Chemical synthesis, biological evaluation and structure-activity relationship analysis of azaisoindolinones, a novel class of direct enoyl-ACP reductase inhibitors as potential antimycobacterial agents

Article abstract of DOI:10.1016/j.bmc.2011.09.017

The synthesis and biological evaluation of azaisoindolinone compounds embedding a lipophilic chain on the framework were performed. These compounds were designed as InhA inhibitors and as anti-Mycobacterium tuberculosis agents. Structure-activity relation

Full text of DOI:10.1016/j.bmc.2011.09.017

Bioorganic & Medicinal Chemistry 19 (2011) 6225–6232
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemical synthesis, biological evaluation and structure–activity relationship
analysis of azaisoindolinones, a novel class of direct enoyl-ACP
reductase inhibitors as potential antimycobacterial agents
Céline Deraeve a, Ioana Miruna Dorobantu a, Farah Rebbah a, Frédéric Le Quéméner a, Patricia Constant c,
a
a
Annaïk Quémard b, Vania Bernardes-Génisson a, , Jean Bernadou , Geneviève Pratviel
⇑
a Laboratoire de Chimie de Coordination du CNRS, 205, Route de Narbonne, F-31077 Toulouse, France
b Département Mécanismes Moléculaires des Infections Mycobactériennes, CNRS, Institut de Pharmacologie et de Biologie Structurale (IPBS), 205 Route de Narbonne,
F-31077 Toulouse, France
c Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2011
Revised 6 September 2011
Accepted 8 September 2011
Available online 14 September 2011
The synthesis and biological evaluation of azaisoindolinone compounds embedding a lipophilic chain on
the framework were performed. These compounds were designed as InhA inhibitors and as anti-
Mycobacterium tuberculosis agents. Structure–activity relationships concerning the length and the loca-
tion of the lipophilic chain around the azaisoindolinone framework, the suppression of the phenyl group,
the bioisosteric substitution of ether link and alkylating of the tertiary hydroxyl and the hemiamidal
nitrogen were also investigated, revealing insightful information and thereby enabling further diversifi-
cation of the azaisoindolinone scaffold for new antitubercular agents.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
fragment upon the phenyl ring (second generation) were planned
as direct bisubstrate inhibitors of InhA enzyme. We hypothesized
Tuberculosis is one of the world’s most important infectious
diseases causing over one million deaths annually. The rapid devel-
opment of strains resistant to existing antitubercular compounds,
mainly isoniazid and rifampicine, underscores the urgent need to
identify new lead molecules with novel properties and mecha-
nisms of action.
Isoniazid (INH), a frontline antitubercular agent, is a pro-drug
that requires activation by the mycobacterial catalase peroxidase
enzyme (KatG) to form the active metabolite (INH-NAD adduct,
Fig. 1), which exerts its lethal effect on intracellular target.1–4
The INH-NAD adduct inhibits the mycolic acid biosynthesis in
Mycobacterium tuberculosis by affecting InhA, an enoyl-ACP reduc-
tase enzyme of the type II fatty acid synthesis (FAS-II) system.5 This
enzyme is recognized and validated as an important drug target in
M. tuberculosis since its homolog in human is absent. It is well
established nowadays that resistance to INH largely results from
mutations in M. tuberculosis KatG that diminish its ability to con-
vert INH into its active form.6–9 Thus, the development of direct
inhibitors of InhA is an attractive strategy.
that the presence of this lipophilic chain would show interest in
establishing new interactions in the deep hydrophobic substrate
cavity and increasing thereby the affinity of the inhibitor for the
InhA target. Indeed, the presence of the alkoxy lipophilic chain in
compound 2a proved necessary for the inhibitory activity towards
InhA since its absence (compound 1) abolished the activity (Fig. 1).
Among the previously synthesized INH-NAD truncated adducts
(Fig. 1), the dihydropyridine (DHP)-based analogs 4, despite of
their inhibitory activity towards InhA, were too unstable (oxida-
tion of DHP ring and loss of the N-substituent (2)) for further devel-
opment. In the pyridinium series, the presence of the positive
charge may represent an obstacle to the membrane crossing, dis-
carding them to be validated as hit molecules. In contrast, the sta-
ble azaisoindolinone compound 2a (chain OC12
) showed, in
addition to its InhA inhibitory activity, a very interesting antimi-
crobial qualitative profile, as compared to isoniazid. As isoniazid,
it was able to inhibit M. smegmatis and M. tuberculosis growth
while it did not inhibit Corynebacterium glutamicum growth (a
strain which does not have InhA homologs) and Escherichia coli
growth (a strain which has an homolog of the enoyl-ACP reductase
InhA called FabI).13 All together these results suggested that in the
case of 2a one of the target might be InhA. Thus, encouraged by
these results, we selected the azaisoindolinone 2a as a hit com-
pound for further studies on targeting InhA.
Our previous work based on the development of INH-NAD
truncated analogs as new antitubercular agents inspired from the
active metabolite of INH led to derivatives exhibiting InhA inhibi-
tory activity (Fig. 1).10–12 These compounds embedding a lipophilic
In this Letter, we described the design, the synthesis of the
modified azaisoindolinones, their evaluation towards InhA potency
⇑
Corresponding author. Fax: +33 (0)5 61 55 30 03.
E-mail address: vaniabg@lcc-toulouse.fr (V. Bernardes-Génisson).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.017

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C. Deraeve et al. / Bioorg. Med. Chem. 19 (2011) 6225–6232
INH-NAD adducts
N
N
O
NH
NH2
N
NH2
N
O
HO
O
O
N
N
NH2
O
O
O
O
P
N
N
N
N
N
N
O P O P O
OH OH
O P O
O
O
OH OH
O
O
OH
OH
OH
OH
OH OH
OH OH
INH-NAD truncated analogs
X
n
X
X
n
O
O
O
HO
NH
NH2
O
HO
HO
NH
NH
N
O
O
N
OEt
Br
OEt
N
N
O
2a X = O , n = 11 at 100µM:
InhA inhibition = 41%
2b X = CH2, n = 10 at 100 µM:
InhA inhibition = 4%
1 No inhibition at 100 µM
3a X = O , n = 11 at 100µM:
InhA inhibition = 35%
3b X = CH2, n = 10 at 100 µM:
InhA inhibition = 91%
4a X = O, n = 11 at 100 µM:
InhA inhibition = 6%
4b X = CH2, n = 10 at 10 µM:
InhA inhibition = 25%
AZAISOINDOLINONE ANALOGS
PYRIDINIUM ANALOGS
DIHYDROPYRIDINE ANALOGS
Figure 1. Structures of INH-NAD adducts and truncated analogs.10
and M. tuberculosis growth and the structure–activity relationship
(SAR) analysis. This work aims at optimizing the potentiating effect
of the alkoxy lipophilic chain, attached to the azaisoindolinone
scaffold, in order to precise the pharmacophore elements neces-
sary to interact with InhA and further develop new and more po-
tent antituberculosis agents.
