Dual IGF-1R/SRC inhibitors based on a N′-aroyl-2-(1H-indol-3-yl)-2- oxoacetohydrazide structure

Article abstract of DOI:10.1016/j.ejmech.2011.03.065

The N′-aroyl-2-(1H-indol-3-yl)-2-oxoacetohydrazide motif was identified as a novel scaffold for the development of kinase inhibitors. Derivatives with a biphenyl element attached to the hydrazide structure proved to be submicromolar dual inhibitors of the cancer-related kinases IGF-1R and SRC. One of the most potent kinase inhibitors of the series produced a selective growth inhibition in a panel of cultivated cancer cell lines.

Full text of DOI:10.1016/j.ejmech.2011.03.065

European Journal of Medicinal Chemistry 46 (2011) 2759e2769
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Dual IGF-1R/SRC inhibitors based on a N0-aroyl-2-(1H-indol-3-yl)-2-
oxoacetohydrazide structure
Stefanie Schmidt a, Lutz Preu a, Thomas Lemcke b, Frank Totzke c, Christoph Schächtele c,
,
Michael H.G. Kubbutat c, Conrad Kunick a
*
a Technische Universität Braunschweig, Institut für Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany
b Universität Hamburg, Institut für Pharmazie, Bundesstraße 45, D-20146 Hamburg, Germany
c ProQinase GmbH, Breisacher Straße 117, 79106 D-Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
The N0-aroyl-2-(1H-indol-3-yl)-2-oxoacetohydrazide motif was identified as a novel scaffold for the
development of kinase inhibitors. Derivatives with a biphenyl element attached to the hydrazide
structure proved to be submicromolar dual inhibitors of the cancer-related kinases IGF-1R and SRC. One
of the most potent kinase inhibitors of the series produced a selective growth inhibition in a panel of
cultivated cancer cell lines.
Article history:
Received 18 January 2011
Received in revised form
29 March 2011
Accepted 30 March 2011
Available online 6 April 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Anticancer
Hydrazides
Indoles
Protein kinase inhibitors
Suzuki reaction
1. Introduction
identify novel multikinase inhibitor chemotypes [9e12] we here
report N0-aroyl-2-(1H-indol-3-yl)-2-oxoacetohydrazides as dual
Because many human tumours are characterized by hyperac-
tivity of protein kinases, the inhibition of these enzymes has been
established as a cancer treatment in recent years. Given the great
success of this therapeutic strategy, the search for additional
protein kinase inhibitors is one of the major research topics in the
oncology drug development area [1,2]. While in the beginning of
the protein kinase inhibitor era selectivity towards a single target
enzyme appeared desirable, it is now generally accepted that
inhibiting more than one tumour-related protein kinase at a time
offers the advantage of hitting the tumour in multiple aspects [3,4].
Among other advantages, the aspecificity of drugs targeting several
kinases may lead to a lower risk of drug-resistance development
[3,4]. Multikinase inhibitors launched recently as anticancer drugs
comprise sunitinib [5], sorafenib [6] and dasatinib [7,8]. Albeit
namely the latter was reported as an inhibitor of human protein
kinases with high promiscuity [7], the undesired side effects of the
drug are tolerable. In the framework of our ongoing studies to
IGF-1R/SRC inhibitors.
The insulin-like growth factor receptor-1 tyrosine kinase (IGF-
1R) constitutes an emerging novel target for anticancer therapy
[13]. There are clear hints pointing to the involvement of IGF-1R in
cancer development. Particularly, activation of the IGF-1R receptor
kinase leads to triggering of intracellular signalling pathways
promoting cell proliferation and survival, angiogenesis, metastasis
and invasion [14]. Mice overexpressing the physiological activator
IGF-II of the IGF-1R develop a variety of tumours, among them
hepatocellular carcinomas and lymphomas [15]. Since over-
expression of the receptor and its physiological ligands have been
observed in several solid human tumours, small molecules inhib-
iting IGF-1R have been developed as potential anticancer drugs and
have already advanced to clinical trials [13,16]. c-SRC is a non-
receptor tyrosine kinase mediating mitogenic signals between IGF-
1R and downstream signalling cascades. A variety of investigations
suggest that SRC plays a prominent role in cancer cell proliferation,
invasion, and motility. SRC hyperactivity has been implicated in the
development and progression of human cancers. Drug develop-
ment research in recent years led to several small molecule SRC
inhibitors currently being evaluated in clinical phases, among them
* Corresponding author. Tel.: þ49 531 391 2751; fax: þ49 531 391 2799.
E-mail address: c.kunick@tu-braunschweig.de (C. Kunick).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.065

