Design, synthesis and evaluation of new GEQ derivatives as inhibitors of InhA enzyme and Mycobacterium tuberculosis growth

ACCEPTED MANUSCRIPT

Graphical abstract

Design, synthesis and evaluation of new GEQ derivatives as inhibitors of InhA enzyme and

Mycobacterium tuberculosis growth

Aurélien Chollet, Giorgia Mori, Christophe Menendez, Frédéric Rodriguez, Isabelle Fabing, Maria Rosalia

Pasca, Jan Madacki, Jana Korduláková, Patricia Constant, Annaïk Quémard, Vania Bernardes-Génisson,

Christian Lherbet, Michel Baltas

ACCEPTED MANUSCRIPT

Design, synthesis and evaluation of new GEQ derivatives as

inhibitors of InhA enzyme and Mycobacterium tuberculosis growth

a,b,c,d

e

a,b

Aurélien Chollet,

Giorgia Mori, Christophe Menendez, Frédéric Rodriguez, Isabelle

a,b

e

f

g,h

Fabing, Maria Rosalia Pasca, Jan Madacki, Jana Korduláková, Patricia Constant, Annaïk

g,h

c,d

,a,b

a,b

Quémard, Vania Bernardes-Génisson, Christian Lherbet,* Michel Baltas,

a

Laboratoire de Synthèse et Physicochimie de Molécules d’Intérêt Biologique (SPCMIB),

Centre National de la Recherche Scientifique (CNRS), 118 Route de Narbonne, 31062,

Toulouse, Cedex 09 France

b

Université de Toulouse, Université Paul Sabatier, LSPCMIB, F-31077, Toulouse, France

c

Laboratoire de Chimie de Coordination, Centre National de la Recherche Scientifique

(

d

CNRS), , 205, Route de Narbonne, BP 44099, F-31077 Toulouse, Cedex 4, France

Université de Toulouse, Université Paul Sabatier, INPT, F-31077 Toulouse, Cedex 4, France

Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, University of Pavia, via

e

Ferrata 1, 27100 Pavia, Italy.

f

Department of Biochemistry, Comenius University in Bratislava, Faculty of Natural

Sciences, Mlynská dolina Ch-1, 842 15 Bratislava, Slovakia

g

Département Tuberculose & Biologie des Infections, CNRS, IPBS (Institut de

Pharmacologie et de Biologie Structurale), UMR5089, 205 route de Narbonne, BP 64182,

3

1077 Toulouse, France

h

Université de Toulouse, Université Paul Sabatier, IPBS, 31077 Toulouse, France

Corresponding author: lherbet@chimie.ups-tlse.fr; (Christian Lherbet)

Keywords: Medicinal chemistry; inhibitors; InhA; Mycobacterium tuberculosis; efflux

pumps.

1

ACCEPTED MANUSCRIPT

ABSTRACT

A series of fluorene-based derivatives was synthesized and evaluated for inhibiting both InhA

and Mycobacterium tuberculosis growth. These compounds were inspired by the previously

reported Genz-10850 molecule, a good InhA inhibitor, but with a poor activity against M.

tuberculosis growth. Structure-activity relationships were performed by introducing the

following chemical modifications: 1) the piperazine ring; 2) the amide group; 3) the aryl

moiety; and 4) the fluorene moiety. Among these new derivatives, one of them was more

effective against both the InhA activity and mycobacterial growth, compared to the hit

compound. Docking studies were also performed to rationalize activities of these derivatives.

Furthermore, we showed for the first time that efflux pump inhibitors potentiated the efficacy

of Genz-10850 (GEQ) derivatives against M. tuberculosis growth, demonstrating that these

compounds could be substrates of some efflux pumps.

2

ACCEPTED MANUSCRIPT

1

. Introduction

Tuberculosis remains one of the leading infectious diseases around the world with 9 million

new cases and 1.5 million deaths in 2013.[1] One-third of the world population has been

estimated as infected by Mycobacterium tuberculosis, the causative pathogen of tuberculosis.

With the resurgence of the disease related to the HIV-coinfection and to resistance to current

clinical treatment, there is a growing need to find new antitubercular drugs.[1,2] In the last

few years, M. tuberculosis enzymes involved in the fatty acid synthase type II (FAS-II)

system have been identified and validated as relevant drug targets.[3-5] Among the FAS-II

enzymes, InhA, a trans-2-enoyl-ACP reductase, is one of the most druggable target in

tuberculosis field. Different classes of direct InhA inhibitors, including 4-hydroxy-2-pyridone

derivatives, have been recently reported in the literature with potent bactericidal activity

against M. tuberculosis strains.[6,7] Among all these inhibitors, Genz-10850 (also called

GEQ) has been identified as a very promising inhibitor of InhA (Figure 1), after in vitro

screening of a library of 500,000 compounds.[8] Later, He et al. synthesized a series of GEQ

derivatives with InhA inhibitory activities ranging from nanomolar to micromolar.[9]

Compound 1 (Figure 1) was one of these derivatives and it exhibited an IC value in the low

50

nanomolar range. These molecules have poor activity against M. tuberculosis, showing a MIC

above 125 µM because of their low permeability or the activation of efflux pumps.[9]

Therefore, chemical modification of these inhibitors was performed in order to improve their

low biological activity.

In the present work, we reported the synthesis and the evaluation of new GEQ analogues with

an improved inhibitory activity against both InhA and M. tuberculosis growth. Furthermore,

we showed that efflux pumps inhibitors potentiate their effectiveness against M. tuberculosis

growth.

Figure 1. GEQ and its analogue 1 as inhibitors of InhA

3

ACCEPTED MANUSCRIPT

2

. Results and discussion

2

.1. Design of the GEQ derivatives. During the development of GEQ analogues by Ortiz

de Montellano et al., a significant improvement was performed by replacing the indole ring

with a phenyl group (Figure 1).[9] The reported IC₅₀toward InhA inhibition for benzoyl

analogue 1 was lower than that of GEQ. Consequently compound 1 remained as our reference

molecule all along this study.

To ensure similar hydrophobic interactions in the binding site of InhA, the global scaffold of

the compound 1 was retained. The proposed modifications were performed in an attempt to

improve both inhibition of InhA and the M. tuberculosis membrane permeability (Figure 2).

Figure 2. Structural modifications proposed from the compound 1

Modification of the central core.

The piperazine central core was replaced with a piperidine in order to overcome the eventual

protonation of the nitrogen atom (Figure 2). We are consequently expected to improve

activity of the lead molecule concerning its inhibitory action toward mycobacterial growth.

GEQ analogues bearing a succinimide central core have been recently reported by our

group.[10] These analogues displayed good inhibitory activity against InhA protein and some

of them displayed a significantly increased activity against M. tuberculosis growth, with a

MIC of 5.4 µM for the best compound. Similarly, the replacement of the piperazine ring with

piperazinone was proposed in order to enhance the interactions with the InhA enzyme.

Modification of the amide group.

Key interaction has been described between the oxygen atom of the piperazine amide of GEQ

and the hydroxyl group of the Tyr158 residue of InhA.[11] Consequently, in an attempt to

strengthen this interaction, investigations were performed around the carbonyl group of the

4

ACCEPTED MANUSCRIPT

lead molecule 1 through introduction of isosteric groups. Then, the carbonyl group of the

amide moiety in compound 1 was replaced by various functional groups to gain insights into

the specificity of the binding site. Then, sulfonyl- , sulfinyl-, phosphonyl- and phosphinamide

were also introduced. In order to gain insights into settled hydrogen bond, urea and thiourea

analogues were also prepared. Furthermore, thioamide compounds could be synthesized and

evaluated. The length of the C=S bond coupled with the larger size of the sulfur atom might

better be accommodated with the hydroxyl group of the tyrosine.[12]

Modification of the aryl moiety.

Previously, succinimide GEQ analogues, bearing a long alkyl chain (C and C ) instead of the

8

9

indole moiety, were found to be efficient against InhA.[10] In a similar manner, derivatives

with a piperazine central core and a fatty alkyl chain were synthesized with either an amide or

an (thio)urea linker.

Modification of the fluorene moiety.

GEQ molecule and the C₁₆fatty acyl substrate analogue displayed a similar binding mode

within the active site of the InhA protein (PDB-1P44 [8] and PDB-1BVR [13]). Indeed,

interactions with the key residue Tyr158 were maintained in both situations and hydrophobic

contacts guide either the fluorene fragment or the long alkyl chain in the upper part of the

cavity. Superposition of both molecules within the binding pocket highlights the possibility of

substitution on the fluorene moiety (Figure 3). In a similar strategy, Tonge and coworkers

have developed analogues of triclosan through introduction of an alkyl chain.[14] In fact, the

3

5

-hexyl substituted diphenyl ether analogue of triclosan exhibits a 10 -fold better activity.

[

15,16] Consequently, we focused our attention on the synthesis of 2- and 3-alkyl-substituted

fluorene derivatives. The length of the chain and the nature of the linker were also

investigated. Kuo and coworkers also evidenced, in a similar fashion, the high potency of

GEQ derivative substituted with a benzyl amide group on the position 2 of the fluorene

moiety.[8] Consequently, a benzyl ether group was also covalently attached to the fluorene in

order to explore the whole cavity.

5

ACCEPTED MANUSCRIPT

Figure 3. Superimposition of GEQ (blue) and substrate analogue (purple) molecules in the InhA binding site

(

PDB IDs respectively 1P44 [8] and 1BVR [13]). Colouring according the kd Hydrophobicity [17] scale for

each amino acid from dodger blue to the most hydrophilic, to white, to orange red for the most hydrophobic.

2.2. Chemistry

The first strategy described in scheme 1 was based upon an N-acylation reaction onto the 1-

(9H-fluoren-9-yl)piperazine 3. The key intermediate 3 was synthesized in a quantitative yield

via the reaction of 9-bromofluorene 2 and piperazine as previously reported.[18] Both

reference compounds GEQ and 1 were de novo synthesized to serve as a positive control for

the subsequent in situ enzyme inhibition. The coupling between the indole carboxylic acid

and the piperazine moiety was performed using peptide coupling agents HOBt, EDC.HCl and

DIPEA as base to afford the GEQ molecule in a similar manner as previously reported.[9] To

yield compound 1, the piperazine synthon 3 was coupled to benzoyl chloride reagent in the

presence of TEA as a base, in a relatively good yield (72%). Similarly, valeryl chloride and

nonanoyl chloride were engaged in the same coupling reaction to introduce an alkyl chain and

provide compounds 4 and 5. Compound 1 was subsequently involved in a thionation reaction

with Lawesson’s reagent as described by Coppola et al. to introduce the thioamide function in

compound 6.[19] To gain insights into the specificity of the binding site, compounds 7-10

were prepared in a similar fashion with respectively benzenesulfinic chloride, benzene

sulfonyl chloride, [20] phenylphosphinic dichloride and methylphenylphosphinic chloride. To

get the phosphonamide 10, commercially available phenyl phosphonic dichloride, was

coupled to compound 3 then, after overnight stirring at room temperature, methanol was

6

ACCEPTED MANUSCRIPT

added to quench the reaction and to substitute the last chlorine atom with a methoxy

group.[21] The benzenesulfinic chloride reagent was obtained after activation of the

corresponding benzenesulfinic acid sodium salt with thionyl chloride.[22] Compounds 7, 9

and 10 were obtained as racemic mixture of R- and S- derivatives and were directly engaged

in enzymatic assay to evaluate their inhibitory potency. The ureas 11, 13-15 and thiourea 12

were synthesized in a one-step reaction from compound 3 and the corresponding isocyanate

[

23] or isothiocyanate.[24]

Scheme 1. General synthetic procedure for replacement of the benzamide moiety. Reagents and reaction

conditions: a. TEA, piperazine, THF, reflux 6 h; b. RCOCl, TEA, DCM (for GEQ compound: RCOOH, HOBt,

DIPEA, DMF); c. From compound 1, Lawesson's reagent, toluene; f. RNCX, CH CN, 18 h; d. PhXCl, TEA,

3

DCM (for compound 8, MeOH was added in a second time); e. RCNX, CH CN, 18 h.

