Synthesis and modulation properties of imidazo[4,5-b]pyridin-7-one and indazole-4,7-dione derivatives towards the Cryptosporidium parvum CpABC3 transporter

Article abstract of DOI:10.1016/j.ejmech.2010.02.033

The syntheses of new N-polysubstituted imidazo[4,5-b]pyridine-7-one (IP, 5 and 8a-8f) and indazole-4,7-dione (ID, 9 and 10) derivatives are described. The binding affinity of IP and ID towards the recombinant Nucleotide Binding Domain NBD1 of Cryptosporidium parvum CpABC3 was evaluated by intrinsic fluorescence quenching. IP induced a moderate quenching of the intrinsic fluorescence of H6-NBD1 whereas IDs 9 and 10 showed a binding affinity comparable to the ATP analogue TNP-ATP. In addition, 8d, 8e and 10 were shown to be competitive inhibitors of the ATPase activity, but with low affinity. These compounds could thus act like some flavonoid derivatives, which can partly overlap both the nucleotide-binding site and the adjacent hydrophobic steroid-binding region of mammalian P-glycoproteins.

Full text of DOI:10.1016/j.ejmech.2010.02.033

European Journal of Medicinal Chemistry 45 (2010) 2480e2488
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and modulation properties of imidazo[4,5-b]pyridin-7-one and
indazole-4,7-dione derivatives towards the Cryptosporidium parvum
CpABC3 transporter
,
a
a
Waël Zeinyeh a, Hexue Xia b, Philippe Lawton b, Sylvie Radix a , Christelle Marminon , Pascal Nebois ,
*
,
Nadia Walchshofer a
*
a Université de Lyon, Université Lyon 1, ISPB-Faculté de Pharmacie, EA 4443 (B2C) « Biomolécules, Cancer et Chimiorésistances», 8, av. Rockefeller, F-69373, Lyon Cedex 08, France
b Université de Lyon, Université Lyon 1, ISPB-Faculté de Pharmacie, Laboratoire de Parasitologie, 8, av. Rockefeller, F-69373, Lyon Cedex 08, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The syntheses of new N-polysubstituted imidazo[4,5-b]pyridine-7-one (IP, 5 and 8ae8f) and indazole-
4,7-dione (ID, 9 and 10) derivatives are described. The binding affinity of IP and ID towards the
recombinant Nucleotide Binding Domain NBD1 of Cryptosporidium parvum CpABC3 was evaluated by
intrinsic fluorescence quenching. IP induced a moderate quenching of the intrinsic fluorescence of H6-
NBD1 whereas IDs 9 and 10 showed a binding affinity comparable to the ATP analogue TNP-ATP. In
addition, 8d, 8e and 10 were shown to be competitive inhibitors of the ATPase activity, but with low
affinity. These compounds could thus act like some flavonoid derivatives, which can partly overlap both
the nucleotide-binding site and the adjacent hydrophobic steroid-binding region of mammalian
P-glycoproteins.
Received 27 October 2009
Received in revised form
11 February 2010
Accepted 13 February 2010
Available online 18 February 2010
Keywords:
ABC transporter
Alkylation
Cryptosporidium parvum
Imidazo[4,5-b]pyridinone
Quinone
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
mammalian ABC transporter P-glycoprotein belongs [6]. The
structural analysis of CpABC3 showed a typical structure of a P-
Cryptosporidium parvum is an emerging infectious enteric
protozoan [1]. It is the causative agent of waterborne intestinal
infections and as such, may impair development in early infected
children or be life-threatening to immunocompromised patients
[2]. Since there is no effective drug treatment for cryptosporidi-
osis [3], the identification of chemotherapeutic targets could lead
glycoprotein with two Trans-Membrane Domains (TMDs) and two
cytosolic Nucleotide-Binding Domains (NBD1 and NBD2), the
most conserved feature of the MDR sub-family of ABC trans-
porters [7].
We recently described the overexpression in Escherichia coli of
an hexahistidine-tagged recombinant protein encompassing the N-
terminal NBD1 of C. parvum CpABC3 containing a single ATP-
binding site [8]. We have shown that this recombinant C. parvum
NBD is fully functional towards known reference molecules acting
on mammalian P-glycoproteins, such as TNP-ATP, quercetin,
progesterone. On the other hand, we are involved in a program
based on the synthesis of azaheterobicyclic molecules, especially
benzimidazolediones [9], imidazopyridinones [10], and purine
derivatives [11], designed to target parasitic enzymes that accept
purine nucleosides as substrates. Thus, in order to identify scaffolds
suitable for preparation of CpABC3 ligands, the binding affinity of
such azaheterocycles as imidazo[4,5-b]pyridin-7-ones (IP) and
indazole-4,7-diones (ID) towards the recombinant NBD1 of CpABC3
was assessed (Fig. 1).
to new therapeutic approaches and may be
a key for the
discovery of really effective drugs. It has been suggested that the
intrinsic resistance of C. parvum to most of the available anti-
parasitic molecules was partly due to natural drug efflux
involving ATP-Binding Cassette transporters (ABC proteins), which
couple the energy of ATP hydrolysis to the transport of various
substances through cell membranes [4]. Three types of ABC
proteins have been described in C. parvum [5] and one of them,
CpABC3, is related to the MDR sub-family to which the
* Corresponding authors. Tel.: þ33 478777253; fax: þ33 478777158.
E-mail address: sylvie.radix@univ-lyon1.fr (S. Radix).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.033

W. Zeinyeh et al. / European Journal of Medicinal Chemistry 45 (2010) 2480e2488
2481
Fig. 1. Structures of N-1 and N-3-substituted imidazo[4,5-b]pyridin-7-one derivatives (N1-IP and N3-IP) and N-1 and N-2-substituted indazole-4,7-diones (N1-ID and N2-ID).
Numbering system for NMR data.
2. Results and discussion
experiments of hydrolysis of 3a in acidic conditions (aqueous conc.
HCl) only gave a yield of 52% of the desired IP 5 after one week.
Unfortunately, a higher yield was not achieved using HCl in MeOH
[14]. The mechanism of this latter reaction consists in a nucleophilic
aromatic substitution of chloride by methoxide group and
a subsequent SN2 attack of chloride to yield the hydroxypyridine
moiety and methyl chloride, which is converted into dimethylether
[15]. In an analogous way, we developed a two-step sequence in
basic conditions from 3a to IP 5 via 7-methoxylated derivative 4.
The 7-chloroimidazo[4,5-b]pyridine 3a was subjected to aromatic
nucleophilic substitution using sodium methoxide to obtain the
compound 4 with a very good yield. Subsequently, demethylation
was easily performed using sodium ethyl thiolate in DMF as
nucleophilic reagent. This convenient two-step procedure from 3a
to 5 provided a better global yield than the direct acidic hydrolysis
of 3a.
2.1. Chemistry
The first step was to prepare imidazo[4,5-b]pyridine derivatives
(IP) whose synthesis has surprisingly, not been yet described in the
literature. The route we have developed to synthesise the main
intermediate, 1-benzyl-1,4-dihydroimidazo[4,5-b]pyridin-7-one 5,
is illustrated in Scheme 1.