2.1.1. Enantiomers separation by chiral supercritical fluid
chromatography (SFC)
The racemic sample of 2a was submitted to a chiral chromatog-
raphy analysis in order to separate the two enantiomers. A solution
of (RS)-2a in methanol was injected in a SFC chain and each
enantiomer (Rt = 6.36 and 8.98 min) was collected individually
and re-injected after 20 h for control. The re-injection of each
isomer showed the apparition of a second peak in a 1:1 ratio (see
Supplementary data), which corresponded to the opposite enantio-
mer. These analytical results clearly indicate that a racemisation
step occurs in solution probably through an open intermediate
structure (keto-amide, Scheme 1) that cannot be detected by 1H
and 13C NMR analysis. Thereby, in this work all compounds were
analyzed as a racemate mixture.
2. Results and discussion
2.1. Chemistry
The target compounds synthesized in this study were designed
for a SAR study. The modifications concerning the length and the
location of the lipophilic chain around the azaisoindolinone
framework, the suppression of the phenyl group, the bioisosteric
substitutions of the ether link and alkylation of the tertiary hydro-
xyl C-7OH, (R1) and the hemiamidal N-8H (R2) were performed
(Fig. 2). We were also interested in investigating further structural
influence of each enantiomer over InhA activity. To extract perti-
nent information from the SAR exploration, only one parameter
was changed each time.
2.1.2. Synthesis
The chemical synthetic routes of target compounds are pre-
sented in Scheme 2. The new compounds studied in this work con-
tain lipophilic chains with different lengths, positions and linker
atoms (2c, 2d, 2e, 2f, 7a and 7b). They have been synthesized
according to our previously described procedure using the com-
mercial 3,4-pyridine-dicarboximide reagent (5) as starting mate-
rial (Scheme 2).10 The various substituted aryl halide compounds
employed in these synthesis were obtained directly by treating
corresponding halogenophenols/thiols with halogenoalkanes in
the presence of base (see Supplementary data). Compounds 2a
and 2b have already been reported by us in the literature.10 For
preparation of compound 9, containing a NH linker, a protection/
deprotection step of the amine function with BOC group was re-
quired. The alkylation of the tertiary hydroxyl (C-7OH) of 2a and
13 was performed in the presence of SOCl2 in the appropriate
refluxing alcohol to provide 6 and 14, respectively. Compound 12
X
X = CH2, O, S, NH
H
R2
n = 7, 11, 17
N8
HO
7
N8
O
R1
7
R
1 = H, CH3 or C12H25
O
O
R2 = H or C12H25
N
N
Figure 2. Modifications effected around azaisoindolinone framework. The num-
bering does not follow the IUPAC nomenclature.

C. Deraeve et al. / Bioorg. Med. Chem. 19 (2011) 6225–6232
6227
O
O
11
O
11
11
O
O
NH
HO
NH
HO
NH2
O
O
N
N
N
(S)-2a
(
R)-2a
Scheme 1.
O
11
11
O
n
b
HO
NH
MeO
NH
HO
NH
O
O
O
N
N
6
N
2b
2a meta n = 11
2c meta n = 17
2d meta n = 7
2e para n =11
2f ortho n = 11
O
O
H
N
a
j
N
11
11
11
11
O
O
HO
N
NH
N
HO
NH
HO
NH
O
e
O
O
d
f
c
O
O
N
10
N
N
11
N
9
5
N
8
g
i
S
n
R
N
h
O
NH
HO
NH
11
O
O
HO
NH
O
N
14
R = C14H29
R = phenyl
N
12
13
7a n = 11
7b n = 17
Scheme 2. Reagents and conditions: (a) BrC6H4OC12H25 or BrC6H4OC18H37 or BrC6H4C8H17, n-BuLi, THF, À78 °C; (b) SOCl2, CH3OH, reflux; (c) BrC6H4N(C12H25)COOC(CH3)3), n-
BuLi, À78 °C/1 h; (d) CF3COOH, CH2Cl2, 0 °C; (e) C12H25I, K2CO3, DMF, 50 °C; (f) PhMgBr, THF, À78 °C; (g) C14H29MgBr or PhLi, THF, À78 °C; h) SOCl2, C12H25OH, reflux; (i)
BrC6H4SC12H25 or BrC6H4SC18H37, n-BuLi, À78 °C/1 h; (j) n-dodecylboronic acid, Pd(dppf)Cl2, Ag2O, K2CO3, THF, 80 °C.
was obtained by reaction of the corresponding alkyl Grignard re-
agent upon 5. Nucleophilic substitution reaction of dicarboximide
5 with iodododecane, carried out in DMF and in the presence of
K2CO3 at 50 °C afforded 10, which followed by a reaction with
PhMgBr and a purification step gave 11.
molecule 2a at 100 lM. At 30 lM, 7b and 14 also exhibited
superior InhA inhibitory activity when compared to 2a.
2.2.2. M. tuberculosis growth inhibition
Compounds 2a, 2b, 2c, 2d, 6, 7a, 7b, 9, 11, 12 and 14 were
evaluated in vitro with regards to bacterial growth inhibition of
pathogenic mycobacterial species M. tuberculosis. The minimum
inhibitory concentrations (MIC) of the newly and previously syn-
thesized compounds were determined against M. tuberculosis by
using twofold serial dilutions technique and using a colorimetric
test involving 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra-
zolium bromide (MTT) reduction measured at 570 nm. These com-
pounds were compared to isoniazid and ciprofloxacine as
standards antituberculosis drugs. The MICs were defined as the
concentration of the compounds required to give complete inhibi-
tion of mycobacterial growth. The IC50 were only determined for
2.2. Biology
2.2.1. InhA activity inhibition
The target compounds 2a, 2b, 2c, 2d, 6, 7a, 7b, 9, 11, 12 and 14
were initially screened in vitro for their ability to inhibit InhA
activity. The InhA inhibition percentage was determined according
to the method previously described14 measuring the decrease of
the absorbance at 340 nm. The molecules were tested at 100
and/or 30 lM in case of solubility problems. Triclosan was tested
as positive control. The data are summarized in Table 1.
