichloromethane using TEA as the base at 0-25 °C for 16 h to
obtain 10 with 15-87% yield. It is noteworthy that reactions were accomplished in one-
pot procedures when benzoyl chlorides were generated from the respective acids.
Furthermore, benzenesulfonyl chloride was reacted with amine 9 as previously
described for piperazine 5 to afford compound 11 with 15% yield. Finally, piperazine
1
2 was obtained after chloride generation from cinnamic acid with concomitant
acylation of 1-(diphenylmethyl)-piperazine. Compound 12 was isolated with 33% yield.
Spectroscopic and mass spectrometry analyses of all of the synthesized compounds are
consistent with the proposed chemical structures. As we sought to first evaluate any
MtInhA inhibitory activity by the synthesized compounds, the reaction conditions were
not yet optimized.
2
.1.1. Structure
The crystal structure of 9H-fluoren-9-yl-piperazine 4d was determined by X-ray
diffraction [31]. Carbocyclic 1-(9H-fluoren-9-yl) and 4-(4-fluorbenzoyl) moieties are
attached to the piperazine ring (Figure 2). The crystal data and details of the data
collection and structure refinement are summarized in Table S1 (Supporting
Information). Both the 9H-fluoren-9-yl and the 4-fluorbenzoyl moieties adopt a U-like
conformation around the heterocyclic scaffold with the carbonyl group pointing out
from the mean plane of the 4-fluorbenzyl substituent, as observed in the crystal
8

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structure. The dihedral angle formed between C(24)-C(19)-C(18)-O is 44.5(2)°,
whereas the angle observed for O-C(18)-N(2) is 121.3°, which results in near planarity
of the amide group. As expected, the 9H-fluoren-9-yl ring exhibits a nearly planar
structure inferred by the sum of the internal angles of the five-membered carbocycle
[
C(13)-C(12)-C(7)-C(6)-C(1)]. This sum was found to be 539.98° for compound 4d and
deviates only slightly from the ideal value of 540° for a regular pentagon.
To investigate the structural requirements of pharmacologically active compounds,
utilization of computational methods that are able to reproduce the experimental data in
good agreement is crucial. Thus, the geometrical parameters observed in piperazine 4d
were compared with semi-empirical AM1 [32] and PM3 [33-34] quantum chemical
calculations. Incidentally, these methods have been used to analyze the conformational
landscape of heterocyclic compounds, resulting in calculated values that are in good
agreement with experimentally determined values [35]. The simple linear regressions of
the experimental bond lengths and bond and dihedral angles from X-ray diffraction with
semi-empirical calculated values exhibited good correlation coefficients. For bond
lengths, both semi-empirical methods exhibited a similar correlation with the following
experimental values: r = 0.947 for PM3 and r = 0.954 for AM1. These values account
for, respectively, 89.8% and 91.0% of the variability in the bond lengths in the 4d
structure. The bond angles calculated from semi-empirical methods were also in good
agreement with the experimentally determined bond angles, r = 0.935 and r = 0.945 for
the PM3 and AM1 methods, respectively. The PM3 method accounts for 87.4% of the
variability in the bond angles, whereas the AM1 approach accounts for 89.3% of this
variation. However, the correlation between experimental and calculated values for the
dihedral angles exhibited a significant difference between the two semi-empirical
calculations. For the PM3 method, r = 0.891, which accounts for only 79.5% of the
9

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variability in the dihedral angles in 4d, whereas for the AM1 method, r = 0.988, which
accounts for 97.7% of this variability. Thus, according to the magnitude of the
correlation coefficients, the AM1 method is more suitable than the PM3 method to
estimate the experimental geometrical parameters of the piperazines under study. In
addition, this result represents another example in which the crystal structure is at or
close to the global minimum energy conformation.
2
.2. Inhibitory activity measurements
The reduction reaction of 2-trans-dodecenoyl-CoA (DD-CoA) catalyzed by
+
MtInhA was spectrophotometrically measured monitoring NADH oxidation to NAD
[
36-37]. The enzyme inhibition assays were carried out in the presence of the
synthesized compounds in a solution containing a final concentration of 1% dimethyl
sulfoxide (DMSO) as the solvent. This concentration did not interfere with the assay
conditions.
The synthesized lead-like 2 was assayed and exhibited an IC (the concentration of
5
0
inhibitor that reduces the enzyme velocity by half) of 0.18 µM, which is 2-fold higher
than the previously reported value of 0.09 µM [26] (Table 1). Modification of the
hydrogen bond acceptor character from a carbonyl group to a sulfonyl moiety in
piperazine 5a resulted in a compound with an IC value of approximately 20 µM. This
5
0
result may be attributed to a reduced hydrogen-bond basicity of sulfonamide compared
to amide groups [38]. Notwithstanding, electron-donating groups tend to improve this
basicity, compound 5b containing a methyl group at the C2 position of the phenyl
moiety exhibited reduced inhibition compared with the non-substituted counterpart (5a);
IC > 50 µM. In an attempt to increase the hydrogen-bond basicity of the carbonyl
5
0
1
0