3

For substituted fluorene derivatives, the key fluorenone intermediates (17, 18, 20, 21, 23-25,

2

7) were synthesized according to various routes presented in Scheme 2. 2-Bromo-fluorenone

was engaged in a Suzuki coupling reaction with 1-hexene and 1-octene after hydroboration of

those last two reagents with 9-BBN to afford 2-alkyl-fluorenone intermediates 17 and 18 in

yields similar to those previously described.[25] Intermediate 20 was obtained using a

pallado-catalyzed reaction in the presence of cesium carbonate and tBuXPhos, with propan-1-

ol as coupling partner.[26] The 3-methoxy-fluorenone 21 was prepared according to a

previously reported method which involved a Suzuki coupling reaction between ethyl-2-

bromobenzoate and 3-methoxyphenylboronic acid followed by methanesulfonic-acid-

7

ACCEPTED MANUSCRIPT

catalyzed ring closure.[27] The 3-methoxy intermediate was partially engaged in a methyl

ether deprotective reaction using the couple HBr/HOAc to give the corresponding 3-hydroxyl-

fluorenone 22.[28] O-alkylation of the hydroxyl intermediate with benzyl, propyl and hexyl

bromide permitted us to achieve the 3-substituted fluorenones with an ether linker (23-25). In

a second time, conversion of intermediate 22 to its triflate derivative 26 allowed the

introduction of a hexyl chain in the position C2 after a Suzuki reaction, as previously

described by Spencer et al.[25] These fluorenone intermediates were respectively engaged in

a two-steps reaction implying a ketone reduction using sodium borohydride followed by a

specific conversion of the alcohols to the alkyl bromides using phosphorus tribromide. The

intermediates were obtained in good to excellent yields without purification. Commercially

available 1-benzoylpiperazine was finally coupled to the different 9-bromofluorenes to afford

the final products 28-35.

Scheme 2. Synthesis of fluorene substituted derivatives. Reagents and reaction conditions: f. (1) 1-Hexene or 1-

octene, 9-BBN, THF, 0 °C, 2 h then RT, 3 h; (2) Cs CO , AsPh , Pd(dppf)Cl ,THF, DMF, H O, 85 °C, 18 h; g.

2

3

2

Pd(OAc) , tBuXPhos, Cs CO , propan-1-ol, toluene, 80 °C, 18 h; h. HBr HOAc, 6 h, reflux; i. RBr, K CO ,

2

3

2

3

DMF; j. Tf O, 2.6-di-tert-butyl-4-methylpyridine, CH Cl , -78 °C, 1 h, 0 °C, 1.5 h; k. (1) 1-Hexene, 9-BBN,

2

8

ACCEPTED MANUSCRIPT

THF, 0 °C, 2 h, RT, 3 h (2) K PO , Pd(dppf)Cl , THF, 16 h, reflux; l. MeOH, NaBH , 0.5 h, RT; m. DCM, PBr ,

3

4

2

4

3

0

°C, 2 h; n. 1-benzoylpiperazine, K CO , DMF.

2 3

The synthesis of the derivatives 39 and 40 bearing a piperidine as a central core is described

in Scheme 3. Firstly, the decarbonylation reaction of the O-alkylated fluorenone 25 in the

presence of hydrazine led to the fluorene intermediate 36.[29] Then, this compound and

commercially available fluorene 37 were engaged in a deprotonation reaction with nBuLi and

were coupled with N-benzoyl-4-bromopiperidine. The piperazinone core was also introduced

to replace the piperazine central ring by using the previously synthesized N-Boc protected

intermediate 41. Boc deprotection with TFA, followed directly by a coupling reaction with 9-

bromofluorene in the presence of triethylamine afforded the target compound 42.[30]

O

5

O

5

o

O

25

36 (98%)

X

39 X = H (46%)

⁰^X⁼^O^C6^H (49%)

O

4

p

13

+

N

Br

N

O

3

6

38

7 X = H (fluorene)

Scheme 3. Replacement of the piperazine central core by piperidine and piperazinone. Reagents and reaction

conditions: o.(1) H NNH , diethylene glycol, 10 min; (2) KOH, reflux, 4 h; p. nBuLi, THF, -78 °C ꢀ RT; q. (1)

2

TFA, DCM, 0 °C to RT, 18 h; (2) 9-bromo-fluorene, TEA, DCM, 18 h, RT.

2

.3. Inhibitory InhA activities

The new compounds were tested for their capacity to inhibit the reduction of the substrate

double bond by NADH in the presence of InhA. The assays were performed in triplicate in the

presence of the substrate analogue 2-trans-dodecenoyl-CoA and the percentage of InhA

inhibition was determined by measuring the conversion of the NADH cofactor to its oxidized

+

form NAD by means of the decreasing of the absorbance at 340 nm. The molecules were

9

ACCEPTED MANUSCRIPT

firstly tested at 50 µM and 10 µM for some of them due to solubility problem. For the more

potent compounds, IC were determined using the 4-parameter curve-fitting software XLFit

5

0

(

IDBS) with at least six points. The results are reported in Tables 1 and 2.

As references, GEQ compound and its phenyl analogue 1 were firstly tested on InhA protein

to confirm their activity and for comparison (Table 1). Both compounds showed a complete

InhA inhibition at 50 µM and IC in the nanomolar range, as expected [9,11]. Compound 1

5

0

demonstrated the most potent inhibitory activity by comparison with GEQ.

Investigations around the piperazine amide bond revealed an impressive decrease of activity

for these derivatives. Indeed, replacing the oxygen atom with sulfur completely dropped the

activity of the molecule (compound 6, Table 1). Additionally, no InhA inhibitory activities

were observed for sulfinamide 7, sulfonamide 8, phosphinamide 9 and phosphonamide 10

derivatives. These results demonstrate that the amide bond is critically required for good

inhibitory activity by its interaction with the hydroxyl group of Tyr158. Nonetheless, the

derivative 11 containing a urea functional group instead of the amide bond showed a

relatively good InhA inhibitory activity with about 84% inhibition at 50 µM and IC = 1.18

5

0

µM. An additional nitro substituent in the phenyl group at the para position totally abolished

the activity (compound 15, Table 1). This result highlighted either the possibility of steric

hindrance or the necessity of an electron-rich aromatic ring to maintain interaction with the

pyrophosphate moiety of the NADH molecule. Moreover, compound 12 bearing a thiourea

linker, demonstrated a relatively good activity at 50 µM with 57% inhibition but still far from

the reference compound 1.

Alkyl chains were also introduced to replace the phenyl ring maintaining either the amide or

the urea as a linker to the piperazine ring. Firstly, we could observe a decrease in activity

correlated with a longer chain length (compounds 13, 14 and 4, 5; Table 1). The poor activity

noticed for long alkyl chain derivatives is in accordance with Tonge and coworkers binding

site description.[6] They described this region of the binding site as size-limited and

surrounded by both polar and non polar groups. Indeed, both compounds 13 and 4 exhibited

about 70% of activity at 50 µM (Table 1) toward InhA protein.

Efforts were also furnished to replace the piperazine ring by either piperidine (compound 39,

Table 1) or piperazinone (compound 42, Table 1). The additional keto bond in compound 42

abolished the inhibitory activity and comforted the suggested steric clash in the case of carbon

substitution of the piperazine ring.[8] Removal of the nitrogen atom in the piperazine ring into

compound 39 led to a potent InhA inhibitor with similar potency at 50 µM than the reference

1

0

ACCEPTED MANUSCRIPT

compound 1. IC was determined and revealed a sub-micromolar activity for the piperidine

5

0

derivative.

Table 1. Inhibitory potencies of compounds 1, 3-5, 6-15, 39, 42 against InhA

InhA inhibition (%)

Compound

W

X

Y

Z

IC (µM)

50

5

0 µM

87

94

7

10 µM

GEQ

N

CH

2

CO

H

CO

CS

SO

Indole

Ph

-

81

94

-

0.86 ± 0.16

0.49 ± 0.06

1

3

4

5

6

7

8

9

-

C

H

75

47

31

14

12

17

84

57

68

34

33

93

26

45

22

-

79

32

42

-

4

9

H

8

17

Ph

SO

PO(OCH

PO(CH

CONH

CSNH

CONH

CO

2

)

3

1

3

4

0

1

2

3

4

5

9

2

3

)

1.18 ± 0.15

-

C

H

4

9

H

8

17

pNO

Ph

-

88

-

2

CH CH

CO

0.94 ± 0.11

-

N

CO

Ph

Substitutions on the fluorene have already been performed and revealed that, generally,

halogen and nitro substituents and even some bulky groups were well tolerated concerning the

InhA inhibitory activity.[8] We subsequently investigated compounds bearing fluorene

substituted with alkyl chains on either C-2- or C-3 position to mimic the substrate and we

introduced for some of them, an ether linker. The biological activities against the InhA protein

are reported in Table 2.

Hexyl and octyl chains were subsequently attached on the C-2 position of fluorene and the

corresponding derivatives revealed around 50% inhibition at 50 µM. However, no

enhancement was observed compared to the reference compound (compounds 28 and 29,

Table 2). A shorter chain seems to be privileged in this position. Lately, compound 27 holding

a similar 6-carbons length alkyl chain on the C-3 position of the fluorene revealed a real gain

of activity compared to compounds 28 and 30 at the C-2 position. Ether-linked derivatives

have been particularly studied. First results on the C-3 position of the fluorene revealed a

significant improvement of the activity with the increasing length of the alkyl chain

(

compounds 31-33, Table 2). The rank order of InhA inhibitory potency was 31 (OC ) < 32

1

ACCEPTED MANUSCRIPT

(

OC ) < 33 (OC ). The hexyloxy derivative 33 exhibited an IC of 0.20 µM, about 2.5 times

3

6

50

lower than reference compound 1. The additional alkyl chain partly mimicking the substrate

would enhance the activity of the molecule and would probably ensure constructive

hydrophobic interactions within the active site. The C3-alkyl chain substitution attached via

an ether linkage was also investigated at position 2 of the fluorene. No significant difference

was observed concerning the substituted position for ether-linked derivatives with such a

carbon length (compounds 32 and 34, Table 2). With respectively IC of 1.00 and 1.20 µM,

5

0

compounds substituted in positions C-2- and C-3- appear equivalent as inhibitors.

In order to explore the whole cavity and to strengthen hydrophobic interactions with the

substrate binding loop, a benzyl substituent was covalently attached through an ether linker to

the C-3 position of the fluorene. Enzymatic assays also revealed good activity with an IC₅₀

estimated at 1.0 µM (compound 35 Table 2).

Then compound 40 bearing both a piperidine central core and a 3-hexyloxy substituent, was

tested. Compared to the unsubstituted derivative 39, no difference of activity was observed

(

compounds 39 Table 1 and 40 Table 2). Both compounds exhibited IC in the micromolar

50

range. Nevertheless, it is important to note that compound 40 is in a racemic mixture due to

the asymmetric C-9 on the fluorene moiety. We can subsequently envision that only one of

the two enantiomers is biologically active.

Table 2. Inhibitory potencies of compounds 28-35, 40 against InhA

InhA inhibition (%)

Compound

X

Y

Z

IC (µM)

50

5

0 µM

87

10 µM

81

GEQ

1

-

H

-

N

0.86 ± 0.16

0.49 ± 0.06

-

H

94

2

3

4

8

9

0

1

2

3

4

5

0

C

6

H

13

17

48

45

54

27

-

8

C

H

60*

92

52

74

≈ 1.0*

6

13

OCH

OC

2.69 ± 0.20

1.01 ± 0.14

0.20 ± 0.04

1.30 ± 0.10

≈ 1.0*

3

H

91

84

3

7

H

79*

88

70

83

6

13

H

OC

H

3

7

OCH

Ph

H

N

CH

63*

64*

57*

71

2

OC

H

1.05 ± 0.19

6

13

*

Due to solubility problem. IC50 was estimated by direct inhibition measurement to obtain about 50% inhibition

1

2

ACCEPTED MANUSCRIPT

2

.4. Enantiomeric separation by chiral supercritical fluid chromatography

Each substituted fluorene derivatives present an asymmetric carbon at the 9-position of the

fluorene. Consequently, two compounds bearing two different alkyl chains, namely 31 and the

more potent 33 were engaged into enantiomeric separation to analyze the contribution of both

enantiomers according to their activity against the InhA protein. Chiral chromatography was

performed on a Supercritical Fluid Chromatography (SFC) equipment (Prep80Q from Waters)

with the AD-H column (from Chiral Technologies) and methanol as co-solvent. Compounds

3

3a (rt = 10.4 min)/33b (rt = 16.4 min) and 31a (rt = 10.5 min)/31b (rt = 12.6 min) were

obtained with an excellent enantiopurity (> 98.5%). However, none of the isolates were able

to crystallize, so the absolute configuration of the chiral carbon was not determined.

