Synthesis of 3-heteryl substituted pyrrolidine-2,5-diones via catalytic Michael reaction and evaluation of their inhibitory activity against InhA and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2013.10.069

In the present paper, we report the synthesis via catalytic Michael reaction and biological results of a series of 3-heteryl substituted pyrrolidine-2,5-dione derivatives as moderate inhibitors against Mycobacterium tuberculosis H37Rv growth. Some of them present also inhibition activities against InhA.

Full text of DOI:10.1016/j.ejmech.2013.10.069

Accepted Manuscript
Synthesis of 3-heteryl substituted pyrrolidine-2,5-diones via catalytic Michael reaction
and evaluation of their inhibitory activity against InhA and Mycobacterium tuberculosis
Tetiana Matviiuk, Giorgia Mori, Christian Lherbet, Frédéric Rodriguez, Maria Rosalia
Pasca, Marian Gorichko, Brigitte Guidetti, Zoia Voitenko, Michel Baltas
PII:
S0223-5234(13)00720-4
10.1016/j.ejmech.2013.10.069
EJMECH 6532
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 July 2013
Revised Date: 16 October 2013
Accepted Date: 28 October 2013
Please cite this article as: T. Matviiuk, G. Mori, C. Lherbet, F. Rodriguez, M.R. Pasca, M. Gorichko, B.
Guidetti, Z. Voitenko, M. Baltas, Synthesis of 3-heteryl substituted pyrrolidine-2,5-diones via catalytic
Michael reaction and evaluation of their inhibitory activity against InhA and Mycobacterium tuberculosis,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.10.069.
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ACCEPTED MANUSCRIPT
Synthesis of 3-heteryl substituted pyrrolidine-2,5-diones via catalytic Michael reaction
and evaluation of their inhibitory activity against InhA and Mycobacterium tuberculosis
Tetiana Matviiuk, Giorgia Mori, Christian Lherbet, Frédéric Rodriguez, Maria Rosalia Pasca, Marian
Gorichko, Brigitte Guidetti, Zoia Voitenko, Michel Baltas

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Synthesis of 3-heteryl substituted pyrrolidine-2,5-diones via catalytic Michael reaction
and evaluation of their inhibitory activity against InhA and Mycobacterium tuberculosis
Tetiana Matviiuk,a,b Giorgia Mori,d Christian Lherbet,*,a,c Frédéric Rodriguez,a,c Maria Rosalia
Pasca,d Marian Gorichko,b Brigitte Guidetti,a Zoia Voitenko,*,b Michel Baltas*,a,c
a Université de Toulouse; UPS; Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, LSPCMIB; 118 route de
Narbonne, F-31062 Toulouse cedex 9, France.
b Taras Shevchenko National University of Kyiv, Department of Chemistry, 64 str. Volodymyrska, Kyiv, 01601, Ukraine. Fax: 38 044 239
3204; Tel: 38 044 239 3315; E-mail: z_voitenko@mail.univ.kiev.ua
c CNRS; Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, LSPCMIB, UMR-5068; 118 Route de Narbonne, F-
31062 Toulouse cedex 9, France.
d University of Pavia; Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, via Ferrata 1, 27100 Pavia, Italy
Fax: 33 (0)5 61556011; Tel: 33 (0)5 61556289; E-mail:lherbet@chimie.ups-tlse.fr; baltas@chimie.ups-tlse.fr
Keywords : succinimide, Michael addition, tuberculosis, InhA.
Abstract
In the present paper, we report the synthesis via catalytic Michael reaction and biological
results of a series of 3-heteryl substituted pyrrolidine-2,5-dione derivatives as moderate
inhibitors against M. tuberculosis H37Rv growth. Some of them present also inhibition
activities against InhA.
1. Introduction
Tuberculosis (TB) is the leading cause with malaria and AIDS of worldwide mortality [1].
The effectiveness of current anti-tuberculosis drugs to combat this infection is severely
compromised by the emergence of multi- and extensively drug-resistant tuberculosis
(MDR-TB [2,3] and XDR-TB [4]). Therefore, new drugs are required to raise the
probability to shortly stop all forms of drug-resistant TB. In this context many studies are

