Development of isoniazid-NAD truncated adducts embedding a lipophilic fragment as potential bi-substrate InhA inhibitors and antimycobacterial agents

Article abstract of DOI:10.1016/j.ejmech.2010.07.016

Isoniazid-NAD truncated adducts embedding a lipophilic fragment were designed, synthesized and evaluated as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (InhA) of Mycobacterium tuberculosis and as antimycobacterial agents. These compounds, planned as bi-substrate inhibitors and inspired from the active metabolite of isoniazid, combine both the nicotinamide moiety of the cofactor NAD and a lipophilic hydrocarbon chain mimic of the InhA substrate. The lipophilic fragment was introduced using either Suzuki-Miyaura cross-coupling or a classical nucleophilic substitution reaction. Several compounds developed in this work were indeed able to inhibit the InhA activity and showed promising antimycobacterial activities. However a direct correlation between the expressed activity and the bi-substrate mode of action could not yet be unambiguously demonstrated.

Full text of DOI:10.1016/j.ejmech.2010.07.016

European Journal of Medicinal Chemistry 45 (2010) 4554e4561
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of isoniazideNAD truncated adducts embedding a lipophilic
fragment as potential bi-substrate InhA inhibitors and antimycobacterial agents
,
b
c
Tamara Delaine a, Vania Bernardes-Génisson a , Annaïk Quémard , Patricia Constant ,
*
,
Bernard Meunier d, Jean Bernadou a
*
a Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse, Cedex 4, France
b Département Mécanismes Moléculaires des Infections Mycobactériennes, CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne,
F-31077 Toulouse, France
c Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France
d Palumed, 3 rue de l’Industrie e Zone industrielle de Vic 31320 Castanet-Toulousan, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoniazid-NAD truncated adducts embedding a lipophilic fragment were designed, synthesized and
evaluated as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (InhA) of Mycobacterium
tuberculosis and as antimycobacterial agents. These compounds, planned as bi-substrate inhibitors and
inspired from the active metabolite of isoniazid, combine both the nicotinamide moiety of the cofactor
NAD and a lipophilic hydrocarbon chain mimic of the InhA substrate. The lipophilic fragment was
introduced using either SuzukieMiyaura cross-coupling or a classical nucleophilic substitution reaction.
Several compounds developed in this work were indeed able to inhibit the InhA activity and showed
promising antimycobacterial activities. However a direct correlation between the expressed activity and
the bi-substrate mode of action could not yet be unambiguously demonstrated.
Received 20 April 2010
Received in revised form
7 July 2010
Accepted 9 July 2010
Available online 15 July 2010
Keywords:
Bi-substrate inhibitor
Enoyl-ACP reductase
Truncated isoniazideNAD adduct
Antitubercular agent
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
substrates using NADH cofactor as a hydrogen donor (Scheme 1)
[9,10]. The inhibition of this enzymatic activity blocks the fatty acid
Resistance of Mycobacterium tuberculosis (MTB) (pathogen agent
of tuberculosis) strains to the two main drugs, isoniazid (INH) and
rifampicin, used in therapy regimen of tuberculosis [1] is a worry
problem nowadays. Indeed, no new first-line antibiotics have been
introduced into the pharmaceutical arsenal against this disease in
the last thirty years. Thus it is well recognized that there is a pressing
need for the development of more effective antimicrobial agents.
Isoniazid [2] is a pro-drug that requires oxidative activation by
the MTB catalaseeperoxidase KatG [3e5]. The activated form of
INH, probably an isonicotinoyl radical, [6e8] is capable of reacting
with NAD cofactor to form the covalent adduct INHeNAD (Fig. 1)
which inhibits the InhA enzyme of MTB.
elongation controlled by the Fatty Acid Synthase II system (FAS-II)
necessary for the biosynthesis of mycolic acids, specific and
essential components of the mycobacterial envelope [11,12].
It is well established nowadays that resistance to INH largely
results from mutations in MTB KatG that diminish its ability to
convert INH into its active form [13e16]. This phenomenon of
resistance is also attributed to a lesser degree to mutations in the
InhA enzyme, more precisely at the site of interaction with the
diphosphate moiety of the NADH [17]. Based on these consider-
ations, we can assume that truncated INHeNAD analogues (absence
of the adenosine diphosphate ADP motif) able to directly inhibit the
InhA target without requiring the KatG dependent activation step
might have tremendous promise as novel drugs for fighting drug
resistance related to KatG or InhA. In addition, the truncated
analogues would be more useful since diphosphate-linked mole-
cules are readily cleaved by enzymes such as phosphodiesterases
and their ionic nature prevents them from penetrating the cell
membrane [18]. Another important factor to take into account in
developing new drugs is the criterion of selectivity. Although it is
todaya debated question [19], enzymes of the FAS-II systems such as
InhA is an enoyl-ACP reductase that catalyzes the reduction of
the trans double bond conjugated to the carbonyl group of fatty acyl
* Corresponding authors. Tel.: þ33 (0)5 61 33 31 50/33 (0)5 61 33 31 17; fax: þ33
(0)5 61 55 30 03.
E-mail addresses: vaniabg@lcc-toulouse.fr (V. Bernardes-Génisson), bernadou@
lcc-toulouse.fr (J. Bernadou).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.016

T. Delaine et al. / European Journal of Medicinal Chemistry 45 (2010) 4554e4561
4555
2. Results and discussion
N
O
2.1. Synthesis
NH2
O
H
N
N
N
NH2
For the preparation of the pyridine, pyridinium and 1,4-dihy-
dropyridine compounds containing a lipophilic group attached to
the corresponding aroyl moiety, two different procedures previ-
ously developed in our laboratory [21,22] were extended to provide
access to the novel functionalized hemiamidal intermediates 6a
and 6c (method a in Scheme 2) as precursors of pyridinium and
1,4-dihydropyridine bi-substrate-type compounds.
O
P
O
P
N
N
O
O
O
O
O
OH OH
OH OH
OH
OH
O
The functionalized intermediate 6a can also be prepared by
a more concise route using 3,4-pyridinedicarboximide (7) as
substrate in the presence of 1,3-dibromobenzene and BuLi. In view
of more extensive functionalization of the basic hemiamidal
structure, intermediate 6c was also prepared using 3,4-pyr-
idinedicarboximide in the presence of the protected bromophenol
8a. The deprotection of the allyl protecting group of 6b was readily
achieved by reacting with tetrakis(triphenylphosphine)palladium
(0) in basic medium (Scheme 2, method b).
Introduction of the lipophilic residue of 9, 10, 11 through CeC
bond formation was performed by a palladium-catalyzed reaction,
typically known as SuzukieMiyaura cross-coupling, in the presence
of Ag2O [23]. The reaction of the aryl halide 6a with the appropriate
commercially available alkyl and arylboronic acid reagents led to
target compounds 9, 10, 11 as shown in Scheme 3.
The ether linker containing compound 6d bearing a long linear
hydrocarbon chain (C12) was obtained through a simple nucleophilic
substitution reaction of 6c with iodododecane, along with a by-
product arising from a doublealkylationproduct (m/z579 [Mþ þ 1]). In
ordertoimprove 6dpreparation,weoptedtoobtainthiscompoundby
a direct synthetic sequence described in Scheme 2 (method b, ways h
and i) starting from alkylation of 3-bromophenol with iodododecane.
By this alternative route, formation of the impurity was overcome and
deprotection and protection steps were no longer necessary.
Synthesis of pyridiniums 12, 14, 15 and the corresponding keto-
amide 1,4-dihydropyridines 13, 16, 17 was accomplished by the
procedure previously described (Schemes 3 and 4) [21,22,24]. The
alkylating agent 3-hydroxypropyl bromoacetate was synthesized
by esterification of bromoacetate bromide with 1,3-propanediol in
very good yield (85%).