It was prepared in six steps from commercially available pyri-
dine-2,3-diamine with an overall yield of 27%. N-benzylation of
imidazo[4,5-b]pyridine-4-oxide 1 was carried out according to
a previously reported method [10]. Classical conditions of nucleo-
philic substitution, with freshly prepared benzyl iodide [12] as
alkylating agent, mainly led to the N-1 regioisomer 2a.
Regioisomers 2a and 2b were easily separated by column chro-
matography on silica gel. Treatment of N-1 benzylated 4-oxide 2a
with phosphoryl chloride at reflux in chloroform for 24 hours
predominantly yielded 7-chloro derivative 3a [13]. Examination of
1H NMR spectra of the crude reaction mixtures and of the
remaining column fractions showed only traces of N-1 benzylated
5-chloro regioisomer 3b. The structure of 3a was confirmed by the
N-4-alkylation of IP 5 was then carried out to obtain poly N-
substituted imidazo[4,5-b]pyridin-7-ones 8. The alkylating agents
6 [16] and 7 [17] were first synthesised according to literature
procedures (Scheme 2).
Reaction of IP 5 with 1.1 to 6 equiv. of various alkylating reagents
(MeI, commercially available
u-halo alkyl esters and N-Boc pro-
presence of a doublet at lower field (
d
8.37 ppm) for H-5 with
tected alkyl amines 6 and 7) in KOH methanol solution at 90 ꢀC led
to a single N-4-alkylated regioisomer with moderate to excellent
yields (Scheme 3, Table 1).
a fairly small coupling constant (J5,6 ¼ 5.3 Hz). The structure of the
isomer 3b was assigned on the basis of the presence of a larger
doublet (J6,7 ¼ 8.5 Hz) for H-7 at 8.07 ppm. Surprisingly, chlorina-
tion of the N-3 benzylated 4-oxide 2b under comparable conditions
only underwent reduction of the N-oxide group to provide 3-ben-
zylimidazo[4,5-b]pyridine 3c with a yield of 70%. Preliminary
The regioselectivity of the N-4-alkylation was controlled by
NMR. 1D NOE difference spectrum of the O-methylated compound
4 showed the coupling through-space between CH3 protons and
both H-6 of the imidazopyridine moiety and the phenyl ortho
Scheme 1. Synthesis of 1-benzyl-1,4-dihydroimidazo[4,5-b]pyridin-7-one 5. Reagents and conditions: a) i) HC(OEt)3, reflux, 3h30 ii) 37% HCl, reflux, 1 h (83%); b) m-CPBA 90%,
AcOH, 50 ꢀC, 16 h (78%); c) i) K2CO3, DMF, RT, 1 h ii) PhCH2I, RT, 4 h; d) POCl3, CHCl3, 50 ꢀC, 19 h; e) MeONa, MeOH, 70 ꢀC, 48 h; f) EtSH, NaH 60%, DMF, reflux, 3h30; g) 37% HCl,
reflux, one week.

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W. Zeinyeh et al. / European Journal of Medicinal Chemistry 45 (2010) 2480e2488
Scheme 2. Synthesis of N-Boc-protected alkylamine derivatives 6 and 7.
protons (Fig. 2A). On the other hand, 1D NOE data registered from
8a showed interactions of CH3 protons only with H-5 of the imi-
dazopyridine nucleus (Fig. 2B).
Indazole-4,7-dione derivatives 9 and 10 were then prepared
(Fig. 3). The most useful method to obtain indazole-4,7-dione
skeleton is the reaction between diazomethane or its derivatives
and 1,4-benzoquinone substituted at C-2 or/and C-3 position(s)
[18]. Owing to the hazardous nature of these reagents, we used
a previously reported method to synthesise N-1- and N-2-ID from
7-nitroindazole [19].
ID 10 was synthesised according to the previously reported two-
step procedure used to prepare ID 9 (Scheme 4) [19]. The synthesis
started with the N-alkylation of commercially available 7-nitro-
indazole using 4-(chloromethyl)-2-methylthiazole as alkylating
reagent. The improved alkylation conditions (NaH 1.2eq. in DMF
used as a base) led to the highest yield, with a smooth selectivity in
favour of the N-1-regioisomer 11a (11a: 11b ¼ 65: 35).
a characteristic emission peak at 328 nm due to buried Trp residues
[8]. 20-30-O-(2,4,6-trinitrophenyl) adenosine 50-triphosphate (TNP-
ATP) and progesterone were used as reference molecules. TNP-ATP
is a classical competitive inhibitor of ATPase activity [22]. Proges-
terone is a known steroidal P-glycoprotein modulator [23], and the
steroid-interacting hydrophobic region is postulated to be located
close to the ATP-binding site of P-glycoproteins [24,25].
The analogues 5 and 8a did not bind to the recombinant H6-
NBD1, indicating the great importance of the intracyclic nitrogen
substitution. The results obtained for 8be8f, 9 and 10 are described
in Fig. 5 and Table 2. Compounds 8b, 8c, 8d, 8f induced a moderate
quenching of the intrinsic fluorescence of H6-NBD1, similar to this
caused by progesterone. Compound 8e was not significantly
different from the control. Conversely, compounds 9 and 10 were
much more efficient and were comparable to the ATP analogue
TNP-ATP.
The recombinant H6-NBD1 produced a measurable ATPase
activity weaker than that of a purified P-glycoprotein, as already
reported for a yeast NBD1 [26]. Nevertheless, the ability to inhibit
the recombinant H6-NBD1 ATPase activity was evaluated for a set
of four molecules, 8d (IP derivative with an ester group), 8e, 8f (IP
derivatives with a Boc-amino group), and 10 (ID) (Table 2,
Fig. 5BeD). Compound 8f was not further tested due to its lack of
inhibitory activity. Compounds 8d, 8e and 10 were competitive
inhibitors. However, compound 8e was as bad an inhibitor as
a quencher. The N-1-alkylated indazole-4-7-dione 10 caused the
best inhibition of ATPase activity, about four times less than TNP-
ATP, but twice as much than compound 8d. It is worthy to note that
the compounds 8d, 8e, 10 required rather high concentrations to
inhibit the ATPase activity, whereas compound 10 showed an
intrinsic fluorescence quenching similar to TNP-ATP. It could be
hypothesised that the tested compounds could act like some
flavonoid derivatives, which can partly overlap both the nucleotide-
binding site and an adjacent hydrophobic steroid-binding region of
P-glycoprotein [24,25].
Unambiguous assignment of the structure of both N-1 and N-2
regioisomers 13a and 13b (obtained by N-benzylation of 7-nitro-
indazole) has been previously reported [19]. The comparison of 1H
NMR spectra allowed to easily assign the structure of N-1 and N-2
regioisomers (Fig. 4). Indeed, the H-4 proton signals of N-2 products
(11b and 13b) were significantly deshielded compared with the
same protons in N-1 compounds (11a and 13a): Dd ¼ þ0.11 or
þ0.16 ppm.
The synthesis was completed via reduction of the nitro group of
11a by catalytic hydrogenation in acidic medium [20] followed by
oxidation of the amino derivative 12 with phenyliodine(III) bis
(trifluoroacetate) (PIFA) [21] to provide quinone 10.
