Design, chemical synthesis of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives and biological activity against enoyl-ACP reductase (InhA) and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2013.09.041

We report here the discovery, synthesis and screening results of a series of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives as a novel class of potent inhibitors of Mycobacterium tuberculosis H37Rv strain as well as the enoyl acyl carrier protein re

Full text of DOI:10.1016/j.ejmech.2013.09.041

Accepted Manuscript
Design, chemical synthesis of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives
and biological activity against enoyl-ACP reductase (InhA) and Mycobacterium
tuberculosis
Tetiana Matviiuk, Frédéric Rodriguez, Nathalie Saffon, Sonia Mallet-Ladeira, Marian
Gorichko, Ana Luisa de Jesus Lopes Ribeiro, Maria Rosalia Pasca, Christian Lherbet,
Zoia Voitenko, Michel Baltas
PII:
S0223-5234(13)00622-3
10.1016/j.ejmech.2013.09.041
EJMECH 6444
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 May 2013
Revised Date: 18 September 2013
Accepted Date: 20 September 2013
Please cite this article as: T. Matviiuk, F. Rodriguez, N. Saffon, S. Mallet-Ladeira, M. Gorichko, A.L. de
Jesus Lopes Ribeiro, M.R. Pasca, C. Lherbet, Z. Voitenko, M. Baltas, Design, chemical synthesis of 3-
(
(
9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives and biological activity against enoyl-ACP reductase
InhA) and Mycobacterium tuberculosis, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.09.041.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, chemical synthesis of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives and
biological activity against enoyl-ACP reductase (InhA) and Mycobacterium tuberculosis
Tetiana Matviiuk, Frédéric Rodriguez, Nathalie Saffon, Sonia Mallet-Ladeira, Marian Gorichko, Ana
Luisa de Jesus Lopes Ribeiro, Maria Rosalia Pasca, Christian Lherbet, Zoia Voitenko, Michel Baltas

ACCEPTED MANUSCRIPT
Highlights
‹
‹
‹
A series of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives were synthesized.
Several compounds displayed good inhibitory activity against InhA.
Some of them exhibited promising activities against M. tuberculosis and multi-drug resistant
M. tuberculosis strains.