2

.5.

Biological activities for enantiopure compounds against the InhA protein

First enzymatic assays were performed for compounds 31a/31b and 33a/33b respectively at 5

µM and 250 nM to evaluate their inhibitory activity against InhA protein. IC was measured

5

0

for the most potent molecules (Table 3). For the methoxy derivative 31, the racemic mixture

exhibits 60% inhibitory activity at 5 µM. While the first isolated enantiomer (rt = 10.5 min)

did not inhibit InhA activity (compound 31a Table 3), the second one (rt = 12.6 min,

compound 31b, Table 3) was much more potent (IC₅₀estimated at 2.07 µM) and was

responsible for the overall activity.

The most interesting result was observed for the hexyloxy derivative. Indeed, the first

enantiomer 33a was not successfully efficient on InhA inhibitory activity whereas the other

one 33b inhibited at 71%. Consequently, IC of 102 nM was measured for compound 33b,

5

0

which displays the best inhibitory activity for this series of molecules.

Table 3 inhibitory activities of compounds 31a, 31b, 33a and 33b after enantiomeric separation

Ligand

concentration

-

Compound

X

Y

Z

InhA Inhibition (%)

IC (µM)

50

GEQ

1

-

H

OCH

-

0.86 ± 0.16

0.49 ± 0.06

2.69 ± 0.20

-

2.07 ± 0.29

0.20 ± 0.04

-

H

N

-

3

1

3

5 µM

250 nM

60

11

68

53

0

3

1a

3

1b

3

OC

6

H

13

3

3a

3

3b

71

0.102 ± 0.004

1

3

ACCEPTED MANUSCRIPT

2

.6.

Computational study

Molecular docking was performed in an attempt to determine the interaction network between

the newly synthesized compounds and the receptor, the InhA protein. To model the binding

mode of these compounds, we performed a docking study with the InhA protein that was

crystallized with GEQ ligand (PDB ID: 1P44, chain a) using Molegro Virtual Docker

(MVD). For compounds (7, 10, 28, 29, 30, 31, 32, 33, 34, 35, 39, 40) bearing an asymmetric

carbon atom, each enantiomer was docked individually. The final (filtered) results were

expressed as ligand efficiency (LE) indices [31] rather than RAW docking scores. The

docking and filtering procedures gives at least two LE descriptor values per compound: two

values for the same molecule (if not asymmetric) or one value per enantiomer. These results

are given in the Table S1 of supplementary materials. In order to compare experimental

results and theoretical LE values, the InhA inhibition percent at 50 µM (PI50) was used, PI50

values being available for each compound.

In the case of InhA, the protein is characterized by i) a wide and flexible binding site; ii) some

structural transitions giving opening/closing of a minor and major portal; iii) a cofactor

+

(

NAD /NADH) to be taken into account in calculations. These elements (not exhaustive)

reduce the production of correlations between descriptors issued directly (not using a QSAR

approach) from docking studies vs. experimental data, even for derivatives that keep an

essential feature (i.e. fluorene group) of a single compound.

According to this context, only a coarse-grained approach seems to be relevant for the

analysis of this GEQ-focused ligand collection. The PI50 results were classified in 3 groups:

group1 for compounds giving upper than 75% of inhibitory activity; group3 for compounds

that exhibits PI50 values lesser than 30% and group2 for intermediate compounds and the

docking results were analyzed using this classification. Then, these tree classes were

confronted to LE descriptors, and we found that group1 was clearly related to best scores and

group3 to worst scores (Figure S1 of supplementary materials). This result is limited but

interesting because it gives insights to improve post-docking filter results. Especially since we

found that the calculated values of LogP (octanol/water) for each compound do not seem to be

correlated with LE descriptors.

Then, analysis of selected poses after docking study allowed us to understand the biological

activities of our compounds regarding their structure.

Sulfinamide derivative 7 showed poor inhibitory activity against InhA (PI50 = 14%) and

docking study revealed, for compound with S configuration, a disfavored orientation (figure

1

4

ACCEPTED MANUSCRIPT

4

A) as well as its enantiomer R. To maintain the hydrogen bond with the Tyr158 residue, the

sulfinamide tetrahedral linker forced the piperazine ring to shift 1.1 Å which induced the

fluorene group to rotate 110° and to clash with Met199 residue. For thioamide derivative 6,

the overall binding mode is quite similar to the reference molecule 1, even if its enzymatic

activity at 50 µM was evaluated at 31%. Indeed, for thioamide 6, the C-S bond is longer and

the sulfur atom bigger than the corresponding carbonyl in GEQ which induced steric

hindrance with the Tyr158 side chain (Figure 4B).

Compounds bearing an alkyl chain on the fluorene moiety (28, 29 and 30) demonstrated a

modest activity on the InhA protein up to IC (InhA) value of 1.0 µM. Their corresponding R

5

0

enantiomers displayed a consistent orientation within the InhA binding site wherein the

additional alkyl chain is oriented onto the major portal mimicking, as predicted, the substrate

analogue. Interestingly, in the case of compound 32, bearing a 3-propoxy substituent on the

fluorene moiety, both enantiomers disclose a consistent orientation. Indeed, similar to the

latter compounds, the R enantiomer orientated the alkyl chain within the hydrophobic

substrate cavity surrounding with Met103, Ile202 and Leu207 residue (Figure 4C). On the

other hand, the S enantiomer oriented the alkyl chain upon the minor portal with close

interaction with hydrophobic residues Met155, Leu218 and Trp222 (Figure 4D).

For benzyloxy derivative 35, the best docking pose allows us to envisage the possibility of

binding only for compound with configuration R (Figure 4E) with the benzyl moiety

sandwiched between Met103 and Ile202 residues. Nonetheless, the binding site of limited size

seems to disfavor such interaction which leads consequently to an increase of docking score

values and correlates with the lower activity compared to the reference molecule 1.

Concerning the best molecule 33, only the R-enantiomer compound displayed a consistent

orientation within the InhA binding site wherein the additional alkyl chain is oriented onto the

major portal (Figure 4F). This result supports the biological activity found for only

enantiomer 33b. Even though the geometry of the enantiopure compound 33b was not

determined, we can suggest, with caution, that C-9 carbon atom is of R configuration.

1

5

ACCEPTED MANUSCRIPT

A. 7S

C. 32R

E. 35R

B. 6

D. 32S

F. 33R

Figure 4. Selected docking conformations of compounds 7 (A.), 6 (B.), 32 (C. with R configuration and D. with

S configuration), 35 (E. R-enantiomer) and 33 (F. R-enantiomer). The best pose for each compound (green) was

depicted in the InhA binding site, as well NADH cofactor molecule (black), GEQ (grey) for comparison and

flexible residues as defined in the experimental part.

2

.7.

Inhibition of Mycobacterium tuberculosis (H37Rv) growth

1

6

ACCEPTED MANUSCRIPT

The activities of the synthesized compounds were evaluated by measuring the inhibition of M.

tuberculosis (H37Rv) growth. Isoniazid and GEQ molecules were used as control. Results are

reported in Table 4.

Table 4 : MIC determination for M. tuberculosis H37Rv strain

Compound

MIC (µM)

Compound

MIC (µM)

GEQ

50.1

14.1

> 39.9

29.9

12.8

13.5

> 26.7

> 25.6

> 24.7

> 25.7

> 27.1

> 25.9

14.3

> 24.7

12.1

22.8

INH

29

30

0.4

> 21.4

22.8

1

3

4

5

6

7

8

9

31

26.0

31a

31b

32

> 26.0

24.2

22.0

>22.0

11.0

> 24.2

> 21.7

28.3

88.2

> 27.1

33

33a

33b

34

35

39

1

2

0

1

2

3

4

5

8

40

42

GEQ molecule did not display any substantial inhibitory activity against M. tuberculosis

growth (MIC > 40 µM). Consistent with this, GEQ molecule has been evaluated against M.

tuberculosis growth through CFU counting method and using the Alamar Blue Assay, giving

an MIC value above 125 µM.[8] Furthermore, we measured a MIC value of 14.1 µM for the

benzoyl derivative 1, while He et al. reported an MIC above 125 µM.[9]

The majority of the compounds displayed better activities than GEQ. The first family of

compounds related to central core modifications revealed poor MIC values for piperidine

derivative 39 and piperazinone derivative 42 as well (MIC values: 39, 28.3 µM; 42, > 27.1

µM).

As shown in Table 4, investigation around the amide bond showed modest antimycobacterial

activity. The thioamide analogue 6 displayed MIC in a similar range (13.5 µM) as compound

1

. The sulfin-, sulfon-, phosphin- and phosphonamide derivatives (compounds 7-10)

presented MICs values above 25.0 µM. The urea and thiourea derivatives 11 and 12 did not

demonstrate better activities against M. tuberculosis growth. Interestingly, compound 15

bearing an additional nitro group on the phenyl ring exhibited MIC value in a similar range as

compound 1 (MIC = 12.1 µM).

1

7

ACCEPTED MANUSCRIPT

All the derivatives possessing an alkyl chain instead of the benzoyl moiety showed MIC

values ranged from 12.8 to above 24.9 µM; among them, compound 5 was the most active

(MIC = 12.8 µM).

The class of racemic compounds bearing an alkyl chain on the fluorene moiety (28-35)

displayed weak in vitro activities against M. tuberculosis with MIC values superior to 20 µM.

In addition, the racemic mixture 33 exhibited MIC value of 22.0 µM. Interestingly, the

corresponding enantiopure compound 33b, showing the best inhibition of InhA enzymatic

activity and the lowest MIC (11.0 µM). Interestingly, the other enantiomer showed a MIC

superior to 22.0 µM. These results eventually suggest a binding specificity of this enantiomer

as observed with InhA binding assays.

2

.8.

Biological activity in the presence of efflux pump inhibitors

In some bacteria, such as in M. tuberculosis, efflux pumps have been described as a possible

mechanism for intrinsic and acquired drug resistance.[32] While He et al. suggested the

possibility that GEQ compounds are actively extruded out of the bacterial cell by efflux, no

studies have been performed to confirm this resistance mechanism.[9] In order to elucidate

the role of the efflux pumps in the poor effectiveness of the GEQ compound and its

derivatives 1, 33b and 40, their activity was determined in the presence of already known

efflux inhibitors (reserpine, verapamil or carbonyl cyanide m-chlorophenylhydrazone

(CCCP)). Reserpine is a calcium channel blocker and plant alkaloid that inhibits P-

glycoprotein in eukaryotic cells.[33] Moreover, reserpine reduced resistance to isoniazid in

some M. tuberculosis strains.[34] Verapamil is a calcium channel blocker that inhibits P-

glycoprotein and also several bacterial ABC efflux pumps. CCCP is uncoupler of the proton

motive force that inhibits the efflux of several drugs[35], but it is described as a substrate of

some efflux pumps.[36] The efflux pump inhibitors, at the used concentration, did not inhibit

M. tuberculosis growth. Results are reported in Table 5.

Table 5 : MIC determination for some compounds against M. tuberculosis H37Rv in the presence of efflux

pump inhibitors

a

Efflux pump inhibitors

ø

Reserpine

Verapamil

CCCP

Compounds

MIC (µM)

GEQ

1

50.1

14.1

11.0

25.4

56.5

< 2.7

< 12.7

56.5

11.0

< 12.7

< 3.5

< 2.7

3

3b

1

8

ACCEPTED MANUSCRIPT

4

0

88.2

22.1

44.1

nd

a

Reserpine, verapamil and CCCP were respectively added at 3.0, 40.0 and 7.5 µg/mL final concentration.

nd for not determined

b

The activity of GEQ compound increased more than four-fold in combination with verapamil

and CCCP, underlying the possibility that some transporter could extrude it out of the cells, in

agreement with its poor activity.

CCCP increased the effectiveness of the N-benzoyl derivative 1 more than five-fold.

Reserpine and verapamil decreased the sensitivity to this compound at the used concentration;

it could be hypothesized that these combination influenced either the permeability of the

compound or its binding with the target.