O
HO
NH
NH
H
HO
NH2
O
O
N
HO
O
N
OH
N
HO
O
OH
OH
OH
Fig. 1. Structures of INHeNAD adduct and of some truncated BH-NAD adducts of the
first generation (in the pyridine and pyridinium salt structures the -keto-amide motif
is found solely in the cyclised hemiamidal form. In contrast, 1,4-dihydropyridine
derivatives mainly exist in solution as the open -keto-amide tautomer).
g
g
InhA homologs, which are present in bacteria but not in mammalian
cells, are prime targets for the discovery of drugs with low potential
toxicity to the human host [20].
We started some years ago a program aimed at the discovery of
new antitubercular compounds based on the development
of truncated INHeNAD analogues (bearing a benzoyl ring in place
of the isonicotinoyl moiety and the ADP moiety been lacking) as
direct inhibitors of the enoyl-ACP reductase InhA [21]. Unfortu-
nately, the first generation of the BH-NAD truncated analogues
previously synthesized (see Fig. 1, bottom) and tested in our lab
inhibited InhA insufficiently [22]. This indicated that InhA affinity
for the truncated adducts lacking the ADP moiety was too low. To
overcome this loss of affinity by InhA, we hypothesized that the
design of new bi-substrate-type inhibitors based on a covalent
association between molecules mimicking the InhA substrate and
the NADH cofactor might provide compounds with higher affinities
and selectivities for InhA catalytic site (Scheme 1).
In the present work, we report the synthesis of a second
generation of truncated BH-NAD analogues bearing a lipophilic
residue linked to the aroyl group, mimicking the substrate. Some of
these molecules display very significant inhibitory activities against
InhA enzyme and mycobacterial growth.
2.2. Biology
2.2.1. InhA inhibition assay
In view to realize
compounds as direct InhA inhibitors, the InhA inhibition
a brief screening of the synthesized
InhA
n
n
enzyme
n
n
n
substrate
site
Strategy
Strategy
O
O
or
S-ACP
S-ACP
O
O
HO
NH
HO
NH
H
H
O
O
H
O
O
NH2
NH2
NH2
N
R
N
N
R
N
N
NADH/NAD
co-factor site
ADP-ribose
ADP-ribose
dihydropyridine type
pyridine type
inhibitor
pyridinium type
inhibitor
inhibitor
Scheme 1.

4556
T. Delaine et al. / European Journal of Medicinal Chemistry 45 (2010) 4554e4561
O
Method a
NH
Method b
O
7
CH3
O
N
Br
Br
X
Br
OH
OCH3
3
5
4
c) d) e)
2
N
b)
a)
f)
i)
Br
N
O
O
OH
NH
HO
+
+
O
O
O
Br
O
O
OR
N
N
OH
N
N
N
N
N
1
g) or h)
Br
6a X = Br
8a R= CH2CH=CH2
6b X = OCH2CH=CH2
6c X = OH
6d X = OC12H25
j)
8b R = C12H25
Scheme 2. Reagents: a) LDA/ether/2, ꢀ78 ꢁC, 28%, b) NaBH4/ethanol, 85%, c) HCOOH, reflux, d) NH3/MeOH 7 N, e) HCl 2 M, 85%, f) SOCl2 then NH4OH 80%, g) allyl bromide, HON
(C4H9)4, CH2Cl2, 93%, h) C12H25I, K2CO3, 94%, i) 1,3-dibromobenzene, n-BuLi, THF, ꢀ78 ꢁC, 32% or 8a, t-BuLi, THF, ꢀ78 ꢁC, 23% or 8b, t-BuLi, THF, ꢀ78 ꢁC, 8%, j) tetrakis(triphenyl)
phosphinepalladium(0), K2CO3, methanol, 77%.
7
10
Br
11
11
11
O
NH
HO
8
b)
O
e)
d)
O
a)
NH
NH
NH
HO
HO
HO
O
O
O
N
c)
NH
HO
NH2
13
O
N
R
N
R
N
N
6a
9
12
11
N
R = CH2COOEt
Scheme 3. Reagents:a) n-dodecylboronic acid, Pd(dppf)Cl2, Ag2O, K2CO3, THF, 80 ꢁC, 24%, b) neoctylboronic acid, Pd(dppf)Cl2, Ag2O, K2CO3, THF, 80 ꢁC,15%, c) 4-n-nonylbenzeneboronic
acid, Pd(dppf)Cl2, Ag2O, K2CO3, THF, 80 ꢁC, 26%, d) BrCH2COOEt, THF, reflux, 78%, e) NaBH(OAc)3, ethanol:acetic acid (3:1), 33%.
percentage was calculated according to the method previously
described (Table 1) [7]. The molecules were tested at 100 M. A pool
of INHeNAD adducts was prepared and purified as previously
described [7] and it was used as a positive control giving strong
inhibition of InhA activity (Table 1).
In the 1,4-dihydropyridine series that is structurally closer to
INHeNAD adduct than the two previous series, the ether linker
containing derivatives 16 and 17 (O-C12 substituent) were not
active even though the incubation time was of 2 h (data not shown).
In contrast, 1,4-dihydropyridine 13 (C12 substituent) was able to
m
Concerning the synthesized products, several molecules of the
pyridine series displayed inhibitory potency towards InhA activity.
It is noteworthy that the best inhibitor was the compound 6d with
an O-C12 ether link. This compound, first incubated for 5 min with
InhA, was shown to inhibit enzymatic activity by 41%. (Table 1). The
inhibition was much greater (80%) for longer incubation time (2 h).
Neither the phenolic hemiamidal 6c (OH substituent) nor
compound 6b with a short O-allyl chain exhibited significant
inhibitory activity towards InhA (Table 1). These results suggest
that the introduction of the medium C12 alkyl chain substituent in
compound 6d was responsible for the increase of the inhibitory
activity. The presence of the oxygen atom as a linker also seemed to
be critical for inhibition since a simple suppression of the oxygen
atom led to a significant loss of activity (compound 9, C12
substituent).
inhibit 25% of the InhA activity at 10 mM. The assays carried out at
a higher concentration of inhibitor 13 were not conclusive because
of solubility problems. Interestingly, the inhibition rate observed
for 13 at 10
mM was comparable to that obtained for the pyridinium
analogue 12 (C12 substituent) at the same concentration (19%).
OH
NH
O
11
a)
HO
NH
HO
O
O
N
N
6d
6c
In the more elaborate bi-substrate systems, the pyridinium
series, the best activity was observed for 12 (C12 substituent),
O
O
11
O
11
which was able to inhibit 91% of InhA activity at 100
mM and 40% at
20 M with a 5 min pre-incubation time (Table 1). The longer
m
b)
c)
NH
O
HO
incubation time (2 h) was not efficient in this case. Curiously, here,
the introduction of an ether link at the meta position of the aroyl
group (compounds 14 and 15, O-C12 substituent) reduced the effect
on InhA activity, in contrast to the situation in the pyridine series
O
NH2
N
R
N
R
16
(see above). The inhibitory activities of 14 and 15 at 100 mM were
14
R = CH2COOEt
R = CH2COO(CH2)3OH
comparable (30e35%). It is interesting to note that these pyr-
idinium salts 14, 15 and especially 12 display InhA inhibitory
activity while the pyridinium salts of the first generation of BH-
NAD analogues that lack the lipophilic substituent (Fig. 1), at the
same concentration, were inactive [22].
15
17
Scheme 4. Reagents: a) C12H25I, K2CO3, 61%, b) BrCH2COOEt THF, reflux, 54% or
BrCH2COOCH2CH2CH2OH, THF, reflux, 35%, c) NaBH(OAc)3, ethanol:acetic acid (3:1),
51% for 16 and 44% for 17.

