Evaluation of the inhibitory activity of (aza)isoindolinone-type compounds: toward in vitro InhA action, Mycobacterium tuberculosis growth and mycolic acid biosynthesis

Article abstract of DOI:10.1111/cbdd.12804

Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure–InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.

Full text of DOI:10.1111/cbdd.12804

Received Date : 18-Feb-2016
Revised Date : 06-Jun-2016
Accepted Date : 11-Jun-2016
Article type : Research Article
Evaluation of the inhibitory activity of (aza)isoindolinone-type
compounds: Towards in vitro InhA action, Mycobacterium
tuberculosis growth and mycolic acid biosynthesis
Short running title: (Aza)isoindolinone-type derivatives: evaluation towards in vitro InhA
action, Mycobacterium tuberculosis growth and mycolic acid biosynthesis
Keywords: (aza)isoindolinone, InhA inhibition, docking, Mycobacterium tuberculosis,
structure-activity relationship, mycolic acid biosynthesis.
Aurélien Chollet,a,b,c,d Jean-Luc Stigliani,a,b Maria Rosalia Pasca,e Giorgia Morie, Christian
Lherbet,c,d Patricia Constant,f,g Annaïk Quémard,f,g Jean Bernadou,a,b Geneviève Pratviel,a,b
Vania Bernardes-Génisson*a,b
aLaboratoire de Chimie de Coordination (LCC), Centre National de la Recherche Scientifique
(CNRS), BP 44099, 205 Route de Narbonne, F-31077, Toulouse, Cedex 4, France
bUniversité de Toulouse, Université Paul Sabatier, INPT, F-31077, Toulouse, Cedex 4, France
cLaboratoire de Synthèse et Physicochimie de Molécules d’Intérêt Biologique (SPCMIB),
Centre National de la Recherche Scientifique (CNRS), 118 Route de Narbonne, 31062,
Toulouse, Cedex 9, France
dUniversité de Toulouse, Université Paul Sabatier, LSPCMIB, F-31077, Toulouse, France
eDipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, via Ferrata 1, 27100 Pavia,
Italy.
fInstitut de Pharmacologie et de Biologie Structurale (IPBS), Centre National de la Recherche
Scientifique (CNRS), BP 64182, 205 Route de Narbonne, F-31077, Toulouse, France
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
'Accepted Article', doi: 10.1111/cbdd.12804
This article is protected by copyright. All rights reserved.

gUniversité de Toulouse, Université Paul Sabatier, IPBS, F-31077, Toulouse, France
Corresponding author: Vania Bernardes-Génisson, vania.bernardes-genisson@lcc-
toulouse.fr
Abstract
Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as
potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that
azaisoindolinone-type compounds displayed, in vitro, inhibitory activity towards InhA.
Herein we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15
new compounds and their evaluations toward the in vitro InhA activity. Based on these
results, a structure–InhA inhibitory activity relationship analysis and a molecular docking
study, using the conformation of InhA found in the 2H7M crystal structure, were carried out
in order to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors
with InhA in vitro. Then, the work was extended toward evaluations of these compounds
against Mycobacterium tuberculosis (Mtb) growth and finally some of them were also
investigated in respect with their ability to inhibit mycolic acid biosynthesis inside
mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb
growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.
Introduction
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, disease that is
responsible for the death of 1.5 million people in the world annually (1). Over the years, some
strains of mycobacteria have acquired simultaneous resistance to the two most used anti-
tubercular drugs: isoniazid (INH) and rifampicin (Multi Drug Resistant strains, MDR). In
addition, some strains have even developed resistance not only to the first-line drugs but also
to quinolones and a second-line drug (Extremely Drug Resistant strains, XDR). Importantly,
the emergence of some strains resistant to all drugs used in anti-tubercular therapeutic
regimen (Total Drug Resistant strains, TDR) has also been reported. In this context,
tuberculosis represents a serious world health problem. Without effective correct treatments,
the concern is that the occurrence of infections caused by MDR-strains will increase and
become out of control. Thereby, the need for new anti-tubercular agents with new chemical
structure and new mechanism of action appears evident and urgent.
This article is protected by copyright. All rights reserved.

Isoniazid (INH) (Figure 1), one of the most efficient anti-tubercular medicines, is a pro-drug
that requires activation by the mycobacterial catalase peroxidase enzyme (KatG) to form, after
reaction with the cofactor NAD, the active metabolite (INH-NADH adduct, Figure 1). This
adduct exerts its action by inhibiting InhA, an enoyl-ACP-reductase enzyme of Mtb, that is
implicated in the mycolic acid biosynthesis, essential constituent of the mycobacterial cell
wall. Moreover, it was shown that INH-resistant Mtb strains are frequently correlated to a
default of KatG that is unable to activate INH. Direct targeting of InhA appears thus an
attractive approach for the discovery of new antibiotic drugs because i) InhA is the main
target of the confirmed anti-tubercular pro-drug INH, ii) enzymes belonging to the Fatty Acid
Synthase II (FAS II) system, such as InhA, do not have a human ortholog and iii) InhA
inhibitor, non KatG dependent, would allow to bypass resistance problems.
Lately, we have been interested in the azaisoindolinone family, designed from the core of the
active metabolite of INH (Figure 1), as a promising scaffold for direct InhA inhibition. Our
previous work about structure-activity relationship study on azaisoindolinone derivatives has
essentially implicated modifications focusing on the hydrocarbon chain (length, position and
nature of the linker of the lipophilic chain) and on the tertiary alcohol at C7 position (2, 3).
The thiododecyl chain placed in the meta position of the phenyl ring was optimal and the free
hydroxyl at C7 revealed to be fundamental for InhA inhibition in vitro (compound 2a, Figure
1).
Figure 1. Structures of isoniazid, INH-NADH adduct and azaisoindolinone compound 2a.
In the continuity with this program, we describe in this paper a study about the structural
evolution of azaisoindolinone scaffold 2a, targeting specific regions of the molecule, as
This article is protected by copyright. All rights reserved.

shown in Figure 2, in order to explore new structure-activity elements and get insights into
important structural features to improve inhibitory enzymatic activity. The synthesis of
modified azaisoindolinones, their evaluation, as direct inhibitors of the InhA activity in vitro,
and the simulation of their mode of interaction with InhA by molecular docking experiments
were performed. Then the compounds were tested for inhibition of whole cell Mtb growth and
an investigation whether the observed inhibition might be correlated to the blockage of
mycolic acid biosynthesis was also carried out.
Methods and Materials
Material
All chemicals were obtained from Aldrich or Acros Organics and used without further
purification. The melting points were determined on an Electrothermal 9300 capillary melting
point apparatus and were not corrected. TLC chromatography (silica gel F254 nm). Infra-red
spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR Spectrometer. 1H NMR spectra
were recorded on a Bruker spectrometer at 400 and 500 MHz using CDCl3, DMSO-d6 or
1
MeOD-d4 as the solvent. For H NMR the residual proton signal of the deuterated solvent
was used as an internal reference: CDCl3 δ= 7.29 ppm, DMSO = 2.50 ppm and MeOD = 3.31
13
ppm. C NMR spectra were recorded on a Bruker spectrometer at 100 and 125 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. Flash column
chromatography was performed on CombiFlash Companion with silica Redisep columns from
12 g to 80 g. All reagents were obtained from commercial suppliers and were used without
further purification. Anhydrous solvents were freshly distilled before use or were obtained
from the M.Braun Solvent Purification System (MB-SPS-800). The enzymatic evaluation was
performed on a Cary Bio 100.
General protocol for nucleophilic addition of the dodecylthiophenyl moiety
To a solution of 1-bromo-3-dodecylthiobenzene (2 eq) in dry tetrahydrofuran, under argon
atmosphere and at -40 °C was added 2.0 M solution of n-butyllithium in cyclohexane (2 eq).
After addition, the temperature was maintained at -50 °C for 60 min. Then the temperature
was decreased at -78 °C and upon this solution was added a solution of the corresponding
dione (1 eq) in dry tetrahydrofuran. The reaction mixture was stirred for 30 minutes at -78 °C
and was warmed to room temperature for 30 minutes more. The reaction was quenched with a
This article is protected by copyright. All rights reserved.

saturated ammonium chloride solution and the mixture was extracted with ethyl acetate. The
organic phases were combined, dried over sodium sulfate and concentrated under vacuum.
The residue was chromatographed as indicated in each case to furnish the entitled compound.
(RS)-1-[3-(Dodecylsulfanyl)phenyl]-1-hydroxy-1,2-dihydro-3H-pyrrolo[3,4-c]pyridin-3-
one (2a)
Reagents: 1-bromo-3-dodecylthiobenzene (860 mg, 2.4 mmol), dry tetrahydrofuran (10 mL),
n-butyllithium solution (1.7 mL 2.0 M solution in cyclohexane; 3.4 mmol) and 3,4-
pyridinedicarboximide (178 mg, 1.2 mmol). The crude product was purified by flash
chromatography (gradient from 100% dichloromethane to 80/20 dichloromethane) to afford a
white solid (297 mg, 58%, mixture of para/meta regioisomers 80/20), mp: 86 °C. TLC Rf:
0.30 (dichloromethane/methanol 95/5). IR (cm-1): 3159, 2917, 2849, 1698, 1614, 1587, 1464,
1347, 1205, 1067, 964, 784, 698. 1H NMR (400 MHz, MeOD) δ (ppm): 8.94 (s, 1H); 8.75 (d,
J = 5.0 Hz, 1H), 7.55 (s, 1H); 7,46 (d, J = 4.6 Hz, 1H); 7.33–7.30 (m, 3H); 2.94 (t, J = 7.0 Hz,
2H); 1.63–1.59 (m, 2H); 1.50–1.25 (m, 18H); 0.91 (t, J = 6.0 Hz, 3H).13C NMR (100 MHz,
MeOD) δ (ppm): 168.3 (C); 159.0 (C); 152.7 (CH); 150.0 (C); 144.5 (CH); 140.0 (C); 138.0
(C); 128.9 (CH); 128.4 (CH); 125.2 (CH); 122.5 (CH); 118.0 (CH); 87.6 (C); 70.1 (CH2);
32.6 (CH2); 31.7 (CH2); 29.4–28.8 (6 x CH2); 28.4 (CH2); 22.2 (CH2); 13.0 (CH3). MS (ESI)
m/z: 449.2 [M+Na+]; 427.3 [M+H+]. HRMS (ESI): for C25H35N2O2S [M+H+]: calcd:
427.2419; found: 427.2459.
(RS)-3-[3-(Dodecylsulfanyl)phenyl]-3-hydroxy-2,3-dihydro-1H-isoindol-1-one (2b)
Reagents: 1-bromo-3-dodecylthiobenzene (2.43 g, 6.8 mmol), tetrahydrofuran (5 mL), n-
butyllithium 2.0 M (3.4 mL solution in cyclohexane; 6.8 mmol) and phtalimide (500 mg, 3.4
mmol). The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 80/20 dichloromethane/methanol) to afford a white solid (696 mg, 48%),
mp: 122 °C. TLC Rf: 0.59 (dichloromethane/methanol 90/10). IR (cm-1): 3285, 2918, 2850,
1
1708, 1665, 1614, 1588, 1466, 1434, 1349, 1192, 1115, 1056, 962, 790, 721, 696. H NMR
(400 MHz, DMSO-d6) δ (ppm): 9.25 (s, 1H); 7.64 (d, J = 6.8 Hz, 1H); 7.56-7.46 (m, 2H);
7.40 (s, 1H); 7.33-7.21 (m, 4H); 6.95 (s, 1H); 2.92 (t, J = 7.2 Hz, 2H); 1.56–1.49 (m, 2H);
1.45–1.22 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 168.8
(C); 151.0 (C); 143.5 (C); 137.0 (C); 132.8 (CH); 131.0 (C); 129.5 (CH); 129.4 (CH); 127.5
(CH); 125.2 (CH); 123.2 (CH); 123.1 (CH); 123.0 (CH); 87.6 (C); 32.4 (CH2); 31.7 (CH2);
29.5–29.3 (4 x CH2); 29.2 (CH2); 29.0 (CH2); 28.9 (CH2); 28.6 (CH2); 22.5 (CH2); 14.4
This article is protected by copyright. All rights reserved.