Among the various evaluated azaisoindolinones, 2c and 7a dis-
played better inhibitory potencies towards InhA than the parent
the best compounds (with MIC inferior to 100
lM). The results
are presented in Table 1. These data reported in Table 1 showed

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C. Deraeve et al. / Bioorg. Med. Chem. 19 (2011) 6225–6232
Table 1
Effects of modifications upon the azaisoindolinone framework on enzyme-inhibitory and antibacterial activities
R1
R3
R2O
N
O
N
Entry
Compound
R1
R2
R3
InhA inhibition (%)
MIC (
lM) MTB
IC50 (lM) MTB
100
lM
30 lM
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
Ph-mOC12
Ph-mC12
Ph-mOC18
Ph-mOC8
Ph-pOC12
Ph-oOC12
Ph-mOC12
Ph-mSC12
Ph-mSC18
Ph-mNHC12
Ph
H
H
H
H
H
H
Me
H
H
H
H
H
C12
H
H
H
H
H
H
H
H
H
H
C12
H
H
27
4
67
12
16
0
12
—
28
—
1
156
9.8
25
—
7
12
—
—
250
>100
>100
25
12.5
>100
>100
12.5
>100
25
0
—
6
0
—
15
26
1
10
6.5
—
—
4.5
—
7a
7b
9
11
12
14
66
9
ND*
10
11
12
13
14
15
16
9 InhA activation (%)
13
0
C14
Ph
10 InhA activation (%)
—
20
ND*
9.5
Triclosana
Isoniazid
Ciprofloxacin
37a
—
—
0.6
2.5
43.1b
—
—
—
—
—
*
a
b
ND not determined due to solubility problems.
Tested at 10
Ref. 10.
lM.
that some new synthesized compounds exhibited significant anti-
bacterial activity against M. tuberculosis with MIC values inferior to
that of 2a. In addition, some of them were also inhibitors of the
InhA activity. Among the assayed compounds, 2b, 7a and 11 were
found to be the three most active derivatives with IC50 values in
suggests that the substituent located at this position could help
orienting the chain towards better interaction within the substrate
site.
In the second time, the possibility of introducing the lipophilic
chain on the hemiamidal region (C-7OH and N-8H) was also inves-
tigated. When the lipophilic chain (C12) was attached at the hydro-
xyl group of the C-7 position of the hemiamidal ring, the
the range 4.5–7.0 lM.
2.3. Structure–activity relationships analysis
compound could not be tested at 100
in the enzymatic reaction medium (entry 13). However, the enzy-
matic percentage of inhibition obtained for 14 (C-7OC12) at 30 lM
lM due to its poor solubility
First, it was investigated if the aryl fragment bearing the lipo-
philic chain was essential for the enzymatic inhibitory activity or
if the lipophilic moiety could be directly attached to the hemiami-
dal core. When an aliphatic chain (C14) (entry 12) was connected
directly on the C-7 position with suppression of the phenyl ring,
no InhA inhibitory activity and no inhibition of M. tuberculosis
growth were observed. This shows the critical requirement of the
aryl ring in maintaining the inhibitory activity towards InhA and
M. tuberculosis growth. We concluded that the aryl group, as a
spacer between hemiamidal cycle and the lipophilic chain, is fun-
damental for the observed inhibitory enzymatic activity. Probably
the phenyl ring imposes a more favorable rigid conformation
allowing the hydrocarbon chain to insert into the hydrophobic
substrate cavity of InhA.
By retaining the phenyl ring intact, we tried to ascertain the
optimal effect of the chain around the azaisoindolinone scaffold.
Firstly, the substitution pattern of the phenyl ring was examined.
The synthesized ortho, meta and para derivatives exhibited differ-
ent behaviors towards InhA activity. It was found that when the
dodecyloxyl substituent was moved to the para position (2e), the
InhA inhibitory activity decreased, when compared to the hit mol-
ecule 2a (entries 1 and 5). Shifting the chain to the ortho position
(2f) completely abolished the activity (entries 1 and 6).
is slightly better than the hit molecule 2a at the same concentra-
tion (entries 1 and 13). Furthermore, this analog is also a more effi-
cient inhibitor of M. tuberculosis growth as compared with 2a.
Compound 11, holding a C12 chain at N-8 position of the hemia-
midal cycle, showed a poor InhA inhibitory activity. However, it
exhibited one of the best activities against M. tuberculosis strains
indicating that 11 has probably an other target than InhA. Com-
pared with the hit molecule 2a, the inhibitory activity of 11 upon
M. tuberculosis growth was remarkably improved by a factor 12
(entries 1 and 11).
The strategy of bioisosteric replacement is a powerful and
highly productive tool in analog design. On the basis of this con-
cept, the oxygen atom link of the chain in 2a was changed to
nitrogen (9), sulfur (7a) or suppressed (2b, C12 chain) to ascertain
whether the oxygen link atom is implicated in the binding inter-
action with the target. One can observe that the replacement of
the oxygen linker by a NH group completely abolished the activ-
ity (entries 1 and 10) whereas the replacement of the O-atom by
S-atom improved the inhibitory activity towards InhA (entries 1
and 8). On the other hand, the C12 analog 2b is not an efficient
inhibitor of InhA.10 These results allow us to think that the pres-
ence of an electronegative heteroatom (O or S) is essential for
binding affinity.
When compounds 2e and 2f were evaluated against M. tubercu-
losis growth, no significant improvement of antibacterial activity
was observed with respect to 2a. Their antimycobacterial MIC val-
SAR exploration was also carried out by modifying the length of
the lipophilic aliphatic chain (OC8 and OC18) in order to explore the
ues were superior to 100
lM (entries 1, 5 and 6).
presumed hydrophobic binding cavity. Thus, compounds 2c (OC18)
From the enzymatic data, the best place for the lipophilic ali-
phatic chain is the meta position of the phenyl group. This result
and 2d (OC8) were prepared to establish the optimal length. The
rank order of InhA inhibitory potency was 2c (OC18) > 2a

C. Deraeve et al. / Bioorg. Med. Chem. 19 (2011) 6225–6232
6229
(OC12) > 2d (OC8). Increasing the length of the alkyl chain resulted
in significant improvement of the activity (entries 1, 3 and 4). For
the sulfur derivative, the C12 chain was also replaced by a C18 chain.
In this case, the benefic effect of a more lipophilic chain towards
InhA inhibition was also significant (entries 8 and 9). However,
for 7b this positive effect was not reflected in inhibition of M.
tuberculosis growth.
Next, methylation of the tertiary alcohol at C-7 (2a) dramati-
cally decreased the InhA inhibitory activity (entries 1 and 7). On
the one hand, it may suggest that the hydroxyl group is important
to establish binding interactions with InhA target. On the other
hand, this free OH group may play a fundamental role in hemiami-
dal opening process (as evidenced by us by the chiral HPLC exper-
iments) towards a keto-amide form, which may be the key species
for interaction with InhA.15 Surprisingly, this protection resulted in
an improved activity of 6 against M. tuberculosis growth.
spectra were recorded on a Perkin-Elmer spectrometer GX 2000.
1H NMR spectra were recorded on a Bruker spectrometer at 250,
300, 400 and 500 MHz using CDCl3, DMSO-d6 or MeOD-d4 as the
solvent. For 1H NMR the residual proton signal of the deuterated
solvent was used as an internal reference: CDCl3 d = 7.29 ppm,
DMSO = 2.50 ppm and MeOD = 3.31 ppm. 13C NMR spectra were
recorded on a Bruker spectrometer at 63, 75, 100 and 126 MHz.