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based on targeting the cell wall of Mycobacteria and more particularly components
essential to its survival. The fatty acid synthase system of M. tuberculosis contains unique
signature fatty acid, the mycolic acid, which is a central constituent of the mycobacterial
cell wall. Mycolic acid biosynthesis is carried out by several successive enzymatic cycles
corresponding to two related but distinct Fatty Acid Synthase (FAS) systems, FAS I and II
[5]. InhA protein (ENR, EC number: 1.3.1.9) is a key enzyme of FAS II and shows a
NADH-dependent enoyl-ACP reductase activity. InhA is a good target as the FAS II
system is present in bacteria but is absent in humans. It has already been validated as the
primary molecular target of the frontline antitubercular drug isoniazid [6]. Moreover
recent studies demonstrated that InhA is also the target for the second line drug
ethionamide (ETA) [7].
Michael conjugate addition is a well known effective method in the elaboration of
pharmaceutical intermediates, peptide analogues, antibiotics and other drugs [8–10]. In
the continuation of our previous study on Michael reaction of heterocyclic substrates with
maleimides as electron-deficient dienophiles [11–13], we were interested in expanding not
only the boundaries of this reaction but also in applying it for the synthesis of novel
pharmaceutical substances. Maleimides are excellent dienophiles/dipolarophiles as well as
Michael acceptors. In most cases they usually form succinimide derivatives. This
fragment is frequently employed in drug design and defined as promising pharmacophore
moiety. Indeed a large number of reported pharmaceutical substances bear a pyrrolidine-
2,5-dione core [14,15] including some approved drugs [16] and clinical drug candidates
[17].
Pyrrolidine-2,5-dione structural units were also found in numerous natural products.
Moiramide B and Andrimide (Fig. 1) have been described as new highly specific
antibiotics [18] exhibiting potent antibacterial activity against methicillin-resistant

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Staphylococcus aureus and a range of other antibiotic-resistant human pathogens. These
natural products were identified to target FAS system that is also the primary target for
antitubercular drugs [19]. Furthermore, Hirsutellones A–E (Fig. 1), a family of novel
alkaloids, were reported to display significant growth inhibitory activity against
Mycobacterium tuberculosis H37Rv strain [20].
Herein we report complementary results on synthesis of selected 3-heteryl substituted
succinimide derivatives via catalytic conjugated addition of nucleophilic heterocycles to
maleimides. We also report our first findings showing the encouraging activities against
M. Tuberculosis H37Rv growth and/or inhibitory ones against InhA of some of the
compounds synthesized.
2. Results and discussion
2.1. Chemistry
N-Aryl maleimides were used as electron-deficient substrates in conjugate addition with
nucleophilic heterocycles.
The first heterocycle in our investigation was 4H-1,2,4-triazole-3-thiole (1) (Scheme 1).
This substrate was selected because a large number of reported molecules bearing this
structural unit possess antibacterial, [21], antimicrobial [22] and antitubercular properties
[23]. In the Michael addition with maleimides, as we have very recently reported [24], the
ambident nucleophile 1 gives the conjugated products via a more nucleophilic exocyclic
mercapto group as a single type of products (Scheme 1). The highest yields of the adducts
2a,b were obtained in the presence of catalytic amount of AlCl3 (1.5%) (Table 1). Lithium
perchlorate has shown lower catalytic efficacy than aluminium chloride for the formation
of products 2a,b.

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Scaffold 3 was then selected for the synthesis of pyrrolidine-2,5-dione derivatives 4a,b.
These compounds were designed as analogues of known nanomolar inhibitor of InhA
protein GEQ (Fig. 2) [25]. The 1-(9H-fluoren-9-yl)-piperazine group of this inhibitor has
been shown to be an important pharmacophore and is responsible for extensive
hydrophobic interactions in the binding site of InhA protein [26]. Compounds 4a and 4b
were synthesized via C–N Michael addition of 1-(9H-fluoren-9-yl)piperazine (3) to N-
phenyl and N-benzyl maleimides (Scheme 2). The best results were obtained when
catalytic amount of lithium perchlorate was used (Table 1). Aluminium chloride appears
to be not an effective catalyst in this case.
Thereafter, Michael addition of N-aryl substituted maleimides was carried out with
1,2,3,4-tetrahydroisoquinoline (THIQ) (5) (Scheme 2). Simple THIQs were found in
several plants and mammalian species. Moreover, THIQs are the constituents of several
drugs and they exhibit antibacterial, [27] antitubercular [28] and antimicrobial [29]
activities. As for compound 3, reaction with 1,2,3,4-tetrahydroisoquinoline (THIQ)
afforded the best yields of the products 6a,b when lithium perchlorate (LiClO4) was added
as catalyst to the reaction mixture in dry dioxane at room temperature (method A).
Another synthetic approach using Hünig’s base (Method B) was applied for the synthesis
of the adducts 4a,b and 6a,b but the yields were lower than in the case of LiClO4 catalysis
(Table 1).
1H-Imidazole (7), a substrate with well-known range of bioactivity, was selected for
further C–N addition studies. The reaction proceeds in excellent yield as was recently
reported by us [24].
Finally, Michael addition was carried out between N-substituted maleimides and two