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group and improve inhibitory activity, the α,β-unsaturated amide 6 was synthesized and
evaluated. Unfortunately, the conjugated system was unable to inhibit MtInhA activity
and exhibited an IC50 > 50 µM. These findings suggest that steric factor was
determinant on electronic feature of the proposed systems. Displacement of the position
of the carbonyl through insertion of a methylene group resulted in drastically reduced
inhibition by piperazine 7. Notably, the amide was maintained while the insertion of the
methylene group altered the conformational possibilities of the carbonyl moiety. This
result suggests that the position of the carbonyl group does not tolerate even small
spatial variations. Finally, the synthesis of compound 8 lacking an amide group afforded
a piperazine that did not inhibit MtInhA at 50 µM, emphasizing the importance of this
hydrogen bond acceptor for MtInhA inhibition.
An open system of the 9H-fluoren-9-yl ring was also evaluated to assess its
influence (10-12) on MtInhA enzyme inhibition. From the results summarized in Table
1
, substitution of the planar system in 2 for the freely rotatable diphenylmethyl moiety
in 10a produced a compound that is nearly 88-fold less potent (IC = 16.0 µM) as
5
0
compared to compound 2. Indeed, the hydrophobic 9H-fluoren-9-yl moiety has been
described as important for inhibition and mimics the fatty acid substrate within the
substrate binding pocket [25]. This moiety is also present in the structure of recently
described MtInhA inhibitors [39-40]. Substitution at the C4 position of the aryl ring
with a methyl group yields piperazine 10c, which inhibited MtInhA with an IC of 7.8
5
0
µM. By contrast, substitution at this position with a methoxy group (10b) greatly
reduced inhibition by this compound (IC > 50 µM). Unexpectedly, even bioisosteric
5
0
replacement of the 4-methyl (10c) with 4-Cl (10d) yielded a molecule with an IC > 50
5
0
µM, indicating fine tuning of this system. Indeed, except to 10a and 10c, none of the
modifications on scaffold 10 generated molecules with IC values lower than 50 µM.
5
0
1
1

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Similarly, sulfonyl (11) and cinnamic acid (12) derivatives did not inhibit MtInhA
catalysis at the maximum evaluated concentration of 50 µM.
In the next round of structural investigations, we evaluated substitutions at the C4
position in the phenyl ring of the lead-like compound 2. The results summarized in
Table 1 for piperazines 4a-e and 4k indicated that, in general, electron-donating,
hydrophobic and non-bulky groups exhibited enhanced MtInhA inhibition. These
findings are in accordance with previous reports, which have shown that enzyme
inhibition is highly dependent on the chemical characteristics and size of groups around
the phenyl substituent [26-27,41]. In addition, this solvent-exposed cavity is formed by
polar and nonpolar groups and has been predicted to accommodate small and planar
chemical groups [27,41].
It is noteworthy that from both ground-state and spectroscopic perspectives,
halogens have been described to exhibit electron-donating characteristics from positive
mesomeric effects (M+) [42]. Accordingly, nitro-substituted compound 4e exhibited an
IC > 50 µM. This result can be attributed to the ability of electron withdrawing groups
5
0
to reduce the ability of the carbonyl to act as a hydrogen bond acceptor. Between the
evaluated moieties, the nitro moiety was the most electron withdrawing group evaluated
at the C4 position of the phenyl ring presenting negative mesomeric and inductive
effects (M- and I-). Such features may account for the reduced inhibition by compound
4
e. It is important to mention that nitro is a planar group in the same way as the indole
substituent present in piperazine derivative 1, which was able to produce an inhibitor of
MtInhA with an IC of 0.16 µM [25]. Therefore, for 4e activity, the electronic effect,
5
0
instead of steric effects, seems to be crucial.
1
2

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Piperazine 4k was proposed with the objective of evaluating the effect of a
compound with a molecular volume similar to lead-like 2 but showing distinct
electronic and hydrophobic characteristics. Pyridine derivative 4k showed moderate
inhibition on MtInhA activity with an IC of 9.7 µM, which was nearly 53-fold less
5
0
potent than benzyl-substituted compound 2.
Regarding electron-donating groups, the methoxy-substituted piperazine 4a
exhibited an IC of 2.9 µM, whereas the 4-methyl group in 4b was able to inhibit
5
0
MtInhA with an IC50 of 0.22 µM (0.40 µM [26]), which is submicromolar and
comparable to lead-like compound 2. Once more, classical bioisosteric replacement of a
methyl group with a chloro substituent yielded compound 4c, which was approximately
1
0-fold less effective for MtInhA inhibition than the methyl derivative 4b. By contrast,
fluoro-substituted 4d maintained inhibition in the submicromolar range, with an IC of
5
0
0
.40 µM. This result shows that piperazine 4b (4-methyl) is nearly 2-fold more potent
than 4d (4-fluoro), based on the magnitude of the IC values. Although 4-fluoro and 4-
5
0
methyl share identical mesomeric effects, their inductive effects are opposite: fluoro
substitution reduces the electron density along the sigma bonds, whereas the methyl
group increases this electron density. Thus, 4-methyl (4b) can increase the basicity of
the carbonyl group and maintain a more hydrophobic environment around the benzyl
ring, leading to enhanced inhibition of MtInhA compared to the 4-fluoro-substituted
compound (4d).
In evaluating the position of the methyl in the benzyl ring, the inhibition data
indicated a preference for the C4 and C3 positions in comparison with 2-substituted
derivatives. It is noteworthy that hydrogens at the C2 and C3 positions of the phenyl
ring have been described as having weak hydrogen bonds with NADH [41]. Piperazine
4
f (3-methyl) exhibited an IC of 0.25 µM, whereas compound 4g (2-methyl) inhibited
50
1
3

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MtInhA with an IC of 6.0 µM. Considering the fluorinated molecules 4d and 4h-i, the
5
0
C3 position was slightly preferred for enhanced inhibitory potency in comparison with
the C4 and C2 positions, as 4h (3-fluoro) and 4i (2-fluoro) exhibited IC values of 0.24
5
0
µM and 0.36 µM, respectively. Finally, 2,5-difluoro-substituted 4j exhibited reduced
inhibitory potency on the catalytic activity of MtInhA, with an IC50 of 1.6 µM.
According to the structures of 4g and 4i studied by docking simulations, the presence of
the methyl group at the C2 position of the aryl moiety causes steric hindrance due to the
restricted space in the cavity formed by Met103, Tyr158, and Met161 residues of
MtInhA (Figure 3). It is noteworthy that halogen substituents typically form multiple
interactions, as well as participate in desolvation effects in ligand-protein complexes
[
43]. Based on the observed distances and the sum of the van der Walls radii, the fluoro
group does not appear to form hydrogen bonds with MtInhA or NADH. Therefore,
steric hindrance, halogen-mediated interactions, and desolvation effects may account for
the better activity of compound 4i compared to 4g.
2
.2.1. Determination of K values for select MtInhA inhibitors
i
Using a threshold of 3 µM for the IC values, nine compounds (2, 4a-d, 4f and 4h-
5
0
j) were selected for additional studies on the mode of inhibition (uncompetitive,
noncompetitive or competitive) of MtInhA. It is noteworthy that none of these
molecules exhibited time-dependent inhibitory activity up to 30 min of preincubation
with MtInhA (data not shown), suggesting rapid equilibrium processes. Accordingly,
the mode of inhibition could be determined from Lineweaver-Burk plots and data fitting
to appropriate equations to give values for the inhibition constants (K and/or K ) [44]
ii
is
(Table 2). When the substrate DD-CoA was fixed at a non-saturating concentration and
1
4