2.2. Activity tests
The affinity binding of the synthesised molecules 5, 8ae8f, 9
and 10 was monitored by quenching of the intrinsic fluorescence of
the recombinant C. parvum H6-NBD1, which showed
Scheme 3. N4-Alkylation of 1-benzyl-1,4-dihydroimidazo[4,5-b]pyridin-7-one 5 in basic conditions.

W. Zeinyeh et al. / European Journal of Medicinal Chemistry 45 (2010) 2480e2488
2483
Table 1
CDCl3). Carbon multiplicity was determined by DEPT experiments.
Low- and high-resolution mass spectroscopy was recorded with
a ThermoQuest FINNIGAN MAT 95 XL apparatus operating at 70 eV.
Elemental analyses were performed at the Centre de Microanalyse
du CNRS at Solaize (France). Product purification by flash column
chromatography was performed using Merck Kieselgel 60 A
(40e63 mm). Analytical thin layer chromatography (TLC) was
carried out using Merck commercial aluminium sheets coated
(0.2 mm layer thickness) with Kieselgel 60 F254, with visualization
by ultraviolet. All commercially available chemicals were used as
received. Imidazo[4,5-b]pyridine-4-oxide 1 [13,27], benzyl iodide
Conditions and results of N4-alkylation of 5.
R3X
Product KOH
Time
Yielda
CH3I (1.2 eq.)
EtO2CCH2Br (1.2 eq.)
EtO2C(CH2)3Br (1.2 eq.)
8a
8b
8c
1.1 eq. 5h30
1.1 eq. 5h30
1.1 eq. 22h30 64%
1.1 eq. 22h30 51%
91%
82%
ꢀ
MeCH(Cl)CO2Et (þ/-) NaI (1.2 eq./1.2 eq.) 8d
6 (6 eq.)
7 (6 eq.)
8e
8f
8 eq.
8 eq.
4 days 60%
4 days 62%
a
Isolated yields.
[12], N-Boc-3-bromopropylamine
hexan-1-ol methanesulfonate 7 [17] were prepared according to
6 [16], and N-Boc-6-amino-
3. Conclusion
literature procedures.
We report a useful and short access to original imidazo[4,5-b]
pyridin-7-one derivatives. The binding affinity of these compounds
(IP) and indazole-4,7-diones (ID, 9 and 10) towards the recombi-
nant H6-NBD1 of CpABC3 evidenced that compounds 9-10 were
more active than imidazo[4,5-b]pyridin-7-ones, but for the latter,
the nature of the intracyclic N-4 substitution appears critical and
will be further explored. Aromatic azaheterocycles IP or ID seem
then mandatory for an efficient binding to the H6-NBD1, but with
a moderate inhibition of ATPase activity. IP and ID, which could be
substituted by different side-chains, should be suitable scaffolds as
CpABC3 ligands. Further studies are necessary to determine if these
compounds represent a new class of bifunctional derivatives such
as flavonoids, which are known to bind simultaneously to adjacent
ATP and steroid sites of mammalian P-glycoproteins.
4.1.2. N-alkylation of 3H-imidazo[4,5-b]pyridine-4-oxide (1)
K2CO3 (0.370 g, 2.66 mmol) was added under argon to
a suspension of compound 1 (0.300 g, 2.22 mmol) in DMF (5 mL).
After stirring at room temperature for 1 h, freshly prepared
benzyl iodide (0.400 mL, 3.20 mmol) was added. The mixture was
stirred for 4 h and concentrated to give dark red oil which was
partitioned between CH2Cl2 and water. Aqueous phase was
extracted by CH2Cl2 and the combined organic extracts were
dried (Na2SO4), filtered and concentrated. The residue was puri-
fied by flash chromatography on silica gel (CH2Cl2/methanol,
95:5) to give a mixture of regioisomers 2a as a white solid
(0.295 g, 59%, Rf ¼ 0.08) and 2b as a light yellow solid (0.125 g,
25%, Rf ¼ 0.22).
4.1.2.1. 1-Benzyl-1H-imidazo[4,5-b]pyridine-4-oxide (2a). M.p. 174e
4. Experimental
176 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.64 (s, 1H, H-2), 8.20 (d,
3J(H,H) ¼ 6.3 Hz, 1H, H-5), 7.60 (d, 3J(H,H) ¼ 8.3 Hz, 1H, H-7),
7.40e7.25 (m, 5H, H-phenyl), 7.21 (dd, 3J(H,H) ¼ 6.3 and 8.3 Hz, 1H,
4.1. Chemistry
H-6), 5.54 ppm (s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6):
d
¼ 147.5
4.1.1. General methods and materials
(C), 146.0 (C), 136.8 (CH, C-2), 134.1 (CH, C-5), 131.2 (C, C-phenyl
ipso), 129.7 (2 ꢁ CH, C-phenyl), 129.2 (CH, C-phenyl para), 128.4
(2 ꢁ CH, C-phenyl), 120.0 (CH, C-7), 110.7 (CH, C-6), 49.4 ppm (CH2);
All air- and moisture-sensitive reactions were carried out under
an argon atmosphere. All melting points were measured on
a Barnstead Electrothermal 9200 apparatus and were uncorrected.
Infrared spectra (IR) were recorded using KBr pellets on a Per-
kineElmer FT-IR SPECTRUM ONE spectrometer. 1H spectra were
recorded with Bruker ALS300 and Bruker DRX300 Fourier trans-
form spectrometers, using an internal deuterium lock, operating at
300 MHz. 13C NMR spectra were recorded with a Bruker DRX300
Fourier transform spectrometer, using an internal deuterium lock,
operating at 75 MHz. All spectra were recorded at 25 ꢀC. Chemical
shifts are reported in parts per million (ppm) relative to residual
IR (KBr):
n
¼ 3034, 2946, 1578, 1442, 1256, 1216,1007, 729 cmꢂ1; MS
(70 eV, EI): m/z (%): 225 (4) [Mþ], 209 (54) [Mþ-O], 208 (96) [Mþ-
OH], 195 (42), 119 (16), 91 (100) [C7Hþ7 ], 77 (14), 65 (16); HRMS (CI):
m/z: calcd for C13H11N3O þ H: 226.0980; found: 226.0977.
4.1.2.2. 3-Benzyl-3H-imidazo[4,5-b]pyridine-4-oxide (2b). M.p. 101e
103 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.39 (d, 3J (H,H) ¼ 6.8 Hz,
1H, H-5), 8.37 (s,1H, H-2), 8.32(d, 3J (H,H) ¼ 7.7 Hz,1H, H-7), 7.51-7.39
(m, 5H, H-phenyl), 7.17 (dd, 3J (H,H) ¼ 6.8 and 7.7 Hz, 1H, H-6),
protons or carbons thirteen of deuterated solvent (
d
¼ 2.54 ppm for
5.72 ppm (s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6):
d
¼ 160.2 (CH,
1H NMR and
d
¼ 40.45 ppm for 13C NMR for DMSO-d6 and
(d
¼ 7.26 ppm for 1H NMR and
d
¼ 77.36 ppm for 13C NMR for
C-2), 146.3 (C), 146.1 (C), 133.0 (C, C-phenyl ipso), 129.9 (2 ꢁ CH,
Fig. 2. Establishment of the regioselectivity of N-4-methylation of 5 by 1D NOE difference spectroscopy. 1H NMR spectra were registered in DMSO-d6. A) Irradiation of O-CH3
resonance ( ¼ 3.75 ppm) in compound 8a.