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bulky C-H active heterocycles – 2-phenylindolizine (9) and 7,9-dinitropyrido[2,1-
a]isoindole (10) (Scheme 3). Lithium perchlorate appears to be not effective as catalyst in
the case of C–C bond formation; only traces of the desired products were isolated.
Different other Lewis acids have been then tested without success (ZnCl2, SnCl4, TiCl4,
BiCl3). The products corresponding to C–C conjugated addition 11a,b and 12 were
obtained in good yields only when anhydrous aluminium chloride was used as catalyst
(Scheme 3, Table 1). The structure of these 3-substituted indolizine compounds was
confirmed by NOESY experiments (See supporting information). A correlation could be
found between the alpha hydrogen atom of the succinimide and the H-5 of indolizine.
Thereby, in the course of this study, we have found that lithium perchlorate is an efficient
catalyst for C–N conjugate addition when aluminium chloride demonstrates high efficacy
for C–C addition reaction.
2.2. Biology
2.2.1. Antimycobacterial activities
All synthesized compounds were evaluated by determining the minimal inhibitory
concentration (MIC) on M. tuberculosis H37Rv strain [30,31]. They were also evaluated
in vitro as potential InhA inhibitors at 50 µM by applying a commonly used method [32]
Recombinant M. tuberculosis InhA was expressed in E. coli and subsequently purified
according to a previous reported procedure [32]. The results are shown in Table 2. Well
known InhA inhibitors Triclosan [33], GEQ [25] and Isoniazid as H37Rv strain inhibitors
were used for comparison. The latter (Genz-10855) has been reported to display
significantly high binding affinity and selectivity to the InhA protein but in the same time
poor growth inhibitory activity on Mycobacterium tuberculosis.
Compounds 4a and 4b present the best activities on InhA protein among the tested
derivatives. Nevertheless the values are still too modest compared to that of GEQ. In the
same time, unlike GEQ, MIC values of compounds 4a and 4b are much better (Table 2).

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Molecular docking evaluation was performed to understand the differences in binding of
reported nanomolar inhibitor and our analogues with pyrrolidine-2,5-dione substituent.
Two enantiomers (R and S) of the compound 4a and GEQ were docked into the binding
site of InhA by applying the same calculation algorithm and procedure. The best binding
modes of investigated molecules are represented in Fig. 2. Docking was carried out taking
into account flexibility of 11 amino acid residues from the lateral chains (including
Tyr158) and cofactor NAD+ as a set of features in the binding pocket of the protein. The
RMSD value between crystallographic conformation (PDB entry 1P44, chain A) [26] and
the best docking mode of GEQ (with the best scoring data) is 0.6 Å, that could be a proof
of the prediction ability of applying screening procedure.
Some predicted differences in the mode of binding of 4a (R and S) are observed (Fig. 2)
compared to GEQ. From the alignment of binding complexes (obtained from the docking
studies and crystallographic data for InhA complex with GEQ inhibitor), we could
summarize that 4a could be inserted in the active site but through a different geometry
depending on the enantiomer used. Indeed, the fluorenyl ring could not exactly match the
large hydrophobic pocket of InhA protein. It is also worth mentioned that electrostatic
interactions and hydrogen bonding between 4a and cofactor NAD+ are missing (at least
for one enantiomer), relative to the corresponding crystallographic inhibitor GEQ (see
supporting information: 2D ligand-protein interaction map). Neverless, docking results for
both enantiomers are similar (See supporting information) and show lower score values by
comparison with GEQ. These results are in accordance with the biological results.
Compound 12 shows an inhibitory activity similar to compounds 4a and 4b. It is
noteworthy that both compounds 12 and 4 contain rigid tricyclic structural fragments,
while compounds 6a,b and 8a,b have more flexible and non-planar moieties.

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Interestingly, compound 6a presents high inhibition activity on bacteria growth while
derivative 6b shows a reduced one. These inhibitor structures are interesting for further
investigation. Due to their low inhibition of InhA, compounds 6a and 6b might present a
different mechanism of action and target other proteins involved in bacteria growth.
Synthesized derivatives of 4H-1,2,4-triazole-3-thiole 2a and 2b have shown also good
inhibition values on bacteria but as for compounds 6a, 6b they have presented poor
activity on the protein.
Finally, the MIC evaluations of synthesized compounds have shown that in their
majority, they displayed higher activities than GEQ (Table 2). The best results were obtained
for isoquinoline (6a, 6b) and 4H-1,2,4-triazole-3-thiol (2a and 2b) derivatives. These two
groups of structurally different functionalized succinimides might constitute an attractive
starting point for further development of antituberculosis agents. It is also noteworthy that
GEQ analogues 4a,b show much higher inhibition of M. tuberculosis than GEQ itself.
Finally, moderate values of MIC were determined for the derivatives of 2-phenylindolizine
11a,b, 7,9-dinitropyrido[2,1-a]isoindole 12 and imidazole 8a,b.
2.2.2. Cytotoxicities of the compounds
The cytotoxicity of different 3-heteryl substituted pyrrolidine-2,5-dione derivatives was
evaluated on two human cell lines, the human colon cancer cell line HCT116 and the human
fibroblast cell line GM637 (Table 2). The data showed that the IC50 of the different
compounds tested were above 100 µM, indicating that these compounds are not cytotoxic
against human cell lines. Only one exception was found for compound 2a bearing a 4H-1,2,4-
triazole-3-thiole moiety. The results show that this compound is clearly cytotoxic against
human cell lines in the micromolar range.