T. Delaine et al. / European Journal of Medicinal Chemistry 45 (2010) 4554e4561
4557
Table 1
Compounds tested as inhibitors of InhA. Results are expressed as a percentage of InhA inhibition. *Pool of a mixture of INHeNAD adducts [7].
Type of compound
Pyridine
Pyridinium
DHP
Pool*
6a
6b
6c
6d
9
10
11
12
14
15
13
16
17
Conc
m
M
Incubation time
% of InhA inhibition
20 23 17
100
5 min (2 h)
5 min (2 h)
5 min (2 h)
5 min (2 h)
41 (80)
4
9
17
91
40 (28)
19
35
30
ND
ND
25
6
3
ND
ND
92 (97)
67 (87)
20
10
1
ND: not determined due to precipitation; DHP: 1,4-dihydropyridine. Mean values of at least three different measurements. Errors were in the range of ꢂ3% of the obtained
value.
Table 2
Compounds tested as inhibitory agents of M. smegmatis and M. tuberculosis growth.
Type of compound
Pyridine
Pyridinium
DHP
Isoniazid
6a
6b
6c
6d
68
83
9
10
4.5
4.6
ꢂ1
11
12
13
M. smegmatis
50% inhibition (
IC50 ( M)
mM)
>5000
1300
300
50
47
ꢂ7
1800
3.2
5.7
ꢂ0.9
5.4
5.3
ꢂ0.6
8
7.8
m
ꢂ5
ꢂ0.6
MIC (
m
M)
M)
156
156
78
9.8
19.5
19.5
9.8
39
9.8
9.8
39
<0.3
M. tuberculosis
MIC (
m
DHP: 1,4-dihydropyridine. Mean values of at least three different measurements.
In summary, bi-substrate-type analogues 6d, 12, 13, 14 and 15
are able to inhibit InhA activity whereas none of the first generation
truncated non-bi-substrate adducts [22] were active. These results
suggest that there might be space for additional hydrophobic
interactions in the substrate binding pocket of InhA and that the
introduction of the alkyl chain led to a better interaction of the
molecules with the active site.
In compounds of the bi-substrate-type in the pyridine series,
compound 11 was the only one that lacks of inhibitory activity on
M. smegmatis growth. This lack of activity may be related to the
rigidity and the bulk of the bi-aryl system.
The compounds active on M. smegmatis (6d, 9, 10, 12, 13) were
selected for further evaluation against the growth of M. tuberculosis
pathogen. Susceptibility of M. tuberculosis H37Rv to these
compounds and to isoniazid was determined by measuring the
minimum inhibitory concentration (MIC) (Table 2). All compounds
were active. Compounds 6d (O-C12 substituent), 10 (C8 substit-
uent) and 13 (C12 substituent) showed the same MIC as those
found for M. smegmatis. Pyridinium derivative 12 (C12 substituent)
was less active against M. tuberculosis than M. smegmatis. In
contrast, 9 (C12 substituent) was 8-fold more active on M. tuber-
culosis growth (Table 2).
2.2.2. Bacterial growth inhibition experiments
The next step consisted of evaluating compounds 6ae6d and
9e13 with regards to bacterial growth inhibition of the non-path-
ogenic mycobacterial species Mycobacterium smegmatis.
The bacterial growth was followed by using a colorimetric test
involving 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazo-
lium bromide (MTT) reduction measured at 570 nm. Isoniazid was
tested as a positive control of inhibition. The concentration of
6ae6d and 9e13 able to inhibit 50% of M. smegmatis growth was
determined (Table 2). For the most potent compounds, the IC50 and
the minimum inhibitory concentration (MIC) were also established.
Compounds 6a (Br substituent), 6b (O-allyl substituent), 6c (OH
substituent) and 11 (PhC9 substituent) did not exhibit significant
The lack of inhibition of InhA activity for compound 9 (C12
substituent) (Table 1), in contrast with the low MIC values for M.
tuberculosis suggests here also that its activity could originate from
a mode of action different from InhA inhibition.
inhibition (50% inhibition in the range 300 to >5000
mM). In
3. Conclusion
contrast the bi-substrate-based analogues 6d (O-C12 substituent),
9 (C12 substituent), 10 (C8 substituent), 12 (C12 substituent) and 13
In this work we successfully synthesized three series (pyridine,
pyridinium and 1,4-dihydropyridines) of potential inhibitors of
MTB enoyl-ACP reductase (InhA) that are structurally simplified
analogues of INHeNAD adduct, the active form of isoniazid. The
incorporation of a lipophilic component into the nicotinamide
hemiamidal framework provided more active derivatives.
Compounds 6d, 12, 13, 14 and 15 showed a significant InhA inhib-
itory activity as compared to the first generation of truncated BH-
NAD adducts which were not active. These data showed that the
presence of an alkyl substituent partially compensated the loss of
affinity linked to the truncation of the ADP-ribose moiety of the
adduct. During the design of these compounds we speculated that
the hydrophobic substituent, embedded to the bi-substrate inhib-
itor, would be recognized by the fatty acid substrate binding site of
InhA and that the nicotinamide moiety would interact with the
cofactor pocket. Thus, these derivatives would act as dual inhibi-
tors. Although the results of this work do not provide information
about the mode of interaction of the bi-substrate-based inhibitors
(C12 substituent) showed IC50s varying from 4.6 to 83
mM and MICs
varying from 9.8 to 156 M. Compounds 12 and 13, which
m
respectively represent truncated oxidized and reduced BH-NAD
adducts substituted on the aryl ring (C12 substituent) were the
most active compounds (MIC of 9.8
MIC is 8-fold lower than that of the parent pyridine-based deriva-
tive 9 (MIC of 78 M) and their activities were higher than the
reference antitubercular agent isoniazid (MIC of 39 M in the
mM for both compounds). Their
m
m
conditions used). So, consistently, 12 and 13 which were the most
potent inhibitors of InhA activity among all the molecules tested
(Table 1) appeared also as the most potent inhibitors of M. smeg-
matis growth.
However, compound 10 bearing a C8-chain as a substituent did
not show any significant InhA inhibitory activity while it was one of
the most efficient inhibitory compounds towards M. smegmatis
growth. So we cannot rule out the possibility of existing of other
targets, different from InhA, or of other unspecified mechanisms.

4558
T. Delaine et al. / European Journal of Medicinal Chemistry 45 (2010) 4554e4561
with InhA, they strongly suggest that the addition of a lipophilic
residue is valuable for their effect on InhA activity.
Tested for the antimycobacterial activity, some compounds (10,
12, 13) exhibited higher inhibition of M. smegmatis growth than the
reference compound isoniazid, although they appeared less active
toward M. tuberculosis growth.
In summary, this work provides key information for the devel-
opment of further optimized direct InhA inhibitors presenting
a lipophilic fragment on INHeNAD scaffold [25]. A structureeactivity
relationship study of the effects of the lipophilic substituent on the
affinity for InhA of these bi-substrate-type compounds will be
performed.
temperature over 1 h. Water was added (40 mL) and the product
was extracted with ethyl acetate (4 ꢃ 50 mL). The combined
organic phases were dried over sodium sulfate and the solvent was
evaporated in vacuo. The residue was purified by recrystallization in
acetone to yield 670 mg (32%) of hemiamidal 6a as beige solid. Mp
211 ꢁC. IR (KBr, nmax/cmꢀ1) 3296, 3059, 1693, 1609, 1444, 1291, 647.