Mass spectra (EI) were obtained on an MS-Nermag R10-10 spec-
trometer. For the MS–ESI a Perkin-Elmer SCIEX API 365 spectrom-
eter was used and for the MS–CI a Thermo electron TQS 7000 was
employed. Column chromatography was performed on silica gel
(Merck 0.063–0.200 mm). The purity of compounds was assessed
by microanalysis and/or by thin layer chromatography coupled to
13C NMR. All reagents were obtained from commercial suppliers
and were used without further purification. Anhydrous solvents
were freshly distilled before use. THF and ether were dried with so-
dium in the presence of benzophenone. Dimethylformamide was
dried on calcium hydride.
2.4. Cytotoxicity evaluation on eukaryotic cells
The syntheses of intermediates are described in the Supplemen-
tary data. Compounds 2a and 2b are described in the literature.10
Compounds 2c, 2d, 2e, 2f, 7a, 7b and 8 were synthesized according
to the representative protocol described below for 2c and the spec-
tral characterization is described below.
The most efficient anti M. tuberculosis compounds (2a, 2b, 2c, 6,
7a, 11 and 14) were submitted to in vitro cytotoxicity assay. Their
cytotoxicity was determined over THP-1 cells and expressed by
MIC values. The MIC values of these compounds towards THP-1
cell cytotoxicity (see Supplementary data) were higher than
MICM.tuberculosis values but the difference could not satisfy the
drug-like properties.
Compound 11 was prepared as previously described16 using 10
as starting material. Compounds 12 and 13 were synthesized
adapting this same protocol.
The protocols for the preparation of the new compounds 6, 9,
10 and 14 are described below.
3. Conclusion
4.1.1. Chiral chromatography
In the present study, it was demonstrated, by the first time, that
aryl azaisoindolinone framework undergoes epimerization at the
C-7 carbon probably via a cycle opening pathway. A series of aza-
isoindolinones embedding a lipophilic chain was synthesized and
the structure–activity relationship analysis, carried out around
the parent azaisoindolinone scaffold 2a, permitted to precise some
pharmacophore elements. The best molecular interaction with the
InhA enzyme was achieved by the following modifications: (i) the
lipophilic chain must be attached through the phenyl ring to
the azaisoindolinone scaffold, (ii) 12 or 18 carbons long chain
bound to oxygen or sulfur atom to the meta position of the phenyl
ring and (iii) a free tertiary hydroxyl at C-7 position.
The improvements in the InhA inhibitory activity brought by
these structural modifications were also correlated with an increase
of the inhibition of the growth of M. tuberculosis. Compounds 2c and
7a showed superior InhA inhibitory property and better activity
against M. tuberculosis growth than the parent molecule 2a.
In contrast, yet interesting, some structural modifications led to a
loss of InhA inhibitory activity (compounds 2b, 6 and 11) still asso-
ciated to a capacity to inhibit M. tuberculosis growth. The mechanism
of action of these compounds does not involve InhA as a target.
Although the initial strategy of targeting the hydrophobic sub-
strate cavity of InhA enzyme with azaindolinones bearing long
hydrocarbon chain led to compounds with better affinity for InhA
and better activity against M. tuberculosis, their cytotoxicity to-
wards eukaryote cells precludes their development. With the con-
tribution of molecular computational modeling we plan to modify
the chemical structure in order to gain more in InhA selectivity and
thereby compensate the toxicity.
Supercritical fluid chromatography (SFC) was performed with
an OD–H 10 mm  250 mm column eluted with 15% MeOH at
12 mL/min (P 120 bar, oven temperature = 40 °C) for purification.
An OD–H 4.6 mm  250 mm column eluted with 15% MeOH at
4 mL/min (P 100 bar, oven temperature = 35 °C) was used for ana-
lytical chromatography.
4.2. (RS)-1-hydroxy-1-[3-(octadecyloxy)-phenyl]-1,2-dihydro-
3H-pyrrolo[3,4-c]pyridin-3-one (2c)
To a solution of the suitable bromoether (1.00 g; 0.6 mmol) in
dry tetrahydrofurane (18 mL), under argon atmosphere and at
À40 °C was added n-butyllithium 2.5 M (1.7 mL; 2.0 mmol). After
addition, the temperature was maintained at À50 °C for 30 min.
Then the temperature was decreased at À78 °C and this solution
was added upon
a
solution of 3,4-pyridinedicarboximide
(175 mg, 1.2 mmol) in dry tetrahydrofurane (15 mL). The reaction
mixture was stirred for 1 h at À50 °C. Then the reaction was
quenched with a saturated ammonium chloride solution (30 mL)
and the mixture was extracted with ethyl acetate (3 Â 20 mL).
The organic phases were combined, dried over sodium sulfate
and concentrated under vaccum. The residue (860 mg) was puri-
fied by column chromatography (SiO2) (eluent: dichloromethane/
methanol: 100/0 to 93/7) to furnish 2c as a mixture of para/meta
compounds. The para regioisomer was isolated by a recrystalliza-
tion in acetone/methanol (3 days).
Yield 32% (187 mg – mixture of para/meta 2:1 regioisomers).
White solid, mp 113 °C. TLC Rf: 0.35 (dichloromethane/methanol
95:5, v/v). IR (cmÀ1): 3141, 3059, 2920, 2851, 1708, 1607, 1578,
1286, 1258, 1142, 1096, 1082, 1045, 1030, 841, 700. 1H NMR
(500 MHz, MeOD) d (ppm): 8.93 (s, 1H); 8.73 (d, J = 5.4 Hz, 1H);
7.46 (dd, J = 1.0 and 5.1 Hz, 1H); 7.27 (t, J = 8.0 Hz, 1H); 7.17 (m,
1H); 7.07 (dd, J = 1.0 and 8.8 Hz, 1H); 6.91 (dd, J = 8.3 Hz and J no
mesurable, 1H); 4.00–3.97 (m, 2H); 1.81–1.75 (m, 2H); 1.48–1.31
(m, 30H); 0.92 (t, J = 6.8 Hz, 3H). 13C NMR (126 MHz, MeOD) d
(ppm): 168.5 (C); 159.6 (C); 159.4 (C); 152.6 (CH); 144.5 (CH);
4. Experimental part
4.1. General
The melting points were determined on an Electrothermal 9300
capillary melting point apparatus and are not corrected. Infra-red

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C. Deraeve et al. / Bioorg. Med. Chem. 19 (2011) 6225–6232
141.1 (C); 129.4 (CH); 126.4 (C); 117.9 (CH); 117.4 (CH); 114.5
(CH); 111.8 (CH); 67.9 (CH2); 31.6 (CH2); 29.3–29.2 (10 Â CH2);
28.98 (CH2); 28.94 (CH2); 28.91 (CH2); 25.7 (CH2); 22.2 (CH2);
12.9 (CH3), 1C missing. MS (ESI) m/z: 495.5 [M+H+]. HRMS (ESI):
for C31H47N2O3 calcd: 495.3587; found: 495.3618. Anal. Calcd for
(CH); 111.6 (CH); 85.6 (C); 67.8 (CH2); 31.7 (CH2); 29.4
(3 Â CH2); 29.21 (CH2); 29.16 (CH2); 29.12 (CH2); 28.7 (CH2);
25.7 (CH2); 22.4 (CH2); 13.1 (CH3). MS (ESI) m/z: 433.5 [M+Na+];
411.5 [M+H+]; 393.4 [M+H+ÀH2O]. HRMS (ESI) for C25H35N2O3:
calcd: 411.2648; found: 411.2681. Anal. Calcd for C25H34N2O3Á0.5
CH3OH: C, 71,80; H, 8.51; N, 6.57. Found: C, 71.59; H, 8.26; H, 6.64.