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NADH was varied in the presence of fixed-varying concentrations of the compounds,
the double reciprocal plots yielded parallel lines (Supporting Information). These results
indicated that all of the evaluated compounds are uncompetitive inhibitors with respect
to NADH. Fitting the data to Eq. 2 yielded the values for the ternary complex inhibition
constants (K ), with values ranging from 0.147–1.95 µM. (Table 2). The inhibitory
ii
profile suggests that these inhibitors exhibit low affinity to the free enzyme and a nearly
complete binding preference for the enzyme-substrate (ES) complex to yield an inactive
enzyme-substrate-inhibitor (ESI) complex [44,45]. Moreover, the binding of these
compounds to the MtInhA:NADH binary complex corroborate the proposed hydrogen
bonding network between the pharmacophore-like carbonyl group, hydroxyl group of
the nicotinamide ribose and Y158 in the formation of the ESI complex [25-26,42].
Furthermore, when NADH was fixed at a non-saturating concentration and the DD-CoA
substrate was varied in the presence of fixed-varying concentrations of the piperazines
2
, 4a, 4c, 4f, and 4i-j, a noncompetitive inhibition profile was obtained, as indicated by
the lines that intercept on left of y-axis in the double reciprocal plots. Fitting the data to
Eq. 3 yielded K values ranging from 0.100–2.77 µM and K values ranging from
is
ii
0
.348–7.22 µM (Table 2). Except for compound 4c, inhibitors exhibited K < K ,
is ii
indicating a binding preference for the free enzyme in comparison to binding to the
MtInhA-DD-CoA binary complex. These results suggested that the compounds could
interact with the DD-CoA binding site even with a noncompetitive signature [44].
Aiming to shed light on this issue, saturating concentrations of DD-CoA and NADH
were used to assay lead-like compound 2. In these assays, when the DD-CoA
concentration was maintained at approximately 135 µM (K ≅ 45 µM [36]) and the
m
NADH concentration was varied from 10–120 µM, the uncompetitive inhibition with
respect to NADH was maintained (Supporting Information). However, when the NADH
1
5

ACCEPTED MANUSCRIPT
concentration was saturating at 240 µM (Km ≅ 60 µM [36]) and the DD-CoA
concentration was varied from 15-200 µM, a competitive inhibition profile with respect
to the DD-CoA substrate was observed for piperazine 2 (Supporting Information).
Taken together kinetics results and crystallographic studies of the analogous compound
1
strengthened the hypothesis that compound 2 interacts with the enzyme at the fatty
acid binding site [25].
Inhibition assays for 4b, 4d and 4h indicated that these compounds are competitive
inhibitors with respect to the DD-CoA substrate, as the lines intersected at a single value
on the y-axis of the double-reciprocal plots (Supporting Information). Fitting the data to
Eq. 4 yielded K values ranging from 0.101–0.338 µM (Table 2). As proposed for 2,
is
these compounds likely interact with the fatty acid binding site of MtInhA.
Finally, MIC determination for the most potent MtInhA inhibitors was
accomplished in vitro using the M. tuberculosis H37Rv strain (Supporting Information).
Although the poor solubility of the evaluated compounds precluded a more accurate
MIC determination, piperazines 4b and 4d exhibited moderate antimycobacterial
activity, with a MIC of 25 µg/mL (≈ 67.8 µM).
3
. Conclusion
The inhibition of MtInhA activity by piperazine derivatives was assessed using
kinetic assays. Additionally, insights into the structural requirements for effective
enzyme inhibition and the determination of the mode of inhibition of the synthesized
compounds emphasized the pharmacophore role of the amide carbonyl group in the
formation of the ESI complex. The results presented herein suggest that modulation of
the hydrogen bond acceptor may be a critical step in the optimization of MtInhA
1
6

ACCEPTED MANUSCRIPT
inhibitors. Future challenges will include the design of novel piperazine derivative
compounds for the fatty acid binding site maintaining the drug-like properties that allow
for cellular potency. Finally, our results may provide important details for the design of
novel structures to interact with a clinically validated TB target, thereby prompting the
discovery of novel antituberculosis agents.
4
. Experimental Section
4
.1. Synthesis and structure: apparatus and analysis
All common reactants and solvents were used as obtained from commercial
suppliers without further purification. Melting points were determined using a
1
Microquímica MQAPF-302 apparatus. H NMR spectra were acquired on an Anasazi
1
13
EFT-60 spectrometer ( H at 60.13 MHz) at 30 °C. C NMR spectra were acquired on a
1
3
Varian (Federal University of Rio Grande do Sul, UFRGS/Brazil) spectrometer ( C at
1
00.6 MHz) at 25 °C. CDCl was used as the solvent, and TMS was used as an internal
3
standard in 5-mm samples tubes. Chemical shifts are expressed in ppm, and J values are
given in Hz. High-resolution mass spectra (HRMS) were obtained for all compounds on
an LTQ Orbitrap Discovery mass spectrometer (Thermo Fisher Scientific, Bremen,
Germany). This hybrid system combines the LTQ XL linear ion trap mass spectrometer
with an Orbitrap mass analyzer. The experiments were performed using direct infusion
of the sample in a solution of acetonitrile (50%), methanol (50%), and formic acid
-1
(
0.1%), with a flow of 5 µL min in positive-ion mode using electrospray ionization.
Elemental composition calculations were performed using a specific tool included in the
Qual Browser module of the Xcalibur (Thermo Fisher Scientific, release 2.0.7)
software. Fourier transform infrared (FTIR) spectra were recorded using a universal
1
7