(CH3). MS (DCI/CH4) m/z: 425.2 [M+]. HRMS (DCI/CH4): for C26H35NO2S [M+]: calcd:
425.2393; found: 425.2389.
(RS)-5-Amino-3-[3-(dodecylsulfanyl)phenyl]-3-hydroxy-2-methyl-2,3-dihydro-1H-
isoindol-1-one (2c)
Reagents: 1-bromo-3-dodecylthiobenzene (1.01 g, 2.8 mmol), dry tetrahydrofuran (5 mL), n-
butyllithium solution (1.1 mL 2.5 M solution in hexanes; 2.8 mmol) and 4-amino-N-methyl-
phtalimide (250 mg, 1.4 mmol). The crude product was purified by flash chromatography
(gradient from 100% dichloromethane to 80/20 dichloromethane) to afford a white solid (254
mg, 40%), mp: 124 °C. TLC Rf: 0.40 (dichloromethane/methanol 95/5). IR (cm-1): 3459,
3353, 3219, 2921, 2851, 1643, 1605, 1500, 1465, 1422, 1396, 1326, 1194, 1171, 1048, 1031,
970, 742, 701. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.35-7.18 (m, 4H); 7.01 (d, J = 7.6
Hz, 1H); 6.82 (s, 1H); 6.56 (dd, J = 2.0 Hz, 8.0 Hz, 1H); 6.35 (d, J = 2.0 Hz 1H); 5.77 (s, 2H);
2.92 (t, J = 7.2 Hz, 2H); 2.59 (s, 3H) 1.56–1.49 (m, 2H); 1.45–1.22 (m, 18H); 0.86 (t, J = 6.8
13
Hz, 3H). C NMR (100 MHz, DMSO-d6) δ (ppm): 167.5 (C); 153.4 (C); 152.1 (C); 142.0
(C); 137.3 (C); 129.7 (CH); 127.3 (CH); 125.2 (CH); 124.0 (CH); 123.2 (CH); 117.7 (C);
114.2 (CH); 107.0 (CH); 89.5 (C); 32.3 (CH2); 31.7 (CH2); 29.5–29.3 (4 x CH2); 29.2 (CH2);
29.0 (CH2); 28.9 (CH2); 28.6 (CH2); 23.7 (CH3); 22.5 (CH2); 14.4 (CH3). MS (DCI/CH4) m/z:
455.3 [M+H+]. HRMS (DCI/CH4): for C27H39N2O2S [M+H+]: calcd: 455.2723; found:
455.2732.
(RS)-1,2-Dihydroxy-1-[3-(dodecylsulfanyl)phenyl]-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridine-3-one (4a)
Reagents: 1-bromo-3-dodecylthiobenzene (435 mg, 1.2 mmol), dry tetrahydrofuran (3 mL), n-
butyllithium solution (0.48 mL 2.5 M solution in hexanes, 1.2 mmol) and of 2-hydroxy-2,5-
diaza-2H-indene-1,3-dione. The crude product was purified by flash chromatography
(gradient from 100% dichloromethane to 80/20 dichloromethane) to afford (159 mg, 30% -
mixture of para and meta regioisomers 70/30). TLC Rf: 0.17 (dichloromethane/methanol
95/5). IR (cm-1): 3104, 2922, 2852, 1702, 1616, 1590, 1465, 1433, 1376, 1259, 1203, 1184,
1135, 1109, 1075, 973, 953, 902, 863, 841, 781, 745, 697, 653, 611. MS (ESI) m/z: 443.3
[M+H+]. HRMS (ESI): for C25H35N2O3S [M+H+]: calcd: 443.2367; found: 443.2368
This article is protected by copyright. All rights reserved.

Separation of p-4a and m-4a by liquid chromatography
Both compounds, para and meta, were isolated using a HPLC preparative chromatography
system (Waters autopurification system), a 2-ethyl-pyridine column 250 x 10.0 mm 5µm and
8% iPrOH / 92% CH2Cl2 as eluant. Isocatric elution was performed at 6.0 mL/min and
absorbance was followed at λ = 254.0 nm. Both regioisomers p-4a and m-4a were separated
with respective retention times of tR1 = 12.4 min and tR2 = 13.2 min.
(RS)-1,2-Dihydroxy-1-[3-(dodecylsulfanyl)phenyl]-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridine-3-one (p-4a)
1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.13 (s, 1H), 8.93 (s, 1H); 8.77 (d, J = 5.1 Hz,
1H), 7.62 (s, 1H); 7,40 (dd, J = 0.9 Hz, 4.6 Hz, 1H); 7.34–7.26 (m, 2H); 7.17–7.11 (m, 1H);
2.94 (t, J = 7.2 Hz, 2H); 1.60–1.48 (m, 2H); 1.40–1.16 (m, 18H); 0.87 (t, J = 6.3 Hz, 3H). 13C
NMR (100 MHz, DMSO-d6) δ (ppm): 161.4 (C); 154.4 (C); 153.8 (CH); 144.4 (CH); 139.2
(C); 137.5 (C); 129.7 (CH); 128.1 (CH); 125.7 (CH); 124.8 (C); 123.7 (CH); 118.1 (CH); 89.7
(C); 32.3 (CH2); 31.8 (CH2); 29.5-29.4 (4 x CH2); 29.2 (CH2); 29.0 (CH2); 28.9 (CH2); 28.6
(CH2); 22.6 (CH2); 14.4 (CH3).
(RS)-2,3-Dihydroxy-3-(3-(dodecylsulfanyl)phenyl)-2,3-dihydro-1H-pyrrolo[3,4-
c]pyridin-1-one (m-4a)
1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.25 (s, 1H), 8.78 (d, J = 5.1 Hz, 1H); 8.60 (s,
1H), 7.73 (d, J = 4.8 Hz, 1H); 7.62 (s, 1H); 7.36–7.25 (m, 3H); 7.18–7.13 (m, 1H); 2.94 (t, J =
7.2 Hz, 2H); 1.61–1.48 (m, 2H); 1.42–1.16 (m, 18H); 0.87 (t, J = 6.6 Hz, 3H). 13C NMR (100
MHz, DMSO-d6) δ (ppm): 160.9 (C); 151.2 (CH); 144.7 (CH); 140.9 (C); 139.6 (C); 137.4
(C); 137.0 (C); 129.7 (CH); 128.1 (CH); 125.7 (CH); 123.7 (CH); 117.0 (CH); 89.8 (C); 32.3
(CH2); 31.8 (CH2); 29.5-29.4 (4 x CH2); 29.2 (CH2); 29.0 (CH2); 28.9 (CH2); 28.6 (CH2); 22.6
(CH2); 14.4 (CH3).
(RS)-2,3-Dihydroxy-3-[3-(dodecylsulfanyl)phenyl]-2,3-dihydro-1H-isoindol-1-one (4b)
Reagents: 1-bromo-3-dodecylthiobenzene (879 mg, 2.45 mmol), dry tetrahydrofuran (5 mL),
n-butyllithium (0.98 mL, 2.5 M solution in hexanes; 2.45 mmol) and N-hydroxyphtalimide
(200 mg, 1.23 mmol). The crude product was purified by flash chromatography (gradient
from 80/20 dichloromethane/methanol to 50/50) to afford a white solid (239 mg, 44%), mp:
145 °C. TLC Rf: 0.17 (hexane/ethyl acetate 60/40). IR (cm-1): 3179, 2953, 2920, 2850, 1686,
1617, 1588, 1467, 1432, 1269, 1230, 1202, 1186, 1141, 1095, 1066, 972, 943, 902, 864, 818,
This article is protected by copyright. All rights reserved.