ACCEPTED MANUSCRIPT
Design, chemical synthesis of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives and biological
activity against enoyl-ACP reductase (InhA) and Mycobacterium tuberculosis
a,b,e
a,b
c
c
e
Tetiana Matviiuk, Frédéric Rodriguez, Nathalie Saffon, Sonia Mallet-Ladeira, Marian Gorichko, Ana Luisa
d
d
a,b*
e
a,b*
5
0
5
0
5
0
5
0
de Jesus Lopes Ribeiro, Maria Rosalia Pasca, Christian Lherbet, Zoia Voitenko, Michel Baltas
a
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
b.
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
1
1
2
2
3
3
4
c.
d.
e.
Université de Toulouse, UPS and CNRS, ICT FR2599, 118 route de Narbonne, 31062 Toulouse cedex 9, France
University of Pavia; Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, via Ferrata 1, 27100 Pavia, Italy.
Taras Shevchenko National University of Kyiv, Department of Chemistry, 64 str. Volodymyrska, Kyiv, 01033,
Ukraine
Fax: 33 (0)5 61556011; Tel: 33 (0)5 61556289; E-mail : lherbet@chimie.ups-tlse.fr; baltas@chimie.ups-tlse.fr
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Keywords: Mycobacterium tuberculosis, InhA, inhibition, 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione, succinimide
Abstract
We report here the discovery, synthesis and screening results of a series of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione
derivatives as a novel class of potent inhibitors of Mycobacterium tuberculosis H37Rv strain as well as the enoyl acyl
carrier protein reductase (ENR) InhA. Among them, several compounds displayed good activities against InhA which is
one of the key enzymes involved in the type II fatty acid biosynthesis pathway of the mycobacteria cell wall. Furthermore,
some exhibited promising activities against M. tuberculosis and multi-drug resistant M. tuberculosis strains.
5
1
. Introduction
1
1
2
2
3
3
0
5
0
5
0
5
Tuberculosis (TB) is the leading cause of worldwide mortality alongside malaria and AIDS with about two million deaths
every year [1]. Today, nearly one third of the global population is infected, their location being mainly in developing
countries. Tuberculosis is caused by Mycobacterium tuberculosis (M. tuberculosis), and current chemotherapeutic
treatments are based on the use of antibiotics, the most important being isoniazid (INH), rifampicin, pyrazinamide,
ethambutol and streptomycin. 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, more new drugs are required to raise the probability of stopping short all forms of drug-resistant TB. It is in this
context that many studies target the cell wall of mycobacteria and in particular mycolic acid biosynthesis which involves
several successive enzymatic cycles and especially two related but distinct Fatty Acid Synthase (FAS) systems, FAS I and
II [5]. The InhA protein (ENR, EC number: 1.3.1.9) is part of FAS II and shows an NADH-dependent enoyl-ACP reductase
activity. InhA is an essential enzyme of M. tuberculosis and thus a good target in addition to the fact that the FAS II system
is present in bacteria but is absent in humans.
We previously described the synthesis of potential inhibitors of InhA, bearing a triazole as a central core to mimick the
phenolic group of triclosan [6,7]. In the course of our studies on the Michael reaction on maleimide [8,9], we focused on
the succinimide fragment as a core structure of newly designed inhibitors. Molecules containing succinimide as a structural
fragment have been employed numerous times in drug design. For example, 2,5-pyrrolidinediones are core structural units
in naturally occurring substances and also in some approved drugs and clinical drug candidates. [10-13]. Since Komura and
co-workers in 1987 reported the isolation of Andrimide as a new and highly specific antibiotic, 1,3-substituted and 3,4-
disubstituted succinimides emerged as a new class of natural products with high biological activity [14]. Andrimide and
Moiramide B (Fig 1) exhibit potent antibacterial activity against methicillin-resistant Staphylococcus aureus and a range of
other antibiotic-resistant human pathogens. These natural antibiotics have been described to target the FAS system that is
also the primary target for antitubercular drugs [15]. Moreover, Hirsutellone A, a natural molecule bearing a succinimide
ring (Fig. 1) displayed a significant growth inhibitory activity against M. tuberculosis H37Rv [16]. Furthermore, the
hydrophobic fluorenyl moiety has received much attention as a pharmacophore for the inhibition of InhA [17,18]. Indeed, it
has already played the role of an anchor in the binding site of InhA, for example in GEQ inhibitor (Genz-10850) and is
responsible for extensive hydrophobic interactions (Fig. 1).
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Based on these data, we first performed in silico screening of the database of virtual compounds based on the succinimide
core fragment as promising pharmacophore moiety. From docking studies, 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione was
identified for the development of new antitubercular agents (Fig. 1). We report herein the synthesis and evaluation of 3-
(
9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives as inhibitors of InhA and M. tuberculosis.
5
2
2
. Results and discussion
.1. Chemistry
1
1
2
2
3
3
0
5
0
5
0
5
The compounds described in Table 1 were prepared by multi-step synthesis through two important intermediates 2 and 9
Schemes 1 and 4). Compound 2 is accessible by three different methods (methods A, B C, Scheme 1). As shown in
(
Scheme 1, the first step is the condensation of fluorenone with succinonitrile in the presence of tBuOK to obtain 3-(9H-
fluoren-9-ylidene)pyrrolidine-2,5-dione 1 in good yield, as described before [19]. Two different methods were then used to
reduce the double bond. Generally, reduction of unsaturated succinimides is accomplished by catalytic hydrogenation in the
presence of 10% Pd/C in high yields or by using the NiCl /NaBH couple [20]. In our hands, reduction of the double bond
2
4
by NiCl (7 eq) and NaBH (8 eq) afforded compound 2 in only 40% yield (method A, Scheme 1). So, a second method
2
4
was applied combining zinc powder and acetic acid under reflux conditions, leading to target compound 2 in excellent yield
97%, method B) [21]. The third method (method C, Scheme 1) used diacid 3 as an intermediate. Compound 3 was
(
synthesized in 74% yield by fusion, from commercially available fluorene condensed with maleic anhydride, followed by
aqueous basic treatment for several hours before acidification and recrystallization of the solid formed [22,23]. Then
succinimide 2 was formed in 73% yield from 3 upon treatment with ammonium hydroxide at high temperature [24].
GEQ analogue 6 bearing a succinimide and an indole moiety was synthesized in three steps. First, compound 5 was
synthesized in two steps starting from 2, using a known procedure [21,25], followed by its condensation with indole-
5-carboxylic acid in the presence of EDC affording 6 in 83% yield [26].
Synthesis of compounds 7a-c and 8a-d was performed as outlined in Scheme 3. Coupling of the succinimide
intermediate 2 with acyl chloride or alkyl bromide derivatives, respectively, afforded compounds 7a-c and 8a-d in
good yields. Note that two methods were applied to obtain compounds 8a-d depending on the base used. When
potassium hydride in DMF was used, higher yields were obtained. Both enantiomers for racemic compounds 7a and
8
a were obtained under supercritical fluid chromatography separation, in order to compare their activities in the
presence of InhA.
Another strategy was used to gain access to succinimide derivatives 11a-d through succinic anhydride 9 (Scheme 4)
obtained by treatment of diacid 3 with acetic anhydride under reflux. Compound 9 was then allowed to react with different
primary amines affording amides 10a-d. When THF is used as solvent at room temperature, good β-regioselectivity was
observed. Indeed, reactions performed with p-F-aniline, 3,4,5-trimethoxyaniline and tryptamine afforded mixtures of α- and
1
β-anilides in ratios of 85:15, 60:40 and 70:30 respectively, as measured by H NMR on the crude product. Pure β-isomers
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were obtained by recrystallization of the mixtures for compounds 10a and 10c and flash chromatography for 10b. The same
reaction was also carried out in CH CN to afford 10a but in this case, a 1/1 mixture (α/β regioselectivity) as measured by
3
1
H NMR was observed. Cyclisation was then performed in acetic anhydride to afford the corresponding succinimide
derivatives 11a-c and 11e in good yields except for hydrazinamide compound 10d which is the result of the condensation
of the front-line antitubercular drug isoniazid with succinic anhydride 9. Attempts to cyclize it afforded a complex mixture.
5
2
.2. Biology
1
0
2.2.1. InhA inhibition assay
Recombinant M. tuberculosis InhA was expressed in E. coli and subsequently purified according to a previously
reported procedure [6]. The synthetic compounds were evaluated in vitro for the inhibition of InhA from M.
tuberculosis at 50 µM by applying a commonly used method [6]. The results are shown in Table 2.
1
5
0
5
0
5
Three different series of molecules can be compared in Table 2, a first series with the succinimide ring bearing an acyl
group (compounds 6, 7a-7c), a second for succinimide substituted with an alkyl or aromatic group (compounds 8a-8d and
1
1a-11e) and a third possessing the β−amide frame (compounds 10a-10d).
In the first series, all compounds tested presented similar activities against InhA, varying between 50% and 73% of
2
2
3
3
inhibition at 50 µM, with the lowest value for the indole derivative 6 and the highest for compound 7b bearing a C -alkyl
8
chain. No notable differences were observed in the activities of each enantiomer of 7a.
The second series presented more promising results. For compounds where an aryl ring is directly attached to the nitrogen
atom of the succinimide (11a, 11b, 11e), the 3,5-dichloroanilide derivative 11b emerges as an excellent InhA inhibitor
(
>95% inhibition at 50 µM), while the two other compounds 11a, 11e are rather weak inhibitors. For compounds where a
methylene group is first attached to the nitrogen atom (8a-8d and 11c) compound 8a is a weak inhibitor while 8b and 8d
present moderate activities (56% and 59% respectively). The last two compounds 8c and 11c emerge as the best inhibitors
of this series with inhibition values of 86% and 79% at 50 µM respectively. It is noteworthy that compound 8c presents a
C -alkyl chain, as does the best active compound (7b) in the first series, while compound 11c presents a tryptamine group
8
in comparison to the 5-indole carbonyl derivative 6 of the first series which is a moderate inhibitor.
Finally, in the third series (β−amide derivatives 10a-10d), only compound 10c possessing the tryptamine group presents a
good inhibition activity (60% at 50 µM) while the three other derivatives are inactive or weak inhibitors.
2
.2.2. Bacterial growth inhibition experiments with M. tuberculosis H37Rv strain
All the synthesized compounds were evaluated by determining the minimal inhibitory concentration (MIC) on M.
tuberculosis H37Rv strain (Table 3). Triclosan and GEQ were used for comparison.
The majority of the compounds displayed higher activities than GEQ. A MIC of above 40 µM was found for GEQ
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which is consistent with the literature (>125 µM) [17]. Ortiz de Montellano et al. suggested that this high MIC could
be due to a poor membrane permeability. Indeed, by replacing the piperazine moiety by a succinimide ring, GEQ
analogue 6 showed a higher inhibition (MIC 39 µM). This could be due to an increased membrane permeability.
In the first series, compounds 6, 7b and 7c presented the same moderate activities while compound 7a strongly
inhibited M. tuberculosis growth (MIC 5.4 µM). In the second series, again most of the compounds are moderately
active only compound 11b, possessing a 3,5-dichloromethyl substitution on the nitrogen atom, presented a good MIC
value of 19.6 µM. Finally, in the third series, compounds 10b bearing the same 3,5-dichlorophenyl pattern and 10d
bearing the isoniazide pharmacophore emerged as very potent with MIC values of 4.7 µM and 10.0 µM respectively.
Considering the biological effects of the compounds evaluated, derivatives 7a and 11b might be considered as
inhibitors affecting both InhA protein (though perhaps not exclusively) and M. tuberculosis growth. Compounds 7b,
5
1
1
2
2
3
3
0
5
0
5
0
5
8
c and 11c are the ones that affected InhA protein most but have weak effects on M. tuberculosis growth.
Finally, for compounds 10b and 10d of the third series the lack of inhibition on InhA combined with low MIC
suggests that InhA is not the target. In addition, when compound 10b was tested against multi-drug-resistant M.
tuberculosis strains, it exhibited the same level of efficiency as for the H37Rv strain (Table 4). These results
confirmed that InhA, inhibited by the adduct INH-NAD, is not the target for 10b [27].
2
.3. Docking
Compound 11b, showing the best InhA inhibition activity, was chosen and studied using molecular docking methods.
The active site of InhA is known to be flexible with states corresponding to major or minor portal opening and loop
reordering (residues 195-210) [28-30]. Recently, methods were developed in the field of ligand or structure based
design, which tried to take InhA’s features into account [31-33] in order to improve the correlation of rankings
obtained by calculation and by experimental methods. Nevertheless, it is still often difficult to correlate simulated
results with relevant biological ones, in particular in the case of screening libraries including a limited number of
compounds. Consequently we used molecular docking to prove the concept of modeling the hypothetical binding
conformation of compound 11b in the active site, relative to the conformation of GEQ in the same protein structure
(
PDB 1P44 entry) [18].
The predicted binding mode of 11b (carbons in violet) with the best scoring results obtained from the docking studies
is shown in Fig. 2 superimposed on the crystallographic conformation of Genz-10855 (carbons in green). The
inhibitor fits the binding pocket of InhA in the same manner as GEQ inhibitor. Hydrogen bonding with Tyr158
residue is highly conserved and other residues of the binding site (i.e. Met161, Phe149) showed minor fluctuations. In
+
contrast to the GEQ position, hydrogen bonds between 11b and cofactor NAD are not observed. This corresponds to
the position of the carbonyl groups of the pyrrolidine-2,5-dione ring (displaced in the direction of the minor portal, on
the right in the figure) and rotation flexibility of Phe149 from the binding pocket of InhA. Finally, while the opposite
+
configuration of 11b is able to form hydrogen bonds with Tyr158 but also with NAD , it exhibits lower docking
scores despite a similar orientation of the fluorene ring.
3
. Conclusion
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ACCEPTED MANUSCRIPT
A series of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives was prepared and assayed for the inhibition of InhA
and M. tuberculosis growth. Among them, three molecules 11b, 11c and 8c displayed interesting activities against
InhA. Furthermore, replacing the piperazine ring of GEQ for a succinimide core led to increased activities against M.
tuberculosis H37Rv strains with activities of up to 5 µM (compound 7a). Finally, β−amide derivative 10b with a MIC
of 4.7 µM, was evaluated against MDR-strains and presented the same level of efficiency. Because of the ability of
these new compounds to strongly affect InhA protein and/or M. tuberculosis growth, they could represent new leads
for the development of candidate drugs.
5
1
0
4. Experimental
4
.1. Material
Kinetic studies were performed on a Cary Bio 100. All chemicals were obtained from Aldrich or Acros Organics and
used without further purification. Nuclear magnetic resonance spectra were recorded on a Bruker AC 300
1
5
1
13
spectrometer ( H and C 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 spectrometer using electrospray ionization (ESI) methods. Optical
rotations were measured using a sodium D line on a P-2000 series Jasco, PTC-262 polarimeter. Melting points were
measured on a Mettler Toledo MP50 melting point system and are uncorrected.
2
0
Crystallographic data for compounds 2, 8a and 10a were collected at a temperature of 193(2)K on a Bruker-AXS Quazar
APEX II diffractometer (2 and 8a) using a 30 W air-cooled microfocus source (ImS) with focusing multilayer optics or on
a Bruker-AXS SMART APEX II diffractometer (10a), with MoKα radiation (wavelength = 0.71073 Å). Phi- and omega-
scans were used. The data were integrated with SAINT [34], and an empirical absorption correction with SADABS [34]
was applied. The structures were solved by direct methods, using SHELXS-97 and refined using the least–squares method
2
5
2
on F [35]. All non-H atoms were treated anisotropically. The H atoms were fixed geometrically and treated as a riding
model. For compound 2, the H atom attached to nitrogen was located on a difference Fourier map and refined without any
restraints. For 10a, H atoms attached to nitrogen and to oxygen atoms were located on difference Fourier map and refined
with distance restraints of N-H 0.88(1) Å and O-H 0.84(1) Å.
3
0
Full crystallographic data for the structures have been deposited with the Cambridge Crystallographic Data Centre
under registration numbers CCCDC 934383 (2), CCDC 934384 (8a) and CCDC 934385 (10a) [36]. GEQ compound
(
genz-10850) was synthesized according to the literature procedure [18].
4
.2. Synthesis and Characterizations
3
5
4.2.1. 3-(9H-Fluoren-9-ylidene)pyrrolidine-2,5-dione (1). To a solution of potassium tert-butoxide (4.70 g, 41.6 mmol) in
tert-butanol at 50° C under nitrogen atmosphere, a solution of 9-fluorenone (5.00 g, 27.7 mmol) and succinonitrile (2.22 g,
2
7.7 mmol) in t-BuOH was added dropwise in 15 minutes. After completion the addition, the resulting mixture was
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ACCEPTED MANUSCRIPT
refluxed under nitrogen atmosphere for 2 h, allowed to cool and acidified with 6M hydrochloric acid (20 mL). The solution
was evaporated under reduced pressure and distilled water (50 mL) was added. The crude product was extracted three times
with EtOAc (3 × 30 mL). The combined organic layers were dried over Na SO , filtered and evaporated under reduced
2
4
pressure. The residue was crystallized using a mixture of iPrOH : EtOH (3:1) to afford the desired product as a lightly
0
1
5
0
5
green powder (6.00 g, 84%). Mp: 178–179 C. H NMR (300 MHz, DMSO-d ): δ = 4.01 (s, 2H), 7.31–7.50 (m, 4H), 7.75–
6
1
3
7
1
.88 (m, 3H), 9.29 (d, J = 7.8 Hz, 1H), 11.68 (br s, 1H); C NMR (75 MHz, DMSO-d ): δ = 38.4, 127.4, 119.6, 120.0,
6
+
27.1, 127.9, 128.9, 130.2, 130.4, 135.3, 137.8, 140.7, 140.9, 141.3, 141.7, 171.2, 174.7; HRMS (TOF MS Cl ) m/z
+
(
M+H ) calculated for C H NO 262.0868; found 262.0869.
1
7
12
2
1
4.2.2. 3-(9H-Fluoren-9-yl)pyrrolidine-2,5-dione (2)
Method A: 3-(9H-Fluoren-9-ylidene)pyrrolidine-2,5-dione 1 (0.52 g, 2 mmol) was added in one portion to a solution of
°
NiCl .6H O (3.32 g, 14 mmol) in MeOH – THF (3:1) 15 mL at 4 C. Then NaBH (0.61 g, 16 mmol) was added
2
2
4
portionwise over 30 min and the resulting black slurry was stirred overnight at room temperature. The reaction mixture
was neutralized by 1M HCl to pH = 7. The black slurry was filtered through a short pad of Florisil® and washed three
times with CH Cl (3 × 15 mL). The solution was dried over MgSO , filtered and evaporated under reduced pressure. The
1
2
2
4
product was further purified by flash chromatography (PE/EtOAc – 8:2) to afford white crystals (211 mg, 40%). Mp: 177-
°
-1
1
1
78 C. IR (ν, cm ) 1783, 1448, 1352; H NMR (300 MHz, CDCl ): δ = 1.45 (dd, J = 5.4 Hz, J = 18.6 Hz, 1H), 2.24 (dd, J
3
=
9 Hz, J = 18.6 Hz, 1H), 3.77 (ddd, J = 3.6 Hz, J = 5.4 Hz, J = 9 Hz, 1H), 4.69 (d, J = 3.6 Hz, 1H), 7.23–7.50 (m, 6H),
1
3
7
.77 (d, J = 7.5 Hz, 2H), 8.54 (br s, 1H); C NMR (75 MHz, CDCl ): δ = 30.6, 44.7, 46.2, 120.4, 120.6, 123.8, 124.9,
3
+
+
2
2
3
0
5
0
127.8, 127.9, 128.2, 128.6, 141.3, 141.4, 142.3, 144.1, 176.1, 179.1. HRMS (TOF MS Cl ) m/z (M+H ) calculated for
C H NO 264.1025; found 264.1031.
1
7
14
2
Method B: To a solution of 3-(9H-fluoren-9-ylidene)pyrrolidine-2,5-dione (1) (300 mg, 1.15 mmol) in acetic acid (10 mL,
66.7 mmol), zinc powder (225.5 mg, 3.45 mmol) was added. The reaction mixture was refluxed for 90 min. After the
1
reaction had cooled to room temperature, the solvent was evaporated under reduced pressure and then water (15 mL) was
added and stirred for 15 min. The precipitate was filtered and purified by flash chromatography (PE/EtOAc – 7:3) to afford
the desired product (294 mg, 97%).
4
.2.3. 2-(9H-Fluoren-9-yl)succinic acid (3). Fluorene (15.00 g, 90 mmol) and maleic anhydride (8.84 g, 92 mmol) were
°
mixed in a round bottom flask fitted with a condenser. The mixture was stirred at 200 C for 6 hours. Then the reaction
mixture was cooled and dissolved in a saturated solution of sodium bicarbonate. Insoluble material was filtered.
Acidification of the filtrate gave 2-(9H-fluoren-9-yl)succinic acid, which was then recrystallized from diluted acetic acid
0
-1
1
(
40% in distilled water). Yield: 18.8 g, 74%. Mp: 186–187 C. IR (ν, cm ) 1701, 1451, 1408, 1240; H NMR (300 MHz,
DMSO-d ): δ = 1.21 (dd, J = 3 Hz, J = 16.8 Hz, 1H), 1.93 (dd, J = 11.1 Hz, J = 16.8 Hz, 1H), 3.65 (ddd, J = 3 Hz, J = 6 Hz,
6
3
5
J = 11.1 Hz, 1H), 4.46 (d, J = 3 Hz, 1H), 7.28–7.44 (m, 5 H), 7.66 (d, J = 7.2 Hz, 1H), 7.86–7.91 (m, 2H), 12.46 (br. s, 2H);
1
3
C NMR (75 MHz, DMSO-d ): δ = 29.8, 42.8, 47.6, 120.0, 120.3, 124.3, 124.6, 127.1, 127.5, 127.7, 140.7, 141.0, 143.1,
6
1
44.5, 172.9, 174.6.
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ACCEPTED MANUSCRIPT
4
.2.4. Method C: Synthesis of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione (2) from 2-(9H-fluoren-9-yl)succinic acid (3). 2-
To a dispersed mixture of (9H-Fluoren-9-yl)succinic acid 5 (3.00 g, 10.63 mmol) in 50 mL of distilled water, 9 mL of 25
%
aqueous solution of ammonia (64 mmol of NH OH) was added gradually. The mixture was heated in an oil bath with
4
0
simultaneous distillation of water. The cyclization reaction was continued at 190 C for 2 h with transmittance of gaseous
ammonia in the reaction mixture. Crude product was dispersed in saturated solution of sodium bicarbonate and extracted 3
5
times with ETOAc (3 × 40 mL). The organic phase was dried with MgSO , filtered and the solvent evaporated. The residue
4
was chromatographed with elution system of EtOAC/PE – 3:7 to afford the desired product (2.00 g, 73 %).
4
.2.5. Synthesis of 3-(9H-Fluoren-9-yl)-1-(1H-indol-5-ylcarbonyl)-2,5-pyrrolidinedione (6).
1
1
2
0
5
0
4.2.5.1. tert-Butyl 3-(9H-fluoren-9-yl)-2,5-dioxopyrrolidine-1-carboxylate (4). To a mixture of amide 2 (158 mg, 0.6 mmol)
°
and DMAP (7.4 mg, 0.06 mmol) in acetonitrile, di-tert-butyl dicarbonate (0.16 mL, 0.7 mmol) was added dropwise at 0 C.
°
The mixture was stirred at 4 C for 30 min and then at room temperature for 3 h (reaction progress monitored by TLC).
After completion of the reaction, the solvent was evaporated and the crude product purified by flash chromatography
1
(
PE/CH Cl – 6:4) to afford light brown crystals (124 mg, 57%). Mp: 156 °C (decomposition). H NMR (300 MHz,
2
2
CDCl ): δ = 1.56 (dd, J = 5.7 Hz, J = 18.6 Hz, 1H) , 1.58 (s, 9H), 2.33 (dd, J = 9.3 Hz, J = 18.6 Hz, 1H), 3.86 (ddd, J = 3.9
3
Hz, J = 5.7 Hz, J = 9.3 Hz, 1H), 4.73 (d, J = 3.9 Hz, 1H), 7.14–7.50 (m, 6H), 7.17 (d, J = 7.5 Hz, 2H).
4
.2.5.2 3-(9H-Fluoren-9-yl)-1-hydroxypyrrolidine-2,5-dione (5). To a solution of N-Boc-succinimide 4 (124 mg, 0.34
mmol) in acetonitrile an excess of aqueous solution of hydroxylamine (0.14 mL, 50% wt. 2.0 mmol) was added. After
stirring the mixture overnight at room temperature the aqueous phase was saturated with brine and extracted three times
with EtOAc (3 × 20 mL). The organic layer was dried with sodium sulfate and then evaporated under reduced pressure. The
crude product was purified by flash chromatography (CH Cl /EtOAc/THF – 90:10:5) to afford compound 5 (81 mg, 85%).
2
2
1
Mp: 149-150 °C (decomposition). H NMR (300 MHz, DMSO-d ): δ = 1.33 (d, J = 18 Hz, 1H), 2.23 (d, J = 18 Hz, 1H),
6
3
.73 (s, 1H), 4.68 (s, 1H), 7.14–7.50 (m, 6H), 7.17 (t, J = 7.5 Hz, 2H), 8.13 (br. s, 1H).
2
5
4
.2.5.3. 3-(9H-Fluoren-9-yl)-1-(1H-indol-5-ylcarbonyl)pyrrolidine-2,5-dione (6). N-Hydroxysuccinimide 5 (81 mg, 0.29
mmol) was added to a suspension of indole-5-carboxylic acid (56 mg, 0.35 mmol) in anhydrous CH Cl , then the mixture
2
2
°
was cooled to 4 C and stirred for 15 minutes. Thereafter N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC, 54 mg,
.35 mmol) was added and the reaction mixture was stirred for 63 h. The reaction progress was monitored by TLC. Then
0
3
0
the solvent was evaporated and the residue was washed with water and filtered. The brown solid was recrystallized from
the mixture of i-PrOH/MeOH (5:1) to afford 6 as a white powder (66 mg, 56 %). Overall yield in 3 steps: 32%. Mp: 236–
-
1
1
2
37 °C (decomposition). IR (ν, cm ) 3401; 1758; 1725, 1615; H NMR (300 MHz, CDCl ): δ = 1.62 (br. s, 2H), 2.38 (dd, J
3
1
3
=
9.3 Hz, J = 18.3 Hz, 1H), 3.91 (br. s, 1H), 4.81 (s, 1H), 6.69 (s, 1H), 7.32–7.98 (m, 10H), 8.54 (s, 2H); C NMR (75
MHz, DMSO-d ): δ = 26.9, 103.2, 112.1, 112.3, 114.5, 120.3, 122.4, 122.8, 124.4, 124.5, 127.6, 127.7, 128.1, 128.2, 128.4,
6
+
+
3
5
139.6, 140.7, 141.2, 141.7, 143.5, 162.6, 169.1, 172.2. HRMS (TOF MS Cl ) m/z (M+H ) calculated for
C H N O 407.1390; found 407.1373.
2
6
18
2
3
4
.2.6. General procedure for the acylation of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione (7) A dispersion of NaH in mineral
oil (0.4 mmol) was added to a solution of amide 2 (0.2 mmol) in THF under argon atmosphere. After 20 minutes, the
-
8 -