ACCEPTED MANUSCRIPT
attenuated total reflectance (UATR) attachment on a PerkinElmer Spectrum 100
-1
-1
spectrometer in the wavenumber range of 650-4000 cm with a resolution of 4 cm .
Crystal data were collected using a Bruker APEX II CCD area-detector diffractometer
and employing graphite-monochromatized MoKα radiation. The structures from the
ligand and the complex were determined by direct methods using SHELXS-97 [46].
Subsequent Fourier-difference map analyses yielded the positions of the non-hydrogen
atoms. Refinements were performed using the SHELXL-97 package [47]. All
2
refinements were performed using full-matrix least-squares on F , with anisotropic
displacement parameters for all non-hydrogen atoms. Hydrogen atoms were included in
the refinement in calculated positions. Finally, the structure was rendered using
ORTEP-3 for Windows [48].
4
.2. General procedure for the synthesis of piperazines 2, 4a-j and 10a-f
Method i: Benzoic acid (1 mmol) was reacted with a solution of thionyl chloride in
CH Cl (1 M, 6 mL) in refluxing toluene (3 mL) for 5 h. After cooling to room
2
2
temperature, the solvent was evaporated under reduced pressure. The obtained benzoyl
chloride was dissolved in dry CH Cl (5 mL), and the resultant solution was cooled to 0
2
2
°
C. To this solution, TEA (4.4 mmol, 0.444 g), 1-(9H-fluoren-9-yl)-piperazine (3) (1.1
mmol, 0.275 g) or 1-(diphenylmethyl)-piperazine (9) (1.1 mmol, 0.277 g) in dry CH Cl
2
2
(5 mL) was added dropwise. After the addition, the reaction mixture was stirred for 16 h
at room temperature. Subsequently, the solution was washed with aqueous HCl (10%,
v/v, 3 x 15 mL), a saturated NaHCO solution (1 x 15 mL), and aqueous NaCl (5%,
3
w/v, 1 x 15 mL). Finally, the organic layer was dried over anhydrous magnesium
sulfate. The mixture was filtered, and the solvent was evaporated under reduce pressure.
When necessary, the products were purified using silica gel chromatography with
1
8

ACCEPTED MANUSCRIPT
CH Cl /methanol (20:5, v/v) as the mobile phase. This protocol was used for the
2
2
synthesis of 9H-fluoren-9-yl-piperazines 2, 4a, 4c-e, 4h-i and 1-(diphenylmethyl)-
piperazines 10a-b and 10d-e. The crystal used for the data collection was obtained by
recrystallization of compound 4d from hexane followed by slow evaporation at room
temperature.
Method ii: Benzoyl chloride (1.0 mmol) was solubilized in dry CH Cl (5 mL), and
2
2
the resultant solution was cooled to 0 °C. To this solution, TEA (4.4 mmol, 0.444 g), 1-
(
9H-fluoren-9-yl)-piperazine (3) (1.1 mmol, 0.275 g) or 1-(diphenylmethyl)-piperazine
9) (1.1 mmol, 0.277 g) in dry CH Cl (5 mL) was added dropwise. After the addition,
(
2
2
the reaction mixture was stirred for 16 h at 25 °C. Subsequently, the solution was
washed with aqueous HCl (10%, v/v, 3 x 15 mL), a saturated NaHCO solution (1 x 15
3
mL), and aqueous NaCl (5%, w/v, 1 x 15 mL). Finally, the organic layer was dried over
anhydrous magnesium sulfate. The mixture was filtered, and the solvent was evaporated
under reduce pressure. When necessary, the products were purified using silica gel
chromatography with CH Cl /methanol (20:5, v/v) as the mobile phase. This protocol
2
2
was used for the synthesis of 9H-fluoren-9-yl-piperazines 4b, 4f-g, 4j and 1-
(diphenylmethyl)-piperazines 10c and 10f.
Method iii: Pyridine-4-carboxylic acid (0.5 mmol, 0.061 g) was reacted with a
solution of HBTU (0.55 mmol, 0.208 g), TEA (2 mmol, 0.202 g), 1-(9H-fluoren-9-yl)-
piperazine (3) (0.5 mmol, 0.125 g) in dry DMF (5 mL). The reaction mixture was stirred
for 4 h at 45 °C. Subsequently, the solution was diluted with aqueous NaCl (5%, w/v,
1
5 mL) and extracted with ethyl acetate (3 x 15 mL). Finally, the organic layer was
dried over anhydrous magnesium sulfate. The mixture was filtered, and the solvent was
evaporated under reduce pressure. The product was purified using silica gel
1
9