1H NMR (500 MHz, DMSO-d6)
d
9.53 (s, 1H), 8.89 (d, J ¼ 0.9 Hz, 1H),
8.75 (d, J ¼ 5.1 Hz, 1H), 7.70 (t, J ¼ 1.8 Hz, 1H), 7.56 (ddd, J ¼ 1.0 Hz,
J ¼ 2.0 Hz and J ¼ 7.9 Hz, 1H), 7.44e7.43 (m, 2H), 7.34 (t, J ¼ 7.9 Hz,
1H), 7.33 (s,1H). 13C NMR (126 MHz, DMSO-d6)
d 167.5 (C), 158.3 (C),
153.6 (CH), 145.2 (CH), 143.8 (C), 131.7 (CH), 131.2 (CH), 128.8 (CH),
126.5 (C), 125.3 (CH), 122.2 (C), 118.3 (CH), 86.6 (C). MS (FAB) m/z
307e305 (M þ Hþ). HRMS (FAB) calcd. for C13H10N2O2Br 304.9926;
found 304.9927. Anal. Calcd for C13H9N2O2Br: C, 51.17; H, 2.97; N,
9.18. Found: C, 51.55; H, 2.68, N, 8.97.
4. Experimental part
4.1. Chemistry
4.1.2. 1-Hydroxy-1-(3-(propen-3-yl)oxyphenyl)-1,2-dihydropyrro-
lo[3,4-c]pyridin-3-one (6b)
General procedure. The melting points were determined on an
Electrothermal 9300 capillary melting point apparatus and are not
corrected. Infra-red spectra were recorded on a PerkineElmer
spectrometer GX 2000. 1H NMR spectra were recorded on a Bruker
spectrometer at 250, 300 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:
To a solution of 3-bromo-1-(propen-3-yloxy)benzene 8a (4.4 g,
0.020 mol) in dry THF (20 mL) under argon and at ꢀ78 ꢁC, was
added tert-butyl lithium (13.5 mL, 0.025 mol, 1.5 M in pentane).
After
1
h
with stirring at ꢀ78 ꢁC,
a solution of 3,4-pyr-
idinedicarboximide 7 (2.0 g, 0.016 mol) in dry THF (80 mL) was
added dropwise at ꢀ78 ꢁC and then the temperature of the reaction
was increased up to room temperature. Water was added (80 mL)
and the product was extracted with ethyl acetate (4 ꢃ 100 mL). The
combined organic phases were dried over sodium sulfate and the
solvent was evaporated in vacuo. The residue obtained was purified
by column chromatography (gradient dichloromethane/methanol
100/0e90/10) to yield 2.50 g (43%) of a mixture of para/meta
isomers (3:1). The solid was purified by recrystallization in acetone
to give hemiamidal 6b as a white solid (0.81 g, 23%). Mp 170 ꢁC. IR
(KBr, nmax/cmꢀ1): 3477, 3413, 3229, 3062, 1709, 1615, 1248. 1H NMR
7.29
d
¼ ppm, DMSO
d
¼ 2.50 ppm and MeOD
d
¼ 3.31 ppm. 13C
NMR spectra were recorded on a Bruker spectrometer at 50, 63, 75
and 126 MHz. Mass spectra (FAB and EI) were obtained on an MS-
Nermag R10-10 spectrometer. For the MS-ESI a PerkineElmer
SCIEX API 365 spectrometer was used and for the MS-CI a Thermo
electron TQS 7000 was used. Column chromatography was per-
formed on silica gel (Merck 0.063e0.200 mm). The purity of
compounds 6a, 6b, 6c, 6d, 9, 10, 11, 12, 15 was assessed by micro
analysis and/or by thin layer chromatography using
a 95:5
dichloromethane:methanol system. The products showed a single
spot on TLC SDS 60F250 silica gel plates (0.2 mm)/UV254 or if
a minor spot appeared, it was no more intense than the principal
spot of a 5% solution of the analyzing product, indicating a purity of
>95%. Owing to the instability of 1,4-dihydropyridine compounds,
13 and 17 were synthesized just before biological assays and
purified by column chromatography to give a single spot by TLC. 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 sodium in the
presence of benzophenone. Dimethylformamide and acetonitrile
were dried on calcium hydride.
(500 MHz, DMSO-d6)
d
9.46 (s, 1H), 8.87 (d, J ¼ 1.4 Hz, 1H), 8.72 (d,
J ¼ 5.1 Hz, 1H), 7.42 (dd, J ¼ 1.2 Hz and J ¼ 4.8 Hz, 1H), 7.27 (t,
J ¼ 8.0 Hz, 1H), 7.16 (s, 1H), 7.11 (t, J ¼ 2.5 Hz, 1H), 7.00 (d, J ¼ 7.8 Hz,
1H), 6.92 (dd, J ¼ 2.4 Hz and J ¼ 8.1 Hz, 1H), 6.03 (m, 1H), 5.39 (dd,
J ¼ 1.5 Hz and J ¼ 17.4 Hz, 1H), 5.25 (dd, J ¼ 1.5 Hz and J ¼ 10.5 Hz,
1H), 4.57 (dd, J ¼ 1.5 Hz and J ¼ 5.1 Hz, 2H). 13C NMR (126 MHz,
DMSO-d6)
d 167.5 (C), 158.8 (C), 158.7 (C), 153.4 (CH), 145.0 (CH),
142.7 (C), 134.1 (CH), 130.0 (CH), 126.5 (C), 118.3 (CH), 118.2 (CH),
117.9 (CH2), 114.7 (CH), 112.7 (CH), 87.8 (C), 68.7 (CH2). MS (FAB) m/z
283 (M þ Hþ), 266 (M þ Hþ ꢀ HO), 242 (M þ Hþ ꢀ CH2CHCH2), 226
(M þ Hþ ꢀ OCH2CHCH2). HRMS (FAB) calcd for C16H15N2O3
283.1083; found 283.1085. Anal. Calcd for C16H15N2O3: C, 68.07; H,
5.00; N, 9.92. Found: C, 67.74; H, 4.91, N, 9.64.
4.1.1. 1-(3-Bromophenyl)-1-hydroxy-1,2-dihydropyrrolo[3,4-c]
pyridine-3-one (6a)
4.1.3. 1-Hydroxy-1-(3-hydroxyphenyl)-1,2-dihydropyrrolo[3,4-c]
pyridin-3-one (6c)
4.1.1.1. Method a. A solution of 5 (331 mg, 1.1 mmol) in SOCl2
(20.0 mL, 300.0 mmol) was stirred at 60 ꢁC under inert atmosphere
for 2 h. The reaction mixture was concentrated, the residue dis-
solved in dichloromethane and the solvent removed in vacuo. This
last operation was repeated twice. The oil obtained was taken up in
acetone (17 mL) and a solution of 32% NH4OH (9.2 mL, 3.0 mmol)
added dropwise. The final mixture was stirred at room temperature
for 1 h, concentrated in vacuo and the residue obtained was purified
by silica gel column chromatography (gradient dichloromethane/
methanol 100/0e95/5) to give 268 mg (80%) of hemiamidal 6a as
a beige solid.
To a solution of hemiamidal 6b (200 mg, 0.71 mmol) in meth-
anol (7.4 mL) was added tetrakis(triphenylphosphine)palladium
(16 mg, 0.03 mmol) under argon. After 5 min with stirring, potas-
sium carbonate (621 mg, 4.50 mmol) was introduced to the reac-
tion mixture. The progress of the reaction was followed by TLC.