The efflux inhibitor verapamil did not have effect with our best derivative 33b, whilst in

combination with the other two efflux inhibitors its activity was increased more than four-

fold. Interestingly, the same behaviour was observed with MmpL7 transporter that pumps out

of the cell, isoniazid. [37] It could be hypothesized that the same transporter effluxes

compound 33b because both molecules have the same target. In the case of compound 40

with a piperidine moiety, MICs improved four-fold in the presence of reserpine and two-fold

with verapamil.

These results confirmed the possibility that GEQ and its derivatives could be effluxed out of

the mycobacterial cell by some efflux pump. The chemical diversity on the fluorene moiety

should be enlarged in order to obtain compounds bearing different substitutions to either

improve uptake by mycobacteria or to avoid efflux pumps.

3

. Conclusion

This work describes the synthesis and the evaluation of twenty five GEQ analogues. All these

compounds were evaluated for the inhibition of InhA enzymatic activity and against

Mycobacterium tuberculosis growth. Thus, we observed that the N-benzoyl-piperazine central

core is of key importance to ensure good inhibition of InhA enzymatic activity. The majority

of the compounds displayed higher activities than GEQ against M. tuberculosis growth.

Among them, compound 33b bearing an additional hexyloxy chain on the fluorene moiety

displayed improved activity against both InhA enzyme (IC₅₀up to 102 nM) and M.

tuberculosis growth (MIC = 11 µM). In addition, its activity improved in combination of

1

9

ACCEPTED MANUSCRIPT

some efflux inhibitors. These results suggest that the poor biological activity against M.

tuberculosis of GEQ and its derivatives could depend on the efflux of these molecules by

some mycobacterial transporters.

Further work will focus on optimization of drug uptake with the aim of producing a candidate

series for the treatment of tuberculosis. Moreover, the research for more effective efflux

inhibitors that could be used in combination with conventional antibiotics could be another

challenge to pursue.

4

. Experimental section

4

.1. General condition

All chemicals were obtained from Aldrich-Sigma or Acros Organics and used without further

purification. Anhydrous solvents were freshly distilled before use or were obtained from the

M.Braun Solvent Purification System (MB-SPS-800). Optical rotations were measured using

a sodium D line on a P-2000 series Jasco, PTC-262 polarimeter. The melting points were

determined on a Mettler Toledo MP50 melting point system and are uncorrected. Infra-red

1

spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR Spectrometer. H NMR spectra

were recorded on a Bruker spectrometer at 300 MHz using CDCl , DMSO-d6 or CD OD as

3

1

the solvent. For H NMR the residual proton signal of the deuterated solvent was used as an

1

3

internal reference: CDCl δ = 7.29 ppm, DMSO δ = 2.50 ppm and CD OD δ = 3.31 ppm. C

3

NMR spectra were recorded on a Bruker spectrometer at 75 MHz. Mass spectra (DCI/NH₃)

were obtained on a DSQ Thermo Fisher Scientific. For the MS–ESI a Dionex ultimate 3000

UPLC system with a ABSciex Q TRAP 4500 was used. High-resolution mass spectra

(

HRMS) were recorded on a UPLC Xevo G2 Q-TOF Waters using electrospray ionization

methods. The desired product was purified by flash column chromatography with puriFlash

30 system using puriFlash® columns from Interchim. The purity of title compounds was

4

evaluated by reverse phase LC-MS on a UPLC Acquity system (from Waters) equipped with

a photodiode array detector and a simple quadripole detector. The Acquity CSH C18 1.7 µm

(2.1 mm × 50 mm) column was used as a stationary phase. MilliQ water (with 0.02%

HCOOH) and acetonitrile (with 0.02% HCOOH) were respectively employed as solvents A

and B with a flow rate of 0.6 mL/min. Purity was estimated at λ = 212 nm and two elution

methods were followed and will be mentioned in each case. For the method 1, gradient was 5

min run from 2% to 98% B and then returned to initial conditions. For method 2, the gradient

was 10% B during 1 min and the percentage of B went from 10 to 30 for 1 to 10 min, and the

2

0

ACCEPTED MANUSCRIPT

percentage of B reached 100% 10 to 12 minutes and then returned to initial conditions. The

enzymatic evaluation was performed on a Cary Bio 100.

4

.2. Chemistry

4

.2.1.

1-(9H-Fluoren-9-yl)piperazine (3). A solution of 9-bromofluorene 2 (8.2 mmol, 2.0 g, 1.0 eq), triethylamine (1.2

mmol, 165 µL, 0.15 eq) and piperazine (61.1 mmol, 5.27 g, 7 eq) in dry THF (50.0 mL) was refluxed under argon for 6

hours. The reaction mixture was then filtered off and the filtrate was concentrated under vacuum pressure. The resulting

crude product was purified by flash chromatography (isocratic 95/5 dichloromethane/methanol) to afford a white powder

-

1

(

2.04 g, 100%). TLC R : 0.12 (dichloromethane/methanol 97/3); mp: 133.6 °C; IR (cm ): 738, 805, 1007, 1138, 1325, 1448,

f

1

2

3

831, 2940, 3294. H NMR (300 MHz, DMSO-d6) δ (ppm): 2.46 (m, 4H); 2.66 (m, 4H); 4.86 (s, 1H); 7.32 (td, J = 1.5 Hz,

13

.5 Hz, 2H); 7.40 (t, J = 7.5 Hz, 2H); 7.63 (d, J = 7.2 Hz, 2H), 7.83 (d, J = 7.5 Hz, 2H). C NMR (75 MHz, DMSO-d6) δ

(

ppm): 46.7 (2 x CH ), 50.4 (2 x CH ), 70.2 (CH), 120.4 (2 x CH), 126.3 (2 x CH), 127.5 (2 x CH), 128.5 (2 x CH), 140.9 (2

2 2

+

x C), 144.4 (2 x C). MS (ESI) m/z: 251.3 [M +H ]. HRMS (ESI): for C H N [M+H ]: calcd: 251.1551; found: 251.1548.

17

19

2

4

.2.2.

(4-(9H-Fluoren-9-yl)piperazin-1-yl)(1H-indol-5-yl)methanone (GEQ). In a round bottom flask submerged in a bath

of ice were added indole-5-carboxylic acid (0.80 mmol, 129 mg, 1.0 eq), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride, EDC.HCl, (0.88 mmol, 168 mg, 1.1 eq) and 1-hydroxybenzotriazole, HOBt, (0.88 mmol, 119 mg, 1.1 eq) in

DMF (5.0 mL). 1-(9H-fluoren-9-yl)piperazine (0.80 mmol, 200 mg, 1.0 eq) and DIPEA (2.00 mmol, 330 µL, 2.5 eq) were

subjoined to the reaction mixture and were stirred overnight at room temperature. HCl 1N was added and the aqueous phase

was neutralized with KOH 2 M and the product was extracted with ethyl acetate (3×). The organic phase was dried over

magnesium sulphate, filtered and concentrated under vacuum pressure. The resulting crude product was purified by flash

chromatography (gradient 80/20 to 20/80 petroleum ether/ethyl acetate) to afford a colorless solid (154 mg, 49%). HPLC:

1

method 1, rt = 2.45 min, purity 95%. H NMR (300 MHz, CDCl ) δ (ppm): 2.30 – 3.05 (bs, 4 H); 3.35 – 3.45 (bs, 4H); 4.92

3

(s, 1 H); 6.51 (t, J = 2.4 Hz, 1 H); 7.16 (td, J = 1.5 Hz, 4.8 Hz, 1 H); 7.20 (t, J = 8.7 Hz, 2 H); 7.34 (td, J = 1.2 Hz, 7.2 Hz, 2

13

H); 7.42 (t, J = 7.2 Hz, 2 H); 7.64 – 7.70 (m, 3 H); 7.72 (d, J = 7.5 Hz, 2 H); 9.13 (s, 1 H). C NMR (75 MHz, CDCl ) δ

3

(

ppm): 49.2 (broad, 4 X CH ); 69.9 (CH); 102.8 (CH); 111.2 (CH); 119.9 (2 x CH); 120.1 (CH); 121.1 (CH); 125.7 (CH);

2

1

3

26.0 (2 x CH); 126.6 (C); 127.2 (2 x CH); 128.4 (2 x CH); 136.5 (C); 141.1 (C); 143.3 (C); 172.2 (C). MS (DCI/CH ) m/z:

4

+

94.19 [M+H ]. HRMS (DCI/CH ): for C H N O [M+H ]: calcd: 394.1919; found: 394.1912.

4

26 24

3

4

.2.3. General procedure for 1, 4, 5, 8, 9 and 10.

1

-(9H-Fluoren-9-yl)piperazine 3 (1.0 eq) was dissolved in dry dichloromethane. Triethylamine (1.2 eq) was added and the

reaction mixture was stirred at room temperature for 10 minutes. Benzoyl chloride (1.1 eq) was slowly added to reaction

mixture previously cooled with an ice bath. After complete addition the reaction mixture was stirred overnight at room

temperature. A saturated aqueous solution of sodium hydrogenocarbonate was added and the product was extracted with

dichloromethane (3×). The organic phase was washed with a large amount of water, dried over magnesium sulphate, filtered

and concentrated under vacuum pressure. The resulting crude product was purified by flash chromatography as indicated in

each case to afford the desired compound.

4

.2.3.1. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(phenyl)methanone (1). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.40

mmol, 100 mg), triethylamine (0.48 mmol, 65 µL) and benzoyl chloride (0.44 mmol, 51 µL). The crude product was purified

2

1

ACCEPTED MANUSCRIPT

by flash chromatography (isocratic 50/50 petroleum ether/ethyl acetate) to afford a white solid (102 mg, 72%). TLC R : 0.82

f

-

1

(

dichloromethane/methanol 97/3); mp: 186 °C; IR (cm ): 709, 741, 1005, 1275, 1426, 1636, 2826, 2880, 2906. HPLC:

1

method 1, rt = 2.72 min, purity 99%. H NMR (300 MHz, DMSO-d6) δ (ppm): 2.55 (m, 4H); 3.59 (m, 4H); 5.00 (s, 1H);

13

7

.31-7.48 (m, 9 H); 7.65 (d, J = 7.2 Hz, 2H), 7.84 (d, J = 7.2 Hz, 2H). C NMR (75 MHz, DMSO-d6) δ (ppm): 48.6 (2 x

CH ), 49.4 (2 x CH ), 69.6 (CH), 120.5 (2 x CH); 126.3 (2 x CH); 127.4 (2 x CH); 127.7 (2 x CH); 128.7 (2 x CH); 128.8 (2

2

+

x CH); 129.9 (CH); 136.3 (C); 140.9 (2 x C); 143.8 (2 x C); 169.4 (C). MS (ESI) m/z: 355.2 [M+H ]. 377.2 [M+Na]. HRMS

+

(

ESI): for C H N O [M+H ]: calcd: 355.1812; found: 355.1810.

24

23

2

4

.2.3.2. 1-(4-(9H-Fluoren-9-yl)piperazin-1-yl)pentan-1-one (4). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.40 mmol,

1

00 mg), triethylamine (0.48 mmol, 65 µL) and valeryl chloride (0.48 mmol, 65 µL). The crude product was purified by flash

chromatography (isocratic 80/20 petroleum ether/ethyl acetate) to afford a white solid (112 mg, 84%). TLC R : 0.32

f

-

1

(

dichloromethane/methanol 95/5); mp: 70 °C; IR (cm ): 672, 741, 999, 1137, 1196, 1203, 1219, 1447, 1637, 2817, 2868,

1

2

927. HPLC: method 2, rt = 2.10 min, purity 96%. H NMR (300 MHz, CDCl ) δ (ppm): 0.93 (t, J = 7.2 Hz, 3 H); 1.33 (sex,

3

J = 7.5 Hz, 2 H); 1.60 (quin, J = 7.5 Hz, 2 H); 2.28 (dd, J = 6.6 Hz, 8.4 Hz, 2 H); 2.49 (t, J = 4.8 Hz, 2 H); 2.78 (t, J = 4.8 Hz,

H); 3.41 (t, J = 4.8 Hz, 2 H); 3.66 (t, J = 4.8 Hz, 2 H); 4.90 (s, 1 H); 7.32 (td, J = 1.2 Hz Hz, 7.2 Hz, 2 H); 7.41 (t, J = 7.5

2

13

Hz, 2 H); 7.64 (d, J = 7.5 Hz, 2 H); 7.72 (d, J = 7.8 Hz, 2 H). C NMR (75 MHz, CDCl ) δ (ppm): 13.9 (CH ); 22.6 (CH );

3

2

7.4 (CH ); 33.0 (CH ); 42.3 (CH ); 46.3 (CH ); 48.4 (CH ); 49.7 (CH ); 69.9 (CH); 119.8 (2 x CH); 125.9 (2 x CH); 127.2

2 2 2 2 2 2

+

(

2 x CH); 128.3 (2 x CH); 141.1 (2 x C); 143.5 (2 x C); 171.7 (C). MS (DCI/CH ) m/z: 363.24 [M+C H ], 335.21[M+H ],

4 2 5

+

1

65.07 [M-169]. HRMS (DCI/CH ): for C H N O [M+H ]: calcd: 335.2123; found: 335.2115.