ACCEPTED MANUSCRIPT
chromatography with CH Cl/methanol (40:1, v/v) as the mobile phase. This protocol
3
was used for the synthesis of 9H-fluoren-9-yl-piperazine 4k.
4
.2.1. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(phenyl)methanone (2)
1
Yellow powder; yield: 0.148 g (42%); m.p. 206-208 °C; H NMR (60 MHz, CDCl ): δ
3
1
3
2
.63 (m, 4H, CH ), 3.52 (br, 4H, CH ), 4.84 (s, 1H, CH), 7.32-7.74 (m, 13H, Ar); C
2 2
NMR (100 MHz, CDCl ): δ 48.3, 67.9, 120.7, 127.2, 128.6, 128.7, 130.6, 131.2, 133.6,
3
-1
1
34.4, 142.1, 170.2; FTIR (UATR, cm ): 3330, 2930, 2817, 1634, 1447, 1425, 740;
+
HRMS (ESI) calcd for C H N O + H: 355.1805, found: 355.1796 (M + H) .
2
4
22
2
3
4
.2.2. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(4-methoxyphenyl)methanone (4a)
1
Brown powder; yield: 0.309 g (87%); m.p. 143-145 °C; H NMR (60 MHz, CDCl ): δ
3
2
6
1
.59 -2.70 (m, 4H, CH ), 3.55-3.71 (m, 4H, CH ), 3.77 (s, 3H, OCH ), 4.84 (s, 1H, CH),
2 2 3
-1
.82 (d, 2H, Ar), 7.25-7.78 (m, 10H, Ar); FTIR (UATR, cm ): 2931, 2798, 1632, 1424,
250, 738; HRMS (ESI) calcd for C H N O + H: 385.1911, found: 385.1900 (M +
2
5
24
2
2
+
H) .
4
.2.3. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(4-tolyl)methanone (4b)
1
White powder; yield: 0.202 g (55%); m.p. 146-147 °C; H NMR (60 MHz, CDCl ): δ
3
2
7
8
.33 (s, 3H, CH ), 2.25-2.67 (m, 4H, CH ), 3.53-3.64 (m, 4H, CH ), 4.86 (s, 1H, CH),
3 2 2
-1
.20-7.675 (m, 14H, Ar); FTIR (UATR, cm ): 2920, 2795, 1624, 1429, 1269, 1005,
+
28, 742; HRMS (ESI) calcd for C H N O + H: 369.1961, found: 369.1960 (M + H)
2
5
24
2
.
4
.2.4. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(4-chlorophenyl)methanone (4c)
1
White powder; yield: 0.027 g (7%); m.p. 133-135 °C; H NMR (60 MHz, CDCl ): δ
3
2
.60-2.71 (m, 4H, CH ), 3.54-3.62 (m, 4H, CH ), 4.86 (s, 1H, CH), 7.25-7.76 (m, 12H,
2 2
2
0

ACCEPTED MANUSCRIPT
-1
Ar); FTIR (UATR, cm ): 2808, 1624, 1431, 1269, 1004, 836, 740, 730; HRMS (ESI)
+
calcd for C H ClN O + H: 389.1410, found: 389.1415 (M + H) .
2
4
21
2
4
.2.5. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(4-fluorophenyl)methanone (4d)
1
Yellow powder; yield: 0.029 g (8%); m.p. 140-142 °C; H NMR (60 MHz, CDCl ): δ
3
2
.57-2.71 (m, 4H, CH ), 3.47-3.61 (m, 4H, CH ), 4.86 (s, 1H, CH), 6.84-7.75 (m, 12H,
2 2
-1
Ar); FTIR (UATR, cm ): 2806, 1625, 1603, 1218, 1149, 1003, 840, 739; HRMS (ESI)
+
calcd for C H N OF + H: 373.1711, found: 373.1732 (M + H) .
2
4
21
2
4
.2.6. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(4-nitrophenyl)methanone (4e)
1
Yellow powder; yield: 0.095 g (24%); m.p. 119-120 °C; H NMR (60 MHz, CDCl ): δ
3
2
.52-2.73 (m, 4H, CH ), 3.29-3.71 (m, 4H, CH ), 4.86 (s, 1H, CH), 7.25-7.76 (m, 10H,
2 2
-1
Ar), 8.21 (d, 2H, Ar); FTIR (UATR, cm ): 3330, 2931, 1630, 1599, 1516, 1439, 740;
+
HRMS (ESI) calcd for C H N O + H: 400.1656, found: 400.1644 (M + H) .
2
4
21
3
3
4
.2.7 (4-(9H-Fluoren-9-yl)piperazin-1-yl)(3-tolyl)methanone (4f)
1
Yellow powder; yield: 0.0856 (77%); m.p. 124-126 °C; H NMR (60 MHz, CDCl ): δ
3
2
.32 (s, 3H, CH ), 2.71 (br, 4H, CH ), 3.62 (br, 4H, CH ), 4.94 (s, 1H, CH), 7.16-7.841
3 2 2
-1
(
m, 12H, Ar); FTIR (UATR, cm ): 3373, 293, 2808, 1632, 1450, 1421, 1275, 1201,
1
005, 810, 737; HRMS (ESI) calcd for C H N O + H: 369.1961, found: 369.1960 (M
25 24 2
+
+
H) .
4
.2.8. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(2-tolyl)methanone (4g)
Yellow powder; yield: 0.0917 (83%); m.p. 63-64 °C; 1H NMR (60 MHz, CDCl ): δ 2.24
3
(
s, 3H, CH ), 2.54 (br, 2H, CH ), 2.91 (br, 2H, CH ), 3.28 (br, 2H, CH ), 3.89 (br, 2H,
3 2 2 2
-1
CH ), 5.96 (s, 1H, CH), 7.16-7.77 (m, 12H, Ar); FTIR (UATR, cm ): 2923, 2817, 1627,
2
2
1