ACCEPTED MANUSCRIPT
mixture was cooled to 4 °C and a solution of acyl chloride (0.3 mmol) in THF was added. The reaction mixture was stirred
for 4 h at room temperature. After evaporation of solvent, the resulting residue was mixed with distilled water (30 mL) and
extracted 3 times with CH Cl (3 × 20 mL). The organic layer was dried over MgSO and then concentrated under reduced
2
2
4
pressure. The product was further purified by flash chromatography.
5
4
.2.6.1. 1-Benzoyl-3-(9H-fluoren-9-yl)- 2,5-pyrrolidinedione (7a) The product was purified by flash chromatography
-
1
(
PE/EtOAc – 7:3) to give a light yellow amorphous solid (35 mg, 47%). Mp: 60-61 °C. IR (ν, cm ) 1787; 1717, 1697;
1
1
448; H NMR (300 MHz, CDCl ): δ = 1.64 (dd, J = 5.7 Hz, J = 18.9 Hz, 1H), 2.40 (dd, J = 9.3 Hz, J = 18.9 Hz, 1H), 3.94
3
1
3
(
ddd, J = 3.6 Hz, J = 5.7 Hz, J = 9.3 Hz, 1H), 4.80 (d, J = 3.6 Hz, 1H), 7.32–7.84 (m, 13H); C NMR (75 MHz, CDCl ): δ
3
1
0
= 30.2, 44.5, 46.5, 120.6, 120.7, 123.9, 125.3, 127.9, 128.0, 128.4, 128.9, 129.1, 130.8, 131.4, 135.3, 141.2, 141.4, 142.5,
+
+
1
43.8, 167.6, 173.9, 176.6. HRMS (TOF MS Cl ) m/z (M+H ) calculated for C H NO 368.1287; found 368.1300.
24 18 3
2
D
0
20
[α
]
= +222.0 (c 0.3, CHCl
3
, ee 100 %), [
α
3
]D = −216.0 (c 0.25, CHCl , ee 97.2%). Chiral preparative SFC, Chiralpak OJ-
H 5µM (10˟ 250 mm) cellulose tris(4-methylbenzoate) with 15% CH CN, 40 °C, P=120 bar, flow rate 12 mL/min t d-
3
R
enantiomer 3.27 min, l-enantiomer 4.40 min.
1
5
4
.2.6.2. 3-(9H-Fluoren-9-yl)-1-nonanoyl-2,5-pyrrolidinedione (7b) The crude product was purified by flash
-
1
chromatography (PE/EtOAc – 9:1) to give the final product as a colorless oil (50 mg, 62%). IR (ν, cm ) 1759, 1721, 1699,
1
1
538, 1448; H NMR (300 MHz, CDCl ): δ = 0.86–0.91 (m, 5H), 1.27–1.36 (m, 5H), 1.58–1.75 (m, 4H), 2.26 (t, J = 9.6
3
Hz, 1H), 2.35 (t, J = 7.5 Hz, 2H), 2.88 (td, J = 0.9 Hz, J = 7.2 Hz, 2H), 3.75 (ddd, J = 3.6 Hz, J = 5.7 Hz, J = 9.6 Hz, 1H),
1
3
2
2
3
3
0
5
0
5
4.75 (d, J = 3.6 Hz, 1H), 7.23–7.50 (m, 6H), 7.78 (dd, J = 2.7 Hz, J = 7.5 Hz, 2H); C NMR (75 MHz, CDCl ) : δ = 14.3,
3
2
1
2.8, 23.9, 24.8, 29.1, 29.7, 31.9, 33.9, 39.3, 43.6, 46.6, 120.5, 120.7, 123.9, 124.8, 127.9, 128.4, 128.8, 141.0, 141.4,
+
43.8, 172.9, 173.5, 176.4. LRMS (DCI/NH ) [M+NH ] calculated for C H N O 421.3; found 421.2.
3
4
26 33
2
3
4
.2.6.3. 3-(9H-Fluoren-9-yl)-1-(2-phenylacetyl)-2,5-pyrrolidinedione (7c). The product was purified by flash
-
1
chromatography (PE/EtOAc – 8:2) to give a colorless amorphous solid (30.5 mg, 40%). Mp: 52 °C. IR (ν, cm ) 1769;
1
1
1
700, 1408; H NMR (300 MHz, CDCl ): δ = 1.44 (dd, J = 6.0 Hz, J = 18.6 Hz, 1H), 2.23 (dd, J = 9.6 Hz, J = 18.6 Hz,
3
H), 3.68 (ddd, J = 3.6 Hz, J = 6.0 Hz, J = 9.6 Hz, 1H), 4.27 (s, 2H), 4.70 (d, J = 3.6 Hz, 1H), 7.07–7.44 (m, 11H), 7.76 (t,
1
3
J = 7.8 Hz, 2H); C NMR (75 MHz, DMSO-d ): δ = 30.6, 41.2, 43.3, 46.1, 120.5, 120.6, 123.9, 124.9, 127.5, 127.8, 128.2,
6
+
+
1
28.6, 129.0, 129.8, 131.2, 133.4, 141.3, 142.7, 167.9, 174.2, 177.5. HRMS (TOF MS Cl ) m/z (M+H ) calculated for
C H NO 382.1438; found 382.1433.
2
5
20
3
4
.2.7. General procedure of alkylation of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione (8) Method A: Anhydrous potassium
carbonate (110 mg, 0.8 mmol) was dispersed in dry acetone, then amide 2 (50 mg, 0.2 mmol) was added. The mixture was
stirred for 20 min at room temperature. Then a solution of alkyl bromide (0.3 mmol) in acetone was added. The reaction
mixture was stirred for 5 h and monitored by TLC. After completion of the reaction, the solution was filtered and the
solvent evaporated under reduced pressure. The resulting residue was purified by flash chromatography. Method B: To a
solution of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione 2 (50 mg, 0.2 mmol) in dry DMF, potassium hydride (50% in
paraffin) (15 mg, 0.3 mmol) was added. Then the reaction mixture was cooled to 4 °C and alkyl bromide (0.3 mmol) was
-
9 -