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3. Conclusion
A series of 3-heteryl substituted pyrrolidine-2,5-dione derivatives were prepared and
assayed for the inhibition of InhA and M. tuberculosis growth. Lewis acids were found to
be effective and easy handling catalysts in Michael conjugated addition of nucleophilic
heterocycles to N-aryl substituted maleimides. Among the synthesized molecules,
compounds 4a, 4b and 12 displayed moderate inhibition activities against InhA that are
the best among compounds evaluated and poor activities on M. tuberculosis. More
interesting, compounds 2a, 2b, 6a and 6b derived from 4H-1,2,4-triazole-3-thiol and
1,2,3,4-tetrahydroisoquinoline respectively, exhibit good inhibition values on bacterial
growth. It is important to know that, while compound 2a is cytotoxic, molecules 6a and
6b are not, so that they can be subjected for optimization in order to gain access to new
leads of antitubercular agents. Furthermore, replacing one of the structural parts of GEQ
for a pyrrolidine-2,5-dione ring led to increased activities against M. tuberculosis H37Rv.
4. Experimental Section
4.1. Material
All chemicals were obtained from Aldrich or Acros Organics and used without further
purification. All solvents were distilled before use. Compounds 3, 9 and 10 have been
synthesized according to the literature procedures [34, 35, 36 respectively] and completely
1
characterized using H and 13C NMR and MS. Nuclear magnetic resonance spectra were
13
recorded on a 'Mercury 400' Varian spectrometer (1H, C NMR; TMS signal was used as
an internal standard for calibration of spectral data) and on a Bruker AC 300 spectrometer
(1H, 13C NMR; solvent residue signals were used for calibration of spectral data). Mass
spectrometry (MS) data were obtained on a ThermoQuest TSQ 7000 spectrometer, high-
resolution mass spectra (HRMS) were recorded on a ThermoFinnigan MAT 95 XL

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spectrometer using chemical ionization (CI; CH4 or NH3) and electrospray ionization
(ESI) methods. Melting points were measured on a Mettler Toledo MP50 melting point
system.
4.2. Chemistry
4.2.1. General procedure for C-S Michael addition (Compounds 2a,b): compounds 2a and 2b
were synthesized as previously reported [24].
4.2.1.1. 1-Phenyl-3-(4H-1,2,4-triazol-3-ylsulfanyl)-2,5-pyrrolidinedione (2a).
Yield 62 %; mp: 154-155 °C. 1H NMR (400 MHz, DMSO-d6): δ = 2.92 (dd, J = 18.0 Hz, J =
4.8 Hz, 1H), 3.42 (dd, J = 18.0 Hz, J = 9.6 Hz, 1H), 4.67 (dd, J = 4.8 Hz, J = 9.6 Hz, 1H),
13
7.26 (d, J = 7.2 Hz, 2Н), 7.40–7.53 (m, 3Н), 8.56 (s, 1Н), 14.08 (br s, 1Н). C NMR (100
MHz, DMSO-d6): δ = 36.5, 41.3, 127.0, 128.5, 128.9, 132.8, 145.8, 155.4, 174.3, 174.8.
HRMS calculated for C12H11N4O2S 275.0603; found 275.0596.
4.2.1.2. 1-(4-Methoxyphenyl)-3-(4H-1,2,4-triazol-3-ylsulfanyl)-2,5-pyrrolidinedione (2b).
Yield 63 %; mp: 162-163 °C. 1H NMR (400 MHz, DMSO-d6): δ = 2.93 (dd, J = 16.8 Hz, J =
2.4 Hz, 1H), 3.40 (dd, J = 16.8 Hz, J = 9.4 Hz, 1H), 3.82 (s, 3Н), 4.54 (dd, J = 2.4 Hz, J = 9.4
Hz, 1H), 6.98 (d, J = 8.2 Hz, 2Н), 7.19 (d, J = 8.2 Hz, 2Н), 8.04 (s, 1Н), 14.17 (br s, 1Н). 13C
NMR (100 MHz, DMSO-d6): δ = 36.3, 41.0, 54.8, 128.1, 128.9, 129.4, 132.8, 140.5, 145.7,
155.4, 158.8, 172.9, 173.5. MS [M+H+] calculated for C13H13N4O3S 305.3; found 305.3.
HRMS: calculated for C13H13N4O3S 305.0708; found 305.0707.
4.2.2. General procedure for C–N Michael addition: compounds 8a and 8b were synthesized
as previously reported [24].
Method A (compounds 4a,b, 6a,b, 8a,b): To a solution of 1-(9H-fluoren-9-yl)piperazine 3,
1,2,3,4-tetrahydroisoquinoline 5 or 1H-imidazole 7 (0.19 mmol) in dry dioxane N-substituted
maleimide (0.22 mmol) was added. The mixture was stirred for 15 minutes to dissolve the
reagents, then catalytic amount of LiClO4ꢀ3H2O (4 mg, 0.02 mmol) was added. The reaction