After 36 h the solvent was evaporated off in vacuo and the residue
was purified by silica gel column chromatography (gradient
dichloromethane/methanol 95/5e80/20) to yield 132 mg (77%) of
hemiamidal 6c as a white solid. Mp decomposition after 150 ꢁC. IR
(KBr, nmax/cmꢀ1) 3232, 2960, 1695, 1614, 1446, 1275. 1H NMR
(500 MHz, DMSO-d6)
d
9.42 (s, 1H), 8.86 (s, 1H), 8.72 (d, J ¼ 5.0 Hz,
4.1.1.2. Method b. To a solution of 3,4-pyridinedicarboximide 7
(1.0 g, 6.8 mmol) in dry THF (50 mL) was added 1,3-dibromo-
benzene (1.7 mL, 13.5 mmol) and butyllithium (7.1 mL, 14.2 mmol,
2 M in cyclohexane) at ꢀ78 ꢁC and under argon. The mixture was
stirred at ꢀ78 ꢁC for 1 h and then allowed to warm up to room
1H), 7.36 (d, J ¼ 5.1 Hz, 1H), 7.16 (t, J ¼ 8.1 Hz, 1H), 7.08 (s broad, 1H),
6.91e6.90 (m, 2H), 6.71 (dt, J ¼ 1.2 Hz and J ¼ 7.0 Hz, 1H), 5.75 (s,
1H). 13C NMR (126 MHz, DMSO-d6)
d 167.5 (C), 159.1 (C), 157.9 (C),
153.4 (CH), 145.0 (CH), 142.5 (C), 129.9 (CH), 127.0 (C), 118.2 (CH),
116.6 (CH), 115.6 (CH), 113.0 (CH), 87.6 (C). MS (FAB) m/z 243

T. Delaine et al. / European Journal of Medicinal Chemistry 45 (2010) 4554e4561
4559
(M þ Hþ), 226 (M þ Hþ ꢀ HO). HRMS (FAB) calcd for C13H11N2O3
(m, J ¼ 7.0 Hz, 2H), 1.30e1.27 (m, 18H), 0.90 (t, J ¼ 6.9 Hz, 3H). 13
C
243.0770; found 243.0777.
NMR (126 MHz, CDCl3) d 168.1 (C), 158.3 (C), 153.2 (CH), 145.4 (CH),
144.0 (C), 138.2 (C), 129.3 (CH), 128.8 (CH), 125.6 (CH), 125.2 (C),
122.7 (CH), 117.9 (CH), 88.1 (C), 36.0 (CH2), 31.5 (CH2), 32.0 (CH2),
29.9 (CH2), 29.8 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2),
29.3 (CH2), 22.7 (CH2), 14.1 (CH3). MS (FAB) m/z 395 (M þ Hþ), 378
(M þ Hþ ꢀ HO), 367 (M þ Hþ ꢀ CH2CH2). HRMS (FAB) calcd for
C25H35N2O2 395.2699; found 395.2712.
4.1.4. 1-(3-Dodecyloxyphenyl)-1-hydroxy-1,2-dihydropyrrolo[3,4-
c]pyridin-3-one (6d)
4.1.4.1. Method a. To a solution of hemiamidal 6c (30 mg, 0.124 mmol)
in dry dimethylformamide (2 mL) was added, under inert atmosphere
and at room temperature, potassium carbonate (26 mg, 0.186 mmol).
After 5 min with stirring, iodododecane (55 mg, 0.186 mmol) was
added. After 24 h stirring, the mixture was filtered, the filtrate was
diluted with water (5 mL) and the product was extracted with ethyl
acetate (3 ꢃ 5 mL). The combined organic phases were dried over
sodium sulfate and concentrated in vacuo. The crude product was
purified by silica gel column chromatography (gradient dichloro-
methane/methanol 100/0e90/10) to yield 31 mg (61%) of hemiamidal
6d as beige solid.
4.1.6. 1-Hydroxy-1-(3-octylphenyl)-1,2-dihydropyrrolo[3,4-c]
pyridin-3-one (10)
Compound 10 was prepared in the same manner as 9, with 6a
(200 mg, 0.66 mmol) and n-octyl boronic acid (116 mg, 0.72 mmol),
to yield 33 mg (15%) of hemiamidal 10 as an orange solid. Mp
decomposition after 150 ꢁC. IR (KBr, nmax/cmꢀ1) 3437, 2957, 2927,
2855, 1713, 1613, 1282. 1H NMR (500 MHz, CDCl3)
d 8.99 (s, 1H), 8.74
(m, 1H), 7.38e7.37 (m, 3H), 7.31 (t, J ¼ 7.8 Hz, 1H), 7.21 (d, J ¼ 7.5 Hz,
1H), 6.76 (s, 1H), 4.05 (s, 1H), 2.61 (t, J ¼ 7.9 Hz, 2H), 1.60 (m, 2H),
1.31e1.28 (m, 10H), 0.90 (t, J ¼ 7.0 Hz, 3H). 13C NMR (126 MHz,
4.1.4.2. Method b. To a solution of 3-bromo-dodecyloxybenzene 8b
(1.90 g, 5.57 mmol) in dry THF (5.5 mL) under argon and at ꢀ78 ꢁC,
was added tert-butyl lithium (7.0 mL, 10.5 mmol, 1.5 M in pentane).
After 40 min with stirring at ꢀ78 ꢁC, this solution was added
dropwise at ꢀ78 ꢁC and under inert atmosphere to a solution of 3,4-
pyridinedicarboximide 7 (550 mg, 3.7 mmol) in dry THF (22 mL)
and then the reaction was left to reach room temperature. Water
was added (15 mL) and the product was extracted with ethyl
acetate (3 ꢃ 25 mL). The combined organic phases were dried over
sodium sulfate and the solvent was evaporated in vacuo. The
residue obtained was purified by column chromatography
(gradient dichloromethane/methanol 100/0e90/10) to yield
346 mg (23%) of a mixture of para/meta isomers (3:1). The solid was
purified by recrystallization in acetone to give hemiamidal 6d as
a white solid (124 mg, 8%). Mp 131 ꢁC. IR (KBr, nmax/cmꢀ1) 3413,
3201, 3066, 2923, 2853,1718,1615,1260. 1H NMR (500 MHz, DMSO-
CDCl3)
d 167.7 (C), 157.8 (C), 153,5 (CH), 145.9 (CH), 144.0 (C), 138.0
(C), 129.4 (CH), 128.9 (CH), 125.3 (CH), 125.1 (C), 122.7 (CH), 117.8
(CH), 87.9 (C), 36.0 (CH2), 31.8 (CH2), 31,5 (CH2), 29.4 (CH2), 29.3
(CH2), 29.2 (CH2), 22.6 (CH2), 14.1 (CH3). MS (ESI) m/z 377 (M þ Kþ),
361 (M þ Naþ), 339 (M þ Hþ). HRMS (ESI) calcd for C21H27N2O2
339.2073; found 339.2060.
4.1.7. 1-Hydroxy-1-(3-(4-nonylphenyl)phenyl)-1,2-dihydropyrrolo
[3,4-c]pyridin-3-one (11)
Compound 11 was prepared in the same manner as 9, with 6a
(200 mg, 0.66 mmol) and 4-n-nonylbenzeneboronic acid (164 mg,
0.72 mmol), toyield 74 mg(26%) ofhemiamidal 11 as an orange solid.