1
779, 768, 736. H NMR (400 MHz, DMSO-d6) δ (ppm): 9.89 (s, 1H), 7.69 (d, J = 7.2 Hz,
1H); 7.55 (td, J = 1.2 Hz, 7.8 Hz, 1H); 7.50 (td, J = 1.2 Hz, 7.2 Hz, 1H); 7.33 (s, 1H); 7.31-
7.21 (m, 4H); 7.10 (dt, J = 1.6 Hz, 7.6 Hz, 1H); 2.91 (t, J = 7.6 Hz, 2H); 1.57-1.47 (m, 2H);
1.39-1.16 (m, 18H); 0.85 (t, J = 6.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm):
163.04 (C); 146.8 (C); 140.8 (C); 137.0 (C); 132.8 (CH); 129.7 (CH); 129.4 (CH); 129.1 (C);
127.7 (CH); 125.8 (CH); 123.8 (CH); 123.1 (CH); 122.8 (CH); 90.0 (C); 32.3 (CH2); 31.7
(CH2); 29.5 (CH2); 29.4 (CH2); 29.3 (CH2); 29.2 (CH2); 29.0 (CH2); 28.9 (CH2); 28.6 (CH2);
22.5 (CH2); 14.4 (CH3). MS (DCI/CH4) m/z: 464.2 [M+Na+]. HRMS (DCI/CH4): for
C25H35NO3S.Na [M+Na+]: calcd: 464.2234; found: 464.2235.
(RS)-1-[3-(Dodecylsulfanyl)phenyl]-1-hydroxy-2-methoxy-1,2-dihydro-3H-pyrrolo[3,4-c]
pyridin-3-one (5a)
Reagents: 1-bromo-3-dodecylthiobenzene (200 mg, 0.56 mmol), dry tetrahydrofuran (1.5
mL), n-butyllithium solution (0.22 mL 2.5 M solution in hexanes; 0.56 mmol) and 2-
methoxy-2,5-diaza-2H-indene-1,3-dione (50 mg, 0.28 mmol). The crude product was purified
by flash chromatography (gradient from 100% dichloromethane to 90/10
dichloromethane/methanol) to afford an oil (45 mg, 35% - mixture of para and meta 70/30).
A little quantity of the para compound was isolated for characterization and biological assay.
TLC Rf: 0.36 (dichloromethane/methanol 95/5). IR (cm-1) for the p/m mixture in a ratio
50/50: 3249, 2922, 2851, 1713, 1612, 1589, 1459, 1427, 1341, 1255, 1201, 1110, 1075, 1004,
960, 897, 856, 781, 743, 694. For the pure para compound 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 8.98 (s, 1H); 8.80 (d, J = 4.8 Hz, 1H) 7.86 (s, 1H); 7.42 (dd, J = 1.0 Hz, 5.0 Hz, 1H);
7.35-7.25 (m, 2H); 7.09 (dt, J = 1.6 Hz, 6.8 Hz, 1H); 3.85 (s, 3H); 2.92 (t ,J = 7.4 Hz, 2H);
1.57-1.48 (m, 2H); 1.37-1.18 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 161.4 (C); 154.4 (CH); 154.4 (C); 144.8 (CH); 138.8 (C); 137.6 (C);
129.8 (CH); 128.3 (CH); 125.4 (CH); 124.0 (C); 126.6 (CH); 118.0 (CH); 90.2 (C); 65.5
(CH3); 32.2 (CH2); 31.7 (CH2); 29.5 (CH2); 29.5 (CH2); 29.4 (CH2); 29.3 (CH2); 29.2 (CH2);
29.0 (CH2); 28.9 (CH2); 28.5 (CH2); 22.5 (CH2); 14.4 (CH3). MS (DCI/CH4) m/z: 456.6 [M+].
HRMS (DCI/CH4): for C26H36N2O3S [M+]: calcd: 456.2452; found: 456.2447.
This article is protected by copyright. All rights reserved.

(RS)-3-[3-(Dodecylsulfanyl)phenyl]-3-hydroxy-2-methoxy-2,3-dihydro-1H-isoindol-1-one
(5b)
Reagents: 1-bromo-3-dodecylthiobenzene (660 mg, 1.8 mmol), dry tetrahydrofuran (5 mL), n-
butyllithium solution (0.74 mL 2.5 M solution in hexanes; 1.8 mmol) and 2-methoxy-2H-
isoindole-1,3-dione (164 mg, 0.92 mmol). The crude product was purified by flash
chromatography (gradient from 80/20 hexane/ethyl acetate to 60/40) to afford a white solid
(101 mg, 24%), mp: 97 °C. TLC Rf: 0.29 (hexane/ethyl acetate 70/30). IR (cm-1): 3223, 2917,
2849, 1694, 1614, 1588, 1467, 1435, 1357, 1205, 1115, 1093, 1061, 1023, 1005, 963, 953,
901, 884, 857, 789, 766, 738, 719, 691. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.75 (d, J =
8.8 Hz, 1H); 7.66-7.50 (m, 3H); 7.36-7.20 (m , 3H); 7.07 (dt, J = 1.8 Hz, 7.2 Hz); 3.85 (s,
1H); 2.90 (t, J = 7.2 Hz, 2H); 1.55-1.46 (m, 2H); 1.35-1.18 (m, 18H); 0.85 (t, J = 6.6 Hz, 3H).
13C NMR (100 MHz, DMSO-d6) δ (ppm): 163.1 (C); 146.8 (C); 140.4 (C); 137.2 (C); 133.7
(CH); 130.0 (CH); 129.6 (CH); 128.2 (C); 127.8 (CH); 125.5 (CH); 123.6 (CH); 123.2 (2 x
CH); 90.5 (C); 65.3 (CH3); 32.3 (CH2); 31.7 (CH2); 29.5 (CH2); 29.5 (CH2); 29.4 (CH2); 29.3
(CH2); 29.2 (CH2); 29.0 (CH2); 28.9 (CH2); 28.5 (CH2); 22.5 (CH2); 14.4 (CH3). MS
(DCI/CH4) m/z: 455.2 [M+]. HRMS (DCI/CH4): for C27H37NO3S [M+]: calcd: 455.2493;
found: 455.2494.
(RS)-1-[3-(Dodecylsulfonyl)phenyl]-1-hydroxy-1,2-dihydro-3H-pyrrolo[3,4-c]pyridin-3-
one 5-oxide (3)
To a solution of (RS)-1-[3-(dodecylsulfanyl)phenyl]-1-hydroxy-1,2-dihydro 3H-pyrrolo[3,4-
c]pyridin-3-one (63 mg, 0.15 mmol, 1 eq) in dichloromethane (5 mL) was added at room
temperature 3-chloro-peroxobenzoic acid (mCPBA). The mixture was stirred for 3 hours then
10 mL of brine were added and the entitled product was extracted with dichloromethane (3 x
10 mL). The organic phases were combined, dried over sodium sulfate and concentrated
under vacuum. The residue was purified by flash chromatography (gradient from 100%
dichloromethane to 80/20 dichloromethane) to furnish a white solid (51 mg, 72%) mp: 175
°C. TLC Rf: 0.21 (dichloromethane/methanol 95/5). IR (cm-1): 3117, 3036, 2957, 2922, 2852,
2806, 1708, 1445, 1396, 1323, 1301, 1220, 1136, 1095, 1058, 1022, 864. 1H NMR (400 MHz,
DMSO-d6) δ (ppm): 9.84 (s, 1H); 8.45 (s, 1H); 8.30 (dd, J = 1.6 Hz, 6.4 Hz, 1H); 8.05 (t, J =
1.6 Hz); 7.89 (d, J = 8.0 Hz, 1H); 7.83 (d, J = 8.4 Hz, 1H); 7.67 (t, J = 7.8 Hz, 1H); 7.52 (s,
1H); 7.42 (d, J = 6.8 Hz, 1H); 3.30 (t, J = 8.4 Hz, 2H); 1.57-1.47 (m, 2H); 1.32-1.15 (m,
13
18H); 0.85 (t, J = 6.8 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ (ppm): 165.2 (C); 145.5
(C); 143.0 (CH); 142.3 (C); 140.0 (C); 134.1 (CH); 131.5 (CH); 130.9 (C); 130.3 (CH); 128.3
This article is protected by copyright. All rights reserved.

(CH); 125.1 (CH); 121.1 (CH); 87.0 (C); 54.9 (CH2); 31.7 (CH2); 29.4 (2 x CH2); 29.3 (CH2);
29.1 ( 2 x CH2); 28.8 (CH2); 27.8 (CH2); 22.5 (CH2); 14.4 (CH3). MS (ESI) m/z: 475.2
[M+H+]. HRMS (ESI): for C25H35N2O5S: calcd: 475.2267; found: 475.2269.
General protocol for the synthesis of pyridazinone containing derivatives
Corresponding N-methoxyisoindolinone type compound (1 eq), ethanol (10 mL), and
hydrazine hydrate (15 eq) were added into a round-bottom flask equipped with a condenser. A
nitrogen balloon was attached to the top of the condenser. After refluxing overnight, the
reaction was cooled to room temperature. An off-white solid precipitated out of the solution.
After cooling to 0 °C, water was added slowly and the compound was extracted with
dichloromethane (3 x 10 mL). The residue was purified by flash chromatography to furnish
the entitled compound.
1-[3-(Dodecylsulfanyl)phenyl]-pyrido[3,4-d]pyridazin-4(3H)-one (6a)
Reagents: (RS)-1-[3-(dodecylthio)phenyl]-1-hydroxy-2-methoxy-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridin-3-one (5a) and its meta regioisomer (in a ratio para/meta 60/40, 64 mg, 0.14 mmol),
ethanol (10 mL) and hydrazine hydrate (0.1 mL, 2.09 mmol).
The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (57 mg, 96% -
mixture of para and meta compounds 60/40). The para compound was isolated for
characterization and biological activity evaluation. TLC Rf: 0.56 for the para compound and
0.66 for the meta compound (dichloromethane/methanol 95/5). IR (cm-1) for the mixture of
para/meta 60/40: 2918, 2850, 1707, 1666, 1599, 1566, 1466, 1410, 1333, 1318, 1277, 1217,
1176, 1134, 1101, 1082, 1044, 998, 885, 853, 806, 783, 701, 681, 662, 610, 557. For the pure
para compound 1H NMR (400 MHz, DMSO-d6) δ (ppm): 13.19 (s, 1H); 9.52 (s, 1H); 9.01 (d,
J = 5.2 Hz, 1H); 7.54 (d, J = 5.6 Hz, 1H); 7.53-7.43 (m, 3H); 7.42-7.37 (m, 1H); 3.01 (t, J =
7.2 Hz, 2H); 1.65-1.55 (m, 2H); 1.44-1.15 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100
MHz, DMSO-d6) δ (ppm): 158.8 (C); 153.5 (CH); 149.9 (CH); 145.2 (C); 138.0 (C); 135.1
(C); 134.3 (C); 129.7 (CH); 128.9 (CH); 128.3 (CH); 126.7 (CH); 122.3 (C); 119.1 (CH); 32.2
(CH2); 31.7 (CH2); 29.5 (CH2); 29.4 (CH2); 29.4 (CH2); 29.3 (CH2); 29.1 (CH2); 28.9 (CH2);
28.9 (CH2); 28.5 (CH2); 22.5 (CH2); 14.4 (CH3). MS (ESI) m/z: 424.2 [M+H+]. HRMS (ESI):
for C25H34N3OS [M+H+]: calcd: 424.2421; found: 424.2423.
This article is protected by copyright. All rights reserved.