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mixture was stirred overnight at room temperature. The reaction progress was controlled by
TLC. After completion, the solvent was evaporated and the crude product was purified by
flash chromatography.
Method B (compounds 4a,b, 6a,b): To a solution of amine (0.28 mmol) in iPrOH, N-phenyl
maleimide (0.34 mmol) was added. After 10 min stirring, N,N’-diisopropylethylamine was
added (0.15 mmol). The reaction mixture was stirred overnight at room temperature. Then the
solvent was evaporated under reduced pressure and the corresponding crude product was
purified by flash chromatography.
4.2.2.1. 3-[4-(9H-Fluoren-9-yl)-1-piperazinyl]-1-phenyl-2,5-pyrrolidinedione (4a).
The crude product was purified by flash chromatography (silica, CH2Cl2:EtOAc = 8:2) to give
1
light yellow crystals. Yield: 88% (method A), 70% (method B); mp: 191-192 °C. H NMR
(300 MHz, CDCl3): δ = 2.61–3.04 (m, 10H), 3.92 (dd, J = 4.8 Hz, J = 9.0 Hz, 1H), 4.85 (s,
1H), 7.24–7.49 (m, 9H), 7.63–7.10 (m, 4H). 13C NMR (75 MHz, CDCl3): δ = 32.1, 48.9, 49.9,
62.6, 69.9, 119.8, 126.1, 126.5, 127.1, 128.3, 128.8, 129.2, 131.6, 141.1, 143.7, 174.2, 175.1.
HRMS: calculated for C27H26N3O2 424.2020; found 424.2017.
4.2.2.2. 1-Benzyl-3-[4-(9H-fluoren-9-yl)-1-piperazinyl]-2,5-pyrrolidinedione (4b).
1
Yield: 83% (method A), 58% (method B); mp: 168-169 °C. H NMR (300 MHz, DMSO-
d6): δ = 2.52–2.99 (m, 10H), 3.81 (dd, J = 4.5 Hz, J = 9.0 Hz, 1H), 4.53 (s, 2H) 4.77 (s,
1H), 7.25–7.67 (m, 13H). 13C NMR (75 MHz, CDCl3): δ = 31.7, 48.8, 49.8, 50.5, 62.4,
69.8, 120.0, 126.1, 126.7, 127.0, 128.2, 128.9, 129.3, 131.5, 140.9, 143.2, 174.6, 179.8.
HRMS: calculated for C28H28N3O2 438.2176; found 438.2180.
4.2.2.3. 3-(3,4-Dihydroisoquinolin-2(1H)-yl)-1-phenylpyrrolidine-2,5-dione (6a).
The crude product was purified by flash chromatography (silica, PE:EtOAc = 7:3) to give a
1
white powder. Yield: 87%; mp: 149-150 °C. H NMR (300 MHz, DMSO-d6): δ = 2.79–3.10
(m, 6H), 3.72 (d, J = 14.7 Hz, 1H), 4.09 (d, J = 14.7 Hz, 1H), 4.27 (dd, J = 5.7 Hz, J = 8.7 Hz,

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1H), 7.06–7.12 (m, 4H), 7.29–7.32 (m, 2H), 7.40–7.53 (m, 3H). 13C NMR (75 MHz, DMSO-
d6): δ = 29.0, 31.4, 46.5, 50.9, 62.2, 125.5, 126.0, 126.4, 127.2, 128.4, 128.6, 128.9, 132.3,
133.9, 134.5, 174.6, 175.7. HRMS calculated for C19H19N2O2 307.1447; found 307.1453.
4.2.2.4. 1-Benzyl-3-(3,4-dihydroisoquinolin-2(1H)-yl)pyrrolidine-2,5-dione (6b).
The crude product was purified by flash chromatography (silica, PE:EtOAc = 3:2) to give
1
creamy crystals. Yield: 79%; mp: 106-107 °C. H NMR (300 MHz, DMSO-d6): δ = 2.64–
2.99 (m, 6H), 3.57 (d, J = 14.7 Hz, 1H), 3.98 (d, J = 14.7 Hz, 1H), 4.18 (dd, J = 5.4Hz, J
= 8.4 Hz, 1H), 4.59 (s, 2H), 6.97–7.12 (m, 4H), 7.25–7.37 (m, 5H). 13C NMR (75 MHz,
DMSO-d6): δ = 29.0, 31.1, 41.2, 46.4, 50.8, 61.9, 125.5, 126.0, 126.3, 127.4, 127.5, 128.4,
128.5, 133.8, 134.3, 136.2, 175.3, 176.5. HRMS calculated for C20H21N2O2 321.1603;
found 321.1610.
4.2.2.5. 3-(1H-Imidazol-1-yl)-1-phenyl-2,5-pyrrolidinedione (8a).
Yield 95 %; mp: 161-163 °C. 1H NMR (400 MHz, DMSO-d6): δ = 3.16 (dd, J = 16.8 Hz, J =
6.4 Hz, 1H), 3.40 (dd, J = 16.8 Hz, J = 9.2 Hz, 1H), 5.68 (dd, J = 9.2 Hz, J = 6.4 Hz, 1H),
6.92 (s, 1Н), 7.36–7.49 (m, 5Н), 7.57 (s, 1Н), 7.80 (s, 1Н). 13C NMR (100 MHz, DMSO-d6):
δ = 36.9, 54.8, 118.6, 127.3, 128.6, 128.9, 129.3, 132.5, 138.0, 172.9, 173.4. HRMS:
calculated for C13H12N3O2 242.0924; found 242.0918.
4.2.2.6. 3-(1H-Imidazol-1-yl)-1-(4-methoxyphenyl)-2,5-pyrrolidinedione (8b).
Yield 92 %; mp: 154-155 °C. 1H NMR (400 MHz, DMSO-d6): δ = 3.18 (dd, J = 16.8 Hz, J =
6.8 Hz, 1H), 3.37 (dd, J =16.8 Hz, J = 4.2 Hz, 1H), 3.83 (s, 3Н), 5.64 (dd, J = 4.2 Hz, J = 6.8
13
Hz, 1H), 6.94 (s, 1Н), 7.00–7.26 (m, 4Н), 7.40 (s, 1Н), 7.83 (s, 1Н). C NMR (100 MHz,
DMSO-d6): δ = 37.9, 53.8, 55.2, 114.9, 120.0, 127.5, 127.6, 132.8, 137.9, 159.9, 170.1, 172.7.
HRMS: calculated for C14H14N3O3 272.1030; found 272.1035.