C31H46N2O3ÁCH3OHÁ0.2 CH3COCH3: C, 72.73; H, 9.59; N, 5.20.
Found: C, 72.32; H, 9.53; N, 4.92.
4.6. (RS)-1-[3-(dodecyloxy)phenyl)]-1-methoxy-1,2-dihydro-3H-
pyrrolo[3,4-c]pyridin-3-one (6)
4.3. (RS)-1-hydroxy-1-[3-(octyloxy)phenyl]-1,2-dihydro-3H-
pyrrolo[3,4-c]pyridin-3-one (2d)
To a solution of 2a (100 mg; 0.24 mmol) in methanol (6 mL)
was added 0.5 mL of thionyl chloride. The mixture was heated at
reflux under argon for 16 h. Then the reaction was treated with a
saturated solution of NaHCO3 (5 mL) and extracted with ethyl ace-
tate (3 Â 8 mL). The combined organic layers were dried over
anhydrous sodium sulfate and, after filtration, concentrated under
reduced pressure to furnish 93 mg of a yellow oil. The residue was
purified by column chromatography (dichloromethane/methanol
95:5).
Yield 31% (32 mg). White solid. TLC Rf: 0.60 (dichloromethane/
methanol 95:5, v/v). IR (cmÀ1): 2922, 2852, 1726, 1603, 1438,
1287, 1051, 698. 1H NMR (250 MHz, CDCl3) d (ppm): 9.12 (bd s,
1H); 8.80 (bd s, 1H); 7.31–7.25 (m, 2H); 7.13 (s, 1H); 7.09 (t,
J = 7.0 Hz, 1H); 6.91–6.87 (m, 2H); 3.97 (t, J = 6.4 Hz, 2H); 3.19 (s,
3H); 1.79 (t, J = 5.5 Hz, 2H); 1.45–1.28 (m, 18H); 0.91 (t,
J = 5.9 Hz, 3H); 13C NMR (63 MHz, CDCl3) d (ppm): 168.1 (C);
159.6 (C); 154.9 (C); 153.2 (CH); 146.0 (CH); 139.7 (C); 130.0
(CH); 118.0 (CH); 117.5 (CH); 114.9 (CH); 112.1 (CH); 91.9 (C);
68.2 (CH2); 50.9 (CH3); 31.9 (CH2); 29.7–29.3 (7 Â CH2); 26.0
(CH2); 22.7 (CH2); 14.1 (CH3); 1Cq missing. MS (ESI) m/z: 425.6
[M+H+]. HRMS (ESI) for C26H37N2O3: calcd: 425.2804; found:
425.2847.
Yield 16% (100 mg – mixture of para/meta isomers). The para
regioisomer was obtained after a column chromatography (dichlo-
romethane to dichloromethane/methanol 95: 5, v/v). White solid,
mp 97 °C. TLC Rf: 0.35 (dichloromethane/methanol 95:5, v/v). IR
(cmÀ1): 3350, 3060, 2918, 2849, 1714, 1613, 1465, 1340, 1207,
1067, 727. 1H NMR (500 MHz, MeOD) d (ppm): 8.93 (s, 1H); 8.73
(d, J = 5.0 Hz, 1H); 7.48 (dd, J = 0.8 Hz and 5.1 Hz, 1H); 7.29 (t,
J = 8.0 Hz, 1H); 7.18 (d, J = 2.0 Hz, 1H); 7.07 (d, J = 8.0 Hz, 1H);
6.91 (dd, J = 1.8 and 7.3 Hz, 1H); 4.00–3.96 (m, 2H); 1.77 (q,
J = 6.5 Hz, 2H); 1.49–1.45 (m, 2H); 1.41–1.33 (m, 8H); 0.93 (t,
J = 6.8 Hz, 3H). 13C NMR (126 MHz, MeOD) d (ppm): 168.4 (C);
159.5 (C); 159.4 (C); 152.6 (CH); 144.4 (CH); 141.0 (C); 129.4
(CH); 126.4 (C); 118.0 (CH); 117.3 (CH); 114.3 (CH); 111.7 (CH);
87.8 (C); 67.7 (CH2); 31.6 (CH2); 29.1 (CH2); 29.0 (CH2); 28.9
(CH2); 25.8 (CH2); 22.3 (CH2); 12.9 (CH3). MS (ESI) m/z: 355.4
[M+H+]. HRMS (ESI): for C21H27N2O3 calcd: 355.2022; found:
355.2050. Anal. Calcd for C21H26N2O3Á0.5 CH3OH: C, 69.73; H,
7.56; N, 7.56. Found: C, 69.40; H, 7.24; N, 7.41.
4.4. (RS)-1-[4-(dodecyloxy)phenyl]-1-hydroxy-1,2-dihydro-3H-
pyrrolo[3,4-c]pyridin-3-one (2e)
Yield 21% (46.5 mg – mixture of para/meta 2:1 regioisomers).
The para regioisomer was isolated by recrystallization in acetone.
White solid, mp 118–119 °C. TLC Rf: 0.84 (dichloromethane/
methanol 92.5:7.5, v/v). IR (cmÀ1): 3302, 3060, 2919, 2852,
1688, 1605, 1510, 1475, 1242, 1174, 1081, 1035, 931, 826,
716. 1H NMR (500 MHz, DMSO) d (ppm): 9.42 (s, 1H,); 8.85 (s,
1H); 8.72 (d, J = 5.0 Hz, 1H); 7.39 (d, J = 10.0 Hz, 2H); 7.38 (d,
J = 5.0 Hz, 1H); 7.07 (s, 1H); 6.90 (d, J = 10.0 Hz, 2H); 3.93 (t,
J = 7.5 Hz, 2H); 1.70–1.67 (m, 2H); 1.40–1.37 (m, 2H); 1.27–
1.20 (m, 16H); 0.86 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, DMSO)
d (ppm): 167.5 (C); 159.2 (C); 159.1 (C); 153.4 (CH); 145.0 (CH);
132.8 (C); 127.3 (2 Â CH); 126.5 (C); 118.2 (CH); 114.7 (2 Â CH);
87.6 (C); 67.9 (CH2); 31.8 (CH2); 29.5–29.1 (7 Â CH2); 25.9 (CH2);
22.5 (CH2); 14.4 (CH3). MS (ESI) m/z: 433 [M+Na+], 411 [M+H+].