Mp 131 ꢁC. IR (KBr, nmax/cmꢀ1) 3412, 3230, 3062, 2924, 2853, 1713,
1614, 1435, 1340, 1288. 1H NMR (500 MHz, MeOD-d4)
d 8.96 (d,
d6)
d
9.46 (s, 1H), 8.86 (d, J ¼ 1.0 Hz, 1H), 8.72 (d, J ¼ 5.1 Hz, 1H), 7.42
J ¼ 0.9 Hz,1H), 8.73 (d, J ¼ 5.2 Hz,1H), 7.86 (t, J ¼ 1.7 Hz,1H), 6.61 (dt,
J ¼ 1.6 Hz and J ¼ 7.5 Hz, 1H), 7.52 (d, J ¼ 8.3 Hz, 2H), 7.51e7.48 (m,
2H), 7.45 (t, J ¼ 7.6 Hz, 1H), 7.26 (d, J ¼ 8.3 Hz, 2H), 2.66 (t, J ¼ 7.7 Hz,
2H), 1.66 (m, J ¼ 7.4 Hz, 2H), 1.36e1.30 (m, 12H), 0.91 (t, J ¼ 7.0 Hz,
(dd, J ¼ 1.1 Hz and J ¼ 5.1 Hz, 1H), 7.26 (t, J ¼ 8.0 Hz, 1H), 7.15 (s, 1H),
7.06 (t, J ¼ 2.1 Hz, 1H), 6.98 (dd, J ¼ 0.9 Hz and J ¼ 7.8 Hz, 1H), 6.89
(dd, J ¼ 2.0 Hz and J ¼ 8.0 Hz, 1H), 3.94 (t, J ¼ 6.6 Hz, 2H), 1.69 (q,
J ¼ 7.4 Hz, 2H), 1.39 (m, 2H), 1.30e1.25 (m, 16H), 0.86 (t, J ¼ 6.9 Hz,
3H). 13C NMR (126 MHz, MeOD-d4)
d 168.5 (C), 159.5 (C), 152.6 (CH),
3H). 13C NMR (126 MHz, DMSO-d6)
d
167.5 (C), 159.1 (C), 158.8 (C),
144.5 (CH), 142.2 (C), 141.6 (C), 140.1 (C), 138.0 (C), 128.6 (CH), 128.3
(2 ꢃ CH),126.9 (CH),126.5 (2 ꢃ CH),126.4 (C),123.9 (CH),123.6 (CH),
118.1 (CH), 87.9 (C), 35.1 (CH2), 31.6 (CH2), 31.2 (CH2), 29.3 (CH2), 29.2
(CH2), 29.0 (CH2), 28.9 (CH2), 22.3(CH2),13.0 (CH3). MS (ESI) m/z 467
(M þ Kþ), 451 (M þ Naþ), 429 (M þ Hþ), 411 (M þ Hþ ꢀ H2O). HRMS
(ESI) calcd for C28H33N2O2 429.2542; found 429.2569.
153.4 (CH), 145.0 (CH), 142.7 (C), 131.9 (CH), 126.5 (C), 118.3 (CH),
118.0 (CH), 114.5 (CH), 112.4 (CH), 87.6 (C), 67.9 (CH2), 31.7 (CH2),
29.5e29.1 (7 ꢃ CH2), 26.0 (CH2), 22.5 (CH2), 14.4 (CH3). MS (ESI) m/z
433 (M þ Naþ), 411 (M þ Hþ), 393 (M þ Hþ ꢀ H2O). HRMS (ESI)
calcd for C25H35N2O3 411.2648; found 411.2653.
4.1.5. 1-(3-Dodecylphenyl)-1-hydroxy-1,2-dihydropyrrolo[3,4-c]
pyridin-3-one (9)
4.1.8. 1-(3-Dodecylphenyl)-5-[2-(ethyloxy)-2-oxoethyl]-1-hydroxy-
3-oxo-1,2-dihydropyrrolo[3,4-c]pyridinium bromide (12)
General procedure for SuzukieMiyaura cross-coupling. To
a solution of hemiamidal 6a (400 mg, 1.32 mmol) in dry THF
(24 mL) was added under argon n-dodecylboronic acid (308 mg,
1.44 mmol), 1,10-bis(diphenylphosphino) ferrocene palladium(II)-
chloride (192 mg, 0.26 mmol), silver(I) oxide (760 mg, 3.20 mmol)
and potassium carbonate (542 mg, 4.00 mmol). The tube was closed
hermetically and heated at 80 ꢁC for 48 h. The solution was diluted
in dichloromethane (20 mL) and a solution of H2O2 (30%)/NaOH
(10%) was added. The mixture was stirred for 2 h and then the
product was extracted with dichloromethane (3 ꢃ 50 mL). The
combined organic phases were dried over sodium sulfate and
concentrated in vacuo. The residue obtained was purified by silica
gel column chromatography (gradient dichloromethane/methanol
100/0e90/10) to yield 125 mg (24%) of hemiamidal 9 as an orange
solid. Mp 108 ꢁC. IR (KBr, nmax/cmꢀ1) 3478, 3413, 3064, 2925, 2851,
General procedure for pyridinium salt synthesis. To a solution of
hemiamidal 9 (100 mg, 0.25 mmol) in dry THF (3.2 mL) under reflux,
was added dropwise and under inert atmosphere ethyl bromoace-
tate (113 mL,1.00 mmol). After 48 h, ether (10 mL) was added and the
solid formed was filtered, washed with ether and dried in vacuo to
yield 142 mg (78%) of pyridinium salt 12 as a brown solid. Mp
decomposition. IR (KBr, nmax/cmꢀ1) 3436, 3150, 2824, 2853, 1718,
1655,1437,1217.1H NMR (500 MHz, DMSO-d6)
d 10.27 (s,1H), 9.54 (s,
1H), 9.13 (d, J ¼ 6.2 Hz,1H), 8.31 (d, J ¼ 6.2 Hz,1H), 7.75 (s,1H), 6.61 (s,
1H), 7.36e7.34 (m, 2H), 7.25 (d, J ¼ 7.3 Hz,1H), 5.69 (s, 2H), 3.79e3.78
(m, 2H), 2.58 (t, J ¼ 7.8 Hz, 2H),1.55 (m, 2H),1.29e1.25 (m, 21H), 0.86
(t, J ¼ 6.8 Hz, 3H).13C NMR (126 MHz, DMSO-d6)
d 167.2 (C),166.3 (C),
164.5 (C), 163.5 (C), 150.8 (CH), 143.6 (CH), 138.4 (C), 130.2 (C), 129.6
(CH), 129.2 (CH), 126.1 (CH), 123.7 (CH), 122.3 (CH), 88.7 (C), 60.9
(CH2) 53.7 (CH2), 35.7 (CH2), 31.7 (CH2), 31.5 (CH2), 29.6e29.2
(7 ꢃ CH2 and CH3), 22.6 (CH2),14.4 (CH3). MS (ESI) m/z 481 (Mþ), 467
(Mþ ꢀ CH2), 453 (Mþ ꢀ CH2CH2), 439 (Mþ ꢀ CH2CH2CH2). HRMS
(ESI) calcd for C29H41N2O4 481.3066; found 481.3039.
1706, 1615, 1280. 1H NMR (500 MHz, CDCl3)
d 8.82 (s, 1H), 8.62 (d,
J ¼ 4.5 Hz, 1H), 7.40 (s, 1H), 7.37e7.33 (m, 2H), 7.31e7.28 (m, 2H),
7.19 (d, J ¼ 7.5 Hz, 1H), 5.43 (s broad, 1H), 2.60 (t, J ¼ 7.8 Hz, 2H), 1.59

4560
T. Delaine et al. / European Journal of Medicinal Chemistry 45 (2010) 4554e4561
4.1.9. Ethyl 3-aminocarbonyl-[4-(3-dodecylbenzoyl)-1,4-dihydro
pyridin-1-yl]acetate (13)
General procedure for 1,4-DHP synthesis. To a solution of pyr-
idinium salt 12 (20 mg, 0.036 mmol) in ethanol (1.4 mL) was added
under inert atmosphere and at 0 ꢁC, a solution of sodium tri-
29.6 (CH2), 29.5 (4 ꢃ CH2), 29.3 (CH2), 29.2 (CH2), 29.1 (CH2), 26.0
(CH2), 22.6 (CH2), 14.4 (CH3). MS (ESI) m/z 527 (Mþ), 483
(Mþ ꢀ CH2CH2O), 469 (Mþ ꢀ CH2CH2CH2O). HRMS (ESI) calcd for
C30H43N2O6 527.3121; found 527.3148.
acetoxyborohydride (12 mg, 0.053 mmol) in acetic acid (424
m
L).