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4.2.3. General procedure for C–C Michael addition, (compounds 11a and 11b ): Compounds
11a, 11b were synthesized as previously reported [24].
4.2.3.1. 1-Phenyl-3-(2-phenyl-3-indolizinyl)-2,5-pyrrolidinedione (11a).
The crude product was purified by flash chromatography (EtOAc : PE / 2 : 8) to afford the
1
desired compound as light green crystals. Yield: 84%; mp: 210-211 °C. H NMR (300 MHz,
DMSO-d6): δ = 3.06 (dd, J = 6.9 Hz, J = 17.7 Hz, 1H), 3.32–3.37 (m, 1H), 5.13 (t, J = 8.4 Hz,
1H), 6.60–7.54 (m, 14H), 7.97 (d, J = 6.6 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): δ = 33.6,
37.4, 99.6, 111.3, 114.5, 117.5, 119.1, 122.8, 126.9, 127.1, 128.4, 128.7, 128.9, 129.0, 129.8,
132.3, 132.5, 135.7, 174.5, 175.8. HRMS: calculated for C24H19N2O2 367.1441; found
367.1436.
4.2.3.2. 1-Benzyl-3-(2-phenyl-3-indolizinyl)-2,5-pyrrolidinedione (11b).
The crude product was purified by flash chromatography (EtOAc : PE / 2 : 8) to afford the
desired compound as light grey powder. Yield: 71%; mp: 192–194 °C. 1H NMR (300 MHz,
DMSO-d6): δ = 3.14 (dd, J = 6.3 Hz, J = 16.8 Hz, 1H), 3.33–3.37 (m, 1H), 4.51 (s, 2H) 5.07
(t, J = 9.0 Hz, 1H), 6.67–7.53 (m, 14H), 7.95 (d, J = 6.9 Hz, 1H). 13C NMR (75 MHz, DMSO-
d6): δ = 33.6, 37.5, 46.6, 99.8, 111.5, 114.3, 117.1, 118.9, 123.0, 126.4, 127.1, 128.4, 128.7,
129.1, 129.3, 129.8, 132.3, 132.4, 135.7, 173.8, 176.1. HRMS: calculated for C25H21N2O2
381.1598; found 381.1605.
4.2.3.3. 3-(7,9-Dinitropyrido[2,1-a]isoindol-6-yl)-1-phenyl-2,5-pyrrolidinedione (12).
7,9-Dinitropyrido[2,1-a]isoindole 10 (100 mg, 0.39 mmol) was dispersed in dry dioxane.
Then N-substituted maleimide (0.4 mmol) was added. The mixture was stirred for 15 minutes.
Thereafter anhydrous aluminium chloride (3 mg, 0.022 mmol) was quickly added. The
reaction mixture was refluxed over 6 h. The progress of the reaction was monitored by TLC.
After reaction completion, the solvent was evaporated under reduced pressure and the crude