4 22 27 2

4

.2.3.3. 1-(4-(9H-Fluoren-9-yl)piperazin-1-yl)nonan-1-one (5). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.40 mmol, 100

mg), triethylamine (0.48 mmol, 65 µL) and nonanoyl chloride (0.48 mmol, 86 µL). The crude product was purified by flash

chromatography (isocratic 80/20 petroleum ether/ethyl acetate) to afford a white solid (114 mg, 73%). TLC R : 0.62

f

-

1

(

dichloromethane/methanol 95/5); mp: 88 °C; IR (cm ): 673, 740, 1001, 1137, 1196, 1228, 1320, 1448, 1639, 2818, 2851,

1

2

1

4

7

919. HPLC: method 2, rt = 5.04 min, purity 95%. H NMR (300 MHz, CDCl ) δ (ppm): 0.90 (t, J = 7.2 Hz, 3 H); 1.20 –

3

.42 (m, 10 H); 1.35 – 1.67 (m, 2 H); 2.28 (t, J = 7.2 Hz, 2 H); 2.49 (t, J = 4.8 Hz, 2 H); 2.77 (t, J = 4.8 Hz, 2 H); 3.41 (t, J =

.8 Hz, 2 H); 3.66 (t, J = 4.8 Hz, 2 H); 4.90 (s, 1 H); 7.32 (td, J = 1.5 Hz, 7.5 Hz, 2 H); 7.41 (t, J = 7.5 Hz, 2 H); 7.64 (d, J =

13

.5 Hz, 2 H); 7.72 (d, J = 7.2 Hz, 2 H). C NMR (75 MHz, CDCl ) δ (ppm): 14.1 (CH ); 22.7 (CH ); 25.4 (CH ); 29.2

3

2

(

CH ); 29.4 (CH ); 29.5 (CH ); 31.8 (CH ); 33.3 (CH ); 70.0 (CH); 119.8 (2 x CH); 125.9 (2 x CH); 127.1 (2 x CH); 128.3 (2

2 2 2 2 2

+

x CH); 141.1 (2 x C); 143.5 (2 x C); 171.7 (C). MS (DCI/CH ) m/z: 419.31 [M+C H ], 391.27 [M+H ], 165.07 [M-225].

4

2

5

+

HRMS (DCI/CH ): for C H N O [M+H ]: calcd: 391.2749; found: 391.2751.

4

26 35

2

4

.2.3.4. 1-(9H-Fluoren-9-yl)-4-(phenylsulfonyl)piperazine (8). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.32 mmol, 80

mg), triethylamine (0.38 mmol, 52 µL) and benzene sulfonyl chloride (0.38 mmol, 49 µL). The crude product was purified by

flash chromatography (isocratic 85/15 petroleum ether/ethyl acetate) to afford a white solid (115 mg, 92%), TLC R : 0.39

f

-

1

(

petroleum ether/ethyl acetate 80/20); mp: 161 °C; IR (cm ): 575, 646, 669, 690, 736, 939, 1001, 1113, 1128, 1171, 1292,

1

2

320, 1348, 1447, 2836, 2887, 2942. HPLC: method 1, rt = 3.66 min, purity 98%. H NMR (300 MHz, CDCl ) δ (ppm):

3

.68-2.78 (m, 4 H); 2.99-3.10 (m, 4 H); 4.82 (s, 1 H); 7.28 (td, J = 1.2 Hz, 7.5 Hz, 2 H); 7.40 (t, J = 7.5 Hz, 2 H); 7.51-7.70

13

(

m, 5 H); 7.71 (d, J = 7.5 Hz, 2 H); 7.75-7.81 (m, 2 H). C NMR (75 MHz, CDCl ) δ (ppm): 46.8 (2 x CH ); 48.1 (2 x CH );

3

2

6

1

9.8 (CH); 119.9 (2 x CH); 125.8 (2 x CH); 127.2 (2 x CH); 127.8 (2 x CH); 128.4 (2 x CH); 129.1 (2 x CH); 132.8 (CH);

+

36.0 (C); 141.0 (2 x C); 143.2 (2 x C). MS (DCI/CH ) m/z: 391.15 [M+H ], 249.14 [M-141 (PhSO )], 165.07 [M-225

4

2

+

(

fluorene)]. HRMS (DCI/CH ): C H N O S [M+H ]: calcd: 391.1480; found: 391.1491.

4 23 23 2 2

4

.2.3.5. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(phenyl)phosphinate (10). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.80

mmol, 100 mg), triethylamine (0.80 mmol, 188 µL) and phenylphosphinic dichloride (0.96 mmol, 132 µL). Tetrazole (0.04

mmol, 3 mg) was also put in the reaction mixture and methanol (1.0 mL) was added after overnight stirring to quench the

2

ACCEPTED MANUSCRIPT

reaction and formed the desired product. The crude product was purified by flash chromatography (isocratic 95/5 petroleum

-

1

ether/ethyl acetate) to afford a yellow oil (92 mg, 57%). TLC R : 0.75 (dichloromethane/MeOH 90/10). IR (cm ): 551, 561,

f

6

1

7

43, 669, 695, 730, 798, 910, 968, 999, 1034, 1131, 1231, 1300, 1438, 1450, 1675, 1715, 2845, 2945, 3060. HPLC: method

1

, rt = 2.23 min, purity 92%. H NMR (300 MHz, CDCl ) δ (ppm): 2.61 (t, J = 5.1 Hz, 4 H); 3.09 - 3.18 (m, 4 H); 3.76 (d, J =

3

1.1 Hz, 3 H); 4.83 (s, 1 H); 7.30 (t, J = 7.2 Hz, 2 H); 7.40 (t, J = 7.5 Hz, 2 H); 7.42 – 7.56 (m, 3 H); 7.56 – 7.63 (m, 2 H);

13

.67 – 7.78 (m, 4 H). C NMR (75 MHz, CDCl ) δ (ppm): 44.8 (d, J = 2.2 Hz, 2 x CH ); 49.3 (d, J = 5.8 Hz, 2 x CH ); 51.0

3

2

(

d, J = 6.1 Hz, CH ); 70.3 (CH); 119.7 (CH); 119.8 (CH); 125.8 (CH); 125.9 (CH); 127.0 (CH); 127.1 (CH); 128.2 (2 x CH);

3

1

28.4 (d, J = 14.1 Hz, 2 CH); 130.2 (d, J = 172.4 Hz, C); 131.3 (d, J = 9.4 Hz, 2 x CH); 131.7 (d, J = 3.0 Hz, CH); 141.0 (C);

31

41.1 (C); 143.6 (C); 143.7 (C). P{H} NMR (121.5 MHz, CDCl ): 23.0 ppm. P{H} NMR (121.5 MHz, CDCl ): 23.0

3

+

ppm. MS (DCI/CH ) m/z: 405.176 [M+H ], 239.10 [M-165], 165.07 [M-239]. HRMS (DCI/CH ): for C H N O P [M+H ]:

4

24 26

2

calcd: 405.1732; found: 405.1730.

4

.2.4.

(4-(9H-Fluoren-9-yl)piperazin-1-yl)(phenyl)methanethione (6). A mixture of (4-(9H-fluoren-9-yl)piperazin-1-

yl)(phenyl)methanone 1 (0.28 mmol, 100 mg, 1.0 eq) and Lawesson’s reagent (0.14 mmol, 57 mg, 0.5 eq) in 10 mL of

toluene was refluxed for 8 hours. Another half equivalent of Lawesson’s reagent (0.5 eq) was added and reaction mixture was

refluxed overnight. Then, the solvent was removed under vacuum pressure and the residue was dissolved in dichloromethane.

The solution was washed with 8% aqueous sodium hydrogenocarbonate and was dried over magnesium sulphate. The solvent

was removed under reduced pressure and the residue flash chromatographed (100% dichloromethane) to afford a yellow solid

-

1

(

77 mg, 74%). TLC R : 0.60 (petroleum ether/ethyl acetate 80/20); mp: 204 °C; IR (cm ): 618, 670, 700, 738, 762, 919, 928,

f

1

9

32, 1003, 1040, 1140, 1224, 1254, 1290, 1435, 1469, 1488, 2755, 2880. HPLC: method 1, rt = 3.87 min, purity 97%. H

NMR (300 MHz, CDCl ) δ (ppm): 2.46 (t, J = 5.4 Hz, 2 H); 3.02 (t, J = 5.4 Hz, 2 H); 3.53 (t, J = 5.4 Hz, 2 H); 4.49 (t, J = 5.4

3

Hz, 2 H); 4.94 (s, 1 H); 7.22 – 7.28 (m, 2 H); 7.29 – 7.33 (m, 3 H); 7.34 (td, J = 1.5 Hz, 7.5 Hz, 2 H); 7.43 (t, J = 7.2 Hz, 2

13

H); 7.65 (d, J = 7.2 Hz, 2 H); 7.72 (d, J = 7.5 Hz, 2 H). C NMR (75 MHz, CDCl ) δ (ppm): 48.3 (CH ); 49.3 (CH ); 50.1

3

2

(

CH ); 52.7 (CH ); 69.5 (CH); 119.9 (2 x CH); 125.8 (2 x CH); 125.9 (2 x CH); 127.3 (2 x CH); 128.4 (2 x CH); 128.5 (2 x

2 2

+

CH); 128.6 (CH); 141.1 (2 x C); 142.9 (2 x C); 143.1 (C); 200.4 (C). MS (DCI/CH ) m/z: 399.19 [M+Na], 371.16 [M+H ],

4

+

1

65.01 [fluorene]. HRMS (DCI/CH ): for C H N S [M+H ]: calcd: 371.1582; found: 371.1567.

4

24 23

2

4

.2.5.

1-(9H-Fluoren-9-yl)-4-(phenylsulfinyl)piperazine (7). To a suspension of benzenesulfinic acid sodium salt (0.86

mmol, 158 mg, 1 eq) in anhydrous dichloromethane (5.0 mL), was added dropwise thionyl chloride (1.15 mmol, 83 µL, 1.2

eq) at 4 °C. After the addition was finished, stirring was continued for an additional 2 hours at room temperature. The

mixture obtained was then directly added into a cooled solution of 1-(9H-fluoren-9-yl)piperazine 3 (7.99 mmol, 200 mg, 1.0

eq) and triethylamine (0.96 mmol, 129 µL, 1.2 eq) in anhydrous dichloromethane (5.0 mL). Once the addition was finished,

the reaction mixture was stirred overnight at room temperature. Brine was added and the aqueous phase was extracted with

dichloromethane. The organic phase was dried over magnesium sulphate, filtered and concentrated under vacuum pressure.

The resulting crude product was purified by flash chromatography (isocratic 60/40 petroleum ether/ethyl acetate) to afford a

-

1

white solid (147 mg, 49%). TLC R : 0.39 (petroleum ether/ethyl acetate 60/40); mp: 156 °C; IR (cm ): 560, 640, 669, 689,

f

1

7

01, 742, 756, 906, 1003, 1056, 1086, 1122, 1321, 1442, 2823, 2841, 2941. HPLC: method 1, rt = 2.72 min, purity 95%. H

NMR (300 MHz, CDCl ) δ (ppm): 2.58-2.80 (m, 4 H); 2.92-3.05 (m, 2 H); 3.10-3.25 (m, 2 H); 4.86 (s, 1 H); 7.32 (dd, J = 1.2

3

Hz, 5.7 Hz, 1 H); 7.34 (dd, J = 1.2 Hz, 5.4 Hz, 1 H); 7.41 (td, J = 3.0 Hz, 6.6 Hz, 2 H); 7.46-7.56 (m, 3 H); 7.61 (d, J = 7.2

13

Hz, 1 H); 7.63-7.69 (m, 3 H); 7.71 (d, J = 7.5 Hz, 2 H). C NMR (75 MHz, CDCl ) δ (ppm): 46.5 (2 x CH ); 49.1 (2 x CH );

3

2

6

1

9.9 (CH); 119.7 (2 x CH); 125.8 (2 x CH); 126.1 (2 x CH); 127.1 (2 x CH); 128.2 (2 x CH); 128.8 (2 x CH); 130.8 (CH);

+

41.0 (2 x C), 142.8 (2 x C); 143.6 (C). MS (DCI/CH ) m/z: 375.15 [M+H ], 249.14 [M-125 (PhSO)], 165.07 [M-209

4

+

(

fluorene)]. HRMS (DCI/CH ): for C H N O [M+H ]: calcd: 375.1531 found: 375.1536.