ACCEPTED MANUSCRIPT
added. The reaction mixture was stirred for 3 h and filtered. The precipitate was washed with CH Cl . The filtrate was
2
2
concentrated under reduced pressure and the crude product was purified by flash chromatography.
4
.2.7.1. 3-(9H-Fluoren-9-yl)-1-(phenylmethyl)- 2,5-pyrrolidinedione (8a). The desired product was synthesized according
to Method A and was purified by flash chromatography (PE/EtOAc – 8:2) to give 8a as white crystals (49 mg, 69%). Mp:
-
1
1
5
159 °C. IR (ν, cm ) 1769, 1701, 1491, 1401; H NMR (300 MHz, CDCl ): δ = 1.37 (dd, J = 4.8 Hz, J = 18.6 Hz, 1H), 2.22
3
(
dd, J = 9.0 Hz, J = 18.6 Hz, 1H), 3.67 (ddd, J = 3.6 Hz, J = 4.8 Hz, J = 9 Hz, 1H), 4.64–4.81 (m, 3H), 6.70–6.86 (m, 2H),
1
3
7
1
.29–7.49 (m, 9H), 7.69–7.75 (m, 2H); C NMR (75 MHz, CDCl ): δ = 29.4, 43.6, 44.2, 46.8, 100.5, 107.2, 120.3, 123.8,
3
+
24.9, 127.6, 127.7, 128.1, 128.3, 137.7, 141.2, 141.3, 142.1, 144.2, 161.1, 176.0, 178.9; HRMS (TOF MS Cl ) m/z
+
(
M+H ) calculated for C H NO 354.1481; found 354.1487.
2
4
20
2
2
D
0
20
D
1
0
[α
] = +249.0 (c 0.3, CHCl
, ee 100 %), [
α
]
= −256.0 (c 0.25, CHCl , ee 98.5%). Chiral preparative SFC, Chiralpak OJ-
3
3
H 5µM (10˟ 250 mm) cellulose tris(4-methylbenzoate) with 25% CH CN, 40 °C, P=120 bar, flow rate 12 mL/min t d-
3
R
enantiomer 2.50 min, l-enantiomer 5.27 min.
4
.2.7.2. 1-(3,5-Dimethoxybenzyl)-3-(9H-fluoren-9-yl)-2,5-pyrrolidinedione (8b). The desired product was synthesized
1
5
according to Method B and was purified by flash chromatography (PE/CH Cl – 7:3) to give 8b as a lightly yellow solid
2
2
-
1
1
(
60 mg, 73%). Mp: 140 °C. IR (ν, cm ) 1773; 1699, 1607, 1597, 1432; H NMR (300 MHz, CDCl ): δ = 1.37 (dd, J = 4.8
3
Hz, J = 18.6 Hz, 1H), 2.22 (dd, J = 9.3 Hz, J = 18.6 Hz, 1H), 3.67 (ddd, J = 3.6 Hz, J = 4.8 Hz, J = 9.3 Hz, 1H), 3.79 (s,
H), 4.58 (d, J = 13.7 Hz, 1H), 4.67 (d, J = 3.6 Hz, 1H), 4.71 (d, J = 13.7 Hz, 1H), 6.45 (t, J = 2.4 Hz, 1H), 6.60 (d, J = 2.4
6
1
3
Hz, 2H), 6.79–6.83 (m, 1H), 6.92 (td, J = 1.2 Hz, J = 7.5 Hz, 1H), 7.28–7.49 (m, 4H), 7.72 (t, J = 7.8 Hz, 2H); C NMR
2
2
3
0
5
0
(75 MHz, CDCl ): δ = 29.4, 42.8, 42.9, 46.6, 55.5, 100.5, 107.2, 120.3, 123.8, 124.9, 127.6, 127.7, 128.1, 128.3, 137.7,
3
+
+
1
41.2, 141.3, 142.1, 144.2, 161.1, 176.0, 178.9. HRMS (TOF MS Cl ) m/z (M+H ) calculated for C H NO 414.1705;
26 24 4
found 414.1721.
4
.2.7.3. 3-(9H-Fluoren-9-yl)-1-nonyl-2,5-pyrrolidinedione (8c). The desired product was synthesized according to Method
-
1
A and was purified by flash chromatography (PE/EtOAc – 9:1) to give 8c as lightly yellow oil (37 mg, 71%). IR (ν, cm )
1
2
922, 2851, 1770, 1699, 1689, 1409; H NMR (300 MHz, CDCl ): δ = 0.86–0.95 (m, 5H), 1.24–1.41 (m, 9H), 1.50–1.65
3
(
m, 4H), 2.19 (dd, J = 9.1 Hz, J = 18.6 Hz, 1H), 3.55 (t, J = 7.5 Hz, 1H), 3.67 (ddd, J = 3.6 Hz, J = 5.1 Hz, J = 9.1 Hz, 1H),
1
3
4
1
.73 (d, J = 3.6 Hz, 1H), 7.17–7.24 (m, 2H), 7.31–7.51 (m, 4H), 7.76 (d, J = 7.5 Hz, 2H); C NMR (75 MHz, CDCl ): δ =
3
4.3, 22.8, 27.1, 27.9, 29.38, 29.41, 29.6, 32.0, 39.2, 43.2, 46.4, 120.4, 120.5, 123.9, 124.8, 127.6, 127.8, 128.1, 128.5,
+
141.3, 141.5, 142.3, 144.4, 176.5, 179.3; LRMS (DCI/NH ) [M+NH ] calculated for C H N O 407.5; found 407.2.
3
4
26 35
2
2
4
.2.7.4. 3-(9H-Fluoren-9-yl)-1-(2-oxo-2-phenylethyl)-2,5-pyrrolidinedione (8d). The product was synthesized according to
Method B and was purified by flash chromatography (PE/EtOAc – 7:3) to afford 8d as a white powder (72 mg, 94%). Mp:
-
1
1
1
86 °C. IR (ν, cm ) 2923, 1775, 1707, 1699, 1420; H NMR (300 MHz, CDCl ): δ = 1.57 (dd, J = 5.7 Hz, J = 18.6 Hz,
3
3
5
1H), 2.36 (dd, J = 9.3 Hz, J = 18.6 Hz, 1H), 3.90 (ddd, J = 3.6 Hz, J = 5.7 Hz, J = 9.3 Hz, 1H), 4.79 (d, J = 3.6 Hz, 1H),
1
3
4
4
.97 (d, J = 17.1 Hz, 1H), 5.05 (d, J = 17.1 Hz, 1H), 7.30–8.03 (m, 13H); C NMR (75 MHz, CDCl ): δ = 29.6, 43.6, 44.9,
3
6.2, 120.4, 123.9, 125.3, 127.8, 127.9, 128.2, 128.3, 128.5, 129.1, 134.2, 134.6, 141.4, 142.2, 144.4, 175.8, 178.7, 190.1;
+
+
HRMS (TOF MS Cl ) m/z (M+H ) calculated for C H NO 382.1443; found 382.1453.
2
5
20
3
-
10 -