4-[3-(Dodecylsulfanyl)phenyl]phthalazin-1(2H)-one (6b)
Reagents: (RS)-3-[3-(Dodecylsulfanyl)phenyl]-3-hydroxy-2-methoxy-2,3-dihydro-1H-
isoindol-1-one (5b) (57 mg, 0.14 mmol), ethanol (10 mL) and hydrazine hydrate (0.11 mL,
2.13 mmol).
The crude product was purified by flash chromatography (isocratic 80/20 hexane/ethyl) to
afford a white solid (15 mg, 26%). TLC Rf: 0.72 (dichloromethane/methanol 95/5). IR (cm-1):
2914, 2849, 1683, 1567, 1339, 1223, 1155, 995, 896, 782, 721, 697, 685. 1H NMR (500 MHz,
CDCl3) δ (ppm): 12.86 (s, 1H); 8.39-8.31 (m, 1H); 7.90 (t, J = 5.2 Hz, 2H); 7.68-7.63 (m,
1H); 7.51-7.44 (m, 3H); 7.38 (d, J = 7.2 Hz, 1H); 3.01 (t, J = 7.2 Hz, 2H); 1.61 (m, 2H); 1.42-
1.35 (m, 2H); 1.30-1.15 (m, 16H); 0.84 (t, J = 6.8 Hz, 3H). 13C NMR (125 MHz, DMSO-d6)
δ (ppm): 159.7 (C); 146.5 (C); 137.7 (C); 136.3 (C); 134.1 (CH); 132.2 (CH); 129.6 (CH);
129.4 (C); 128.7 (CH); 128.6 (CH); 128.3 (C); 126.9 (CH); 126.8 (CH); 126.6 (CH); 32.2
(CH2); 31.7 (CH2); 29.5 (3 x CH2); 29.4 (CH2); 29.3 (CH2); 29.1 (CH2); 28.9 (CH2); 28.5
(CH2); 22.5 (CH2); 14.4 (CH3). MS (DCI/CH4) m/z: 423.6 [M+H+]. HRMS (DCI/CH4): for
C26H34N2OS [M+H+]: calcd: 423.2480; found: 423.2470.
General protocol for addition of ethynyl-TMS group
To a solution of ethynyl-trimethylsilane (2 eq) in dry tetrahydrofuran, under argon atmosphere
and at -40 °C was added n-butyllithium 2.5M solution (2 eq). After addition, the temperature
was maintained at -50 °C for 60 min. Then the temperature was decreased at -78 °C and upon
this solution was added a solution of the corresponding (aza)-isoindolinone starting material
(1 eq) in dry tetrahydrofuran. The reaction mixture was stirred for 30 minutes at -78 °C and
was warmed to room temperature for 30 minutes more. The reaction was quenched with a
saturated ammonium chloride solution and the product was extracted with ethyl acetate. The
organic phases were combined, dried over sodium sulfate and concentrated under vacuum.
The residue was purified by flash chromatography to furnish the entitled compound.
(RS)-1-Hydroxy-1-[2-(trimethylsilyl)ethynyl]-1,2-dihydro-3H-pyrrolo[3,4-c]pyridine-3-
one (7a)
Reagents: ethynyltrimethylsilane (663 mg, 0.95 mL, 6.65 mmol), dry tetrahydrofuran (10
mL), n-butyllithium solution (2.7 mL 2.5M solution in hexanes; 6.65 mmol) and 3,4-
pyridinedicarboximide (500 mg, 3.37 mmol). The crude product was purified by flash
chromatography (gradient from 100% dichloromethane to 90/10 dichloromethane/methanol)
to afford a white solid (407 mg, 49%), mp: 170 °C. TLC Rf: 0.31 (dichloromethane/methanol
This article is protected by copyright. All rights reserved.

90/10). IR (cm-1): 3203, 3067, 2958, 2897, 2735, 1710, 1690, 1616, 1556, 1488, 1459, 1436,
1381, 1338, 1286, 1213, 1168, 1125, 1049, 837, 798, 759. 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 9.70 (s, 1H); 8.89-8.86 (m, 2H); 7.70 (dd, J = 1.2 Hz, 5.2 Hz, 1H); 7.39 (s, 1H); 0.15
13
(s, 9H). C NMR (100 MHz, DMSO-d5) δ (ppm): 166.6 (C); 156.2 (C); 154.1 (CH); 145.1
(CH); 125.9 (C); 118.1 (CH); 103.0 (C); 88.5 (C); 79.3 (C); 0.0 (3 x CH3). MS (DCI/CH4)
m/z: 247.1 [M+H+]. HRMS (DCI/CH4): for C12H14N2O2Si [M+H+]: calcd: 247.0905; found:
247.0903.
(RS)-3-Hydroxy-3-[2-(trimethylsilyl)ethynyl]-2,3-dihydro-1H-isoindol-1-one (7b)
Reagents: ethynyltrimethylsilane (133 mg, 0.19 mL, 1.36 mmol), dry tetrahydrofuran (2.5
mL), n-butyllithium solution (0.68 mL 2.5 M solution in hexanes, 1.36 mmol) and phtalimide
(100 mg, 0.68 mmol).
The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 85/15 dichloromethane/methanol) to afford a white solid (97 mg, 58%),
mp: 168 °C. TLC Rf: 0.26 (dichloromethane/methanol 95/5). IR (cm-1): 3310, 3219, 2959,
2900, 2799, 1708, 1689, 1615, 1471, 1425, 1347, 1321, 1272, 1249, 1210, 1161, 1128, 1094,
1
1061, 1047, 1020, 839, 756, 694. H NMR (400 MHz, CDCl3) δ (ppm): 7.80 (d, J = 7.6 Hz,
1H); 7.74-7.66 (m, 2H); 7.58 (t, J = 7.2 Hz, 1H); 0.20 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
(ppm): 169.3 (C); 146.9 (C); 133.5 (CH); 130.4 (CH); 129.3 (C); 123.8 (CH); 122.8 (CH);
100.5 (C); 90.2 (C); 53.4 (C); 0.46 (3 x CH3). MS (DCI/CH4) m/z: 246.1 [M+H+]. HRMS
(DCI/CH4): for C13H15NO2Si [M+H+]: calcd: 246.0947; found: 246.0950.
General protocol for the Huisgen type cycloaddition reaction
To a solution of the corresponding (trimethylsilyl)ethynylisoindolinone intermediate (1.0 eq)
and alkyl azide with a defined number of carbons (1.1 eq) in methanol were added copper(II)
sulfate pentahydrate (0.2 eq), sodium ascorbate (0.4 eq) and finally tetrabutylammonium
fluoride (TBAF) solution (1.0 eq) to start the click chemistry reaction. The mixture was
stirred for 16 hours and then was concentrated under vacuum and directly purified by flash
chromatography to furnish the entitled compound.
For starting compounds with a pyridine moiety, an additional washing step with a saturated
solution of ethylenediaminetetraacetic acid (EDTA) was needed to remove copper.
This article is protected by copyright. All rights reserved.

(RS)-1-Hydroxy-1-(1-octyl-1H-1,2,3-triazol-4-yl)-1-hydroxy-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridin-3-one (8a)
Reagents: (RS)-3-hydroxy-3-[2-(trimethylsilyl)ethynyl]-2,3-dihydro-1H-isoindol-1-one (65
mg, 0.32 mmol), 1-octyl-azide (55 mg, 0.36 mmol), methanol (10 mL), copper(II) sulfate
pentahydrate (16 mg, 0.04 mmol), sodium ascorbate (26 mg, 0.13 mmol, 0.4 eq) and
tetrabutylammonium fluoride solution (0.32 mL of a 1.0 M solution in tetrahydrofuran, 0.32
mmol). The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 90/10 dichloromethane/methanol) to afford (86 mg, 82%), mp: 175 °C.
TLC Rf: 0.42 (dichloromethane/methanol 90/10). IR (cm-1): 3120, 3072, 2946, 2917, 2849,
2776, 1725, 1616, 1492, 1464, 1436, 1395, 1377, 1345, 1288, 1260, 1230, 1156, 1141, 1081,
1052, 1034, 929, 839, 795, 734. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.53 (s, 1H); 8.87
(s, 1H); 8.78 (d, J = 5.2 Hz, 1H); 8.25 (s, 1H); 7.53 (dd, J= 1.0 Hz, 5.0 Hz, 1H); 7.21 (s, 1H);
13
4.34 (t, J = 7 Hz, 2H); 1.84-1.75 (m, 2H); 1.30-1.17 (m, 10H); 0.85 (t, J = 6.8 Hz, 3H). C
NMR (100 MHz, DMSO-d6) δ (ppm): 167.2 (C); 157.6 (C); 153.4 (CH); 147.9 (C); 144.9
(CH); 126.8 (C); 123.4 (CH); 118.6 (CH); 84.1 (C); 49.9 (CH2); 31.6 (CH2); 30.1 (CH2); 29.0
(CH2); 28.8 (CH2); 26.3 (CH2); 22.5 (CH2); 14.4 (CH3). MS (DCI/CH4) m/z: 330.2 [M+H+].
HRMS (DCI/CH4): for C17H24N5O2 [M+H+]: calcd: 330.1941; found: 330.1930.
(RS)-1-(1-Decyl-1H-1,2,3-triazol-4-yl)-1-hydroxy1-1,2-dihydro-3H-pyrrolo [3,4c]pyridin-
3-one (9a)
Reagents: (RS)-3-hydroxy-3-[2-(trimethylsilyl)ethynyl]-2,3-dihydro-1H-isoindol-1-one (70
mg, 0.32 mmol), 1-decyl-azide (65 mg, 0.36 mmol), methanol (10 mL), copper(II) sulfate
pentahydrate (16 mg, 0.04 mmol), sodium ascorbate (26 mg, 0.13 mmol) and
tetrabutylammonium fluoride solution (0.32 mL of a 1.0 M solution in tetrahydrofuran, 0.32
mmol).
The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (107 mg, 94%),
mp: 168 °C. TLC Rf: 0.44 (dichloromethane/methanol 90/10). IR (cm-1): 3120, 3072, 2916,
2849, 1725, 1615, 1492, 1462, 1435, 1345, 1284, 1156, 1140, 1082, 1052, 1034, 930, 839,
1
8223, 796, 758, 736. H NMR (400 MHz, DMSO-d6) δ (ppm): 9.53 (s, 1H); 8.87 (s, 1H);
8.78 (d, J = 5.2 Hz, 1H); 8.25 (s, 1H); 7.53 (dd, J= 1.0 Hz, 5.0 Hz, 1H); 7.21 (s, 1H); 4.34 (t, J
= 7 Hz, 2H); 1.84-1.75 (m, 2H); 1.30-1.17 (m, 14H); 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100
MHz, DMSO-d6) δ (ppm): 167.2 (C); 157.6 (C); 153.4 (CH); 147.9 (C); 144.9 (CH); 126.8
(C); 123.4 (CH); 118.6 (CH); 84.1 (C); 49.9 (CH2); 31.6 (CH2); 30.1 (CH2); 29.4 (CH2); 29.3
This article is protected by copyright. All rights reserved.