4

23 23

2

3

ACCEPTED MANUSCRIPT

4

.2.6. (4-(9H-Fluoren-9-yl)piperazin-1-yl)(methyl)(phenyl)phosphine oxide (9). Reagents: 1-

(9H-fluoren-9-yl)piperazine 3 (0.80 mmol, 200 mg), triethylamine (0.96 mmol, 129 µL) and

methylphenylphosphinic chloride (0.96 mmol, 132 µL). The crude product was purified by

flash chromatography (isocratic 95/5 petroleum ether/ethyl acetate) to a white solid (204 mg,

-

1

6

2

4%). TLC R : 0.19 (dichloromethane/MeOH 95/5); mp: 178 °C; IR (cm ): 620, 639, 666,

f

92, 713, 737, 883, 901, 965, 998, 1117, 1138, 1186, 1300, 1319, 1377, 1437, 1449, 2839,

1

908, 2931. HPLC: method 1, rt = 1.91 min, purity 94%. H NMR (300 MHz, CDCl ) δ

3

(

ppm): 1.68 (d, J_p_,_H= 13.8 Hz, 3 H); 2.63 (t, J = 4.5 Hz, 4 H); 2.94-3.16 (m, 4 H); 4.84 (s, 1

H); 7.31 (tt, J = 1.2 Hz, 7.5 Hz, 2 H); 7.39 (t, J= 7.5 Hz, 1 H); 7.43-7.55 (m, 3 H); 7.63 (t, J =

.3 Hz, 1 H); 7.69 (d, J = 7.5 Hz, 1 H); 7.74 (dd, J = 1.8 Hz, 12.0 Hz, 1 H); 7.76 (dd, J = 1.5

6

13

Hz, 12.0 Hz, 1H). C NMR (75 MHz, CDCl ) δ (ppm): 14.1 (d, J = 92.3 Hz, CH ); 44.8 (2 x

3

CH ); 49.3 (d, J = 7.7 Hz, 2 x CH ); 70.2 (d, J = 1.0 Hz, CH); 119.8 (2 x CH); 125.8 (d, J =

2

4

.1 Hz, 2 x CH); 127.1 (2 x CH); 128.1 (2 x CH); 128.4 (d, J = 12.3 Hz, 2 x CH); 131.1 (d, J

=

9.6 Hz, 2 x CH); 131.6 (d, J = 2.7 Hz, CH); 131.7 (C); 133.4 (C); 140.9 (2 x C); 143.5 (C) .

3

1

+

P NMR (121.5 MHz, CDCl ) δ (ppm): 35.32. MS (DCI/CH ) m/z: 389.18 [M+H ]. HRMS

3

4

+

(

DCI/CH ): for C H N OP [M+H ]: calcd: 389.1783; found: 389.1784.

4 24 26 2

4

.2.7.

General procedure for 11-15. The corresponding isocyanate or isothiocyanate (1.0 eq) was dissolved in dry

acetonitrile under argon. A solution of 1-(9H-fluoren-9-yl)piperazine 3 (1.0 eq) in dry acetonitrile was added to the reaction

mixture. After overnight stirring, a precipitate was isolated and washed with acetonitrile. The resulting crude product was

purified by flash chromatography to afford the title compound.

4

.2.7.1. 4-(9H-Fluoren-9-yl)-N-phenylpiperazine-1-carboxamide (11). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.40

mmol, 100 mg) and phenyl isocyanate (0.40 mmol, 48 µL). The crude product was purified by flash chromatography (dry-

load, gradient 100% petroleum ether to 100% ethyl acetate in 15 minutes) to afford a white solid (67 mg, 45%). TLC R : 0.63

f

-

1

(

dichloromethane/MeOH 90/10); mp: 223 °C; IR (cm ): 527, 691, 742, 872, 944, 972, 995, 1144, 1199, 1209, 1246, 1300,

1

312, 1325, 1365, 1439, 1504, 1521, 1596, 1654, 2821, 2947, 3408. HPLC: method 1, rt = 2.25 min, purity 95%. H NMR

(300 MHz, DMSO-d6) δ (ppm): 2.55 (t, J = 5.1 Hz, 4 H); 3.43 (t, J = 4.8 Hz, 4 H); 5.01 (s, 1 H); 6.93 (tt, J = 1.2 Hz, 7.2 Hz,

1

H); 7.18 – 7.26 (m, 2 H); 7.34 (td, J = 1.2 Hz, 7.5 Hz, 2 H); 7.29 – 7.46 (m, 4 H); 7.65 (d, J = 7.5 Hz, 2 H); 7.86 (d, J = 7.5

13

Hz, 2 H); 8.44 (s, 1 H). C NMR (75 MHz, DMSO-d6) δ (ppm): 44.9 (2 x CH ); 49.0 (2 x CH ); 69.6 (CH); 120.0 (2 x CH);

2

1

20.5 (2 x CH); 122.1 (CH); 126.3 (2 x CH); 127.7 (2 x CH); 128.7 (2 x CH); 128.8 (2 x CH); 140.9 (2 x CH); 141.0 (C);

+

43.9 (2 x C); 155.3 (C). MS (DCI/CH ) m/z: 370.19 [M+H ], 251.15 [M-119], 165.07 [M-205]. HRMS (DCI/CH ): for

4

+

C H N O [M+H ]: calcd: 370.1919; found: 370.1913.

24

3

4

.2.7.2. 4-(9H-Fluoren-9-yl)-N-phenylpiperazine-1-carbothioamide (12). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.40

mmol, 100 mg) and phenyl isothiocyanate (0.40 mmol, 48 µL). The crude product was purified by flash chromatography

dry-load, gradient 100% petroleum ether to 100% ethyl acetate in 15 minutes) to afford a white solid (71 mg, 46%). TLC R_f:

(

2

4

ACCEPTED MANUSCRIPT

-

1

0

1

.71 (dichloromethane/methanol 90/10); mp: 206 °C; IR (cm ): 525, 701, 737, 746, 997, 1037, 1123, 1219, 1261, 1301,

1

315, 1399, 1439, 1496, 1518, 1591, 2814, 3306. HPLC: method 2, rt = 2.86 min, purity 98%. H NMR (300 MHz, DMSO-

d6) δ (ppm): 2.60 (t, J = 4.8 Hz, 4 H); 3.88 (t, J = 4.5 Hz, 4 H); 5.04 (s, 1 H); 7.05 – 7.13 (m, 1 H); 7.23 – 7.32 (m, 4 H); 7.36

1

3

(td, J = 1.2 Hz, 7.5 Hz, 2 H); 7.43 (t, J = 7.5 Hz, 2 H); 7.66 (d, J = 7.2 Hz, 2 H); 7.87 (d, J = 7.5 Hz, 2 H); 9.24 (s, 1 H).

C

NMR (75 MHz, DMSO-d6) δ (ppm): 48.8 (2 x CH ); 49.2 (2 x CH ); 69.3 (CH); 120.6 (2 x CH); 124.7 (CH); 125.6 (2 x

2

CH); 126.3 (2 x CH); 127.7 (2 x CH); 128.4 (2 x CH); 128.8 (2 x CH); 141.0 (2 x CH); 141.5 (C); 143.8 (2 x C); 181.8 (C).

+

MS (DCI/CH ) m/z: 386.17 [M+H ], 251.15 [M-134], 165.07 [M-220], 136.02 [M-249]. HRMS (DCI/CH ): for C H N S

4

24 24

3

+

[

M+H ]: calcd: 386.1691; found: 386.1690.

4

.2.7.3. N-Butyl-4-(9H-fluoren-9-yl)piperazine-1-carboxamide (13). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.40 mmol,

1

00 mg) and butyl isocyanate (0.48 mmol, 54 µL). The crude product was purified by flash chromatography (isocratic,

petroleum ether/ethyl acetate 70/30) to afford a white solid (72 mg, 51%). TLC R : 0.83 (dichloromethane/methanol 90/10);

f

-

1

mp: 174 °C; IR (cm ): 737, 758, 848, 939, 1001, 1141, 1208, 1264, 1304, 1327, 1411, 1447, 1538, 1613, 2822, 2861, 2928,

1

2

–

956, 3353. HPLC: method 1, rt = 2.05 min, purity 98%. H NMR (300 MHz, CDCl ) δ (ppm): 0.93 (t, J = 7.2 Hz, 3 H); 1.26

3

1.41 (m, 2 H); 1.41 – 1.54 (m, 2 H); 2.63 (t, J = 4.8 Hz, 4 H); 3.17 – 3.26 (m, 2 H); 3.34 (t, J = 4.8 Hz, 4 H); 4.45 (t, J = 5.7

Hz, 1 H); 4.88 (s, 1 H); 7.31 (td, J = 1.2 Hz, 7.5 Hz, 2 H); 7.41 (t, J = 7.5 Hz, 2 H); 7.63 (d, J = 7.2 Hz, 2 H); 7.71 (d, J = 7.5

13

Hz, 2 H). C NMR (75 MHz, CDCl ) δ (ppm): 13.9 (CH ); 20.1 (CH ); 32.4 (CH ); 40.7 (CH ); 44.5 (2 x CH ); 48.8 (2 x

3

2

CH ); 70.0 (CH); 119.8 (2 x CH); 126.0 (2 x CH); 127.1 (2 x CH); 128.2 (2 x CH); 141.0 (2 x C); 143.6 (2 x C); 158.0 (C).

2

+

MS (DCI/CH ) m/z: 350.22 [M+H ], 251.16 [M-98], 184.15 [M-165], 165.07 [M-184], 143.12 [M-206]. HRMS (DCI/CH ):

4

+

for C H N O [M+H ]: calcd: 350.2232; found: 350.2228.

22

28

3

4

.2.7.4. 4-(9H-Fluoren-9-yl)-N-octylpiperazine-1-carboxamide (14). Reagents: 1-(9H-fluoren-9-yl)piperazine 3 (0.40

mmol, 100 mg) and octyl isocyanate (0.48 mmol, 85 µL). The crude product was purified by flash chromatography (isocratic,

petroleum ether/ethyl acetate 70/30) to afford a white solid (85 mg, 52%). TLC R : 0.83 (dichloromethane/methanol 90/10);

f

-

1

mp: 139 °C; IR (cm ): 618, 738, 844, 1006, 1127, 1143, 1177, 1206, 1260, 1301, 1326, 1406, 1444, 1466, 1540, 1616, 2851,

1

2

1

2

925, 3298. HPLC: method 1, rt = 3.12 min, purity 99%. H NMR (300 MHz, CDCl ) δ (ppm): 0.86 – 0.95 (m, 3 H); 1.22 –

3

.38 (m, 10 H); 1.49 (quint, J = 6.6 Hz, 2 H); 2.64 (t, J = 4.8 Hz, 4 H); 3.16 – 3.25 (m, 2 H); 3.35 (t, J = 4.8 Hz, 4 H); 4.36 (s,

H); 4.89 (s, 1 H); 7.31 (td, J = 1.2 Hz, 7.2 Hz, 2 H); 7.41 (t, J = 7.2 Hz, 2 H); 7.64 (d, J = 7.5 Hz, 2 H); 7.72 (d, J = 7.5 Hz,

13

H). C NMR (75 MHz, CDCl ) δ (ppm): 14.1 (CH ); 22.7 (CH ); 27.0 (CH ); 29.2 (CH ); 29.3 (CH ); 30.3 (CH ); 31.8

3

2

(

CH ); 41.0 (CH ); 44.5 (2 x CH ); 48.7 (2 x CH ); 70.0 (CH); 119.8 (2 x CH); 126.0 (2 x CH); 127.1 (2 x CH); 128.2 (2 x

2 2 2 2

+

CH); 141.0 (2 x C); 143.6 (2 x C); 157.7 (C). MS (DCI/CH ) m/z: 406.28 [M+H ], 251.16 [M-154], 240.21 [M-165], 199.18

4

+

[

M-206], 165.07 [M-240]. HRMS (DCI/CH ): for C H N O [M+H ]: calcd: 406.2858; found: 406.2840.