ACCEPTED MANUSCRIPT
4
.2.8. General procedure for the synthesis of fluorene succinamic acid derivatives (10a-d). 2-(9H-Fluoren-9-yl)succinic
acid 3 200 mg (0.71 mmol) was dissolved in 5 mL of acetic anhydride (49 mmol) and refluxed for 2 h 30 min. Then solvent
was evaporated and residue dried under oil vacuum pump. Dried 2-(9H-fluoren-9-yl)succinic anhydride 9 was dissolved in
7-10 mL of dry THF and 1 equivalent of amine was added. The mixture was stirred at room temperature overnight.
Thereafter solvent was evaporated and residue dissolved in saturated solution of sodium hydrocarbonate, filtered and
acidified with 1M HCl. Precipitate was filtered and purified by flesh chromatography or recrystalized from ethanol to
afford the product.
5
4
.2.8.1. 3-(9H-fluoren-9-yl)dihydro-2,5-furandione (9). The product was dried under vacuum and used without further
1
0
purification. A small amount was recrystallized for analysis from glacial acetic acid to give a white powder (187 mg,
1
quantitative yield). Mp: 168°C. H NMR (300 MHz, CDCl ): δ = 1.69 (dd, J = 6.3 Hz, J = 19.2 Hz, 1 H), 2.47 (dd, J = 9.9
3
Hz, J = 19.2 Hz, 1 H), 3.99 (ddd, J = 3.6 Hz, J = 6.3 Hz, J = 9.9 Hz, 1 H), 4.70 (d, J = 3.6 Hz, 1 H), 7.30–7.51 (m, 6 H),
1
3
7
1
.77–7.81 (m, 2H). C NMR (75 MHz, DMSO-d ): δ = 29.2, 44.1, 46.2, 120.5, 120.6, 123.7, 124.6, 127.9, 128.3, 128.5,
6
29.0, 140.2, 142.2, 143.1, 143.8, 169.3, 173.2.
1
5
4.2.8.2. 2-(9H-Fluoren-9-yl)-4-(4-fluoroaniline)-4-oxobutanoic acid (10a). The product was crystallized from EtOH to give
-
1
1
a white powder (113 mg, 86%). Mp: 171 °C decomposition. IR (ν, cm ) 3317, 1702, 1669, 1620, 1557, 1510; H NMR
(
300 MHz, DMSO-d ): δ = 1.28 (d, J = 15.6 Hz, 1 H), 2.15 (dd, J = 11.4 Hz, J = 15.9 Hz, 1 H), 3.85 (d, J = 10.5 Hz, 1
6
1
H), 4.52 (br. s, 1 H), 6.90–8.05 (m, 12 H), 9.70 (br. s, 1 H), 12.86 (br. s, 1 H). H NMR (300 MHz, CD OD): δ = 1.41 (dd, J
3
=
3.6 Hz, J = 15.9 Hz, 1 H), 2.21 (dd, J = 10.5 Hz, J = 15.9 Hz, 1 H), 3.94 (ddd, J = 3.9 Hz, J = 6.6 Hz, J =
1
3
2
0
10.5 Hz, 1 H), 4.60 (d, J = 3.6 Hz, 1 H), 6.87–7.86 (m, 12 H) ; C NMR (75 MHz, CDCl ): δ = 32.6, 44.3, 49.4, 115.8,
3
1
2
16.1, 120.9, 121.1, 122.8, 122.9, 125.4, 126.1, 128.1, 128.6, 128.8, 128.9, 136.0, 142.6, 143.1, 144.7, 146.0, 160.0 (d, J =
+
+
19
41.8 Hz), 172.5, 177.2. HRMS (TOF MS Cl ) m/z (M+H ) calculated for C H NO F 376.1349; found 376.1360.
F
2
3
19
3
NMR (300 MHz, DMSO-d ): δ = -116.0.
6
2
3
3
5
0
5
4.2.8.3. 4-(3,5-Dichloroanilino)-2-(9H-fluoren-9-yl)-4-oxobutanoic acid (10b). The crude product was crystallized from
-
1
EtOAc to afford light brown crystals (140 mg, 69%). Mp: 98–99 °C (decomposition). IR (ν, cm ) 1705, 1670, 1586, 1539,
1
1
1
7
447, 1410; H NMR (300 MHz, DMSO-d ): δ = 1.29 (dd, J = 3.0 Hz, J = 16.5 Hz, 1H), 2.11 (dd, J = 11.1 Hz, J = 16.5 Hz,
6
H), 3.82 (ddd, J = 3.0 Hz, J = 6.3 Hz, J = 11.1 Hz, 1H), 4.52 (d, J = 3.0 Hz, 1H), 7.17 (t, J = 1.9 Hz, 1H), 7.31–7.47 (m,
1
3
H), 7.63–7.68 (m, 1H), 7.90 (t, J = 7.2 Hz, 2H), 9.98 (s, 1H), 12.93 (br. s, 1H); C NMR (75 MHz, DMSO-d ): δ = 31.8,
6
42.2, 47.5, 116.8, 120.1, 122.1, 124.3, 124.7, 127.2, 127.6, 127.7, 133.9, 140.7, 141.1, 141.3, 143.3, 144.5, 170.2, 174.8;
+
+
HRMS (TOF MS Cl ) m/z (M+H ) calculated for [C H NO Cl ] 426.0664; found 426.0656.
2
3
18
3
2
4
.2.8.4. 2-(9H-Fluoren-9-yl)-4-{[2-(1H-indol-3-yl)ethyl]amino}-4-oxobutanoic acid (10c). The product was purified by
crystallization from mixture i-PrOH /EtOH – 3:1 to give brown crystals (160 mg, 83%). Mp: 123 °C decomposition. IR (ν,
-
1
1
cm ) 1709, 1618, 1535, 1448; H NMR (300 MHz, CDCl ): δ = 1.20 (dd, J = 2.4 Hz, J = 15.9 Hz, 1H), 1.94 (dd, J = 10.8
3
Hz, J = 15.9 Hz, 1H), 2.76 (t, J = 6.6 Hz, 2H), 3.26–3.45 (m, 2H), 3.68–3.74 (m, 1H), 4.67 (d, J = 2.1 Hz, 1H), 5.32 (t, J =
1
3
6
2
.0 Hz, 1H), 6.80 (s, 1H), 6.99–7.55 (m, 10H), 7.69 (d, J = 7.5 Hz, 2H), 8.14 (br. s, 1H); C NMR (75 MHz, CDCl ): δ =
3
4.9, 31.6, 40.0, 44.0, 48.0, 111.4, 112.2, 118.6, 119.6, 120.0, 122.3, 122.4, 124.3, 125.9, 127.2, 127.5, 127.6, 127.9, 136.4,
-
11 -