¼ 3.92 ppm) in compound 4. B) Irradiation of N-CH3 resonance (
d
d

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W. Zeinyeh et al. / European Journal of Medicinal Chemistry 45 (2010) 2480e2488
37
91 (100), 65 (8%); HRMS (CI): m/z: calcd for C13
H ClN3: 244.06410;
10
found: 244.06419.
4.1.3.2. 1-Benzyl-5-chloro-1H-imidazo[4,5-b]pyridine (3b). Traces;
1H NMR (300 MHz, DMSO-d6):
d
¼ 8.74 (s, 1H, H-2), 8.07 (d, 3J(H,
H) ¼ 8.5 Hz, 1H, H-7), 7.38e7.11 (m, 6H, H-6, H-arom), 5.55 ppm
(s, 2H, CH2).
4.1.3.3. 3-Benzylimidazo[4,5-b]pyridine (3c). Colourless oil (0.70 g,
70%). 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.70 (s, 1H, H-2), 8.41 (d, 3J
Fig. 3. Structure of N-1-alkylated indazole-4-7-diones 9 and 10. Numbering system for
NMR data.
(H,H) ¼ 4.7 Hz, 1H, H-5), 7.98 (d, 3J(H,H) ¼ 8.0 Hz, 1H, H-7),
7.34e7.311 (m, 5H, H-arom), 7.24 (dd, 3J(H,H) ¼ 4.7 and 8.0 Hz, 1H,
H-7), 5.55 ppm (s, 2H, CH2); MS (70 eV, ESI): m/z (%): 441 (40)
[2 Mþ þ Na], 232 (9) [Mþ þ H], 210 [MHþ].
C-phenyl),129.5 (CH, C-5),129.4 (CH, C-phenyl para),128.9 (CH, C-7),
128.6 (2 ꢁ CH, C-phenyl), 112.0 (CH, C-6), 80.1 ppm (CH2); IR (KBr):
n
¼ 3088, 2923,1394,1297,1185, 843, 747, 645 cmꢂ1; MS (70 eV, ESI):
4.1.4. 1-Benzyl-7-methoxy-1H-imidazo[4,5-b]pyridine (4)
m/z (%): 226 (100) [MHþ], 210 (13); HRMS (EI): m/z: calcd for
Compound 3a was added to a freshly prepared solution of
MeONa (1.25 M) in MeOH (75 mL) under argon and the mixture
was refluxed for 48 hours. After cooling down to room tempera-
ture, the solvent was evaporated and the residue was partitioned
between CH2Cl2 and water. Aqueous phase was extracted by
CH2Cl2. The combined organic extracts were washed once by water,
dried over Na2SO4, filtered and concentrated. The residue was
purified by flash chromatography on silica gel (CH2Cl2/
MeOH ¼ 95:5) to give compound 4 as a white solid (0.90 g, 82%,
Rf ¼ 0.20). M.p. 169e170 ꢀC (dec.); 1H NMR (300 MHz, DMSO-d6):
C13H11N3O: 225.0902; found: 225.0903.
4.1.3. Chlorination of imidazo[4,5-b]pyridine-4-oxide derivatives
(2a) and (2b)
POCl3 (2.4 mL, 25.8 mmol) and 15 pieces of 4Å molecular sieves
were added to a solution of compounds 2a or 2b (1.1 g, 4.8 mmol) in
chloroform (30 mL) and the mixture was heated at 50 ꢀC for
24 hours. The mixture was cooled to room temperature and
quenched with aqueous NaHCO3 (0.6 M, 100 mL). Aqueous phase
was extracted by chloroform and the combined organic extracts
were dried over Na2SO4, filtered and concentrated. The residue was
purified by flash chromatography on silica gel (CH2Cl2/
MeOH ¼ 95:5).
d
¼ 8.50 (s, 1H, H-2), 8.26 (d, 3J(H,H) ¼ 5.5 Hz, 1H, H-5), 7.36e7.22
(m, 5H, H-phenyl), 6.87 (d, 3J(H,H) ¼ 5.5 Hz, 1H, H-6), 5.55 (s, 2H,
CH2), 3.92 ppm (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
d
¼ 157.8
(C, C-3a), 153.3 (C, C-7), 145.9 (CH, C-5), 145.3 (CH, C-2), 137.8 (C, C-
phenyl ipso), 128.6 (2 ꢁ CH, C-phenyl meta), 127.6 (CH, C-phenyl
para), 127.1 (2 ꢁ CH, C-phenyl ortho), 115.6 (C, C-7a), 101.1 (CH, C-6),
4.1.3.1. 1-Benzyl-7-chloro-1H-imidazo[4,5-b]pyridine
(3a). White
solid (1.15 g, 95%, Rf ¼ 0.45). M.p. 162e164 ꢀC; 1H NMR (300 MHz,
55.9 (CH3), 49.9 ppm (CH2); IR (KBr):
n
¼ 3081, 3026, 2924, 2848,
DMSO-d6):
d
¼ 8.78 (s, 1H, H-2), 8.37 (d, 3J(H,H) ¼ 5.3 Hz, 1H, H-5),
1618, 1495, 1455, 1322, 1297, 1184, 1098, 743 cmꢂ1; MS (70 eV, EI):
m/z (%): 239 (100) [Mþ], 224 (6) [Mþ-CH3], 209 (8), 91 (60), 65 (6);
HRMS (CI): m/z: calcd for C14H13N3O þ H: 240.11370; found:
240,11385.
7.36 (d, 3J(H,H) ¼ 5.3 Hz, 1H, H-6), 7.34e7.25 (m, 3H, 2 ꢁ H-meta, H-
para), 7.12 (d, 3J(H,H) ¼ 7.2 Hz, 2H, 2 ꢁ H-ortho), 5.75 ppm (s, 2H,
CH2); 13C NMR (75 MHz, DMSO-d6):
d
¼ 158.5 (C, C-3a), 149.6 (CH,
C-5), 145.7 (CH, C-2), 138.4 (C, C-7), 129.6 (2 ꢁ CH, C-phenyl), 128.4
(CH, C-phenyl para), 127.1 (2 ꢁ CH, C-phenyl), 126.6 (C, C-phenyl
4.1.5. Synthesis of 1-benzyl-1,4-dihydroimidazo[4,5-b]pyridin-7-
one (5)
4.1.5.1. Hydrolysis of (3a). Compound 3a (0.070 g, 0.28 mmol) was
dissolved in concentrated hydrochloric acid (37%, 2.7 mL).
ipso), 123.3 (C, C-7a), 120.1 (CH, C-6), 50.2 (CH2); IR (KBr):
n
¼ 3073,
2926, 1604, 1551, 1495, 1362, 1315, 1177, 733 cmꢂ1; MS (70 eV, EI):
m/z (%): 245 (13) [(37Cl)Mþ], 243 (92) [(35Cl)Mþ], 242 (41) [Mþ-H],
Scheme 4. Synthesis of 1-[(2-methylthiazol-4-yl)methyl]-1H-indazole-4,7-dione 10. Reagents and conditions: a) i) NaH, DMF, RT, 5 mn ii) 4-chloromethyl-2-methylthiazole, RT,
15 h; b) H2 (4 bar), Pd/C, AcOEt, AcOH, RT, 5 h; c) PhI(OCOCF3)2, MeCN, H2O, 0 ꢀC then RT, 20 h.