ACCEPTED MANUSCRIPT
1
426, 1255, 1001, 729; HRMS (ESI) calcd for C H N O + H: 369.1691, found:
25
24
2
+
3
69.1966 (M + H) .
4
.2.9. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(3-fluorophenyl)methanone (4h)
1
Brown powder; yield: 0.029 g (8%); m.p. 165-167 °C; H NMR (60 MHz, CDCl ): δ
3
2
.53-2.69 (m, 4H, CH ), 3.41-3.66 (m, 4H, CH ), 4.85 (s, 1H, CH), 6.99-7.78 (m, 13H,
2 2
-1
Ar); FTIR (UATR, cm ): 3063, 2824, 1639, 1582, 1439, 1277, 1004, 740; HRMS (ESI)
+
calcd for C H FN O + H: 373.1773, found: 373.1747 (M + H) .
2
4
21
2
4
.2.10. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(2-fluorophenyl)methanone (4i)
1
White powder; yield: 0.119 g (32%); m.p. 169-171 °C; H NMR (60 MHz, CDCl ): δ
3
2
2
1
3
.37-2.53 (m, 2H, CH ), 2.71-2.85 (m, 2H, CH ), 3.18-3.34 (m, 2H, CH ), 3.70-3.90 (m,
2 2 2
-1
H, CH ), 4.85 (s, 1H, CH), 6.99-7.76 (m, 12H, Ar); FTIR (UATR, cm ): 3342, 2835,
2
631, 1610, 1451, 1431, 1276, 1007, 741; HRMS (ESI) calcd for C H FN O + H:
2
4
21
2
+
73.1773, found: 373.1747 (M + H) .
4
.2.11. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(2,5-difluorophenyl)methanone (4j)
1
White powder; yield: 0.339 g (87%); m.p. 167-168 °C; H NMR (60 MHz, CDCl ): δ
3
2
.64-2.99 (m, 4H, CH ), 3.41-3.59 (m, 2H, CH ), 3.89-4.04 (m, 2H, CH ), 5.10 (s, 1H,
2 2 2
1
3
CH), 6.93-7.87 (m, 11H, Ar); C NMR (100 MHz, CDCl ): δ 48.6, 69.2, 115.8 (dd, J =
3
2
5.2, J = 8.4), 117.0 (dd, J = 24.4, J = 8.4), 118.1 (dd, J = 24.0, J = 8.8), 120.1, 126.5,
-
1
27.6, 129.2, 141.3, 153.9 (d, J = 244.9), 158.6 (d, J = 244.9), 163.6; FTIR (UATR, cm
1
)
: 3061, 2929, 1634, 1611, 1449, 1008, 737; HRMS (ESI) calcd for C H F N O + H:
24 20 2 2 3
+
3
91.1616, found: 391.1616 (M + H) .
4
.2.12. (4-(9H-fluoren-9-yl)piperazin-1-yl)(pyridin-4-yl)methanone (4k)
2
2

ACCEPTED MANUSCRIPT
1
White powder; yield: 0.148 g (83%); m.p. 156-157 °C; H NMR (400 MHz, CDCl ): δ
3
2
2
.42-2.44 (m, 2H, CH ), 2.83-2.85 (m, 2H, CH ), 3.27-3.29 (m, 2H, CH ), 3.78-3.80 (m,
2 2 2
H, CH ), 4.87 (s, 1H, CH), 7.23 (d, J = 6.3, 2H, Ar), 7.30 (t, J = 7.8, 2H, Ar) 7.39 (t, J
2
=
7.4, 2H, Ar), 7.60 (d, J = 7.4, 2H, Ar), 7.69 (d, J = 7.4, 2H, Ar), 8.63 (d, J = 5.9, 2H,
1
3
Ar). C NMR (100 MHz, CDCl ): δ 42.8, 48.2, 48.5, 49.4, 69.8, 119.9, 121.2, 125.8,
3
-
1
1
1
3
27.2, 128.4, 141.0, 143.1, 143.4, 150.1, 167.6; FTIR (UATR, cm ): 2815, 1636, 1440,
279, 1000, 832, 751; HRMS (ESI) calcd for C H N O + H: 356.1757, found:
23
21
3
+
56.1756 (M + H) .
4
.2.13. (4-Benzhydrylpiperazin-1-yl)(phenyl)methanone (10a)
1
White powder; yield: 0.309 g (87%); m.p. 145-146 °C; H NMR (60 MHz, CDCl ): δ
3
2
.33-2.47 (m, 4H, CH ), 3.47-3.68 (m, 4H, CH ), 4.26 (s, 1H, CH), 7.61-7.35 (m, 15H,
2 2
-1
Ar); FTIR (UATR, cm ): 3024, 2808, 1631, 1446, 1265, 996, 706; HRMS (ESI) calcd
+
for C H N O + H: 357.1961, found: 357.1937 (M + H) .
2
4
24
2
4
.2.14. (4-Benzhydrylpiperazin-1-yl)(4-methoxyphenyl)methanone (10b)
1
White powder; yield: 0.142 g (37%); m.p. 64-66 °C; H NMR (60 MHz, CDCl ): δ
3
2
6
1
3
.34-2.49 (m, 4H, CH ), 3.63-3.63 (m, 4H, CH ), 3.78 (s, 3H, OCH ), 5.27 (s, 1H, CH),
2 2 3
-1
.78-6.92 (d, 2H, Ar), 7.21-7.43 (m, 12H, C H ); FTIR (UATR, cm ): 3421, 2808,
5
6
605, 1427, 1230, 996, 704; HRMS (ESI) calcd for C H N O + H: 387.2120, found:
2
5
26
2
+
87.2146 (M + H) .
4
.2.15. (4-Benzhydrylpiperazin-1-yl)(4-tolyl)methanone (10c)
1
White powder; yield: 0.203 g (55%); m.p. 62-64 °C; H NMR (60 MHz, CDCl ): δ 2.33
3
(
s, 3H, CH ), 2.35-2.49 (m, 4H, CH ), 3.73-3.79 (m, 4H, CH ), 4.36 (s, H, CH), 7.20-
3 2 2
2
3