(CH2); 29.1 (CH2); 28.8 (CH2); 26.3 (CH2); 22.5 (CH2); 14.4 (CH3). MS (DCI/CH4) m/z:
358.2 [M+H+]. HRMS (DCI/CH4): for C19H28N5O2 [M+H+]: calcd: 358.2243; found:
358.2243.
(RS)-1-(1-dodecyl-1H-1,2,3-triazol-4-yl)-1-hydroxy1-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridin-3-one (10a)
Reagents: (RS)-3-hydroxy-3-[2-(trimethylsilyl)ethynyl]-2,3-dihydro-1H-isoindol-1-one (80
mg, 0.32 mmol), 1-dodecyl-azide (76 mg, 0.36 mmol), methanol (10 mL), copper(II) sulfate
pentahydrate (16 mg, 0.04 mmol), sodium ascorbate (26 mg, 0.13 mmol) and
tetrabutylammonium fluoride solution (0.32 mL of a 1.0 M solution in tetrahydrofuran, 0.32
mmol).
The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (107 mg, 87%),
mp: 168 °C. TLC Rf: 0.46 (dichloromethane/methanol 90/10). IR (cm-1): 3120, 3073, 2917,
2849, 1726, 1615, 1463, 1436, 1344, 1285, 1260, 1231, 1216, 1155, 1141, 1081, 1052, 1034,
929, 821, 796, 759, 734, 661. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.52 (s, 1H); 8.87 (s,
1H); 8.78 (d, J = 5.2 Hz, 1H); 8.25 (s, 1H); 7.53 (dd, J= 1.0 Hz, 5.0 Hz, 1H); 7.20 (s, 1H);
13
4.33 (t, J = 7 Hz, 2H); 1.84-1.75 (m, 2H); 1.30-1.17 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). C
NMR (100 MHz, DMSO-d6) δ (ppm): 167.2 (C); 157.6 (C); 153.4 (CH); 147.9 (C); 144.9
(CH); 126.8 (C); 123.4 (CH); 118.6 (CH); 84.1 (C); 49.9 (CH2); 31.6 (CH2); 30.1 (CH2); 29.5
(CH2); 29.4 (CH2); 29.4 (CH2); 29.3 (CH2); 29.1 (CH2); 28.8 (CH2); 26.3 (CH2); 22.5 (CH2);
14.4 (CH3). MS (DCI/CH4) m/z: 386.2 [M+H+]. HRMS (DCI/CH4): for C21H32N5O2 [M+H+]:
calcd: 386.2552; found: 386.2556.
(RS)-3-Hydroxy-3-(1-octyl-1H-1,2,3-triazol-4-yl)-2,3-dihydro-1H-isoindol-1-one (8b)
Reagents: (RS)-1-hydroxy-1-[2-(trimethylsilyl)ethynyl]-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridin-3-one (52 mg, 0.21 mmol), 1-octyl-azide (36 mg, 0.23 mmol), methanol (5 mL),
copper(II) sulfate pentahydrate (10 mg, 0.04 mmol), sodium ascorbate (17 mg, 0.08 mmol)
and tetrabutylammonium fluoride solution (0.21 mL of a 1.0 M solution in tetrahydrofuran,
0.21 mmol). The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (28 mg, 40%),
mp: 132 °C. TLC Rf: 0.54 (dichloromethane/methanol 90/10). IR (cm-1): 3289, 3174, 3129,
2923, 2853, 1683, 1614, 1468, 1424, 1378, 1353, 1316, 1266, 1222, 1199, 1179, 1143, 1081,
1061, 1048, 1025, 918, 855, 762, 737, 718. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.25 (s,
This article is protected by copyright. All rights reserved.

1H); 8.18 (s, 1H); 7.65-7.45 (m, 4H); 6.91 (s, 1H); 4.32 (t, J = 7.0 Hz, 2H); 1.84 (m, 2H);
1.30-1.20 (m, 10H); 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 168.5
(C); 149.9 (C); 149.1 (C); 132.7 (CH); 131.3 (C); 129.6 (CH); 123.5 (CH); 123.1 (CH); 122.8
(CH); 84.2 (C); 49.8 (CH2); 31.6 (CH2); 30.2 (CH2); 29.0 (CH2); 28.8 (CH2); 26.3 (CH2); 22.5
(CH2); 14.4 (CH3). MS (DCI/CH4) m/z: 329.2 [M+H+]. HRMS (DCI/CH4): for C18H25N4O2
[M+H+]: calcd: 329.1986; found: 329.1978.
(RS)- 3-(1-Decyl-1H-1,2,3-triazol-4-yl)- 3-hydroxy-2,3-dihydro-1H-isoindol-1-one (9b)
Reagents: (RS)-1-hydroxy-1-[2-(trimethylsilyl)ethynyl]-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridin-3-one (100 mg, 0.41 mmol), 1-decyl-azide (75 mg, 0.45 mmol), methanol (10 mL),
copper(II) sulfate pentahydrate (20 mg, 0.08 mmol), sodium ascorbate (32 mg, 0.16 mmol)
and tetrabutylammonium fluoride solution (0.41 mL of a 1.0 M solution in tetrahydrofuran,
0.41 mmol). The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (102 mg, 70%),
mp: 145 °C. TLC Rf: 0.43 (dichloromethane/methanol 90/10). IR (cm-1): 3292, 3179, 3130,
2920, 2851, 1684, 1614, 1470, 1424, 1352, 1315, 1266, 1222, 1190, 1143, 1081, 1048, 1026,
1
917, 880, 854, 803, 761, 737, 720. H NMR (400 MHz, DMSO-d6) δ (ppm): 9.25 (s, 1H);
8.18 (s, 1H); 7.65-7.45 (m, 4H); 6.91 (s, 1H); 4.32 (t, J = 7.0 Hz, 2H); 1.80 (m, 2H); 1.31-1.18
(m, 14H); 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 168.5 (C);
149.9 (C); 149.1 (C); 132.7 (CH); 131.3 (C); 129.6 (CH); 123.5 (CH); 123.1 (CH); 122.8
(CH); 84.2 (C); 49.8 (CH2); 31.7 (CH2); 30.2 (CH2); 29.4 (CH2); 29.3 (CH2); 29.1 (CH2); 28.8
(CH2); 26.3 (CH2); 22.5 (CH2); 14.4 (CH3). MS (DCI/CH4) m/z: 357.2 [M+H+]. HRMS
(DCI/CH4): for C20H28N4O2 [M+H+]: calcd: 357.2304; found: 357.2291.
(RS)-3-(1-Dodecyl-1H-1,2,3-triazol-4-yl)- 3-hydroxy-2,3-dihydro-1H-isoindol-1-one (10b)
Reagents: (RS)-1-hydroxy-1-[2-(trimethylsilyl)ethynyl]-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridin-3-one (100 mg, 0.41 mmol), 1-dodecyl-azide (95 mg, 0.45 mmol), methanol (10
mL), copper(II) sulfate pentahydrate (20 mg, 0.08 mmol), sodium ascorbate (32 mg, 0.16
mmol) and tetrabutylammonium fluoride solution (0.41 mL of a 1.0 M solution in
tetrahydrofuran, 0.41 mmol).
The crude product was purified by flash chromatography (gradient from 100%
dichloromethane to 90/10 dichloromethane/methanol) to afford a yellow solid (85 mg, 54%),
mp: 145 °C. TLC Rf: 0.41 (dichloromethane/methanol 90/10 ). IR (cm-1): 3292, 3175, 3131
2955, 2919, 2851, 1685, 1614, 1470, 1425, 1377, 1351, 1315, 1265, 1221, 1186, 1143, 1081,
This article is protected by copyright. All rights reserved.

1062, 1048, 916, 880, 855, 803, 761, 737, 721. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.25
(s, 1H); 8.18 (s, 1H); 7.65-7.45 (m, 4H); 6.91 (s, 1H); 4.32 (t, J = 7.0 Hz, 2H); 1.85-1.75 (m,
2H); 1.31-1.18 (m, 18H); 0.85 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm):
168.5 (C); 149.9 (C); 149.1 (C); 132.7 (CH); 131.3 (C); 129.6 (CH); 123.5 (CH); 123.1 (CH);
122.8 (CH); 84.2 (C); 49.8 (CH2); 31.7 (CH2); 30.2 (CH2); 29.5 (CH2); 29.4 (CH2); 29.4
(CH2); 29.3 (CH2); 29.2 (CH2); 28.8 (CH2); 26.3 (CH2); 22.5 (CH2); 14.4 (CH3). MS
(DCI/CH4) m/z: 385.2 [M+H+]. HRMS (DCI/CH4): for C22H33N4O2 [M+H+]: calcd: 385.2598;
found: 385.2604.
Biology
InhA expression and purification
The production and purification of the InhA-6xHis protein from a protease-deficient strain of
E. coli BL21(DE3) transformed with the pHAT5/inhA plasmid were performed as followed. 1
mL of the bacteria was grown in 100 mL of LB medium containing ampicillin (100 mg/mL)
at 37 °C. After 4 h, the solution was re-diluted in 1 L of the same medium and re-grown at
37 °C. When the proper concentration (OD595 = 0.6-0.8) was reached, protein expression was
induced by overnight incubation with 1 mM isopropyl-β-D-galactopyranoside (IPTG) at
20 °C. Cells were harvested by centrifugation at 6,000 g for 30 min at 4 °C. The dry pellet
may be kept at -80°C for several months. Thawed cells (1.5 g) were sonicated in 20 mL lysis
buffer (300 mM NaCl, 10 mM imidazole, 50 mM sodium phosphate buffer, pH 8.0). After
centrifugation at 10,000 g for 45 min at 4 °C, the supernatant was applied onto a nickel-
chelated Hi-Trap HP 1 mL column (GE Healthcare) previously equilibrated with the binding
buffer (50 mM NaCl, 10 mM imidazole, 50 mM sodium phosphate buffer, pH 8,0). First, the
unbound proteins were washed out with 10 column volume of binding buffer, and then a
higher imidazole concentration (25 mM) allowed the elution of non-specifically bound
proteins. The His6-tagged InhA protein was eluted with an imidazole gradient from 25 mM to
300 mM over a range of 20 column volume. Fractions containing the target protein were
pooled, concentrated to 2.0 mL and loaded on a HiLoad 16/60 Superdex 200 column (GE
Healthcare) equilibrated with 150 mM NaCl, 30 mM PIPES, pH 6.8. Samples were analyzed
using SDS-PAGE and Coomassie blue staining and then stored at 4 °C for short term storage
or -80 °C with 20% glycerol for long-term storage.
This article is protected by copyright. All rights reserved.