W. Zeinyeh et al. / European Journal of Medicinal Chemistry 45 (2010) 2480e2488
2485
Fig. 4. Assignment of the structure of N-1 and N-2 regioisomers by comparison of 1H NMR spectra. 1H NMR spectra were recorded at 300 MHz in DMSO-d6.
The mixture was refluxed for one week. After cooling down to
room temperature, the mixture was neutralized adding NaOH
pellets to pH 7. Compound 5 partially precipitated after cooling
the mixture in an ice bath. After filtration and extraction of filtrate
with AcOEt, compound 5 was finally obtained as a light yellow
solid (0.033 g, 52%).
4.1.5.2. Demethylation of (4). NaH (60%, 1.36 g, 34 mmol) was
added under argon at 0 ꢀC to a solution of ethanethiol (3.9 mL,
52 mmol) in DMF (35 mL). After 15 mn stirring at 0 ꢀC, this latter
solution was added to compound 4 neat (0.82 g, 3.4 mmol) main-
tained under argon. The mixture was refluxed for 3h30. After
cooling down to room temperature, an aqueous solution of AcOH
Fig. 5. Quenching of the recombinant H6-NBD1 intrinsic fluorescence by some of the compounds (100
mM) in reference to the ATP analogue TNP-ATP and the steroid modulator
progesterone (A) at the same concentration. Competitive inhibition of the ATPase activity by compounds 8d (B), 8e (C) and 10 (D).

2486
W. Zeinyeh et al. / European Journal of Medicinal Chemistry 45 (2010) 2480e2488
7.5 Hz,1H, H-5), 7.40e7.24 (m, 5H, H-phenyl), 6.03 (d,1H, 3J (H,H) ¼
7.5 Hz, H-6), 5.65 (s, 2H, NCH2Ph), 5.03 (s, 2H, NCH2CO2Et), 4.14 (q,
2H, 3J (H,H) ¼ 7.1 Hz, CO2CH2), 1.19 ppm (t, 3H, 3J (H,H) ¼ 7.1 Hz,
Table 2
Interactions of some of the compounds with the recombinant H6-NBD1.
Entry
Compound
Intrinsic fluorescence
ATPase inhibition
Ki ( M)
N.D.b
quenchinga (% of control)
m
CH3); 13C NMR (75 MHz, DMSO-d6):
d
¼ 170.3 (C, COO), 168.9 (C, C-
1
2
3
4
5
6
6
7
8
8b
8c
8d
8e
8f
9
10
85.5 ꢃ 1.8
80.1 ꢃ 8.2
69.3 ꢃ 4,4
96.1 ꢃ 17,4
60.8 ꢃ 18,5
22.9 ꢃ 7.8
21.6 ꢃ 5.9
33.1 ꢃ 7.9
71.7 ꢃ 5.1
7), 147.6 (C, C-3a), 142.0 (CH, C-2), 139.4 (CH, C-5), 138.7 (C, C-
phenyl ipso), 129.5 (2 ꢁ CH, C-phenyl meta), 128.64 (CH, C-phenyl
para), 128.62 (2 ꢁ CH, C-phenyl ortho), 119.7 (C, C-7a), 112.9 (CH, C-
6), 62.1 (CH2, CO2CH2), 51.0 (CH2, NCH2CO2Et), 49.6 (CH2, NCH2Ph),
N.D.
232.0 ꢃ 26.8
538.9 ꢃ 73.4
N.I.c
14.8 ppm (CH3); IR (KBr):
n
¼ 3085, 3063, 3052, 2979, 2939, 1735,
N.D.
116.1 ꢃ 25.3
36.6 ꢃ 4.5
N.D.d
1626, 1585, 1474, 1451, 1395, 1374, 1324, 1231, 1200, 1024, 911, 818,
732; MS (70 eV, EI): m/z (%): 311 (100) [Mþ], 282 (10), 238 (32), 234
(17), 224 (10), 206 (7), 91 (23); HRMS (CI): m/z: calcd for
C17H17N3O3 þ H: 312.1348; found: 312.13461.
TNP-ATP
Progesterone
a
In fluorescence experiments, the compounds were tested at a concentration of
M. Results from 3 separate experiments.
N.D.: not determined.
100
m
b
c
4.1.6.3. Imidazo[4,5-b]pyridin-7-one (8c). Colourless oil (1.110 g,
64%, Rf ¼ 0.52 for CH2Cl2/MeOH ¼ 95:5). 1H NMR (300 MHz, DMSO-
N.I.: not inhibitory.
d
Progesterone is a P-gp modulator but not an inhibitor of the ATPase activity.
d6):
d
¼ 7.60 (s, 1H, H-2), 7.40e7.26 (m, 6H, H-5, 5 ꢁ H-phenyl), 6.23
(d, 1H, 3J (H,H) ¼ 7.5 Hz, H-6), 5.72 (s, 2H, NCH2Ph), 4.22 (t, 3J (H,
H) ¼ 7.1 Hz, 2H, NCH2(CH2)2CO2Et), 4.09 (t, 3J (H,H) ¼ 7.2 Hz, 2H,
CO2CH2), 2.32 (t, 3J (H,H) ¼ 7.1 Hz, 2H, CH2CO2Et), 2.17 (qn, 3J (H,
H) ¼ 7.1 Hz, 2H, CH2CH2CO2Et), 1.21 ppm (t, 3J (H,H) ¼ 7.2 Hz, 3H,
was added drop by drop to pH 7. Aqueous phase was extracted by
CH2Cl2 and the combined organic extracts were dried (Na2SO4),
filtered and concentrated. The residue was recrystallised in AcOEt/
Petroleum ether (PE) to give compound 5 as a white solid (0.690 g,
90%, Rf ¼ 0.22 for CH2Cl2/MeOH ¼ 95:5). M.p. 236e237 ꢀC; 1H NMR
CH3); 13C NMR (75 MHz, DMSO-d6):
d
¼ 172.5 (C, COO), 170.4 (C, C-
7),147.0 (C, C-3a),140.1 (CH, C-2),136.8 (CH, C-5),136.5 (C, C-phenyl
ipso), 129.0 (2 ꢁ CH, C-phenyl meta), 128.4 (2 ꢁ CH, C-phenyl
ortho), 128.3 (CH, C-phenyl para), 120.4 (C, C-7a), 113.3 (CH, C-6),
60.7 (CH2, CO2CH2), 50.1 (CH2, NCH2(CH2)2CO2Et), 49.8 (CH2,
NCH2Ph), 30.8 (CH2), 24.9 (CH2), 14.2 ppm (CH3); MS (70 eV, ESI):
m/z (%): 701 (44) [2 Mþ þ Na], 679 (26) [2 Mþ þ H], 340 (100)
[MHþ]; HRMS (CI): m/z: calcd for C19H21N3O3 þ H: 340.1661; found:
340.16602.