HRMS (DCI-CH4): for C25H35N2O3 calcd: 411.2648; found:
411.2634.
4.7. (RS)-1-[3-(dodecylsulfanyl)phenyl]-1-hydroxy-1,2-dihydro-
3H-pyrrolo[3,4-c]pyridin-3-one (7a)
Yield 42%, (200 mg – mixture of para/meta regioisomers). The
para regioisomer was isolated by recrystallization in acetone.17
White solid, mp 86 °C. TLC Rf: 0.31 (dichloromethane/methanol
95:5, v/v). IR (cmÀ1): 3159, 2917, 2849, 1698, 1614, 1587, 1464,
1347, 1205, 1067, 964, 784, 698. 1H NMR (250 MHz, MeOD) d
(ppm): 8.94 (s, 1H); 8.74 (d, J = 5.0 Hz, 1H), 7.56 (s, 1H); 7,46 (d,
J = 4.6 Hz, 1H); 7.35–7.30 (m, 3H); 2.96 (t, J = 7.0 Hz, 2H); 1.65–
1.56 (m, 2H); 1.53–1.25 (m, 18H); 0.94 (t, J = 6.0 Hz, 3H). 13C
NMR (63 MHz, MeOD) d (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 Â 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: calcd: 427.2419; found: 427.2459.
Anal. Calcd for C25H34N2O2SÁ0.6 CH3COCH3: C, 69.75; H, 8.21; N,
6.07. Found: C, 69.43; H, 7.99; H, 6.33.
4.5. (RS)-1-[2-(dodecyloxy)phenyl]-1-hydroxy-1,2-dihydro-3H-
pyrrolo[3,4-c]pyridin-3-one (2f)
Yield 39% (195 mg – mixture of para/meta regioisomers). The
para regioisomer was isolated by recrystallization in acetone/
methanol. White solid, mp 132 °C. TLC Rf: 0.71 (dichloromethane/
methanol 95:5, v/v). IR (cmÀ1): 3199, 3061, 2920, 2851, 1708,
1619, 1470, 1284, 1243, 1073, 1045, 1022, 847, 798, 748. 1H
NMR (500 MHz, MeOD) d (ppm): 8.90 (d, J = 1.1 Hz, 1H); 8.69 (d,
J = 5.3 Hz, 1H); 8.07 (dd, J = 1.8 and 7.8 Hz, 1H); 7.37 (td, J = 1.8
and 7.5 Hz, 1H); 7.32 (dd, J = 1.4 and 5.1 Hz, 1H); 7.05 (dt, J = 1.0
and 7.7 Hz, 1H); 6.88 (dd, J = 0.7 and 8.3 Hz, 1H); 3.74 (dt, J = 6.3
and 9.2 Hz, 1H); 3.56 (dt, J = 6.3 and 9.1 Hz, 1H); 1.35–1.00 (m,
20H); 0.92 (t, J = 6.8 Hz, 3H). 13C NMR (126 MHz, MeOD) d
(ppm): 169.1 (C); 160.5 (C); 155.9 (C); 152.1 (CH); 143.8 (CH);
130.2 (CH); 128.5 (C); 127.8 (CH); 125.8 (C); 119.8 (CH); 117.3
4.8. (RS)-1-hydroxy-1-[3-(octadecylsulfanyl)phenyl]-1,2-
dihydro-3H-pyrrolo[3,4-c]pyridin-3-one (7b)
Yield 39%, (104 mg – mixture of para/meta regioisomers). The
para regioisomer was isolated by column chromatography (SiO2/
eluent CH2Cl2/MeOH 99:1 to 96:4). White solid, mp: 92 °C. TLC
Rf: 0.42 (dichloromethane/methanol 95:5, v/v). IR (cmÀ1): 3066,
2916, 2851, 1722, 1612, 1591, 1472, 1341, 1200, 1140, 1060,
954, 885, 838, 777, 727, 715, 693. 1H NMR (300 MHz, CDCl3) d
(ppm): 8.64 (s, 1H); 8.49 (d, J = 4.8 Hz, 1H); 8.24 (s, 1H); 7,56 (s,
1H); 7,31–7.24 (m, 4H); 2.91 (t, J = 7.2 Hz, 2H); 1.69–1.59 (m,
2H); 1.29–1.20 (m, 30H); 0.90 (t, J = 5.7 Hz, 3H). 13C NMR
(75 MHz, CDCl3) d (ppm): 168.4 (C); 158.6 (C); 152.7 (CH); 144.9

C. Deraeve et al. / Bioorg. Med. Chem. 19 (2011) 6225–6232
6231
(C); 139.3 (CH); 138.6 (C); 129.3 (C); 128.4 (CH); 125.5 (CH); 125.2
(CH); 122.5 (CH); 118.0 (CH); 88.1 (C); 33.2 (CH2); 31.9 (CH2); 29.7
(8 Â CH2); 29.6 (CH2); 29.5 (CH); 29.4 (CH2); 29.2 (CH2); 29.0
(CH2); 28.9 (CH2); 22.7 (CH2); 14.1 (CH3). HRMS (ESI) for
J = 5.8 Hz, 3H). 13C NMR (63 MHz, CDCl3) d (ppm): 167.2 (C);
166.8 (C); 155.5 (CH); 144.6 (CH); 139.6 (C); 127.2 (C); 116.7
(CH); 38.5 (CH2); 31.9 (CH2); 29.6–29.1 (6 Â CH2); 28.4 (CH2);
26.8 (CH2); 22.7 (CH2); 14.1 (CH3). MS (ESI) m/z: 317 [M+H]. HRMS
(ESI): for C19H29N2O2: calcd: 317.2229; found: 317.2271.
C31H47N2O2S: calcd: 511.3358; found: 511.3355.
4.9. tert-Butyl N-dodecyl-3-[(RS)-1-hydroxy-3-oxo-2,3-dihydro-
1H-pyrrolo [3,4-c]pyridin-1-yl)]phenylcarbamate (8)
4.12. (RS)-2-(dodecyl)-1-hydroxy-1-phenyl-1,2-dihydro-3H-
pyrrolo[3,4-c]pyridin-3-one (11)
Yield 37% (255 mg – mixture of para/meta 2:1 regioisomers).
The para regioisomer was isolated by recrystallization in acetone.