4.1.12. Ethyl 3-aminocarbonyl-[4-(3-dodecyloxybenzoyl)-1,4-
dihydropyridin-1-yl]acetate (16)
Compound 16 was prepared in the same manner as 13, with 14
(27 mg, 0.047 mmol) to yield 12 mg (51%) of 1,4-dihydropyridine 16
as an orange oil. IR (neat, nmax/cmꢀ1) 3357, 2961, 2924, 2854, 1735,
1685, 1648, 1598, 1395, 1261, 1097, 1022, 800. 1H NMR (500 MHz,
After 10 min stirring, acetone (0.4 mL) was added and then water
(10 mL). The reaction mixture was extracted with dichloromethane
(3 ꢃ 10 mL). The combined organic phases were washed with
a saturated NaHCO3 solution (2 ꢃ 15 mL), dried over sodium sulfate
and then concentrated in vacuo. The residue was purified by silica
gel column chromatography (gradient dichloromethane/methanol
: 100/0e90/10) to give 5.6 mg (33%) of 1,4-dihydropyridine 13 as an
CDCl3)
d
7.72 (d, J ¼ 7.5 Hz, 1H), 7.55 (d, J ¼ 2.0 Hz, 1H), 7.39 (t,
J ¼ 8.0 Hz,1H), 7.13 (d, J ¼ 2.4 Hz,1H), 7.11 (d, J ¼ 5.0 Hz,1H), 5.89 (d,
J ¼ 7.9 Hz, 1H), 5.32 (s, 2H), 5.11 (dd, J ¼ 1.1 Hz and J ¼ 4.6 Hz, 1H),
4.91 (dd, J ¼ 4.6 Hz, J ¼ 7.9 Hz, 1H), 4.26 (q, J ¼ 7.1 Hz, 2H), 4.02 (t,
J ¼ 6.6 Hz, 2H), 3.98 (d, J ¼ 3.9 Hz, 2H), 1.80 (q, J ¼ 7.2 Hz, 2H), 1.48
(q, J ¼ 7.4 Hz, 2H), 1.33 (t, J ¼ 7.2 Hz, 3H), 1.31e1.23 (m, 16H), 0.90 (t,
orange oil. 1H NMR (500 MHz, CDCl3)
d 7.96 (m, 1H), 7.87 (s, 1H),
7.43e7.38 (m, 2H), 7.13 (s, 1H), 7.29 (s broad, 2H), 5.89 (d, J ¼ 7.5 Hz,
1H), 5.11 (d, J ¼ 5.1 Hz, 1H), 4.91 (dd, J ¼ 4.5 Hz and J ¼ 7.8 Hz, 1H),
4.26 (q, J ¼ 7.0 Hz, 2H), 3.98 (s, 2H), 2.67 (t, J ¼ 7.5 Hz, 2H), 1.33 (t,
J ¼ 7.1 Hz, 3H), 1.29e1.25 (m, 20H), 0.90 (t, J ¼ 6.5 Hz, 3H). 13C NMR
J ¼ 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3)
d 198.4 (C), 169.4 (C),
(126 MHz, CDCl3)
d
198.8 (C), 169.4 (C), 168.9 (C), 143.5 (C), 138.4
168.9 (C), 159.5 (C), 138.3 (CH), 136.6 (C), 129.7 (CH), 129.6 (CH),
121.6 (CH), 120.3 (CH), 114.1 (CH), 102.8 (C), 102.4 (CH), 68.2 (CH2),
61.8 (CH2), 54.7 (CH2), 44.0 (CH), 31.9 (CH2), 29.7 (CH2), 29.7 (CH2),
29.6 (CH2), 29.6 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 26.0 (CH2),
22.7 (CH2), 14.2 (CH3), 14.1 (CH3). MS (ESI) m/z 521 (M þ Naþ), 499
(M þ Hþ). HRMS (ESI) calcd for C29H43N2O5 499.3172; found
499.3159.
(CH), 135.3 (C), 133.4 (CH), 129.7 (CH), 129.1 (CH), 128.5 (CH), 126.7
(CH), 102.8 (C), 102.5 (CH), 61.8 (CH2), 54.7 (CH2), 44.1 (CH), 35.9
(CH2), 31.9 (CH2), 31.6 (CH2), 29.7e29.3 (7 ꢃ CH2), 22.7 (CH2), 14.2
(CH3), 14.1 (CH3). MS (ESI) m/z 483 (M þ Hþ). HRMS (ESI) calcd for
C29H43N2O4 483.3223; found 483.3215.
4.1.10. 1-(3-Dodecyloxyphenyl)-5-[2-(ethyloxy)-2-oxoethyl]-1-
hydroxy-3-oxo-1,2-dihydropyrrolo[3,4-c]pyridinium bromide (14)
Compound 14 was prepared in the same manner as 12, with 6d
4.1.13. 3-Hydroxypropyl 3-aminocarbonyl-[4-(3-
dodecyloxybenzoyl)-1,4-dihydropyridin-1-yl]acetate (17)
(150 mg, 0.37 mmol) and ethyl bromoacetate (162
m
L,1.46 mmol) to
Compound 17 was prepared in the same manner as 13 with 15
(20 mg, 0.033 mmol) to yield 7.6 mg (44%) of 1,4-dihydropyridine
17 as an orange oil. IR (neat, nmax/cmꢀ1) 3434, 3238, 2924, 2853,
1737, 1731, 1682, 1644, 1604, 1384, 1264, 1054. 1H NMR (500 MHz,
give 114 mg (54%) of pyridinium salt 14 as a brown solid. Mp
decomposition after 170 ꢁC. IR (KBr, nmax/cmꢀ1) 3412, 3152, 3079,
2924, 2853, 1738, 1655, 1601, 1216. 1H NMR (500 MHz, DMSO-d6)
d
10.27 (s, 1H), 9.56 (s, 1H), 9.17 (d, J ¼ 6.3 Hz,1H), 8.34 (d, J ¼ 6.3 Hz,
CDCl3)
d
7.67 (d, J ¼ 7.9 Hz, 1H), 7.54 (d, J ¼ 1.9 Hz, 1H), 7.39 (t,
1H), 7.78 (s, 1H), 7.34 (t, J ¼ 8.1 Hz, 1H), 7.13 (t, J ¼ 2.0 Hz, 1H), 7.07
(d, J ¼ 7.7 Hz, 1H), 6.98 (dd, J ¼ 2.1 Hz and J ¼ 8.1 Hz, 1H), 5.70 (s,
2H), 4.25 (q, J ¼ 7.1 Hz, 2H), 4.00e3.95 (m, 2H), 1.71 (m, J ¼ 7.3 Hz,
2H), 1.41 (q, J ¼ 6.9 Hz, 2H), 1.33e1.23 (m, 16H), 1.27 (t, J ¼ 7.1 Hz,
J ¼ 8.0 Hz, 1H), 7.15 (d, J ¼ 1.4 Hz, 1H), 7.13 (dd, J ¼ 2.2 Hz and
J ¼ 8.3 Hz,1H), 5.88 (d, J ¼ 8.0 Hz,1H), 5.55 (s, 2H), 5.17 (d, J ¼ 1.1 Hz,
1H), 4.88 (dd, J ¼ 4.6 Hz, J ¼ 7.9 Hz, 1H), 4.38e4.32 (m, 2H), 4.01 (t,
J ¼ 6.6 Hz, 2H), 3.98 (d, J ¼ 1.3 Hz, 2H), 3.88 (t, J ¼ 5.2 Hz, OH), 3.72
(q, J ¼ 6.3 Hz, 2H), 1.91 (q, J ¼ 5.9 Hz, 2H), 1.81 (q, J ¼ 6.9 Hz, 2H),
1.49e1.45 (m, 2H), 1.37e1.29 (m, 16H), 0.90 (t, J ¼ 6.9 Hz, 3H). 13C
2H), 0.86 (t, J ¼ 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO-d6)
d 166.7
(C), 166.1 (C), 163.5 (C), 159.5 (C), 150.8 (CH), 143.4 (CH), 140.1 (C),
130.2 (CH), 123.3 (CH), 118.3 (CH), 115.4 (CH), 112.7 (CH), 88.0 (C),
68.1 (CH2), 62.9 (CH2), 60.9 (CH2), 31.7 (CH2), 29.5 (CH2), 29.4
(5 ꢃ CH2), 29.3 (CH2), 29.2 (CH2), 29.1 (CH2), 22.6 (CH3), 14.4 (CH3).