(300 MHz, DMSO-d6):
d
¼ 12.32 (bs,1H, NH or OH), 8.23 (s,1H, H-2),
7.62 (d, 3J (H,H) ¼ 7.3 Hz, 1H, H-5), 7.43e7.26 (m, 5H, H-phenyl),
6.00 (d, 3J (H,H) ¼ 7.3 Hz, 1H, H-6), 5.68 ppm (s, 2H, CH2); 13C NMR
(75 MHz, DMSO-d6):
d
¼ 170.6 (C, C-7), 147.6 (C, C-3a), 142.4 (CH, C-
2), 139.0 (C, C-phenyl ipso), 135.3 (CH, C-5), 129.4 (2 ꢁ CH, C-phenyl
meta), 128.5 (3 ꢁ CH, 2 ꢁ C-phenyl ortho, C-phenyl para), 120.1 (C,
C-7a), 112.4 (CH, C-6), 49.5 ppm (CH2); IR (KBr):
n
¼ 3436, 2962,
2925, 2854, 2525, 1613, 1532, 1517, 1478, 1261, 1217, 1078, 1092,
1026, 803, 731 cmꢂ1; MS (70 eV, EI): m/z (%): 225 (100) [Mþ], 224
(88), 197 (8), 148 (28), 91 (28), 65 (7); HRMS (EI): m/z: calcd for
C13H11N3O þ H: 226.0980; found: 226.0983.
4.1.6.4. Imidazo[4,5-b]pyridin-7-one (8d). Colourless oil (0.083 g,
51%, Rf ¼ 0.65 for CH2Cl2/MeOH ¼ 95:5). 1H NMR (300 MHz, DMSO-
d6):
d
¼ 7.58 (s, 1H, H-2), 7.43 (d, 3J (H,H) ¼ 7.5 Hz, 1H, H-5),
7.39e7.27 (m, 5H, H-phenyl), 6.31 (d, 3J (H,H) ¼ 7.5 Hz, 1H, H-6),
5.71 (d, 2J (H,H) ¼ 14.8 Hz, 1H, AB system, NCHaHb), 5.67 (d, 2J (H,
H) ¼ 14.8 Hz, 1H, AB system, NCHaHb), 5.57 (q, 3J (H,H) ¼ 7.2 Hz, 1H,
NCHCH3), 4.19 (q, 3J (H,H) ¼ 7.2 Hz, 2H, CO2CH2), 1.77 (d, 3J (H,H) ¼
7.2 Hz, 3H, CH3CH),1.21 ppm (t, 3J (H,H) ¼ 7.2 Hz, 3H, CH3); 13C NMR
4.1.6. N-alkylation of 1-benzyl-1,4-dihydroimidazo[4,5-b]pyridin-
7-one (5)
Compound 5 (0.115 g, 0.5 mmol) was added to a solution of KOH
(0.55 mmol or 4 mmol, see Table 1) in EtOH (5 mL). R3X (0.6 mmol
or 3 mmol, see Table 1) was added and the mixture was refluxed
until reaction did not evolve anymore (time reaction indicated in
Table 1). After cooling down to room temperature, EtOH was
evaporated and the residue was partitioned between CH2Cl2 and
water. Aqueous phase was thoroughly extracted by CH2Cl2. The
combined organic extracts were dried over Na2SO4, filtered and
concentrated. The residue was purified by flash chromatography on
silica gel (CH2Cl2/MeOH ¼ 95:5).
(75 MHz, DMSO-d6):
d
¼ 172.8 (C, COO), 170.2 (C, C-7), 147.1 (C, C-
3a), 139.8 (CH, C-2), 136.4 (C, C-phenyl ipso), 134.1 (CH, C-5), 129.0
(2 ꢁ CH, C-phenyl meta), 128.3 (2 ꢁ CH, C-phenyl ortho), 128.7 (CH,
C-phenyl para), 120.0 (C, C-7a), 113.8 (CH, C-6), 62.2 (CH2, CO2CH2),
55.7 (NCH), 50.2 (CH2, NCH2Ph), 17.0 (CH3), 14.1 ppm (CH3,
CO2CH2CH3); MS (70 eV, ESI): m/z (%): 673 (43) [2 Mþ þ Na], 651
(22) [2 Mþ þ H], 326 (100) [MH]; HRMS (CI): m/z: calcd for
C18H19N3O3 þ H: 326.1505; found: 326.15059.
4.1.6.1. Imidazo[4,5-b]pyridin-7-one (8a). White solid (0.109 g, 91%,
Rf ¼ 0.32 for CH2Cl2/MeOH ¼ 95:5). M.p. 165e166 ꢀC; 1H NMR
4.1.6.5. Imidazo[4,5-b]pyridin-7-one (8e). White solid (0.115 g, 60%,
Rf ¼ 0.30 for CH2Cl2/MeOH ¼ 95:5). M.p. 136e137 ꢀC. 1H NMR
(300 MHz, DMSO-d6):
d
¼ 8.26 (s,1H, H-2), 7.66 (d, 3J (H,H) ¼ 7.5 Hz,
(300 MHz, DMSO-d6):
d
¼ 8.24 (s, 1H, H-2), 7.71 (d, 3J (H,H) ¼ 7.5 Hz,
1H, H-5), 7.38e7.22 (m, 5H, H-phenyl), 5.98 (d, 3J (H,H) ¼ 7.5 Hz, 1H,
1H, H-5), 7.32-7.24 (m, 5H, H-phenyl), 6.90 (t, 3J (H,H) ¼ 5.4 Hz, 1H,
NH), 5.98 (d, 3J (H,H) ¼ 7.5 Hz,1H, H-6), 5.66 (s, 2H, NCH2Ph), 4.14 (t,
3J (H,H) ¼ 6.8 Hz, 2H, NCH2(CH2)2NHBoc), 2.91 (td, 3J (H,H) ¼ 5.4
and 6.8 Hz, 2H, CH2NHBoc), 1.87 (qn, 3J (H,H) ¼ 6.8 Hz, 2H,
CH2CH2NHBoc), 1.36 ppm (s, 9H, C(CH3)3); 13C NMR (75 MHz,
H-6), 5.67 (s, 2H, CH2), 3.75 ppm (s, 3H, NCH3); 13C NMR (75 MHz,
DMSO-d6):
d
¼ 169.9 (C, C-7), 147.9 (C, C-3a), 142.1 (CH, C-2), 139.4
(CH, C-5), 138.9 (C, C-phenyl ipso), 129.4 (2 ꢁ CH, C-phenyl meta),
128.5 (3 ꢁ CH, 2 ꢁ C-phenyl ortho, C-phenyl para), 120.1 (C, C-7a),
112.5 (CH, C-6), 49.5 (CH2), 37.6 ppm (CH3); IR (KBr):
n
¼ 3080,
DMSO-d6):
d
¼ 169.9 (C, C-7), 156.4 (C, NHCOtBu), 147.4 (C, C-3a),
2930, 2922, 2852, 1638, 1628, 1573, 1499, 1473, 1456, 1429, 1401,
1301, 1229, 1148, 809, 728, 703 cmꢂ1; MS (70 eV, EI): m/z (%): 239
(100) [Mþ], 224 (8), 162 (30), 133 (5), 91 (26), 65 (5); HRMS (CI):
m/z: calcd for C14H13N3O þ H: 240.1137; found: 240.1135.