ACCEPTED MANUSCRIPT
+
+
1
4
41.5, 141.7, 143.3, 144.6, 173.2, 176.6; HRMS (TOF MS Cl ) m/z (M+H ) calculated for [C H N O ] 425.1865; found
27 25 2 3
25.1869.
4
.2.8.5. 2-(9H-Fluoren-9-yl)-4-(2-isonicotinoylhydrazino)-4-oxobutanoic acid (10d). The product was crystallized from a
-
1
1
5
0
5
mixture of i-PrOH – MeOH (8:2) to give a white powder. (100 mg, 58 %). Mp: 158 °C. IR (ν, cm ) 1704, 1651, 1450; H
NMR (300 MHz, CDCl ): δ = 1.56 (dd, J = 5.4 Hz, J = 18.6 Hz, 1H), 2.40 (dd, J = 9.0 Hz, J = 18.6 Hz, 1H), 3.91 (ddd, J =
3
1
3
3
.6 Hz, J = 5.4 Hz, J = 9.0 Hz, 1H), 4.76 (d, J = 3.6 Hz, 1H), 7.17–7.80 (m, 12H), 8.73 (br. s, 2H), 9.43 (s, 1H); C NMR
(
75 MHz, DMSO-d ): δ = 28.8, 42.1, 47.7, 120.1, 120.4, 122.5, 124.4, 124.7, 127.2, 127.6, 127.8, 140.8, 141.1, 141.7,
6
+
+
1
43.4, 144.6, 148.1, 163.0, 170.0, 174.6; HRMS (TOF MS Cl ) m/z (M+H ) calculated for C H N O 384.1348; found
23 18 3 3
1
384.1343.
4
.2.9. General procedure for cyclization of fluorene succinamic acid derivatives (11a-c-11e). 2-(9H-Fluoren-9-
yl)succinamic acid 10 (0.7 mmol) was dissolved in acetic anhydride (39 mmol, 4 mL). The solution was refluxed for 30
minutes. Then the solvent was evaporated under reduced pressure. To the crude product, a saturated solution of sodium
1
hydrocarbonate (NaHCO , 10 mL) was added and the resulting solution was extracted 3 times with EtOAc (3 × 20 mL).
3
The combined organic layers were dried with magnesium sulfate and the solvent was evaporated under reduced pressure.
Crude products were purified by flesh chromatography.
4
.2.9.1. 3-(9H-Fluoren-9-yl)-1-(4-fluorophenyl)-2,5-pyrrolidinedione (11a) The crude product was purified by flash
-
1
2
0
chromatography (PE/EtOAc – 7:3) to give a white powder (80 mg, 87%). Mp: 231 °C decomposition. IR (ν, cm ) 1778,
1
1
1
705, 1506, 1447, 1391; H NMR (300 MHz, CDCl ): δ = 1.56 (dd, J = 5.1 Hz, J = 18.9 Hz, 1H), 2.38 (dd, J = 9.1 Hz, J =
3
8.9 Hz, 1H), 3.87 (ddd, J = 3.9 Hz, J = 5.1 Hz, J = 9.1 Hz, 1H), 4.80 (d, J = 3.9 Hz, 1H), 7.18–7.55 (m, 10H), 7.78–7.82
1
3
(
m, 2H); C NMR (75 MHz, CDCl ): δ = 29.6, 43.3, 46.7, 116.3, 116.6, 120.5, 120.7, 123.9, 124.7, 127.7, 127.9, 128.3,
3
+
+
1
28.4, 128.5, 128.8, 141.3, 141.4, 142.5, 144.1, 163.5 (d, J = 248.7 Hz), 175.3, 178.1. HRMS (TOF MS Cl ) m/z (M+H )
1
9
2
5
calculated for C H NO F 358.1243; found 358.1259. F NMR (300 MHz, CDCl ): δ = -116.8.
23 17 2 3
4
.2.9.2. 1-(3,5-Dichlorophenyl)-3-(9H-fluoren-9-yl)-2,5-pyrrolidinedione (11b). The product was purified by flash
-
chromatography (PE/EtOAc – 65:35) to give a lightly brown powder (45 mg, 83%). Mp: 196 °C decomposition. IR (ν, cm
1
1
)
1780, 1713, 1704, 1576, 1448, 1381; H NMR (300 MHz, CDCl ): δ = 1.51 (dd, J = 5.1 Hz, J = 18.9 Hz, 1H), 2.32 (dd, J
3
3
0
= 9.3 Hz, J = 18.9 Hz, 1H) 3.79 (ddd, J = 3.9 Hz, J = 5.1 Hz, J = 9.3 Hz, 1H), 4.71 (d, J = 3.9 Hz, 1H), 7.19–7.47 (m, 9H),
1
3
7
1
.71–7.75 (m, 2H); C NMR (75 MHz, CDCl ): δ = 29.6, 43.3, 46.7, 116.4, 116.7, 120.5, 120.7, 123.9, 124.7, 127.7,
3
+
27.9, 128.3, 128.4, 128.5, 128.8, 141.3, 141.4, 142.5, 144.1, 160.8, 164.1, 175.3, 178.1; HRMS (TOF MS Cl ) m/z
+
(
M+H ) calculated for C H NO Cl 408.0558; found 408.0569.
2
3
16
2
2
3
5
4.2.9.3. 3-(9H-Fluoren-9-yl)-1-[2-(1H-indol-3-yl)ethyl]-2,5-pyrrolidinedione (11c). The product was purified by flash
chromatography (CH Cl /EtOAc/THF – 85:15:5) to give purple crystals (39 mg, 75%). Mp: 182 °C decomposition. IR (ν,
2
2
-
1
1
cm ) 3341, 1766, 1687, 1450, 1436, 1333; H NMR (300 MHz, CDCl ): δ = 1.37 (dd, J = 5.1 Hz, J = 18.6 Hz, 1H), 2.17
3
(
dd, J = 9.0 Hz, J = 18.6 Hz, 1H), 3.12 (m, 2H), 3.61 (ddd, J = 3.6 Hz, J = 5.1 Hz, J = 9.0 Hz, 1H), 3.91 (t, J = 7.8 Hz, 2H),
-
12 -