ACCEPTED MANUSCRIPT
-1
7
.47 (m, 14H, Ar); FTIR (UATR, cm ): 2807, 1627, 1426, 1285, 1253, 996, 748, 705;
+
HRMS (ESI) calcd for C H N O + H: 371.2118, found: 371.2131 (M + H) .
2
7
28
2
2
4
.2.16. (4-Benzhydrylpiperazin-1-yl)(4-chlorophenyl)methanone (10d)
1
Brown powder; yield: 0.234 g (60%); m.p. 130-132 °C; H NMR (60 MHz, CDCl ): δ
3
2
.36–2.51 (m, 4H, CH ), 3.53–3.78 (m, 4H, CH ), 4.28 (s, 1H, CH), 7.26-7.45 (m, 15H,
2 2
-1
Ar); FTIR (UATR, cm ): 3027, 2808, 1629, 1431, 1254, 1087, 996, 832, 745, 696;
+
HRMS (ESI) calcd for C H ClN O + H: 391.1572, found: 391.1609 (M + H) .
2
4
23
2
4
.2.17. (4-Benzhydrylpiperazin-1-yl)(4-fluorophenyl)methanone (10e)
1
Brown powder; yield: 0.202 g (54%); m.p. 54-57 °C; H NMR (60 MHz, CDCl ): δ
3
2
.42-2.52 (m, 4H, CH ), 2.92–3.06 (m, 2H, CH ), 3.36-3.60 (m, 2H, CH ), 4.24 (s, 1H,
2 2 2
-1
CH), 6.88-7.51 (m, 14H, Ar); FTIR (UATR, cm ): 2811, 1628, 1603, 1451, 1432, 1254,
1
3
223, 996, 843, 705; HRMS (ESI) calcd for C H N OF + H: 375.1867, found:
24 23 2
+
72.1874 (M + H) .
4
.2.18. (4-Benzhydrylpiperazin-1-yl)(2,5-difluorophenyl)methanone (10f)
1
White powder; yield: 0.321 g (82%); m.p. 78-79 °C; H NMR (60 MHz, CDCl ): δ
3
2
.28-2.52 (br, 4H, CH ), 3.25-3.41 (m, 2H, CH ), 3.70-3.88 (m, 2H, CH ), 4.26 (s, 1H,
2 2 2
-1
CH), 6.93-7.41 (m, 13H, Ar); FTIR (UATR, cm ): 2919, 1627, 1427, 1286, 1253, 996,
+
7
05; HRMS (ESI) calcd for C H F N O + H: 393.1773, found: 393.1747 (M + H) .
2
4
22
2
2
4
.3. General procedure for the synthesis of piperazines 5a-b and 11
Sulfonyl halide (1.2 mmol) was dissolved in dry CH Cl (5 mL), and the resultant
2
2
solution was cooled to 0 °C. To this solution, TEA (2.2 mmol, 0.222 g), 1-(9H-fluoren-
2
4

ACCEPTED MANUSCRIPT
9
-yl)-piperazine (3) (1.1 mmol, 0.275 g) or 1-(diphenylmethyl)-piperazine (9) (1.1
mmol, 0.277 g), in dry CH Cl (5 mL) was added dropwise. After the addition, the
2
2
reaction mixture was stirred for 16 h at room temperature. Extraction and purification
were performed as previously described for piperazines 2, 4 and 10.
4
.3.1. 1-(9H-Fluoren-9-yl)-4-(phenylsulfonyl)piperazine (5a)
1
White powder; yield: 0.238 g (61%); m.p. 152-154 °C; H NMR (60 MHz, CDCl ): δ
3
2
.58-2.70 (m, 4H, CH ), 2.85-3.11 (m, 4H, CH ), 4.91 (s, 1H, CH), 7.13-7.89 (m, 13H,
2 2
-1
Ar); FTIR (UATR, cm ): 3418, 2887, 2835, 1634, 1348, 1171, 735; HRMS (ESI) calcd
+
for C H N O S + H: 391.1475, found: 391.1467 (M + H) .
2
3
22
2
2
4
.3.2. 1-(9H-Fluoren-9-yl)-4-(o-tolylsulfonyl)piperazine (5b)
1
White powder; yield: 0.331 g (82%); m.p. 68-70 °C; H NMR (60 MHz, CDCl ): δ
3
2
.61-2.74 (m, 7H, CH ), 3.09–3.24 (m, 4H, CH ), 4.81 (s, 1H, CH), 7.49-7.75 (m, 13H,
2 2
-1
Ar); FTIR (UATR, cm ): 1447, 1314, 1158, 1133, 940, 722; HRMS (ESI) calcd for
+
C H N O S + H: 405.1631, found: 405.1631 (M + H) .
24
24
2
2
4
.3.3. 1-Benzhydryl-4-(phenylsulfonyl)piperazine (11)
1
White powder; yield: 0.058 (15%); m.p. 153-155 °C (m.p. 157-159 °C [49]); H NMR
(
60 MHz, CDCl ): δ 2.43-2.50 (m, 4H, CH ), 3.03-3.63 (m, 4H, CH ), 4.24 (s, 1H, CH),
3 2 2
-1
7
.25-7.83 (m, 15H, Ar); FTIR (UATR, cm ): 2855, 2811, 1447, 1347, 1331, 1169, 944,
7
41, 689; HRMS (ESI) calcd for C H N O S + H: 393.1631, found: 393.1607 (M +
2
5
26
2
2
+
H) .
4
.4. General procedure for the synthesis of piperazines 6 and 12
2
5