MS (ESI) m/z 497 (Mþ), 483 (Mþ ꢀ CH2), 469 (Mþ ꢀ CH2CH2). HRMS
(ESI) calcd for C29H41N2O5 497.3015; found 497.3015. Anal. Calcd for
C29H41 BrN2O5 þ ½ CH3OH: C, 59.70; H, 7.25; N, 4.72. Found: C,
59.63; H, 6.98, N, 4.73.
NMR (126 MHz, CDCl3)
d 198.8 (C), 169.8 (C), 169.2 (C), 159.5 (C),
138.3 (CH), 136.5 (C), 129.8 (CH), 129.6 (CH), 121.5 (CH), 120.3 (CH),
114.1 (CH), 102.7 (C), 102.1 (CH), 68.3 (CH2), 63.0 (CH2), 59.0 (CH2),
54.9 (CH2), 43.0 (CH), 31.9 (CH2), 31.3 (CH2), 29.8 (CH2), 29.7 (CH2),
29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 26.1 (CH2),
22.7 (CH2), 14.1 (CH3). MS (FAB) m/z 567 (M þ Kþ), 551 (M þ Naþ).
HRMS (ESI) calcd for C30H45N2O6 529.3278, found 529.3284.
4.1.11. 1-(3-Dodecyloxyphenyl)-1-hydroxy-5-[2-(3-
4.2. Biology
hydroxypropyloxy)-2-oxyethyl]-3-oxo-2,3-dihydro-1H-pyrrolo[3,4-
c]pyridin-5-ium bromide (15)
4.2.1. Enzymatic inhibition experiments
Compound 15 was prepared in the same manner as 12, with 6d
(130 mg, 0.32 mmol) and 3-hydroxypropyl 2-bromoacetate
(246 mg, 1.25 mmol) to yield 68 mg (35%) of pyridinium salt 15 as
M. tuberculosis InhA was overexpressed in E. coli and purified as
previously described [26]. Isoniazid, NADH and NADþ were
obtained from SigmaeAldrich. The pool of adduct INHeNAD was
synthesized and purified as previously described [7]. The concen-
tration of the pool INHeNAD was determined on the basis of
e330 ¼ 6900 Mꢀ1 cmꢀ1 [26]. The substrate 2-trans-decenoyl-CoA
was synthesized from 2-trans-decenoic acid using the mixed
anhydride method and purified according to the procedure
described by Goldman and Vagelos [27]. The concentration of the
grey solid. Mp decomposition after 170 ꢁC. IR (KBr, nmax/cmꢀ1
)
3138, 3066, 2923, 2853, 1732, 1655, 1600, 1488,1257, 1213. 1H NMR
(500 MHz, DMSO-d6) 10.27 (s, 1H), 9.56 (s, 1H), 9.16 (d,
d
J ¼ 6.3 Hz, 1H), 8.34 (d, J ¼ 6.2 Hz, 1H), 7.78 (s, 1H), 7.34 (t,
J ¼ 8.0 Hz, 1H), 7.13 (t, J ¼ 2.1 Hz, 1H), 7.07 (d, J ¼ 7.8 Hz, 1H), 6.98
(dd, J ¼ 2.1 Hz and J ¼ 7.9 Hz, 1H), 5.70 (s, 2H), 4.57 (s, 1H), 4.27 (t,
J ¼ 6.6 Hz, 2H), 3.97 (m, 2H), 3.49 (m, 2H), 1.79 (q, J ¼ 6.4 Hz, 2H),
1.71 (q, J ¼ 6.8 Hz, 2H), 1.41 (q, J ¼ 7.4 Hz, 2H), 1.33e1.19 (m, 16H),
substrate was determined on the basis of e260 ¼ 22 600 Mꢀ1 cmꢀ1
.
The absorption of each reaction mixture was determined with
a thermostated Uvikon 923 spectrophotometer (Bio-Tek Kontron
Instruments).
0.86 (t, J ¼ 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO-d6)
d 166.7 (C),
166.0 (C), 163.5 (C), 159.5 (C), 150.8 (CH), 143.4 (CH), 140.0 (C),
130.4 (CH), 130.2 (C), 122.3 (CH), 118.2 (CH), 115.4 (CH), 112.7 (CH),
88.0 (C), 68.1 (CH2), 64.3 (CH2), 60.9 (CH2), 57.5 (CH2), 31.7 (CH2),
For the inhibition assays with InhA the pre-incubation reactions
were performed in 80
ml (total volume) of 30 mM PIPES buffer

T. Delaine et al. / European Journal of Medicinal Chemistry 45 (2010) 4554e4561
4561
solution,150 mM NaCl, pH 6.8 at 25 ꢁC containing 70 nM InhA and 12,
13,14,15,16 or 17 (at 100, 20 or 10 M). DMSO was used as co-solvent
and its final concentration was 0.5%. After 5 min or 2 h of pre-incu-
bation, the addition of 35 M substrate (trans-2-decenoyl-CoA) and
200 M cofactor (NADH) initiated the reactionwhich was followed at
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ther. 46 (2002) 2137e2144.
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m
340 nm (oxidation of NADH) and at 25 ꢁC using a thermostated Uvi-
kon 923 spectrophotometer (Bio-Tek Kontron Instruments). Other
experiments where NADH was added during the pre-incubation
period were performed and identical results were obtained. Control
reactions were carried out under the same conditions as those
described above but without ligands. The pool of INHeNAD adducts
was used as a positive control. The initial rates of the reactions were
calculated. The inhibitory activity of each compound tested was
expressed asthepercentageinhibition of InhAactivity (initialvelocity
of the reaction) with respect to the control experiments. All activity
assays were performed in triplicate.
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4.2.2. Bacterial growth inhibition
The susceptibility of M. smegmatis or of M. tuberculosis H37Rv to
each compound was evaluated by using a colorimetric microassay
based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT, SigmaeAldrich) to formazan by
metabolically active cells [10,28]. Briefly, serial two-fold dilutions of
the inhibitor were prepared in 100
(Difco) and dispensed into 96-well microtiter plates, to each well of
which we added 100 l of M. tuberculosis or M. smegmatis suspen-
sion (diluted in 7H9 broth to an OD600 of 0.02). Plates were incu-
bated for six days at 37 ꢁC, and MTT was added (50
l of a 1 mg/ml
ml Middlebrook 7H9 broth base
m
m
solution). Plates were incubated for a further 24 h and solubilization
buffer [dimethylformamide:SDS 20% (w/v), 1:2] was added to each
well. Absorbance was measured at 570 nm. The MIC was deter-
mined 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 4.02. Isoniazid-
treated cultures were used as positive controls. Each mother solu-
tion was prepared in DMSO at concentrations 100 times greater
than to the highest test concentration.
Appendix. Supplementary material
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a competitive inhibitor of the Mycobacterium tuberculosis enoyl
reductase, J. Org. Chem. 70 (2005) 10502e10510.
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J. Bernadou, Ring-chain tautomerism of simplified analogues of isoniazid-NAD
(P) adducts: an experimental and theoretical study, Eur. J. Org. Chem. (2007)
1624e1630.
Supplementary data associated with article can be found in the
online version, at doi:10.1016/j.ejmech.2010.07.016.
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