4

26 36

3

4

.2.7.5. 4-(9H-Fluoren-9-yl)-N-(4-nitrophenyl)piperazine-1-carboxamide (15). Reagents: 1-(9H-fluoren-9-yl)piperazine 3

(0.40 mmol, 100 mg) and 4-nitrophenylisocyanate (0.40 mmol, 65 mg). The crude product was purified by flash

chromatography chromatography (dry-load, gradient 100% petroleum ether to 100% ethyl acetate in 15 minutes) to afford a

-

1

white solid (82 mg, 82%). TLC R : 0.71 (dichloromethane/methanol 90/10); mp: 263 °C; IR (cm ): 620, 672, 690, 739, 807,

f

8

42, 1001, 1110, 1237, 1299, 1328, 1424, 1497, 1541, 1599, 1611, 1639, 2827, 2913, 3341. HPLC: method 1, rt = 2.68 min,

1

purity 95%. H NMR (300 MHz, DMSO-d6) δ (ppm): 2.57 (t, J = 5.1 Hz, 4 H); 3.47 (t, J = 5.1 Hz, 4 H); 5.02 (s, 1 H); 7.34

(td, J = 1.5 Hz, 7.5 Hz, 2 H); 7.43 (t, J = 7.2 Hz, 2 H); 7.65 (d, J = 7.2 Hz, 2 H); 7.67 – 7.73 (m, 2 H); 7.86 (d, J = 7.5 Hz, 2

13

H); 8.10 – 8.19 (m, 2 H); 9.18 (s, 1 H). C NMR (75 MHz, DMSO-d6) δ (ppm): 45.1 (2 x CH ); 49.0 (2 x CH ); 69.6 (CH);

2

1

18.7 (2 x CH); 120.6 (2 x CH); 125.2 (2 x CH); 126.3 (2 x CH); 127.7 (2 x CH); 128.7 (2 x CH); 141.0 (2 x C); 141.2 (C);

+

1

43.8 (2 x CH); 148.0 (C); 154.3 (C). MS (DCI/CH ) m/z: 415.186 [M+H ]. HRMS (DCI/CH ): for C H N O [M+H ]:

4 4 24 23 4 2

calcd: 415.1770; found: 415.1779.

2

5

ACCEPTED MANUSCRIPT

4

.2.8.

General procedure for compounds 17 and 18. To a solution of the corresponding alkene (1.0 eq), in anhydrous

tetrahydrofuran was added 9-BBN (1.2 eq) at 0°C. The mixture was stirred at 0°C for 2 hours and at RT for 3 hours more,

and then was introduced into a mixture of 2-bromofluorenone 16 (1.25 eq), cesium carbonate (3.75 eq), triphenylarsine (0.25

eq) and Pd(dppf)Cl (0.25 eq) in a mixture of 8 mL of THF, 8 mL DMF and 2 mL H O. The resulting mixture was heated at

2

8

5 °C overnight, cooled and passed through a short pad of silica gel with 4:1 petroleum ether/ethyl acetate. The solvent was

evaporated and the residue was dissolved in ethyl acetate, washed with brine, dried over magnesium sulfate, filtered and

evaporated. The residue was purified by flash chromatography (gradient, 100% petroleum ether to 95:5 petroleum ether/ethyl

acetate in 15 minutes) to afford the title compound.

4

.2.8.1. 2-Hexyl-9H-fluoren-9-one (17). Reagents: 1-hexene (1.7 mmol, 211 µL), 9-BBN (2.05 mmol, 4.1 mL of 0.5 M

solution in THF), 2-bromofluorenone 16 (2.1 mmol, 550 mg), cesium carbonate (6.4 mmol, 2.07 g), triphenylarsine (0.42

mmol, 130 mg) and Pd(dppf)Cl (0.42 mmol, 346 mg). A yellow oil was obtained (257 mg, 57%). TLC R : 0.57 (petroleum

2

f

-

1

ether/ethyl acetate 97/3). IR (cm ): 736, 766, 831, 958, 1106, 1176, 1291, 1457, 1602, 1713, 2854, 2925. H NMR (300

MHz, CDCl ) δ (ppm): 0.85-0.98 (m, 3 H); 1.25-1.45 (m, 6 H); 1.53-1.73 (m, 2 H); 2.66 (t, J = 7.5 Hz, 2 H); 7.25-7.34 (m, 2

3

13

H); 7.44 (d, J = 7.8 Hz, 1 H); 7.47-7.53 (m, 3 H); 7.66 (dt, J = 0.9 Hz, 7.5 Hz, 1 H). C NMR (75 MHz, CDCl ) δ (ppm):

3

1

2

4.1 (CH ); 22.6 (CH ); 28.8 (CH ); 31.2 (CH ); 31.7 (CH ); 35.8 (CH ); 120.0 (CH); 120.2 (CH); 124.3 (CH); 124.4 (CH);

3 2 2 2 2 2

28.6 (CH); 134.4 (C); 134.5 (C); 134.7 (CH); 134.7 (CH); 142.1 (C); 144.5 (C); 144.7 (C); 194.3 (C). MS (DCI/CH ) m/z:

4

+

65.16 [M+H ]. HRMS (DCI/CH ): for C H O [M+H ]: calcd: 265.1592; found: 265.1599.

4

19 20

4

.2.8.2. 2-Octyl-9H-fluoren-9-one (18). Reagents: 1-Octene (1.7 mmol, 267 µL), 9-BBN (2.05 mmol, 4.1 mL of 0.5 M

solution in THF), 2-bromofluorenone 16 (2.1 mmol, 550 mg), cesium carbonate (6.4 mmol, 2.07 g), triphenylarsine (0.42

mmol, 130 mg,) and Pd(dppf)Cl (0.42 mmol, 346 mg). A yellow oil was obtained (170 mg, 34%). TLC R : 0.72 (petroleum

2

f

-

1

ether/ethyl acetate 95/5). IR (cm ): 650, 737, 765, 831, 963, 1106, 1176, 1291, 1457, 1603, 1714, 2853, 2923. H NMR (300

MHz, CDCl ) δ (ppm): 0.91 (t, J = 6.6 Hz, 3 H); 1.22-1.43 (m, 10 H); 1.65 (q, J = 7.5 Hz, 2 H); 2.64 (t, J = 7.5 Hz, 2 H);

3

7

.25-7.34 (m, 2 H); 7.45 (d, J = 7.8 Hz, 1 H); 7.49 (dd, J = 0.9 Hz, 3.3 Hz, 1 H); 7.51 (d, J = 0.9 Hz, 1 H); 7.52 (s, 1 H); 7.67

1

3

(

dt, J = 0.9 Hz, 7.5 Hz, 1 H). C NMR (75 MHz, CDCl ) δ (ppm): 14.1 (CH ); 22.7 (CH ); 29.2 (CH ); 29.3 (CH ); 29.5

3 3 2 2 2

CH ); 31.2 (CH ); 31.9 (CH ); 35.8 (CH ); 120.0 (CH); 120.2 (CH); 124.2 (CH); 124.4 (CH); 128.6 (CH); 134.7 (C); 134.5

2

+

C); 134.6 (CH); 134.7 (CH); 142.0 (C); 144.5 (C); 144.7 (C); 194.1 (C). MS (DCI/CH ) m/z: 293.19 [M+H ]. HRMS

4

+

DCI/CH ): for C H O [M+H ]: calcd: 293.1905; found: 293.1906.

4

21 25

4

.2.9.

2-Propoxy-9H-fluoren-9-one (20). An oven-dried 5 mL round-bottom flask was charged with Pd(OAc) (0.011

2

mmol, 2.6 mg, 0.01 eq), ligand tBuXPhos (0.023 mmol, 9.8 mg, 0.02 mmol) and Cs CO (1.74 mmol, 566 mg, 1.5 eq). The

2

3

round-bottom flask was sealed with septum, evacuated and back-filled with argon. Toluene (2.0 mL), 2-bromo-9-fluorenone

9 (1.16 mmol, 300 mg, 1.0 eq) and propan-1-ol (3.47 mmol, 260 µL, 3.0 eq) were added to the reaction mixture and was

1

stirred overnight at 80 °C. Brine was added and the product was extracted with ethyl acetate (3×). The organic phase was

dried over magnesium sulfate, filtered and concentrated under vacuum pressure. The resulting crude product was purified by

flash chromatography (petroleum ether/diethyl ether 97/3 in 20 minutes) to afford a yellow solid (134 mg, 52%). TLC R_f:

-

1

0

2

.35 (petroleum ether/Et O 97/3); mp: 61 °C; IR (cm ): 741, 766, 837, 996, 1012, 1130, 1247, 1296, 1455, 1489, 1599, 1716,

2

1

875, 2940. H NMR (300 MHz, CDCl ) δ (ppm): 1.08 (t, J = 7.2 Hz, 3 H); 1.85 (sex, J = 7.2 Hz, 2 H); 3.99 (t, J = 6.6 Hz, 2

3

H); 6.99 (dd, J = 2.4 Hz, 8.4 Hz, 1 H); 7.21 (d, J = 2.4 Hz, 1 H); 7.21 (td, J = 1.5 Hz, 7.2 Hz, 1 H); 7.37 – 7.49 (m, 3 H); 7.61

13

(

dt, J = 0.9 Hz, 7.2 Hz, 1 H). C NMR (75 MHz, CDCl ) δ (ppm): 10.5 (CH ); 22.5 (CH ); 70.1 (CH ); 109.9 (CH); 119.5

3 3 2 2

2

6

ACCEPTED MANUSCRIPT

(CH); 120.8 (CH); 121.3 (CH); 124.3 (CH); 127.8 (CH); 134.3 (C); 134.8 (CH); 135.9 (C); 136.8 (C); 145.0 (C); 160.4 (C);

+

1

94.0 (C). MS (DCI/CH ) m/z: 239.11 [M+H ]. HRMS (DCI/CH ): for C H O [M+H ]: calcd: 239.1072; found: 239.1084.

4 4 16 15 2

4

.2.10. 3-Hydroxy-9H-fluoren-9-one (22). A mixture of 3-methoxyfluorenone 21 (1.1 mmol, 226 mg, 1.0 eq), acetic acid

(26 mmol, 1.5 mL, 25 eq), and HBr 47% (2.5 mL) was heated under reflux for 6 hours. After cooling, the reaction mixture

was poured into water (100 mL) and extracted with ethyl acetate. The organic phase was dried over magnesium sulfate,

filtered and concentrated under vacuum pressure. The residue was purified by flash chromatography (petroleum ether:ethyl

acetate 80:20 in 15 minutes) to afford a yellow solid (130 mg, 62%). TLC R : 0.42 (petroleum ether/ethyl acetate 70/30); mp:

f

-

1

2

38 °C; IR (cm ): 670, 736, 829, 870, 926, 1102, 1204, 1298, 1388, 1445, 1586, 1611, 1676, 3082. H NMR (300 MHz,

1

3

CD OD) δ (ppm): 6.69 (dd, J = 2.1 Hz, 8.1 Hz, 1 H); 7.05 (d, J = 2.1 Hz, 1 H); 7.31-7.38 (m, 1 H); 7.48-7.61 (m, 4 H).

C

3

NMR (75 MHz, CD OD) δ (ppm): 107.9 (CH); 114.7 (CH); 120.0 (CH); 123.0 (CH); 125.3 (C); 126.1 (CH); 129.0 (CH);

3

+

1

34.2 (CH); 135.1 (C); 143.4 (C); C H O [M+H ]: calcd: 197.0603; found: 197.0610.

13

9

2

4

.2.11. General procedure for compounds 23-25. 3-Hydroxy-9-fluorenone 22 (1.0 eq) was dissolved in DMF (5.0 mL),

potassium carbonate (1.5 eq) and the corresponding alkyl or aryl bromine reagent (0.95 eq) were added. The mixture was

heated to 80°C and stirred overnight. The reaction mixture was then cooled and 1 N hydrochloric acid (5.0 mL) was added

dropwise thereto in a water bath to quench the reaction. The mixture was extracted with dichloromethane and washed once

with 1 N hydrochloric acid and 4 times with water. The organic layer was dried over magnesium sulfate, filtered and

concentrated under vacuum pressure to afford the title compound. No further purification was needed.