ACCEPTED MANUSCRIPT
The cinnamic acid precursor was synthesized as previously described [28].
Thereafter, this acid (1 mmol, 0.148 g) was reacted with a solution of thionyl chloride in
CH Cl (1 M, 6 mL) for 5 h in refluxing toluene (5 mL). The obtained acyl chloride was
2
2
dissolved in dry CH Cl (5 mL), and the resultant solution was cooled to 0 °C. To this
2
2
solution, TEA (4.4 mmol, 0.444 g), 1-(9H-fluoren-9-yl)-piperazine (3) (1.1 mmol, 0.275
g) or 1-(diphenylmethyl)-piperazine (9) (1.1 mmol, 0.277 g) in dry CH Cl (5 mL) was
2
2
added dropwise. After the addition, the reaction mixture was stirred for 16 h at room
temperature. Finally, ethyl ether (15 mL) was added to the reaction mixture, and the
precipitate was isolated on a filter. The products were purified using silica gel
chromatography with CH Cl /methanol (20:5, v/v) as the mobile phase.
2
2
4
.4.1. (E)-1-(4-(9H-Fluoren-9-yl)piperazin-1-yl)-3-phenylprop-2-en-1-one (6)
1
White powder; yield: 0.098 g (26%); m.p. 169-170 °C; H NMR (60 MHz, CDCl ): δ
3
2
1
3
3
.34-2.50 (m, 4H, CH ), 3.61-3.75 (m, 4H, CH ), 4.86 (s, 1H, CH), 6.77 (d, J = 15 Hz,
2 2
-1
H, CHtrans), 7.25-7.53 (m, 17H, Ar), 7.77 (d, J = 15, CHtrans); FTIR (UATR, cm ):
060, 2813, 1649, 1605, 1410, 1202, 740; HRMS (ESI) calcd for C H N O + H:
2
6
24
2
+
81.1961, found: 381.1967 (M + H) .
4
.4.2. (E)-1-(4-Benzhydrylpiperazin-1-yl)-3-phenylprop-2-en-1-one (12)
1
White powder; yield: 0.126 g (33%); m.p. 141-143 °C; H NMR (60 MHz, CDCl ): δ
3
2
.34-2.50 (m, 4H, CH ), 3.61-3.75 (m, 4H, CH ), 4.26 (s, 1H, CH), 6.80 (d, J = 15 Hz,
2 2
1
H, CHtrans), 7.17-7.36 (m, 18H, Ar), 7.66 (d, J = 15 Hz, 1H, CHtrans); FTIR (UATR,
-1
cm ): 3023, 2809, 1644, 1595, 1452, 1439, 1229, 705; HRMS (ESI) calcd for
+
C H N O + H: 383.2123, found: 383.2118 (M + H) .
26
26
2
4
.5. General procedure for the synthesis of 9H-fluoren-9-yl-piperazine 7
2
6

ACCEPTED MANUSCRIPT
Bromo-N-phenylacetamide was synthesized from 2-bromoacetyl chloride and aniline
as previously described [29]. A solution containing bromo-N-phenylacetamide (1.04
mmol, 0.221 g), potassium carbonate (3.12 mmol, 1.08 g), and 1-(9H-fluoren-9-yl)-
piperazine (3) (1.1 mmol, 0.275 g) in DMF (8 mL) was stirred under argon atmosphere
for 24 h at room temperature. Thereafter, the solution was diluted with distilled water
(
50 mL). The obtained solid was isolated on a filter and dried under reduce pressure.
The product was purified using silica gel chromatography with chloroform/methanol
50:50, v/v) as the mobile phase.
(
4
.5.1. 2-(4-(9H-Fluoren-9-yl)piperazin-1-yl)-N-phenylacetamide (7)
1
White powder; yield: 0.172 g (51%); m.p. 174-175 °C; H NMR (60 MHz, CDCl ): δ
3
2
.60-2.75 (m, 8H, CH ), 3.10 (s, 2H, CH ), 4.86 (s, 1H, CH), 7.03-7.78 (m, 15H, Ar);
2 2
-1
FTIR (UATR, cm ): 2546, 1682, 1447, 1313, 1133, 952, 736; HRMS (ESI) calcd for
+
C H N O + H: 384.2048, found: 384.2070 (M + H) .
25
25
3
4
.6. General procedure for the synthesis of 9H-fluoren-9-yl-piperazine 8
A solution containing (bromomethyl)benzene (1.04 mmol, 0.124 mL), potassium
carbonate (3.12 mmol, 1.08 g), and 1-(9H-fluoren-9-yl)-piperazine (3) (1.1 mmol, 0.275
g) in DMF (8 mL) was stirred under argon atmosphere and at room temperature for 24
h. Thereafter, the mixture was diluted with distilled water (20 mL). The obtained solid
was isolated on a filter and dried under reduce pressure. The product was purified using
silica gel chromatography with chloroform/methanol (50:50, v/v) as the mobile phase.
4
.6.1. 9-(4-Benzylcyclohexyl)-9H-fluorene (8)
1
Yellow powder; yield: 0,055 g (55%); m.p. 129-131 °C; H NMR (60 MHz, CDCl ): δ
3
2
.38 (br, 8H, CH ), 3.48 (s, 2H, CH ), 4.82 (s, 1H, CH), 7.62-7.73 (m, 15H, Ar); FTIR
2 2
2
7

ACCEPTED MANUSCRIPT
-1
(
UATR, cm ): 2807, 1449, 1130, 1007, 736, 697; HRMS (ESI) calcd for C H N + H:
24 24 2
+
3
41.2012, found: 341.2016 (M + H) .
4
.7. Theoretical Calculations and docking simulations
The geometry of the compound was optimized using semi-empirical AM1 and PM3
methods implemented in the HyperChem 7.52 package (2002) [50]. The structures were
fully optimized without fixing any parameter, thus, bringing all geometric variables to
their equilibrium values. The energy minimization protocol employed the Polak-Ribiere
conjugated gradient algorithm. Convergence to a local minimum was achieved when the
.
-1
energy gradient was ≤ 0.01 kcal mol . The RHF method was used in the spin pairing for
the two semi-empirical tools.
Flexible ligand docking, carried out using AutoDock4.2.5.1 and AutoDockTools4
software [51-52], employed the crystal structure of the piperazine 1 (Genz-10850)
bound to MtInhA (PDB ID: 1P44). The NADH coenzyme was treated as part of the
protein receptor in all docking simulations. Water molecules were removed, polar
hydrogens were added, and Gasteiger partial atomic charges for ligands were assigned
by AutoDockTools4 [51]. For the docking simulation, the active site was set on a cubic
grid, spaced at 0.375 Å, with dimensions of 60 points × 60 points × 60 points centered
on the crystallographic bound ligand 1. This grid is large enough to include the
NADH coenzyme as part of the protein receptor, as well as the MtInhA substrate
binding pocket in 1P44. The results of each docking experiment were derived from 25
independent runs, for which a maximum number of 27,000 LGA (Lamarckian Genetic
Algorithm) generations was generated on the initial population of 150 individuals,
6
a maximum number of 2.5 x 10 energy evaluations, with an elitism value of 1, a
2
8