InhA activity inhibition
Triclosan and NADH were obtained from Sigma-Aldrich. 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 of 1 mL for all kinetic reactions. Kinetic assays were performed
using trans-2-dodecenoyl-coenzyme A (DDCoA) and wild type InhA as previously described.
Briefly, pre-incubation reactions were performed in an aqueous buffer (30 mM PIPES and
150 mM NaCl pH 6.8) containing additionally 100 nM InhA, 250 µM cofactor (NADH) and
the tested compound (50 µM or 10 µM). After 5 min of pre-incubation at 25 °C, addition of
50 µM DDCoA initiated the reaction which was followed at 340 nm (oxidation of NADH).
The inhibitory activity of each derivative was expressed as the percentage of inhibition of
InhA activity (initial velocity of the reaction) with respect to the control reaction without
inhibitor. Triclosan was used as a positive control. All activity assays were performed in
triplicate.
Mycobacterium growth inhibition
H37Rv was used as the reference strain. The strains were grown at 37°C in Middlebrook 7H9
broth (Difco), supplemented with 0.05% Tween 80, or on solid Middlebrook 7H11 medium
(Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC). MICs for the new
compounds were determined by means of the micro-broth dilution method. Dilutions of M.
tuberculosis wild-type (about 105-106 cfu/ml) were streaked onto 7H11 solid medium
containing a range of drug concentrations (0.25 µg/mL to 40 µg/mL). Plates were incubated at
37 °C for about 21 days and the growth was visually evaluated. The lowest drug dilution at
which visible growth failed to occur was taken as the MIC value. Results were expressed as
the average of at least three independent determinations.
Acid mycolic biosynthesis inhibition
INH, 2a and m-4a were added to broth cultures of M. tuberculosis H37Rv during the
exponential growth phase at their MIC (1 µM, 25 µM and 12.5 µM respectively). The cultures
were then incubated at 37 °C for 5 h before the addition of [1-14C]acetate (1 µCi, 17 µM) to
follow the biosynthesis of lipid products. Cells were incubated for 18 h and then harvested.
Fatty and mycolic acids were released from whole bacteria through saponification by
incubating the latter with a mixture of 40% KOH (w/v) in methoxyethanol (1/7) for 6 h at 100
°C. After acidification by addition of 20% H2SO4, lipids including mycolic acids were
extracted with diethyl ether. The crude fatty acids were methylated and methyl ester
This article is protected by copyright. All rights reserved.

derivatives were analyzed by TLC on Silica Gel 60 (Macherey-Nagel) run in dichloromethane
(4) followed by phosphorimaging (Variable Mode Imager Typhoon TRIO, Amersham
Biosciences). The relative percentage of radioactivity incorporated in fatty acids (FA), in
alpha-mycolic acids (α-MA) and in methoxy- and keto-mycolic acids (M+K MA, not
separated during TLC) was determined for each condition using Image Quant 5.1 software.
Docking
Molecular graphics were performed with Accelrys Discovery Studio Visualizer (Accelrys
Software Inc., Discovery Studio Modeling Environment, Release 4.0, San Diego: Accelrys
Software Inc., 2013).
The compounds of this study were thoroughly built and fully optimized before the
docking stage using the PM6 Hamiltonian (5) implemented in the MOPAC 2012 quantum
chemistry package (6). In most cases a chiral center was present, both configurations R
and S were taken into account.
The structure 2H7M from the Brookhaven Protein Data Bank was chosen for molecular
docking. The NADH cofactor was kept into the InhA structure although 2H7M ligand and
water molecules were removed. The MolProbity software (7) was used to assign the
position of hydrogen atoms and the protonation state of histidines in the InhA protein.
The docking calculations were performed with the docking program Autodock Vina
v1.1.2 (8). The Autodock graphical interface AutoDockTools (9) was used to keep polar
hydrogens and add partial charges to the proteins using the Kollman United charges. The
search space was included in a box of 24x24x24 Å, centred on the binding site of the
ligands and nicotinamide cofactor. The side chains of the six residues, Met103, Phe149,
Tyr158, Met161, Met199 and Leu218, which surround the catalytic cavity, were allowed
to rotate during the docking. Flexible torsions of side chains and of ligands were assigned
with Autotors, an auxiliary module of AutoDockTools. For each calculation, fifteen poses
ranked according to the scoring-function of Autodock Vina were obtained (VINA score).
The docking procedure was followed by a post-docking optimization with the PM6-DH2
(10) method implemented into the MOPAC 2012 quantum mechanical package (6). For
that purpose, the docking poses were merged into the cavities of the corresponding InhA
and the complexes were submitted to a minimization stage in which the ligand, a set of 12
residues (Met103, Phe149, Met155, Tyr158, Met161, Thr196, Met199, Ile202, Leu207,
Ile215, Ley218 and Glu219) delimiting the binding pocket and the totality of hydrogen
atoms were fully energy-minimized. The remaining residues were kept frozen. The
This article is protected by copyright. All rights reserved.

localized molecular orbital method (keyword MOZYME) which was developed to enable
calculation on large systems such as enzymes was applied (11). The effects of the
solvation interface were simulated with the continuum model COSMO (12) with a
dielectric constant set to 78.4. The interaction energies (Eopt) were calculated with the
equation: ΔE = ΔfH (ligand-receptor complex) - ΔfH (ligand alone) - ΔfH (receptor alone).
Results and discussion
Chemistry
Based on our previous results showing that the C7 hydroxyl group and the lipophilic
thiododecyl chain bound to the ring C of the azaisoindolinone scaffold are essential elements
for the InhA inhibitory activity, the first strategy adopted in this work was to maintain intact
these two elements and to carry out modifications on the azaisoindolinone framework namely
on the ring A and on the hemiamidal cycle B. For this purpose, the pyridine ring (ring A) of
the azaisoindolinone scaffold was substituted by a phenyl, an aminophenyl and a N-oxide
pyridyl cycles (the latter coupled with a sulfur oxidation of the chain giving the corresponding
sulfone). The hemiamidal cycle B was also modified at the N8 position by subsequent
introduction of a hydroxyl or a methoxy group. In a second stage, replacement of the
dodecylthiophenyl motif (ring C) by an alkyl triazole ring was performed via Huisgen-type
cycloaddition. Finally, a ring extension of the hemiamidal 5-membered ring to a 6-membered
pyridazinone-type ring was also performed. These chemical modulations are summarized in
Figure 2.
Figure 2. Structural evolution of azaisoindolinone scaffold 2a.
The chemical synthetic routes of target compounds are presented in Scheme 1. The hit
compound 2a was synthesized according to our previously described procedure (2), which is
This article is protected by copyright. All rights reserved.

based on a nucleophilic addition of the dodecylthiophenyl lithium intermediate over the
commercial 3,4-pyridine-dicarboximide reagent (1a). The introduction of the N-oxide motif
into the hit molecule 2a was carried out using mCPBA as oxidant (13). This reaction was
accompanied by a subsequent oxidation of the sulfur atom leading to the sulfone 3. The
general synthetic strategy to prepare new compounds 2b, 2c and 4b takes advantage of the
addition of dodecylthiophenyl lithium intermediate upon dicarboximide starting materials
embedding suitable functionalities (1b, 1c and 1d respectively). For the preparation of N-
hydroxy or N-methoxy-(aza)isoindolinone derivatives 4a, 5a and 5b, the synthesis initially
consisted in the reaction of hydroxylamine or methoxylamine with (aza)phthalic anhydride 1e
or 1f followed by addition of the aryllithium reagent (14). As previously observed for
compound 1a (15), the addition of the alkylthiophenyl group occurs preferentially on the
a
para carbonyl group and the regioisomers formed are readily separated by flash
chromatography. Except for 4a, whose the separation of isomers (ratio 70:30 para:meta) was
not successful in these conditions and a HPLC preparative chromatography with a 2-ethyl-
pyridine column was necessary. For 1c, the exocyclic amine nitrogen atom oriented the
reaction in a different way and promoted the addition on the meta carbonyl group with respect
to the amino group. On the other hand, the replacement of the pyridine moiety by a phenyl
ring suppresses the dissymmetry of the molecule discarding the formation of regioisomers
during functionalization of the carbonyl group.
For the preparation of triazole compounds, addition of the lithium (trimethylsilyl)acetylide
over the starting material 1a and 1b was carried out. After unsuccessful attempts on
deprotection of the ethynyl group (due to the instability of the products), the intermediates 7a
and 7b were directly submitted to a 1,3-cycloaddition reaction with the corresponding azides,
which were prepared, in their tour, by reaction of NaN3 with the corresponding halogen
alkanes (16). The trimethylsilyl cleavage step was carried out by in situ addition of
tetrabutylammonium fluoride (TBAF) into the reaction mixture (17). This series of click-
chemistry reactions led to a family of six compounds (8a, 8b, 9a, 9b, 10a and 10b) by
varying both aromatic ring A (phenyl /pyridyl) and the length of the alkyl chain (C8, C10 and
C12).
Taking into account that pyridazinone compounds have potential antibacterial activities (18-
21), the ring extension of the hemiamidal core of compounds 5a and 5b into pyridazinone 6a
and 6b, respectively, was performed in good yield by using hydrazine under reflux (96% and
76% yield, respectively) (22, 23). Although this step is associated with the suppression of an
This article is protected by copyright. All rights reserved.

essential substituent (C7 hydroxyl group), this new scaffold has the advantage of deleting the
asymmetric carbon.
S
11
OH
N
HO
O
X
4a: X = N
4b: X = CH
O
O
h,i
S
S
S
N
S
11
O
11
O
11
X
11
OMe
O
R2
R2
HO
HO
NH
e
N
HO
O
a
b
c,d
NH
O
O
R1
X
R1
X
1
N+
O-
X
3
5a: X = N
1a: R1 = H, X = N, R2 = NH
2a: R1= H, X = N, R2 = NH
6a: X = N
5b: X = CH
6b: X = CH
1b
: R1 = H, X = CH, R2 = NH
2b: R1= H, X = CH, R2 = NH
2c: R1= NH2, X = CH, R2 = NCH3
1c: R1 = NH2, X = CH, R2 = NCH3
1d: R1 = H, X = CH, R2 = NOH
1e: R1 = H, X = N, R2 = O
1f: R1 = H, X = CH, R2 = O
f
TMS
N
N
n
N
8a: X = N, n = 7
g
9a: X = N, n = 9
HO
HO
NH
NH
10a: X = N, n = 11
O
O
8b: X = CH, n = 7
9b: X = CH, n = 9
10b: X = CH, n = 11
X
X
7a: X = N
7b: X = CH
Scheme 1. Reagents and conditions: (a) BrC6H4SC12H25, nBuLi, THF, -78 °C, 37-58% (b) mCPBA, 72% (c)
H2NOMe.HCl, PTSA, toluene, reflux (d): BrC6H4SC12H25, nBuLi, THF, -78 °C, 26-36% (e) H2NNH2, EtOH,
reflux, 76-99% (f): ethynyltrimethylsilane, nBuLi, -78 °C, 56-64% (g) C8H17N3, C10H21N3 or C12H25N3,
CuSO4.5H2O, NaAsc, EtOH, TBAF, 28-93% (h) for compound 4a, H2NOH.HCl, pyridine, reflux (i)
BrC6H4SC12H25, nBuLi, THF, -78 °C, 30-44%
Enzymatic tests of InhA inhibition in vitro
The InhA protein was produced and purified according to a previous reported method (24).
The compounds 2a, 2b, 2c, 3, p-4a, m-4a, 4b, 5a, 5b, 6a, 6b, 8a, 8b, 9a, 9b, 10a and 10b
(chiral molecules in racemic form) were evaluated for their InhA inhibitory activity by
measuring the NADH consumption in the enzymatic reaction through the decrease of the
absorbance at 340 nm. Target compounds were tested at 50 µM and for the more potent
molecules also at 10 µM. The result is expressed as the percentage of inhibition of the InhA
This article is protected by copyright. All rights reserved.