142.0 (CH, C-2), 138.8 (C, C-phenyl ipso), 138.7 (CH, C-5), 129.5
(2 ꢁ CH, C-phenyl meta), 128.65 (2 ꢁ CH, C-phenyl ortho), 128.6
(CH, C-phenyl para), 120.1 (C, C-7a), 112.6 (CH, C-6), 78.5 (C, C
(CH3)3), 49.5 (CH2, NCH2Ph), 48.4 (CH2, NCH2(CH2)2NHBoc), 38.0
(CH2, CH2NHBoc), 30.5 (CH2), 29.1 ppm (3 ꢁ C, C(CH3)3); IR (KBr):
4.1.6.2. Imidazo[4,5-b]pyridin-7-one (8b). White solid (0.130 g, 82%,
Rf ¼ 0.64 for CH2Cl2/MeOH ¼ 95:5). M.p. 136e138 ꢀC (Et2O); 1H
n
¼ 3368, 2976, 2960, 1697, 1626, 1571, 1496, 1469, 1393, 1366, 1253,
1168, 1079, 818, 726; HRMS (ESI): m/z: calcd for C21H26N4O3 þ H:
NMR (300 MHz, DMSO-d6):
d
¼ 8.23 (s, 1H, H-2), 7.67 (d, 3J (H,H) ¼
383.2083; found: 383.20823.

W. Zeinyeh et al. / European Journal of Medicinal Chemistry 45 (2010) 2480e2488
2487
4.1.6.6. Imidazo[4,5-b]pyridin-7-one (8f). Colourless oil (0.131 g,
62%, Rf ¼ 0.40 for CH2Cl2/MeOH ¼ 95:5). 1H NMR (300 MHz, CDCl3):
3J (H,H) ¼ 8.0 Hz, 1H, H-4), 7.24 (s, 1H, H-50), 7.04 (dd, 3J (H,H) ¼ 7.3
and 8.0 Hz, 1H, H-5), 6.73 (d, 3J (H,H) ¼ 7.3 Hz, 1H, H-6), 5.98 (s, 2H,
CH2), 5.11 (bs, 2H, NH2), 2.71 ppm (s, 3H, CH3); 13C NMR (75 MHz,
d
¼ 8.22 (s, 1H, H-2), 7.69 (d, 3J (H,H) ¼ 7.5 Hz, 1H, H-5), 7.39e7.24
(m, 5H, H-phenyl), 6.73 (m,1H, NH), 5.97 (d, 3J (H,H) ¼ 7.5 Hz,1H, H-
6), 5.66 (s, 2H, NCH2Ph), 4.14 (m, 2H, NCH2(CH2)5NHBoc), 2.91-2.82
(m, 4H, 2 ꢁ CH2), 1.74 (m, 2H, CH2), 1.37-1.23 ppm (m, 13H, C(CH3)3,
CDCl3)
d
¼ 166.7 (C, C-20), 151.8 (C, C-40), 133.2 (CH, C-3), 132.7 (C, C-
3a), 131.8 (C, C-7a), 125.9 (C, C-7), 121.7 (CH, C-5), 116.5 (CH, C-50),
112.3 (CH, C-6), 111.4 (CH, C-4), 50.2 (CH2), 18.8 ppm (CH3); IR (KBr):
2 ꢁ CH2); 13C NMR (75 MHz, CDCl3):
d
¼ 170.7 (C, C-7), 156.4 (C,
n
¼ 3390, 1586, 1273, 735 cmꢂ1; MS (70 eV, EI): m/z (%): 244 (100)
NHCOtBu), 147.4 (C, C-3a), 140.5 (CH, C-2), 137.1 (CH, C-5), 136.9 (C,
C-phenyl ipso), 129.3 (2 ꢁ CH, C-phenyl meta), 128.8 (2 ꢁ CH, C-
phenyl ortho), 128.6 (CH, C-phenyl para), 120.7 (C, C-7a), 113.4 (CH,
C-6), 79.4 (C, C(CH3)3), 51.1 (CH2, NCH2), 50.5 (CH2, NCH2), 33.0
(CH2, CH2NHBoc), 30.2 (CH2), 30.0 (CH2), 28.8 (3 ꢁ C, C(CH3)3), 26.6
(CH2), 26.5 ppm (CH2); HRMS (ESI): m/z: calcd for C24H32N4O3 þ H:
425.2553; found: 425.25522.
[Mþ], 146 (65), 112 (31), 71 (33); HRMS (CI): m/z: calcd for
C12H12N4S þ H: 245.08; found: 245.08640.
4.1.9. 1-[(2-methylthiazol-4-yl)methyl]-1H-indazole-4,7-dione (10)
A solution of 7-aminoindazole 12 (0.126 g, 0.516 mmol) in
CH3CN/H2O ¼ 4.3:1.5 (mL:mL) was added dropwise to a solution of
PhI(OCOCF3)2 (0.555 g, 1.29 mmol) in CH3CN/H2O ¼ 4.3:1.5 (mL:mL)
cooled at 0 ꢀC. The solution was stirred for 2 h at room temperature.
Then, water and AcOEt (50 mL) were added, and aqueous phase was
extracted by AcOEt. Organic layer was washed with brine to adjust
the pH to about 7, dried (Na2SO4), and concentrated. The residue was
then purified by flash chromatography on silica gel (AcOEt/
PE ¼ 35:65) to give quinone 10 as a red solid (0.119 g, 89%, Rf ¼ 0.49
for AcOEt/PE ¼ 60:40). M.p. 128-129 ꢀC; 1H NMR (300 MHz, CDCl3)
4.1.7. N-alkylation of 7-nitroindazole
To a solution of 7-nitroindazole (0.500 g, 3.06 mmol) in dry DMF
(15 mL), was added NaH (60% in mineral oil, 0.147 g, 3.67 mmol). After
stirring for 5 min, 4-chloro-2-methylthiazole (0.677 g, 3.67 mmol)
was added and stirring was maintained for 15 h. The solution was
then poured into ice-water (200 mL) and diluted HCl was added to
adjust the pH to about 6. This solution was extracted with AcOEt and
the combined organic layers were washed with water and brine, dried
over Na2SO4 and evaporated under vacuum. The residue was purified
by column chromatography (AcOEt/petroleum ether (PE) ¼ 25/75
then 70/30) to give a mixture of regioisomers 11a as a brown solid
(0.252 g, 30%, Rf ¼ 0.54 for AcOEt/PE ¼ 15:85) and 11b as a light
yellow solid (0.459 g, 55%, Rf ¼ 0.14 for AcOEt/PE ¼ 60:40).
d
¼ 7.98 (s, 1H, H-3), 7.12 (s, 1H, H-50), 6.72 (d, 3J (H,H) ¼ 10.4 Hz, AB
system, 2H, H-5 and H-6), 5.85 (s, 2H, CH2), 2.69 ppm (s, 3H, CH3); 13
C
NMR (75 MHz, CDCl3)
d
¼ 181.2 (CO), 177.4 (CO), 166.9 (C, C-20), 149.3
(C, C-40),138.5 (CH, C-3),136.7 (CH, C-5 or C-6),136.6 (CH, C-6 or C-5),
135.5 (C, C-7a), 121.5 (C, C-3a), 117.0 (CH, C-50), 50.6 (CH2), 19.1 ppm
(CH3); IR (KBr):
C12H9N3O2S: 260.0494; found: 260.04931.
n
¼ 1665, 1170, 847 cmꢂ1; HRMS (CI): m/z: calcd for
4.1.7.1. 1-[(2-Methylthiazol-4-yl)methyl]-7-nitro-1H-indazole (11a).