TLC Rf: 0.64 (dichloromethane/methanol 92.5:7.5, v/v). IR (cmÀ1):
3228, 2924, 2854, 1698, 1603, 1454, 1366, 1149, 1071, 1041,
733, 702. 1H NMR (250 MHz, MeOD) d (ppm): 8.94 (s, 1H); 8.73
(d, J = 5.1 Hz, 1H); 7.46–7.36 (m, 4H); 7.22–7.19 (m, 1H); 3.62 (t,
J = 6.9 Hz, 2H); 1.49–1.44 (m, 2H); 1.38–1.25 (m, 27 H); 0.90 (t,
J = 6.2 Hz, 3H). 13C NMR (75 MHz, MeOD) d (ppm): 167.5 (C);
159.7 (C); 153.4 (C); 152.6 (CH); 144.5 (CH); 142.6 (C); 140.5 (C);
128.9 (CH); 127.0 (CH); 126.2 (C); 124.5 (CH); 123.3 (CH); 118.0
(CH); 87.0 (C); 80.3 (C); 49.6 (CH2); 31.7 (CH2); 29.4–29.1
(5 Â CH2); 28.9 (CH2); 28.0 (CH2); 27.2 (3 Â CH3); 26.3 (CH2);
22.3 (CH2); 13.1 (CH3). MS (DCI/NH3) m/z: 527.3 [M+NH4+]; 510.3
[M+H+]. HRMS (DCI–CH4): for C30H44N3O4 calcd: 510.3332, found:
510.3323.
In this case PhMgBr was used in the place of PhLi. However the
reaction can be performed with both reagents.
Yield 68% (42 mg – mixture of para/meta 1.5:1 regioisomers).
The para regioisomer was isolated by recrystallization in acetone.
White solid, mp 80 °C. TLC Rf: 0.66 (dichloromethane/methanol
95:5, v/v). IR (cmÀ1): 3387, 2918, 2850, 1683, 1662, 1449, 1429,
1398, 1371, 1060, 702. 1H NMR (500 MHz, MeOD) d (ppm): 8.97
(s, 1H), 8.71 (d, J = 5.1 Hz, 1H); 7.43–7.36 (m, 6H); 3.51–3.45 (m,
1H); 3.12–3.06 (m, 1H); 1.65–1.23 (m, 20H); 0.91 (t, J = 6.8 Hz,
3H). 13C NMR (126 MHz, MeOD) d (ppm): 166.3 (C); 158.2 (C);
152.5 (CH); 144.0 (CH); 137.9 (C); 128.6 (CH); 128.4 (2 Â CH);
126.8 (C); 125.9 (2 Â CH); 117.9 (CH); 91.2 (C); 39.4 (CH2); 31.7
(CH2); 29.4–29.1 (5 Â CH2); 28.8 (CH2); 28.2 (CH2); 26.8 (CH2);
22.3 (CH2); 13.0 (CH3). MS (ESI) m/z: 417.3 [M+Na+], 395.4
[M+H+]. HRMS (ESI) for C25H35N2O2, calcd: 395.2699; found:
395.2708. Anal. Calcd for C25H34N2O2: C, 76.10; H, 8.69; N, 7.10.
Found: C, 76.09; H, 8.52; N, 6.91.
4.10. (RS)-1-[3-(dodecylamino)phenyl]-1-hydroxy-1,2-dihydro-
3H-pyrrolo[3,4-c] pyridin-3-one (9)
4.13. (RS)-1-hydroxy-1-tetradecyl -1,2-dihydro-3H-pyrrolo[3,4-
c]pyridin-3-one (12)
To a solution of 8 (32 mg, 62.7 mmol) in CH2Cl2 was added
0.11 mL of trifluoroacetic acid under argon and at 0 °C. The reaction
was stirred at 0 °C temperature for 16 h and then quenched with a
saturated solution of NaHCO3 (pH 7–8). The organic phase was sep-
arated, washed by an aqueous NaHCO3 solution, dried over anhy-
drous Na2SO4 and then concentrated under vaccum. The residue
obtained was purified by a column chromatography of silica (elu-
ent: dichloromethane/methanol 4%) to give 9.
Yield 66% (17 mg). TLC Rf: 0.83 (dichloromethane/methanol
95:5, v/v). IR (cmÀ1): 3393, 3194, 2921, 2852, 1704, 1674, 1603,
1466,1433, 1352, 1143, 1098, 1038, 773, 695. 1H NMR (500 MHz,
MeOD) d (ppm): 8.91 (s, 1H); 8.70 (d, J = 5.1 Hz, 1H); 7.47 (dd,
J = 1.0 and 6.3 Hz, 1H); 7.12 (t, J = 8.1, 1H); 6.87 (dd, J = 2.0 and
2.0 Hz, 1H), 6.78 (dd, J = 1.6 and 7.8 Hz, 1H), 6.60 (dd, J = 1.6 and
8.1 Hz, 1H); 3.06 (t, J = 6.9 Hz, 2H); 1.63–1.52 (m, 2H); 1.39–1.20
(m, 18H); 0.92 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, MeOD) d
(ppm): 168.5 (C); 159.8 (C); 152.4 (CH); 149.5 (C); 144.3 (CH);
140.0 (C); 129.0 (CH); 126.4 (C); 118.0 (CH); 113.2 (CH); 112.6
(CH); 109.6 (CH); 89.0 (C); 43.5 (CH2); 31.7 (CH2); 29.4–29.3
(4 Â CH2); 29.2 (CH2); 29.06 (CH2); 28.9 (CH2); 26.9 (CH2); 22.3
(CH2); 13.0 (CH3). MS (DCI) m/z: 438 [M++C2H5], 410 [M+H+]. HRMS
(DCI): for C25H36N3O2 calcd: 410.2808, found: 410.2810.
Yield 54% (505 mg – mixture of para/meta 2:1 regioisomers).
The para isomer was isolated by preparative HPLC (column Sunfire
C18 150 Â 4.6 mm; eluent: TFA 0.1% /CH3CN 40:60; RTpara = 12.4 -
min and RTmeta = 13.2 min). TLC Rf: 0.83 (dichloromethane/metha-
nol 95:5, v/v). White solid, mp 137 °C. IR (cmÀ1): 3313, 3054, 2916,
2850, 1697, 1613, 1595, 1470, 1359, 1154, 1021, 830, 795, 719. 1H
NMR (250 MHz, MeOD) d (ppm): 8.90 (s, 1H); 8.81 (d, J = 5.1 Hz,
1H); 7.68 (d, J = 5.0 Hz 1H); 2.13–2.04 (m, 2H); 1.30–1.27 (m,
24H); 0.94 (t, J = 5.9 Hz, 3H). 13C NMR (63 MHz, MeOD) d (ppm):
167.8 (C); 158.2 (C); 152.4 (CH); 144.2 (CH); 127.5 (C); 117.6
(CH); 88.0 (C); 37.7 (CH2), 31.7 (CH2); 29.4 -29.0 (9 Â CH2); 23.3
(CH2); 22.3 (CH2); 13.0 (CH3). MS (ESI) m/z: 369.4 [M+Na+], 347.5
[M+H+]. HRMS for C21H35N2O2, calcd: 347.2699; found: 347.2694.
4.14. (RS)-1-dodecyloxy-1-phenyl-1,2-dihydro-3H-pyrrolo[3,4-
c]pyridin-3-one (14)
To a solution of hemiamidal 1316 (200 mg; 0.87 mmol) in 1-
dodecanol (34 mL) was added 2 mL of thionyl chloride. The mix-
ture was heated at reflux and under inert atmosphere for 3 h.