activity for the indicated concentration. Triclosan was employed as a positive control and it
induced a complete loss of InhA activity at 10 µM. The data are summarized in Table 1.
The hit compound 2a was re-evaluated and it exhibited 28% inhibition at 50 µM confirming
the magnitude of activity according to the previous reported results (2).
All new synthesized compounds were able to inhibit InhA activity in vitro. Compounds 3, 5b,
6b, 8a, 9b and 10b displayed InhA inhibitory potencies comparable to the hit molecule 2a.
Compounds 2b, 5a, 6a, 9a and 10a were slightly more active than 2a. The best inhibitors
were compounds m-4a and 4b, which displayed approximately the same percentage of InhA
inhibition as 2a for 5-fold lower concentration (entries 1, 7 and 8). In contrast, compounds 2c,
p-4a and 8b showed poor InhA inhibition for this series.
Structure-InhA inhibition relationship
In vitro, the fully oxidized compound 3 bearing an N-oxide pyridine and a sulfone function
displayed the same InhA inhibitory activity as the hit molecule 2a. The presence of extra
heteroatoms in the framework of the azaisoindolinone did not contribute to improve the
interaction of compound 3 within the InhA active site.
The replacement of pyridine by a phenyl ring was well tolerated. In some cases, pyridine
derivatives seem to be slightly better than phenyl derivatives (entries 9 and 10, 11 and 12, 13
and 14, 15 and 16, 17 and 18). This suggests that pyridine nitrogen atom may be engaged in
key interactions within the binding pocket of the protein. Substitution of cycle A by the
amino-phenyl ring led to a deep drop in activity (entry 3). However, no conclusions could be
assessed on the influence of either the extra amino group or the N8-methyl bond. It is not
clear whether this effect was due to the presence of the extra amino group or the presence of
the methyl group (or even to both of them).
Modification of the hemiamidal core was also investigated. Insertion of a hydroxyl group at
N8 revealed a significant enhancement of the inhibitory activity of the molecule toward InhA
in vitro. Indeed, azaisoindolinone-based derivatives 4a (para and meta 70/30 molar ratio)
(entry 5) and 4b (entry 8) exhibited about three times more inhibitory potency toward InhA
than the lead molecule 2a at 50 µM. This encouraging results observed with the mixture of
two regioisomers (4a) led us to separate them by preparative HPLC to evaluate their activities
separately. Surprisingly, the m-4a regioisomer was much more potent (entry 7) than the para
isomer p-4a (entry 6). Both the hydroxylamino group and the position of the nitrogen atom on
the pyridine ring were found to be critical for the activity of the molecule. In accordance with
these observations, the replacement of the hydroxylamino group by methoxylamino group
This article is protected by copyright. All rights reserved.

significantly diminished the activity of 5a and 5b (entries 9 and 10 respectively). This result
strongly supports the proposal that N8-OH may form hydrogen bond interaction into the
active pocket of the InhA.
The replacement of the dodecylthiophenyl (ring C) by a dodecyltriazolyl moiety in the
pyridine series led to a more active compound (10a entry 17) than the reference molecule 2a.
The positive effect exerted by the dodecyltriazolyl group in the place of the
dodecylthiophenyl substituent on this scaffold may be explained by new hydrogen bonding
possibilities afford by the heteroatoms of the aromatic cycle. Structure-InhA inhibitory
activity exploration in the triazole series was also carried out by modifying the length of the
aliphatic chain. As observed previously (2), it was shown that the length of the alkyl chain
plays an important role to ensure efficient inhibitory activity toward the InhA protein (entries
13, 15 and 17).
The pyridazinone-type derivatives 6a and 6b, obtained from the ring expansion of the
hemiamidal moiety, showed a significant improvement of the inhibitory InhA activity when
compared to 2a. In this example, it was also found that pyridine-based compound was more
active than benzene-based compound (entries 11 and 12).
Molecular Docking
Molecular docking calculations were performed in order to simulate the possible binding
modes of azaisoindolinone compounds in the active site of the InhA protein, to rationalize the
structure-InhA inhibition relationship observed in this work, and to define essential structural
features. Although several InhA crystal structures in complex with different inhibitors are
available, we chose the crystal structure PDB ID 2H7M (25) as target for the docking study
because, as previously reported by our group (26), this protein showed good ability to
reproduce different modes of interaction of ligands with respect to the corresponding X-ray
data. In addition, its crystal structure was solved in complex with the arylamide-type inhibitor
d11 with significant inhibitory activity (d11, IC50 InhA = 0.39 µM), and with a high-
resolution 1.6 Å.
It is important to note that each compound with scaffold A, as defined in Table 1, is a chiral
molecule (asymmetric carbon 7) and thus exists as a racemic mixture. Previous studies have
shown that when R2 = NH, a rapid racemization of C7 for both R and S enantiomers occurs in
solution (2), probably through an open intermediate structure in equilibrium (27).
Nevertheless, the cycle form (azaisoindolinone) is the unique form detected by NMR.
Consequently, the enantiomers were not separated and the compounds were used as a
This article is protected by copyright. All rights reserved.

racemate in this work. However, the docking experiments were carried out for each individual
enantiomer.
New compounds 2c(R), 2c(S), m-4a(R), m-4a(S), p-4a(R), p-4a(S), 5a(R), 5a(S), 10a(R),
10a(S) were docked into the substrate binding site of the 2H7M protein in complex with
NADH cofactor by using AutoDock Vina program. Due to their flexibility, these compounds
show several combinations of conformation and orientation within the binding site. For each
compound, the top-Vina score docking poses included into an interval of 2 kcal/mol energy
were selected and were further subjected to an optimization step with the Hamiltonian method
PM6-DH2 as previously described. Among the final possible conformations, we retained the
ones that exhibited the lower optimized energy of interaction.
For comparison, we also show the orientation of the hit molecules 2a(R) and 2a(S). The best
binding mode predicted by docking calculations for different compounds is depicted in Figure
3.
The azaisoindolinone scaffold of 2a(R) (Figure 3A) is located near the NADH cofactor and
forms two hydrogen bonds between the carbonyl oxygen of the hemiamidal moiety and the C-
2’-hydroxyl ribose of the nicotinamide nucleotide fragment and between the tertiary alcohol
and one oxygen atom of the phosphate groups of the cofactor. This conformation shows the
important role played by the tertiary alcohol in the binding of the active site and consequently,
in the inhibitory activity of InhA which strengthens what has been previously revealed by the
structure-activity relationship analysis (2). In the selected conformation of the 2a(S) (Figure
3B), the azaisoindolinone scaffold is also located close of the NADH. However, the carbonyl
group points to the opposite side of the cofactor and does not establish hydrogen bonds with
it. In contrast, the pyridine ring is close to the Tyr158 and a hydrogen bond is observed
between the hydrogen of the phenol function of the tyrosine and the pyridine nitrogen. In this
conformation, the tertiary alcohol is also positioned in such a manner compatible with
hydrogen bonding with one oxygen atom of a phosphate group. In both enantiomers of 2a, the
lipophilic chain occupies the top of the substrate-binding site.
The p-4a(R) and p-4a(S) (Figures 3C and 3D respectively) compounds are located globally
in the same position as 2a(R) and 2a(S), respectively and establish almost the same main
interactions with the cofactor and the protein. It is interesting to note that, for regioisomers p-
4a(R) and p-4a(S), the N-hydroxylamine group was not responsible for additionnal
interactions within the active site of the protein. In contrast, the meta regioisomer, compound
m-4a(R) (Figure 3E), interacts with InhA by the two hydrogen bonds, which involve tertiary
alcohol and carbonyl group, and a supplementary interaction between N8-OH and C2’-
This article is protected by copyright. All rights reserved.

hydroxyl ribose group. The other enantiomer m-4a(S) (Figure 3F) adopts in the active pocket
of InhA an orientation allowing the establishment of four interaction points with the cofactor
and the protein. These interactions take place between the tertiary alcohol at C7 and one of the
phosphate oxygen, between the hydroxyl at N8 position and one of the saturated oxygen of
the phosphate group, between the carbonyl group of the hemiamidal moiety and residue
Thr196 and between the pyridine nitrogen atom and Tyr158 or C2’-hydroxyl ribose of the
cofactor. All together, these interactions can account for a best activity observed for m-4a
when compared to its regioisomer p-4a (entries 6 and 7).
Docking results revealed that compound 5a(R) (Figure 3G) adopts a conformation in the InhA
active site establishing two hydrogen bond type interactions as observed for m-4a(R).
However, different of m-4a(R) the N-OMe group is not able to interact with InhA nor NADH.
On the other hand, the enantiomer 5a(S) (Figure 3H) is positioned in a completely different
manner. It is located far away from the NADH cofactor and hydrogen bonds with neither
NADH nor Tyr 158 could be observed. We noted an unusual interaction between the tertiary
alcohol at C7 and the Met 98. For compounds 10a(R) and 10a(S) (Figure 3I and 3J), the
triazole ring is positioned near the Tyr158 and the ribose moiety. It is worth to note that, even
in this case, the hydrogen bond between the tertiary alcohol at C7 and one of the oxygen
atoms of the phosphate group is preserved. The location of the lipophilic chain is another
difference between these compounds and 2a. For compounds 10a(R) and 10a(S), the chain is
located in the bottom part of the cavity. Finally, the top Vina poses selected for 2c (both R and
S enantiomers), which is one of the less active compounds of the azaisoindolinone series,
showed neither hydrogen bonding with NADH nor with Tyr158 (Figure 3K and 3L). Only the
S enantiomer showed an uncommon interaction between the tertiary alcohol and the residue
Met98. This lack of hydrogen bond interactions with the cofactor may explain the low
inhibitory activity observed for this derivative.
In summary, binding modes allowing interactions with the nicotinamide nucleotide fragment
(ribose and phosphate groups) of the cofactor NADH and with residue Tyr158 of InhA are
always associated with inhibitory InhA activities in vitro.
This article is protected by copyright. All rights reserved.