4.2. Activity tests
M.p. 151 ꢀC; 1H NMR (300 MHz, DMSO-d6)
d
¼ 8.49 (s,1H, H-3), 8.29 (d,
3J (H,H) ¼ 7.7 Hz, 1H, H-6), 8.21 (d, 3J (H,H) ¼ 7.5 Hz, 1H, H-4), 7.40 (dd,
3J (H,H) ¼ 7.5 and 7.7 Hz, 1H, H-5), 7.18 (bs, 1H, H-50), 5.87 (s, 2H, CH2),
4.2.1. Production of the recombinant C. parvum H6-NBD1
The recombinant protein was overexpressed in E. coli BL21(DE3)
pLysS and its medium scale production was carried out under 3.5 L in
a glass bioreactor controlled by the BioXpert 1.3 software (Applikon,
Systeme C Industrie, St-Paul-Trois-Châteaux, France) for 5 hours. The
temperature was set at 37 ꢀC, the pH at 7.0 and the dissolved oxygen
(dO2) at 20%. Three hours after inoculation, induction was performed
with 0.1 mM IPTG for 2 h and the bacterial pellet was stored in
aliquots at ꢂ80 ꢀC. The H6-NBD1 was subsequently affinity-purified
as previously described [8] on Ni-NTA columns and dialyzed over-
night against 50 mM Tris, pH 7.3 with 100 mM KCl, 0.02% HECAMEG
2.50 ppm (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6)
d
¼ 166.0 (C, C-20),
150.8 (C, C-40),135.6 (C, C-7a),135.3 (CH, C-3),129.9 (C, C-7),128.9 (C, C-
3a), 128.7 (CH, C-4), 124.7 (CH, C-5), 120.4 (CH, C-6), 116.2 (CH, C-50),
51.9 (CH2), 18.6 ppm (CH3); IR (KBr):
n
¼ 1524, 1328, 739 cmꢂ1; Anal.
Calcd for C12H10N4O2: C, 52.54; H, 3.67; N, 20.43; O, 11.67; S, 11.69.
Found: C, 52.81; H, 3.68; N, 20.66; O, 11.93; S, 11.44.
4.1.7.2. 2-[(2-Methylthiazol-4-yl)methyl]-7-nitro-1H-indazole (11b).
M.p.168 ꢀC; 1H NMR (300 MHz, DMSO-d6)
d
¼ 8.90 (s,1H, H-3), 8.34
(d, 3J (H,H) ¼ 7.5 Hz, 1H, H-6), 8.32 (d, 3J (H,H) ¼ 8.3 Hz, 1H, H-4),
7.61 (bs, 1H, H-50), 7.29 (dd, 3J (H,H) ¼ 7.5 and 8.3 Hz, 1H, H-5), 5.86
(s, 2H, CH2), 2.63 ppm (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6)
(6-0-[(N-heptylcarbamoyl) methyl] a-D-glucopyranoside) and 20%
glycerol. The protein was used directly for the ATPase inhibition
assay or stored in aliquots at ꢂ80 ꢀC.
d
¼ 166.4 (C, C-20), 149.6 (C, C-40), 139.6 (C, C-7a), 136.6 (C, C-7),
130.2 (CH, C-3), 127.6 (CH, C-4), 125.2 (C, C-3a), 124.9 (CH, C-5),
4.2.2. Fluorescence assays
119.8 (CH, C-6), 118.3 (CH, C-50), 52.7 (CH2), 18.7 ppm (CH3); IR
Fluorescence experiments were performed in triplicates on
a PerkineElmer LS-3B spectrofluorimeter in a 1 cm-path quartz
microcuvette. The recombinant protein was diluted at a concentra-
(KBr):
n
¼ 1633, 1508, 1322, 1291, 1136, 746 cmꢂ1; Anal. Calcd for
C12H10N4O2: C, 52.54; H, 3.67; N, 20.43; O, 11.67; S, 11.69. Found: C,
52.73; H, 3.67; N, 20.86; O, 11.79; S, 11.05.
tion of 0.25e0.5
m
M in 390
ml of 50 mM Tris pH 7.3, 100 mM KCl,
0.02% HECAMEG. The analogues were added in 10
ml and the mixture
4.1.8. 7-Amino-1-[(2-methylthiazol-4-yl)methyl]-7-nitro-1H-
indazole (12)
was incubated for 30 min at 20 ꢃ 2 ꢀC. Intrinsic fluorescence emis-
sion was measured upon excitation at 295 nm and the binding of the
analogues was assayed by quenching of the intrinsic fluorescence at
328 nm. Controls were made in the same conditions with the last
dialysis buffer to eliminate any interference due to the remaining
imidazole. The hydrophobic modulators were dissolved as stock
solutions in DMSO and controls included the same solvent concen-
tration, not exceeding 2.5%. Statistics and curve fitting were made
with the Prism 4 software program from GraphPad (San Diego, CA).
A solution of 7-nitroindazole 11a (1.210 g, 4.42 mmol) in AcOEt
(63 mL) was treated with acetic acid (19 mL) and then added to
a suspension of prereduced 10% Pd/C (0.390 g, 32% w/w) in AcOEt
(15 mL). The mixture was stirred under 5 bar of H2 for 5 h, and then
filtered through celite. The residue was neutralized with diluted
cooled NH4OH and extracted with AcOEt. The combined organic
layers were washed with brine, dried over Na2SO4, filtered and
concentrated. The residue was purified by flash chromatography
(silica gel neutralized with NEt3, AcOEt/PE ¼ 30:70) to give
compound 12 as a purple oil (0.980 g, 91%, Rf ¼ 0.51 for AcOEt/
4.2.3. ATPase inhibition assays
The analogues were serially diluted in various ATP concentra-
tions and their inhibitory activity was measured by incubating
PE ¼ 70:30). 1H NMR (300 MHz, CDCl3)
¼ 8.01 (s, 1H, H-3), 7.26 (d,
d

2488
W. Zeinyeh et al. / European Journal of Medicinal Chemistry 45 (2010) 2480e2488
10 min at 37 ꢀC the purified recombinant H6-NBD1 (2.5
mM final) in
50 mM Tris pH 7.3, 100 mM KCl, 4 mM MgCl2 and 0,02% HECAMEG.
The reaction products were measured in triplicates by HPLC on a 5
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m
m reverse phase Hypersil column (Interchim, Montluçon, France)
with 1,8% methanol (v/v) in 25 mM (NH)4H2PO4 as the mobile
phase. The injection volume was 20 l, the flow rate was 0.5 ml/min
m
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