ACCEPTED MANUSCRIPT
1
3
4
.71 (d, J = 3.6 Hz, 1H), 7.00 (td, J = 1.2 Hz, J = 7.5 Hz, 1H), 7.12–7.49 (m, 9H), 7.73–7.77 (m, 3H), 8.01 (br. s, 1H); C
NMR (75 MHz, CDCl ): δ = 23.6, 29.5, 39.5, 43.1, 46.4, 111.3, 112.4, 118.9, 119.8, 1120.4, 120.5, 122.2, 122.4, 123.9,
3
+
+
1
24.7, 127.6, 127.7, 128.5, 136.0, 136.4, 141.4, 142.2, 144.3, 176.4, 179.2; HRMS (TOF MS Cl ) m/z (M+H ) calculated
for [C H N O ] 407.1760; found 407.1759.
2
7
23
2
2
5
4
.2.9.4. 3-(9H-Fluoren-9-yl)-1-(3,4,5-trimethoxyphenyl)-2,5-pyrrolidinedione (11e). The product was purified by flash
0
-
chromatography (CH Cl /EtOAc – 9 : 1) to give a white powder (49 mg, 83%). Mp: 166–167 C decomposition. IR (ν, cm
2
2
1
1
)
1719, 1658, 1586, 1538, 1441, 1410; H NMR (300 MHz, CDCl ): δ = 1.58 (dd, J = 5.1 Hz, J = 18.9 Hz, 1H), 2.39 (dd, J
3
1
3
=
9.3 Hz, J = 18.9 Hz, 1H), 3.88 (s, 10 H), 4.80 (d, J = 3.9 Hz, 1 H), 6.48 (s, 2H), 7.28–7.83 (m, 8H); C NMR (75 MHz,
1
0
CDCl ): δ = 29.7, 43.2, 46.8, 56.4, 61.0, 104.4, 120.5, 120.8, 123.9, 124.9, 127.4, 127.9, 128.3, 128.8, 141.4, 142.6, 144.0,
3
+
+
1
53.8, 175.5, 178.3; HRMS (TOF MS Cl ) m/z (M+H ) calculated for C H NO 430.1654; found 430.1647.
26 24 5
4
.3. Biology
1
5
4
.3.1. InhA expression and purification.
The production and purification were performed as described [6].
2
0
4.3.2. 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 [6]. 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 (initial velocity of the reaction) with respect to the control reaction without inhibitor. All activity assays were
performed in triplicate.
2
3
3
5
0
5
4
.3.3. Growth conditions
M. tuberculosis H37Rv strain was grown either in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween 80, or
on Middlebrook 7H11 agar (Difco) supplemented with 0.5% glycerol, both supplemented with 10% (vol/vol) OADC. All
compounds were dissolved in dimethyl sulfoxide (DMSO). Mycobacterial cultures were usually grown at 37°C without
shaking.
-
13 -