The reaction was monitored by TLC. After complete transformation
of the starting material, the reaction was stopped. The reaction was
treated with saturated solution of NaHCO3 (30 mL) and extracted
with ethyl acetate (3 Â 25 mL). The combinated organic layers
were dried over anhydrous sodium sulfate and concentrated under
vaccum. The residue obtained as a yellow oil was purified by col-
umn chromatography (eluent: gradient dichloromethane/metha-
nol: 100/0 to 96/4) to obtain 14.
Yield 30% (105 mg). White solid, mp 46 °C. TLC Rf: 0.39 (dichlo-
romethane/methanol 96:4, v/v). IR (cmÀ1): 2922, 1714, 1606, 1449,
1051, 747, 696. 1H NMR (250 MHz, CDCl3) d (ppm): 9.10 (bd s, 1H);
8.79 (bd s, 1H); 7.58–7.51 (m, 2H); 7.43–7.35 (m, 3H); 7.29–7.25
(m, 1H); 6.96 (s, 1H); 3.51 (pseudo q, J = 6.5 Hz, 1H); 3.04 (pseudo
q, J = 6.3 Hz, 1H); 1.65–1.57 (m, 2H); 1.50–1.20 (m, 18H); 0.90 (t,
J = 6.0 Hz, 3H). 13C NMR (63 MHz, CDCl3) d (ppm): 168.5 (C);
155.6 (C); 153.2 (CH); 146.0 (CH); 138.7 (C); 129.0 (CH); 128.8
4.11. 2-Dodecyl-1H-pyrrolo[3,4-c]pyridine-1,3-dione (10)
To
a solution of 3,4-pyridinedicarboximide (5) (200 mg;
1.35 mmol) in DMF (13.5 mL), 1-iodododecane (400 mg;
1.35 mmol) and potassium carbonate (931.5 mg; 6.75 mmol) were
added. The mixture was stirred for 12 h at 50 °C under nitrogen.
Then water (15 mL) was added and the reaction mixture was ex-
tracted with ethyl acetate (3 Â 10 mL). The combined organic
phases were dried over anhydrous sodium sulfate, filtrated and
concentred in vaccum to give 10.
Yield 57% (245 mg). TLC Rf: 0.77 (dichloromethane/metha-
nol 95:5, v/v). Beige solid, mp 56 °C. IR (cmÀ1): 2915, 2847, 1705,
1400, 1065, 1058, 865. 1H NMR (250 MHz, CDCl3) d (ppm): 9.17
(s, 1H); 9.09 (d, J = 4.8 Hz, 1H); 7.78 (d, J = 4.0 Hz, 1H); 3.75 (t,
J = 7.2 Hz, 2H); 1.70–1.64 (m, 2H); 1.34–1.27 (m, 18H); 0.92 (t,

6232
C. Deraeve et al. / Bioorg. Med. Chem. 19 (2011) 6225–6232
(2 Â CH); 126.5 (C); 125.5 (2 Â CH); 117.9 (C); 91.6 (C); 63.4 (CH2);
31.9 (CH2); 29.6–29.3 (7 Â CH2); 26.1 (CH2); 22.7 (CH2); 14.1 (CH3).
MS (ESI) m/z: 395.4 [M+H+]. HRMS: for C25H35N2O2: calcd:
395.2699; found: 395.2693. Anal. Calcd for C25H34N2O2 0.75
CH3OH: C, 73.89; H, 8.91; N, 6.69. Found: C, 74.33; H, 8.71; N, 6.24.
determined to be the lowest concentration of compound that
inhibited visible growth of the bacterial culture (the absorbance
value obtained for untreated bacilli was taken as a growth control).
The IC50, corresponding to the concentration that inhibits 50% of
the bacterial growth, was determined by fitting the data by non-
linear regression using the program GraphPad Prism 5.0. Isoniazid-
and ciprofloxacin-treated cultures were used as positive controls.
Each mother solution was prepared in DMSO at concentrations
100 times greater than the highest test concentration.
4.15. Enzymatic inhibition experiments
M. tuberculosis InhA was overexpressed in E. coli and purified as
previously described.18 Isoniazid and NADH were obtained from
Sigma-Aldrich. The substrate 2-trans-decenoyl-CoA was synthe-
sized from 2-trans-decenoic acid using the mixed anhydride meth-
od and purified according to the procedure described by Goldman
and Vagelos.19 The concentration of the substrate was determined
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.017.
on the basis of
For the enzymatic inhibition assays, pre-incubation reactions
were performed in 90 L (total volume) of 30 mM PIPES buffer
solution, 150 mM NaCl, pH 6.8 at 25 °C containing 70 nM InhA,
200 M cofactor (NADH) and the tested compound (at 100 or
30 M). DMSO was used as co-solvent and its final concentration
was 0.5%. After 5 min of pre-incubation, the addition of 35 M sub-
e .
260 = 22 600 MÀ1 cmÀ1
References and notes
l
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l
l
l
strate (trans-2-decenoyl-CoA) initiated the reaction which was fol-
lowed at 340 nm (oxidation of NADH) and at 25 °C using a
thermostated Uvikon 923 spectrophotometer (Bio-Tek Kontron
Instruments). Control reactions were carried out under the same
conditions as those described above but without ligands, and tri-
6. Saint-Joanis, B.; Souchon, H.; Wilming, M.; Johnsson, K.; Alzari, P.; Cole, S. T.
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Quitugua, T.; Graviss, E. A. Antimicrob. Agents Chemother. 2003, 47, 1241.
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closan (10 lM) was used as a positive control. The initial rates of
the reactions were calculated. The inhibitory activity of each com-
pound tested was expressed as the percentage inhibition of InhA
activity (initial velocity of the reaction) with respect to the control
experiments. All activity assays were performed in triplicate.
4.16. Bacterial growth inhibition
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Org. Chem. 2005, 70, 10502.
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17. Contaminated with 20% of the meta regioisomer.
18. Quémard, A.; Sacchettini, J. C.; Dessen, A.; Vilcheze, C.; Bittman, R.; Jacobs, W.
R., Jr.; Blanchard, J. S. Biochemistry 1995, 34, 8235.
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The susceptibility of M. tuberculosis H37Rv to all synthesized
compounds was evaluated by using a colorimetric microassay
based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT, Sigma–Aldrich) to formazan by
metabolically active cells.20,21 Briefly, serial two-fold dilutions of
the inhibitor solubilized in DMSO were prepared in 100
Middlebrook 7H9 broth base (Difco) and dispensed into 96-well
microtiter plates, to each well of which were added 100 L of M.
tuberculosis (diluted in 7H9 broth to an OD600 of 0.02). Plates were
incubated for six days at 37 °C, and MTT was added (50 L of a
lL
l
l
1 mg/mL solution). Plates were incubated for a further 24 h and
solubilization buffer [DMF/SDS 20% (w/v), 1:2] was added to each
well. Absorbance was measured at 570 nm. The MIC was