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product was recrystallized from DMSO to afford a red powder. Yield: 62%; mp: 290−292
°C. 1H NMR (300 MHz, DMSO-d6): δ = 3.27 (dd, J = 8.2 Hz, J = 18.3 Hz, 1H), 3.79 (dd, J =
9.8 Hz, J = 18.3 Hz, 1H), 5.47 (t, J = 9.0 Hz, 1H), 7.35–7.78 (m, 7H), 8.88 (d, J = 2.1 Hz,
13
1H), 8.99–9.06 (m, 2H), 9.88 (d, J = 2.1 Hz, 1H). C NMR (75 MHz, DMSO-d6): δ = 34.7,
47.8, 110.8, 111.2, 121.4, 127.3, 128.5, 128.9, 131.4, 132.1, 132.6, 135.2, 136.1, 139.0, 147.0,
148.9, 150.5, 175.4, 179.2. HRMS: calculated for C22H15N4O6 431.0986; found 431.0992.
4.3. Biology
4.3.1. Growth conditions and minimum inhibitory concentration (MIC) determination. M.
tuberculosis H37Rv was used as the reference strain. The strains were grown at 37 °C in
Middlebrook 7H9 broth (Difco), supplemented with 0.05% Tween 80, or on solid
Middlebrook 7H11 medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase
(OADC). MICs for the new compounds were determined by means of the micro-broth
dilution method. Dilutions of M. tuberculosis wild-type cultures (about 105–106 cfu/ml) were
streaked onto 7H11 solid medium containing a range of drug concentrations (0.25 µg/mL to
40 µg/mL). Plates were incubated at 37 °C for about 21 days and the growth was visually
evaluated. The lowest drug dilution at which visible growth failed to occur was taken as the
MIC value. Results were expressed as the average of at least three independent
determinations.
4.3.2. Cytotoxicity of the compounds
Human colon cancer cell line HCT116 (ATCC) and human fibroblasts (GM637 cell line)
were cultured in DMEM supplemented with 10% fetal calf serum. For cytotoxicity
evaluation, 3000 cells were seeded per well in 96-wells plates and, 24 h later, were treated
with 9 concentrations ranging from 100 nM to 100 µM (8 replicates for each). After 4 days of

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treatment, the cytotoxicity of each compound was measured by using the WST-1 kit (Roche).
IC50 values were determined by using PRISM software (GraphPad).
4.3.3. InhA expression and purification.
Recombinant M. tuberculosis InhA was expressed in E. coli and subsequently purified.
The production and purification of the protein were performed as described in the
reference [32].
4.3.4. Inhibition Kinetics
Stock solutions of all compounds were prepared in DMSO such that the final
concentration of this co-solvent was constant at 5% v ⁄ v in a final volume 1 mL for all
kinetic reactions. Kinetic assays using trans-2-dodecenoyl-Coenzyme A (DD-CoA) and
wild-type InhA were performed as described [32]. Reactions were initiated by addition of
InhA (100 nM final) to solutions containing DD-CoA (50 µM final), inhibitor, and NADH
(250 µM final) in 30 mM PIPES, 150 mM NaCl, pH 6.8, buffer. Control reactions were
carried out with the same conditions as described above but without inhibitor. The
inhibitory activity of each derivative was expressed as the percentage inhibition of InhA
activity with respect to the control reaction without inhibitor. All activity assays were
performed in triplicate.
4.4. Molecular docking studies
Molecular docking studies were performed using Molegro Virtual Docker 5.5 software
(CLC Bio, Aarhus, Denmark). Molecular graphics and some analyses were performed
with the UCSF Chimera package. Chimera is developed by the Resource for
Biocomputing, Visualization, and Informatics at the University of California, San

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Francisco (supported by NIGMS P41-GM103311). The Protein structures optimization
and preparation (structure checks, rotamers, protonation analysis) were done using
Accelrys Discovery Studio 3.0 client and UCSF Chimera 1.6.2 or 1.7 (Dock Prep without
minimization). The Ligand structures were extracted (SciTE text editor) from aligned
protein structure or sketched using ChemAxon Marvin 5.5 and prepared for the docking
studies (3D structure optimization, hybridization checking, protonation) using Discovery
Studio 3.0 client. After analyzing of known crystallographic structures of InhA, 1P44 pdb
entry (chain A, X-ray, 2.7 Å resolution) [26] from Protein Data Bank [37] includes
NAD+/NADH and ligand structure (GEQ) in the active site was chosen as protein
molecular structure for further docking studies. The cavity detection algorithm
implemented in MVD was used to optimize the definition of a 15 Å (radius) potential
binding site but not for constraining results to the cavity. The corresponding
crystallographic NAD+ structure was taken into account as cofactor using the MVD
feature possibilities. No water molecules were included into the docking procedure. The
side chains around compound 4a (11 residues) were set flexible during the calculation.
Combination of different calculation (Moldock SE, Moldock Optimizer) algorithms and
scoring schemes (Moldock, Plants) were tested [38,39] giving insight similar top binding
modes for GEQ and compound 4a. After calculation, minimization steps (global, lateral
chain, ligands) and optimization of hydrogen bonds were done using MVD default
features followed by clustering. Using these conditions the crystallographic conformation
of GEQ was reproduced with a good accuracy (less than 0.6 Å of RMSD with reference
structure 1P44a) and nearly no fluctuation of flexible residues including Tyr158 was
recorded. Subsequently optimized parameters were applied to the docking procedure. The
enantiomers R and S of compound 4a were screened to understand differences in binding
of our compounds compared to the reference ligand. The results are represented on Fig. 2.