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4
.3.4. MIC determinations
A single colony of M. tuberculosis strain was used to inoculate complete Middlebrook 7H9. The cultures were incubated at
8
3
7°C until exponential growth phase (~10 CFU/mL) was reached, corresponding to an OD
ranging from 0.8 and 1.0.
00nm
6
7
5
0
5
0
5
0
5
Cultures were diluted to the final concentration of about 10 CFU/mL; 1 µL of the diluted cultures was then streaked onto
plates containing two-fold serial dilutions of appropriate compound. MIC values were scored as the lowest drug
concentrations inhibiting bacterial growth. All assays were repeated three times.
M. tuberculosis clinical isolates and drug susceptibility testing. Three M. tuberculosis MDR isolates were collected at the
Sondalo Division of the Valtellina and Valchiavenna, Italy, hospital authority in the 2012. Their resistance profile is shown
in Table 3. All clinical isolates were grown in BACTEC™ MGIT™ 960 and Lowenstein–Jensen slants. Drug susceptibility
testing for all first-line antitubercular drugs was performed with the BACTEC™ MGIT™ 960 System (Becton-Dickinson
Diagnostic Systems, Sparks, Maryland). MIC determination to second-line drugs was also performed by the MGIT™ 960
System.
1
1
2
2
3
3
4
.3.5. Molecular docking studies
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 Francisco
(supported by NIGMS P41-GM103311). The Protein structures were prepared (structure checks, rotamers,
hydrogenation) 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 (hybridization, hydrogenation) using Discovery Studio 3.0 client.
+
The entry 1P44 (chain A, X-ray, 2.7 Å resolution) [18] from Protein Data Bank [37] includes NAD /NADH and GEQ
in the active site and was chosen as InhA molecular structure.
We used Molegro Virtual Docker 5.5 software (CLC Bio, Aarhus, Denmark) for 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 molecules were used as
cofactor using the MVD features and no water molecules were taken into account. The side chains around compound
11b (16 residues) were set flexible in calculation. A combination of different calculation (Moldock SE, Moldock
Optimizer) schemes and scoring schemes (Moldock, Plants) was used [38,39] giving similar best poses for GEQ and
1
1b. After calculation, minimization steps (global, lateral chain, ligands) and optimization of H 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 0.7 Å of RMSD with reference structure 1P44a) and nearly no
fluctuation of flexible residues including Tyr158 was recorded. Subsequently these parameters were applied to
compound 11b and the best (GEQ like, Moldock and Rerank scores) poses were retained for further analysis.
Acknowledgments
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14 -

ACCEPTED MANUSCRIPT
We thank the CNRS and University Paul Sabatier for financial support. T. M. was supported by the French Embassy in
Kiev (Ukraine), the investigations having been performed within the framework of the GDRI (Groupement de recherche
Franco-Ukrainien en Chimie Moléculaire).
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[
1
5
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Table 1. Synthesized compounds.
Cpds
2
Structure
Yield (%)
Cpds
Structure
Yield (%)
40 (Method A) 8c
71
O
97 (Method B)
O
NH
O
N
O
7
3 (Method C)
74
6
3
8d
94
O
O
O
OH
N
OH
O
O
4
5
57
84
10a
10b
86
69
O
O
NBoc
O
OH
F
O
N
H
O
OH
O
Cl
N
OH
O
O
N
H
Cl
6
56
47
62
10c
10d
11a
83
58
87
O
N
O
NH
HN
OH
O
O
O
N
H
7
a
O
N
O
N
O
OH
O
O
O
HN
N
H
7
b
O
N
O
N
O
O
6
O
F
7
8
c
40
11b
83
O
N
O
N
Cl
O
O
O
Cl
a
69
73
11c
11e
75
83
O
O
N
O
N
O
N
H
8
b
OMe
O
O
N
O
N
OMe
OMe
OMe
O
MeO

ACCEPTED MANUSCRIPT
Table 2. Enzyme inhibition values. Results are expressed as a percentage of InhA inhibition.
Compound
% Inhibition
Compound
% Inhibition
at 50 µM
>99
14
at 50 µM
Triclosan
GEQ
8d
>99
59
6
2
3
6
56
10a
10b
10c
50
8
7
a
65
60
7
8
a (l) / 7a(d)
71/62
73
7
b
10d
11a
11b
30
23
7c
63
8
a
25
>95 (31 at 5 µM)
a (l) / 8a(d)
20/27
56
8
b
11c
11e
79
34
8c
86

ACCEPTED MANUSCRIPT
Table 3 Compounds tested as inhibitory agents of M. tuberculosis growth.
Comp
MIC
µg/mL) / (µM)
10 / 34.5
8 / 30.4
Comp
MIC
(µg/mL) / (µM)
>16 / >40.7
>16 / >41.9
16 / 42.6
(
Triclosan
GEQ
8d
2
3
6
8 / 28.3
10a
10b
10c
10d
11a
11b
16 / 39.4
2 / 5.4
2 / 4.7
7a
16 / 37.7
7b
16 / 39.7
16 / 41.9
16 / 45.3
4 / 10.0
7c
>16 / >44.8
8 / 19.6
8a
8
a (l) / 8a(d) 16 (45.3) / 16 (45.3)
8
b
>16 / >38.7
16 / 41.1
11c
11e
>16 / >39.4
16 / 37.3
8
c
Table 4. Activities against multi-drug-resistant M. tuberculosis strains.
MIC
M. tuberculosis
M. tuberculosis clinical isolates
a
a
a
Compound
H37Rv
IC1
IC2
IC3
(
µg/mL / µM)
(
µg/mL / µM)
(µg/mL / µM)
(µg/mL / µM)
10b
2 / 4.7
2 / 4.7
2 / 4.7
2 / 4.7
a
Mtb clinical isolate: IC1 : drug resistance profile: resistant to streptomycin, isoniazid (INH), rifampicin,
ethambutol; IC2 : drug resistance profile: resistant to streptomycin, isoniazid (INH), rifampicin, ethambutol,
pyrazinamide, ethionammide, capreomicin; IC3 : drug resistance profile: resistant to streptomycin, isoniazid
(
INH), rifampicin, ethambutol, pyrazinamide, ethionammide.

ACCEPTED MANUSCRIPT
Figure 1. GEQ, a potent inhibitor of InhA; Structure of natural antibiotics (Moiramide B and Andrimide) and
antimycobacterial alkaloid Hirsutellone A; Structure of inhibitors developed herein.

ACCEPTED MANUSCRIPT
Figure 2. Superimposition of crystallographic conformation of GEQ (carbons in green stick representation)
and simulated by docking binding mode of 11b (carbons in violet stick representation) in the binding
groove of InhA (1P44 PDB entry, chain A). Degree of residue flexibility (side chains of residues around the
site, backbone) was applied in docking procedure. Residues Phe149, Tyr158 and Met161 colored in
magenta (docked protein structure) and green (1P44 protein structure).

ACCEPTED MANUSCRIPT
Scheme 1. Two routes to synthesize succinimide 2; Molecular view of 2. Thermal ellipsoids are drawn at
the 50% probability level. Hydrogen atoms except for that on N1 are omitted for clarity
Scheme 2. Synthesis of derivative 6.

ACCEPTED MANUSCRIPT
Scheme 3. Synthesis of compounds 7a-c and 8a-d; Molecular view of 8a. Thermal ellipsoids are drawn at
the 50% probability level. Hydrogen atoms are omitted for clarity

ACCEPTED MANUSCRIPT
Scheme 4. Synthesis of derivatives 10a-d and 11a-e. Molecular view of compound 10a. Thermal ellipsoids
are drawn at the 50% probability level. Hydrogen atoms are omitted for clarity except for the H on N1 and
O2.

ACCEPTED MANUSCRIPT
Supporting information
Design, chemical synthesis of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives and biological
activity against enoyl-ACP reductase (InhA) and Mycobacterium tuberculosis
a,b,d
a,b
c
c
e
Tetiana Matviiuk,
Frédéric Rodriguez, Nathalie Saffon, Sonia Mallet-Ladeira, Marian Gorichko,
d
d
a,b*
e
Ana Luisa de Jesus Lopes Ribeiro, Maria Rosalia Pasca, Christian Lherbet, Zoia Voitenko, Michel
a,b*
Baltas
a
CNRS; Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, LSPCMIB, UMR-
5
068; 118 Route de Narbonne, F-31062 Toulouse cedex 9, France
b.
c.
d.
e.
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
Université de Toulouse, UPS and CNRS, ICT FR2599, 118 route de Narbonne, 31062 Toulouse cedex 9,
France
University of Pavia; Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, via Ferrata 1, 27100
Pavia, Italy.
Taras Shevchenko National University of Kyiv, Department of Chemistry, 64 str. Volodymyrska, Kyiv,
0
1033, Ukraine
Fax: 33 (0)5 61556011; Tel: 33 (0)5 61556289; E-mail : lherbet@chimie.ups-tlse.fr; baltas@chimie.ups-
tlse.fr
I. NMR data for the compounds
II. Crystallographic data for compounds 2, 8a and 10a
III. SFC separation

ACCEPTED MANUSCRIPT
I. NMR data for the compounds
Compound 1:
SpinWorks 3: scaffold1
PPM
11.0
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
file: ...:\NMR\51_scaffold1_double_bond\fid expt: <zg30>
transmitter freq.: 300.181854 MHz
freq. of 0 ppm: 300.180006 MHz
processed size: 32768 complex points
LB: 0.000 GF: 0.0000
time domain size: 65536 points
width: 6188.12 Hz = 20.6146 ppm = 0.094423 Hz/pt
number of scans: 8
Hz/cm: 112.098 ppm/cm: 0.37344