B.
D.
Vina = -9.6 kcal/mol
A.
C.
Vina = -9.5 kcal/mol
Vina = -8.8 kcal/mol
Vina = -9.6 kcal/mol
E.
Vina = -9.5 kcal/mol
F.
Vina = -9.3 kcal/mol
G.
Vina = -9.3 kcal/mol
H.
Vina = -8.6 kcal/mol
I.
Vina = -9.6 kcal/mol
J.
Vina = -9.5 kcal/mol
K.
Vina = -8.8 kcal/mol
L.
Vina = - 9.4 kcal/mol
Figure 3. The best binding modes of compounds 2a(R) (A.), 2a(S) (B.), p-4a(R) (C.), p-4a(S) (D.), m-4a(R)
This article is protected by copyright. All rights reserved.

(E.), m-4a(S) (F.), 5a(R) (G.), 5a(S) (H.), 10a(R) (I.), 10a(S) (J.), 2c(R) (K.) and 2c(S) (L.) predicted by
docking calculations. Tyr158 residue and NADH cofactor molecule are respectively indicated as dark green and
light green sticks.
Mycobacterium tuberculosis growth inhibition
In order to check whether the improved activity of InhA inhibiting of some compounds could
be retrieved in the inhibition of Mtb growth, compounds 2a, 3, 4a, 4b, 5a, 6a and 10a were
evaluated toward inhibition of Mtb H37Rv growth using the same proceeding as reported in
Menendez et al (28). The minimum inhibitory concentrations (MIC) were determined as the
lowest drug concentration inhibiting bacterial growth. Triclosan was used as a positive
control. The results are presented in Table 1. All tested compounds displayed an inhibitory
activity toward M. tuberculosis growth globally equivalent to 2a. Although compounds m-4a
and 4b were better InhA inhibitors in vitro than 2a, they did not show however, higher
inhibition potency toward Mtb growth.
Table 1. Effects of modifications upon the azaisoindolione framework on enzyme-inhibitory and
antibacterial activities
Scaffold A
Scaffold B
X
InhA inhibition (%)a
MIC
Entry Compound Scaffold
R1
R2
Y
(µM)a
50 µM
10 µM
Mtb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
A
A
A
A
A
A
A
A
A
A
B
B
A
A
A
A
A
A
Ph-SC12
Ph-SC12
Ph-SC12
Ph-S(O2)C12
Ph-SC12
Ph-SC12
Ph-SC12
Ph-SC12
Ph-SC12
Ph-SC12
H
H
CH3
H
OH
OH
OH
OH
OMe
OMe
-
-
H
H
H
H
H
H
N
CH
CH
NO
N/CH
N
CH
CH
N
CH
N
CH
N
CH
CH
CNH2
CH
N/CH
CH
N
CH
CH
CH
-
28.1 1.1
42.4 3.4
12.3 4.6
34.6 2.8
76.1 3.7
11.4 0.6
80.1 3.0
81.6 4.9
42.8 5.1
25.0 5.2
57.8 7.3
30.7 6.9
32.1 0.8
17.0 1.7
45.4 4.7
31.3 3.8
55.6 4.4
25.4 1.2
-
-
-
-
-
-
21
36
-
-
-
-
-
-
-
-
-
23.4
-
-
21.1
22.6
> 22.5
> 22.5
2a
2b
2c
3
4a
p-4a
m-4a
4b
5a
5b
6a
6b
8a
8b
9a
9b
10a
10b
> 22.7
> 21.9
-
Ph-SC12
Ph-SC12
23.6
-
-
-
-
Triaz-C8
Triaz-C8
Triaz-C10
Triaz-C10
Triaz-C12
Triaz-C12
CH
CH
CH
CH
CH
CH
CH
N
CH
N
-
-
26.9
-
CH
-
97.5
0.7
19
-
-
-
-
-
-
43
Triclosan
This article is protected by copyright. All rights reserved.

-
not determined. a. All assays were repeated three times. b. All assays were repeated
three times and standards deviations 0.025 mg/mL.
Inhibition of the mycolic acid biosynthesis
With these results in hands, it was the question whether the inhibition of Mtb growth by the
synthesized compounds comes indeed from the inhibition of the InhA target. To answer to
this question, we tested compounds m-4a, 2a and INH, for comparison, toward the inhibition
of mycolic acid biosynthesis in intact mycobacteria. Bacteria were treated at the MIC of each
compound for 5 h and then incubated with [1-14C]acetate for 18 h. After saponification,
radiolabelled mycolic acids (MA) and fatty acids (FA) were analyzed as methyl esters by
TLC and phosphorimaging. The results showed that, although these compounds inhibit InhA
in vitro, they do not seem to target InhA inside Mycobacterium tuberculosis since they do not
inhibit the mycolic acid biosynthesis in the used conditions. (Figure 4)
100
FA
90
MA
80
a) FA
b)
a MA
70
60
50
40
30
20
10
0
M+K MA
α MA
MA
M+K MA
control
INH
2a
m-4a
control
INH
2a
m-4a
Figure 4. Effect of 2a and m-4b on MA biosynthesis. Bacteria in culture were treated with either INH, 2a or m-
4a at their MIC and the neosynthesis of mycolic acids in vivo was followed by [1-14C]acetate labelling
(duplicates). a) TLC-phosphorimaging analysis of radiolabelled fatty acid and mycolic acid methyl esters. b)
relative incorporation of radioactivity in various molecular species assessed on plate a). FA, fatty acid methyl
esters; αMA, alpha-mycolic acid methyl esters; M+K MA, methoxy- and keto-mycolic acid methyl esters; INH,
isoniazid.
This article is protected by copyright. All rights reserved.

Conclusion
A chemical modulation of the preceding azaisoindolinone-based compound 2a was completed
and 15 new analogues were synthesized and evaluated toward in vitro InhA inhibitory
activity. Two of them, m-4a and 4b, were more effective inhibitors than the hit molecule 2a
and inhibited about 80% of the InhA activity at 50 μM. Furthermore their mode of interaction
with this enzyme in vitro was predicted based on molecular docking calculations and on the
structure-inhibitory activity relationship analysis. This study aided us to define some critical
interactions between possible inhibitors and the receptor. Indeed, the hydrogen bond
interactions of inhibitors with the nicotinamide nucleotide fragment (ribose and phosphate
groups) of the cofactor NADH and with residue Tyr158 of InhA are always associated with an
inhibitory action. These compounds also exhibited an inhibitory activity upon M. tuberculosis
H37Rv growth however, they were not able to inhibit the biosynthesis of mycolic acids in the
studied conditions.
List of abbreviations
ACP = Acyl carrier protein
Asc = Ascorbate
DMSO = Dimethyl sulfoxide
HPLC = High-performance liquid chromatography
INH = Isoniazid
MA = Mycolic acid
m-CPBA = meta-Chloroperoxybenzoic acid
MDR = Multi-drug resistant strains
MIC = Minimum inhibitory concentration
Mtb = Mycobacterium tuberculosis
NADH = Chemical reduced form of nicotinamide adenine dinucletotide
PTSA = para-Toluenesulfonic acid
PIPES = Piperazine-N,N′-bis(2-ethanesulfonic acid)
TBAF = Tetra-n-butylammonium fluoride
TDR = Total drug resistant strains
TLC = Thin layer chromatography
TMS = Trimethylsilane
This article is protected by copyright. All rights reserved.

Acknowledgments
We thank the scientific council of the University Paul Sabatier by the doctoral fellowship of
Aurélien Chollet.
Conflicts of interest
The authors declare no conflict of interests.
References
1. World Health Organization, Global Tuberculosis Report 2015
wwwwhoint/tb/publications/global_report/gtbr2015_executive_summarypdf?ua=1 (Accessed January 2016)
2. Deraeve, C., Dorobantu, I.M., Rebbah, F., Le Quéméner, F., Constant, P., Quémard, A., Bernardes-Génisson,
V., Bernadou, J., Pratviel, G. (2011) Chemical Synthesis, Biological Evaluation and Structure-Activity
Relationship Analysis of Azaisoindolinones, a Novel Class of Direct Enoyl-ACP Reductase Inhibitors as
Potential Antimycobacterial Agents. Bioorg Med Chem;19: 6225–6232.
3. Delaine, T., Bernardes-Génisson, V., Quémard, A., Constant, P., Meunier, B., Bernadou, J. (2010)
Development of isoniazid-NAD truncated adducts embedding a lipophilic fragment as potential bi-substrate
InhA inhibitors and antimycobacterial agents. Eur J Med Chem;45: 4554-4561.
4. Daffé, M., Lanéelle, M.A., Asselineau, C., Lévy-Frébault, V., David, H. (1983) Intérêt taxonomique des
acides gras des mycobactéries: proposition d'une méthode d'analyse. Ann Microbiol;134B: 241–256.
5. Rezac, J., Fanfrlik, J., Salahub, D., Hobza, P. (2009) Semiempirical quantum chemical PM6 method
augmented by dispersion and H-bonding correction terms reliably describes various types of noncovalent
complexes. J Chem Theory Comput;4: 1749-1760.
6. Stewart, J.J.P. Computational Chemistry, Colorado Springs, CO, USA, http://OpenMOPACnet
7. Chen, V.B., Arendall, W B I., Headd, J.J., Keedy, D A., Immormino, R M., Kapral, G.J., Murray, L.W.,
Richardson, J.S., Richardson, D.C. (2010) MolProbity: all-atom structure validation for macromolecular
crystallography. Acta Crystallogr D. Biol Crystallogr;66: 12-21.
8. Trott, O., Olson, A.J. (2010) Autodock Vina: improving the speed and accuracy of docking with a new scoring
function, efficient optimization, and multithreading. J Comput Chem;31: 455-461.
9. Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S., Olson, A.J. (2009)
AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem;30:
2785-2791.
10. Korth, M., Pitonak, M., Rezac, J., Hobza, P.A. (2010) Transferable h-bonding correction for semiempirical
quantum-chemical methods. J Chem Theory Comput;6: 344-352.
11. Stewart, J.J.P. (1996) Application of localized molecular orbitals to the solution of semiempirical self-
consistent field equations. Inter J Quant Chem;58: 133-146.
12. Klamt, A., Schuurmann, G. (1993) Cosmo: a new approach to dielectric screening in solvents with explicit
expressions for the screening energy and its gradient. J Chem Soc Perkin Trans II;799-805.
13. Jana, N.K., Verkade, J.G. (2003) Phase-vanishing methodology for efficient bromination, alkylation,
epoxidation, and oxidation reactions of organic substrates. Org Lett;5: 3787–3790.
14. Nguyen, H.N., Cee, V.J., Deak, H.L., Du, B., Faber, K.P., Gunaydin, H., Hodous, B.L., Hollis, S.L.,
Krolikowski, P.H., Olivieri, P.R., Patel, V.F., Romero, K., Schenkel, L.B., Geuns-Meyer, S.D. (2012) Synthesis
This article is protected by copyright. All rights reserved.