4

.2.11.1. 3-(Benzyloxy)-9H-fluoren-9-one (23). Reagents: 3-Hydroxy-9-fluorenone 22 (0.54 mmol, 105 mg), potassium

carbonate (0.81 mmol, 111 mg) and benzyl bromide (0.64 mmol, 77 µL). A yellow solid was obtained (105 mg, 88%). TLC

-

1

R : 0.35 (petroleum ether/ethyl acetate); mp: 124 °C; IR (cm ): 693, 735, 767, 839, 1023, 1099, 1183, 1204, 1237, 1285,

f

1

306, 1381, 1450, 1491, 1583, 1610, 1734, 2853, 2922, 3305. H NMR (300 MHz, CDCl ) δ (ppm): 5.17 (s, 2 H); 6.82 (dd, J

3

=

2.1 Hz, 8.1 Hz, 1 H); 7.10 (d, J = 2.1 Hz, 1 H); 7.27 – 7.33 (m, 1 H); 7.38 – 7.52 (m, 7 H); 7.62 (d, J = 8.1 Hz, 1 H); 7.64

13

(

dt, J = 0.6 Hz, 7.5 Hz, 1 H). C NMR (75 MHz, CDCl ) δ (ppm): 70.5 (CH ); 108.0 (CH); 113.8 (CH); 120.2 (CH); 123.9

3

2

(CH);126.3 (CH);127.4 (C); 127.6 (2 x CH); 128.4 (CH); 128.8 (2 x CH); 129.3 (CH); 134.2 (CH); 135.3 (C); 136.1 (C);

+

1

43.4 (C); 147.0 (C); 164.5 (C); 192.5 (C). MS (DCI/CH ) m/z: 287.11 [M+H ]. HRMS (DCI/CH ): for C H O [M+H ]:

4 4 20 15 2

calcd: 287.1072; found: 287.1074.

4

.2.11.2. 3-Propoxy-9H-fluoren-9-one (24). Reagents: 3-Hydroxy-9-fluorenone 22 (0.41 mmol, 80 mg), potassium carbonate

(

0.61 mmol, 85 mg) and bromopropane (0.39 mmol, 35 µL). A yellow solid was obtained (95 mg, 98%). TLC R : 0.09

f

-

1

petroleum ether/ethyl acetate 80/20); mp: 55 °C; IR (cm ): 538, 616, 642, 670, 731, 762, 826, 859, 924, 1007, 1013, 1109,

1

203, 1218, 1293, 1370, 1447, 1590, 1603, 1616, 1705, 2879, 2938, 2967. H NMR (300 MHz, CDCl ) δ (ppm): 1.07 (t, J =

3

7

.5 Hz, 3 H); 1.85 (sep, J = 7.2 Hz, 2 H); 4.02 (t, J = 6.6 Hz, 2 H); 6.72 (dd, J = 2.1 Hz, 8.1 Hz, 1 H); 7.01 (d, J = 2.4 Hz, 1

1

3

H); 7.28 (dq, J = 3.3 Hz, 5.1 Hz, 1 H); 7.43-7.48 (m, 2 H); 7.60 (d, J = 8.1 Hz, 1 H); 7.62 (dt, J = 1.2 Hz, 7.2 Hz, 1 H).

C

NMR (75 MHz, CDCl ) δ (ppm): 10.5 (CH ); 22.5 (CH ); 70.1 (CH ); 107.6 (CH); 113.5 (CH); 120.1 (CH); 123.9 (CH);

3

2

1

2

26.3 (CH); 127.0 (C); 129.3 (CH); 134.1 (CH); 135.4 (C); 143.5 (C); 147.0 (C); 165.1 (C); 192.6 (C). MS (DCI/CH ) m/z:

4

+

39.10 [M+H ]. HRMS (DCI/CH ): for C H O [M+H ]: calcd: 239.1072; found: 239.1060.

4

16 15

2

4

.2.11.3. 3-Hexyloxy-9H-fluoren-9-one (25). Reagents: 3-Hydroxy-9-fluorenone 22 (0.41 mmol, 80 mg), potassium

carbonate (0.61 mmol, 85 mg) and bromohexane (0.39 mmol, 54 µL). A brown, solid was obtained (106 mg, 93%). TLC R_f:

-

1

0

.81 (petroleum ether/ethyl acetate 80/20); mp: 84 °C; IR (cm ): 677, 728, 737, 765, 846, 990, 1017, 1097, 1188, 1233,

2

7

ACCEPTED MANUSCRIPT

1

298, 1452, 1587, 1605, 1696, 2861, 2935, 2951. H NMR (300 MHz, CDCl ) δ (ppm): 0.96 (t, J = 6.9 Hz, 3 H); 1.32-1.45

3

(m, 4 H); 1.46-1.58 (m, 2 H); 1.86 (q, J = 6.6 Hz, 2 H); 4.09 (t, J = 6.3 Hz, 2 H); 6.76 (dd, J = 2.4 Hz, 8.4 Hz, 1 H); 7.05 (d, J

=

1

2.1 Hz, 1 H); 7.32 (dq, J = 3.0 Hz, 5.4 Hz, 1 H); 7.45-7.52 (m, 2 H); 7.63 (d, J = 8.1 Hz, 1 H); 7.66 (dt, J = 1.2 Hz, 7.2 Hz,

13

H). C NMR (75 MHz, CDCl ) δ (ppm): 14.1 (CH ); 22.6 (CH ); 25.7 (CH ); 29.1 (CH ); 31.6 (CH ); 68.6 (CH ); 107.6

3

2

(CH); 113.5 (CH); 120.1 (CH); 123.9 (CH); 126.3 (CH); 127.0 (C); 129.3 (CH); 134.1 (CH); 135.4 (C); 143.5 (C); 147.0 (C);

+

1

65.1 (C); 192.6 (C). MS (DCI/CH ) m/z: 281.15 [M+H ]. HRMS (DCI/CH ): for C H O [M+H ]: calcd: 281.1542;

4 4 19 21 2

found: 281.1543.

4

.2.12. 9-Oxo-9H-fluoren-6-yl trifluoromethanesulfonate (26). To a solution of 3-hydroxy-9-fluorenone 22 (2.0 mmol, 300

mg, 1.0 eq) and of 2,6-di-tert-butyl-4-methylpyridine (5.1 mmol, 1.05 g, 2.5 eq) in anhydrous dichloromethane (15.0 mL)

was added triflic anhydride (2.45 mmol, 411 µL, 1.2 eq) dropwise at -78 °C. The mixture was stirred at -78 °C for 1 hour and

at 0 °C for 1.5 hours, and then was evaporated. The yellow residue was directly purified by flash chromatography (isocratic

petroleum ether/ethyl acetate 95/5 in 15 minutes) to afford a yellow solid (491 mg, 73%). TLC R : 0.90 (petroleum

f

1

ether/ethyl acetate 90/10). H NMR (300 MHz, CDCl ) δ (ppm): 7.18 (dd, J = 2.1 Hz, 8.1 Hz, 1 H); 7.34-7.40 (m, 1 H); 7.41

3

(d, J = 2.1 Hz, 1 H); 7.54 (dd, J = 0.9 Hz, 2.4 Hz, 1 H); 7.55 (d, J = 0.9 Hz, 1 H); 7.69 (dt, J = 0.9 Hz, 7.2 Hz, 1 H); 7.73 (d, J

13

=

8.4 Hz, 1 H). C NMR (75 MHz, CDCl ) δ (ppm): 114.1 (CH); 116.8 (C); 121.1 (CH); 121.8 (CH); 124.9 (CH); 126.1

3

(CH); 130.5 (CH); 133.8 (C); 134.4 (C); 135.3 (CH); 142.6 (C); 147.3 (C); 154.0 (C); 191.6 (C). MS (DCI/CH ) m/z: 329.01

4

+

[

M+H ]. HRMS (DCI/CH ): for C H F O S [M+H ]: calcd: 329.0100; found: 329.0095.

4

14

8

3

4

.2.13. 3-Hexyl-9H-fluoren-9-one (27). To a solution 1-hexene (0.34 mmol, 43 µL, 1.0 eq) in anhydrous THF (3.0 mL)

was added 9-BBN (0.41 mmol, 812 µL of a 0.5 M solution in THF, 1.2 eq) at 0 °C. The mixture was stirred at 0°C for 2

hours and at room temperature for 2 hours additional, and then was introduced to a mixture of 9-oxo-9H-fluoren-6-yl

trifluoromethanesulfonate 26 (0.31 mmol, 100 mg, 0.9 eq), K PO (0.47 mmol, 100 mg, 1.4 eq) and Pd(dppf)Cl ,CH Cl

2

3

4

2

(0.02 mmol, 20 mg, 0.07 eq) in anhydrous THF (3.0 mL). The resulting mixture was heated at reflux overnight, cooled and

passed through a short pad of silica gel with petroleum ether:ethyl acetate 4:1. The solvent was evaporated and the residue

was purified by flash chromatography (gradient, 100% petroleum ether to 100% ethyl acetate in 15 minutes) to afford a

-

1

yellow oil (77 mg, 96%). TLC R : 0.59 (petroleum ether/ethyl acetate 95/5). IR (cm ): 677, 737, 764, 838, 919, 1111, 1193,

f

1

297, 1422, 1448, 1601, 1613, 1708, 2855, 2925. H NMR (300 MHz, CDCl ) δ (ppm): 0.88 – 0.98 (m, 3 H); 1.27-1.45 (m, 6

3

H); 1.62 – 1.75 (m, 2 H); 2.69 (t, J = 7.2 Hz, 2 H); 7.12 (dd, J = 1.5 Hz, 7.5 Hz, 1 H); 7.30 (td, J = 1.5 Hz, 7.2 Hz, 1 H); 7.34

7.38 (m, 1 H); 7.49 (td, J = 1.2 Hz, 7.5 Hz, 1 H); 7.52 – 7.54 (m, 1 H); 7.60 (d, J = 7.5 Hz, 1 H); 7.67 (td, J= 1.2 Hz Hz, 7.2

–

13

Hz). C NMR (75 MHz, CDCl ) δ (ppm): 14.1 (CH ); 22.6 (CH ); 29.0 (CH ); 31.2 (CH ); 31.7 (CH ); 36.6 (CH ); 120.1

3

2

(CH); 120.6 (CH); 124.2 (CH); 124.4 (CH); 129.0 (CH); 129.1 (CH); 132.1 (C); 134.4 (CH); 134.8 (C); 144.4 (C); 144.8 (C);

+

+].

+

1

51.0 (C); 193.7 (C). MS (DCI/CH ) m/z: 293.19 [M+C H ], 265.16[M+H HRMS (DCI/CH ): for C H O [M+H ]:

4 2 5 4 19 21

calcd: 265.1592; found: 265.1599.

4

.2.14. General procedure for 28-35. To a mixture of 1-benzoylpiperazine (1.0 eq) and potassium carbonate (2.0 eq) in

dimethylformamide was added dropwise a solution of the corresponding substituted 9-bromo-9H-fluorene (1.0 eq) in

dimethylformamide (The synthesis of 9-bromo-9H-fluorene derivatives is reported in supporting information). After stirring

for 24 hours at room temperature, solvent was removed and the crude residue was dissolved in diethyl ether, washed with

brine, dried over magnesium sulfate, filtered and concentrated under vacuum. The residue was purified by flash

chromatography as indicated in each case to afford the title compound.

4

.2.14.1. (4-(2-Hexyl-9H-fluoren-9-yl)piperazin-1-yl)(phenyl)methanone (28). Reagents: 1-Benzoylpiperazine (0.15 mmol,

9 mg), potassium carbonate (0.30 mmol, 42 mg) and 9-bromo-2-hexyl-9H-fluorene (0.15 mmol, 50 mg). The crude product

2

8

Article Doi

Design, synthesis and evaluation of new GEQ derivatives as inhibitors of InhA enzyme and Mycobacterium tuberculosis growth

DOI: 10.1016/j.ejmech.2015.06.035

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2015.06.035

Full text of DOI:10.1016/j.ejmech.2015.06.035

Products guided by the article

R&D Labs maybe for 1253118-46-5

Relevant to this article

Hot Product