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The graph representation of normalized descriptors LE1 (Molegro’s Moldock Score
divided by heavy atoms number) vs LE3 (Molegro’s Rerank score divided by heavy atoms
number) obtained from the docking studies of compound 4a (R- and S-enantiomers) and
GEQ (60 calculation runs followed by clustering of results) is shown on SI Fig. 1 (See
supporting information). Evidently, the best binding poses (circles colored in red) with the
lowest energy correspond to GEQ structure (the worst values correspond to poses outside
the binding site) that are in correlation with obtained biological results.
Acknowledgments
T. Matviiuk was supported by French Embassy in Kiev, Ukraine. The investigations
having been performed within the framework of the GDRI (Groupement Franco-Ukrainien
en Chimie Moléculaire; between UPS, Toulouse and Taras Shevchenko National
University of Kiev).
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Table 1. Yields determined for each compound.
Cpd
2a
R
Ph
Yield (%)
62
p-MeO-C6H4
63
2b
88 (Method A)
70 (Method B)
83 (Method A)
58 (Method B)
87 (Method A)
55 (Method B)
79 (Method A)
60 (Method B)
95
4a
Ph
Bn
Ph
Bn
4b
6a
6b
Ph
8a
8b
p-MeO-C6H4
92
Ph
Bn
Ph
84
71
62
11a
11b
12

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Table 2. Yields of the synthesized compounds; InhA inhibition values; Minimal
Inhibitory Concentration (MIC) values; Cytotoxicities.
Cpd
R
% Inhibition
at 50 µMa
MICb
(µg/ml) / (µM)
Cytotoxicity
IC50 (µM)
HCT116/GM637H
17 / 15
Ph
15
22
52
56
20
16
9
5 / 18.2
5 / 16.4
2a
2b
4a
4b
6a
6b
8a
8b
p-MeO-C6H4
ND
>100 / > 100
ND
>100 / >100
>100 / >100
>100 / >100
ND
Ph
Bn
Ph
Bn
Ph
40 / 94.4
40 / 91.4
2.5 / 8.2
10 / 31.2
20 / 82.9
15 / 55.3
p-MeO-C6H4
21
Ph
Bn
Ph
22
40 / 109.2
>100 / >100
11a
11b
12
12
20 / 52.6
ND
45
20 / 46.5
>100 / >100
ND
>99
---
10 / 34.5
Triclosan
0.05/0.4
ND
Isoniazid
GEQ
>99
>125 / >317.7
ND
a The results are expressed as a percentage of InhA inhibition.
b Minimal inhibitory concentration on M. tuberculosis H37Rv strain

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O
O
O
NH
N
N
n
H
H
O
O
Moiramide B (n=1), Andrimid (n=2)
O
H
NH
O
O
H
H
H
O
H
H
R
R = H, Hirsutellone A
R = CH3, Hirsutellone D
Fig. 1. Structures of natural antibiotics (Miroamide B and Andrimid) and antimycobacterial
alkaloids Hirsutellone A and D bearing succinimide fragments.
O
N
N
N
H
GEQ (Genz-10855)
Fig. 2. Superposition of GEQ crystallographic bindings (carbons colored in magneta; 1P44
pdb entry) and predicted binding modes of 4a; R-enantiomer U5A (dark grey) and S-
enantiomer D5A (light grey).

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O
N
N
N
R
S
N
N
N
H
O
O
SH
N
N
H
cat. AlCl3
dioxane
R
1
O
2a,b
Scheme 1. Catalytic conjugate addition of maleimides to 4H-1,2,4-triazole-3-thiole 1. 2a R =
Ph; 2b R = p-CH3O-C6H4.
O
N
N
N
R
N
O
4a,b
N
3
5
H
O
N
R
O
N
O
NH
cat. LiClO4
dioxane, r.t.
6a,b
N
R
O
R
O
N
N
N
N
N
H
7
O
8a,b
Scheme 2. Synthesis of C–N Michael adducts. 4a, 6a, 8a R = Ph; 4b, 6b R = Bn; 8b R = p-
CH3O-C6H4.

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N
O
O
N
N
R
N
R
9
O
O
11a,b
cat. AlCl3
dioxane
O2N
O2N
N
N
O
R
NO2
10
NO2
N
O
12
Scheme 3. Catalytic conjugate addition of N-substituted maleimides to 2-phenylindolizine (9)
and 7,9-dinitropyrido[2,1-a]isoindole (10). 11a, 12 R = Ph; 11b R = Bn.

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•
•
•
A series of 3-heteryl substituted pyrrolidine-2,5-dione were synthesized.
They displayed promising inhibitory activity against M. tuberculosis H37Rv.
Some of them present inhibition activities against InhA.

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Synthesis of 3-heteryl substituted pyrrolidine-2,5-diones via catalytic
Michael reaction and evaluation of their inhibitory activity against
InhA and Mycobacterium tuberculosis
Tetiana Matviiuk,a,b Giorgia Mori,d Christian Lherbet,*,a,c Marian Gorichko,b Frédéric
Rodriguez,a,c Maria Rosalia Pasca,d Brigitte Guidetti,a,c Zoia Voitenko,*a,b Michel Baltas*a,c
Supplementary information
I. NMR spectra for the compounds
II. Inhibition values
III. Cytotoxicities of the compounds
IV. Molecular docking studies

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I. NMR spectra of the compounds
Compound 2a