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S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
O
O
O
O
H
(i)
Cl
N
H2N
N
H
+
N
H
R
R
O
O
N
H
N
H
5
6a-q
7a-q
Scheme 1. Synthesis of N0-aroyl-2-(1H-indol-3-yl)-2-oxoacetohydrazides 7a-q. Reagents and conditions: (i) Et3N, MTBE, 0 ꢀC / rt.
compounds XL-999, AZD-0530, KX010107, and Bosutinib, a dual
BCR-ABL and SRC inhibitor [17]. It was reported that in cells
transformed by the src ongogene, IGF-1R was found to be consti-
tutively phosphorylated. This mechanism was suggested to be one
of the events responsible for the transforming property of the src
oncogene [18]. Thus, inhibiting both IGF-1R and SRC by one small
molecule inhibitor could offer the advantage of interfering with
two successive events of the same signalling axis, constituting
a concept potentially lowering the risk for resistance development.
The identification of the dual IGF-1R/SRC inhibitor chemotype
reported here resulted from an observation during a project
directed to discover inhibitors of the protein kinase MET. Hyper-
activity of the MET kinase is related to growth, aggressiveness and
metastasis risk in several solid cancer diseases [19]. When we
evaluated selected commercially available compounds in vitro in
a radiometric protein kinase assay (33PanQinaseÒ Activity Assay),
we identified the 2-(1H-indol-3-yl)-2-oxoacetohydrazide 1 as weak
phenyl ring. While none of 7a-k exhibited MET inhibition with IC50
values below 10 M, 4 congeners (7d, 7e, 7g, 7i) showed IGF-1R
m
inhibition in single-digit micromolar concentrations. Of note,
these compounds have a more or less bulky substituent in the 4-
position of the phenyl ring and inhibited SRC in the same order of
magnitude as IGF-1R. We therefore extended the series to deriva-
tives 7l-q which are characterized by a phenyl substituent in the
indicated
position,
constituting
N0-acylated
biphenyl-4-
carbohydrazides. Actually, all novel congeners with the biphenyl
element inhibited both IGF-1R and SRC in low micromolar or
submicromolar concentrations (Table 1). To get preliminary infor-
mation regarding bioactivity of the novel compounds in a cellular
environment, compound 7o was screened for antiproliferative
activity in the in vitro cell line screening project (IVCLSP) of the
National Cancer Institute (NCI) [26]. Indeed, the cell lines
A549ATCC, NCI-H522 (both non-small cell lung cancer cell lines)
and OVCAR-8 (ovarian cancer) were inhibited impressively
(Table 2). Other cell lines were inhibited in a lower extent, indi-
cating a selective cytotoxicity, as is expected with a protein kinase
inhibitor. Inhibition of A549ATCC by 7o coincides with the fact that
this cancer cell line shows the strongest SRC expression level of all
NCI panel cell lines [27].
MET inhibitor (IC50 c-MET ¼ 69
mM) [20].
2. Chemistry
In an attempt to narrow down the pharmacophore structure
element in 1, the truncated structures 2e4 were synthesized
following published procedures [21e25]. For the synthesis of the
N,N’-diacylated hydrazides 7, suitable benzoic acid hydrazides 6
were reacted with 2-(1H-indol-3-yl)-2-oxoacetyl chloride 5 in
methyl tert-butyl ether (MTBE) in the presence of triethylamine
(Scheme 1). The carbohydrazides 6l-q needed as building blocks for
the synthesis of 7l-q were prepared by means of a microwave-
Docking of the prototype structure 7o into published structures
of IGF-1R (pdb entry 2zm3) [28] and SRC (pdb entry 2bdf) [29]
produced poses showing
a certain degree of similarity and
explaining the gain of activity by introduction of the biphenyl
structural element (Fig. 1). In both poses, elongate conformations of
the inhibitors are aligned below the glycine rich loops (Leu1005-
Phe1010 in IGF-1R and Leu273-Phe278 in SRC) in the cleft between
the N-terminal and the C-teminal lobes of the kinases. The indole
ring systems are located near the hinge area facing the entrances of
the pockets. The carbonyl groups next to the indole elements are
both acting as hydrogen bond acceptors towards the amino acid
residue three positions downstream the gatekeeper (gkþ3,
Met1082 in case of IGF-1R and Met 341 in case of SRC). The benzene
rings of the biphenyl element are involved in hydrophobic contacts
to hydrophobic amino acid residues in the binding cleft. The tert-
butyl phenyl ring of the inhibitor displays hydrophobic interactions
with the phenylalanine residues located at the C-terminal end of
the glycine rich loops (Phe1010 in IGF-1R and Phe 278 in SRC,
respectively). In both poses, the ether oxygen is a hydrogen bond
acceptor towards amino acids of the glycine rich loop, contacting
assisted
Suzuki
coupling
reaction
catalyzed
by
tetrakistriphenylphospane-palladium in the presence of dia-
zabicycloundecene (Scheme 2).
3. Biological evaluation and discussion
While none of the derivatives 2e4 showed improved MET
inhibition, two congeners (3b and 4a) exhibited a modest inhibi-
tion of IGF-1R (Table 1). Because of the overall disappointing results
with 2e4, we postulated that the complete N,N’-diacylated hydra-
zide structure as displayed by 1 is necessary for kinase inhibition.
We continued the series with compounds 7a-k which differ from
the parent structure 1 exclusively by the substitution pattern at the
R1
R2
(HO)2B
O
O
H5C2O2C
H2N
H2N
R1
N
N
H
(i)
9
H
R2
I
(ii)
I
8
6l-q
6g
Scheme 2. Synthesis of biphenyl-4-carbohydazides 6l-q. Reagents and conditions: (i) hydrazine hydrate, EtOH, reflux; (ii) Pd(PPh3)4, DBU, H2O/EtOH, 150 ꢀC,
mW.

S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
2761
M]
Table 1
Protein kinase inhibition by 2-(1H-indol-3-yl)-2-oxoacetohydrazides 1, 2, 7 and 2-(1H-indol-3-yl)-2-oxoacetamides 3, 4.
a,b,c
Entry
Structure
IC50 c-MET [
m
M]
IC50 IGF-1R [
m
M]
IC50 SRC [m
O
O
H
N
N
H
O
1
2
N
H
69
>100
N.T.
NO2
(hit structure from commercial vendor)
R
O
H
N
N
H
O
N
H
2a
2b
2c
2d
R ¼ eH
> 100
> 100
> 100
> 100
>100
> 100
>100
>100
N.T.
N.T.
N.T.
N.T.
R ¼ eCl
R ¼ eBr
R ¼ eI
O
H
R
N
3
O
N
H
3a
3b
3c
3d
R ¼ eH
>100
>100
>100
>100
>100
63
>100
>100
N.T.
N.T.
N.T.
N.T.
R ¼ 4-CH3
R ¼ 4-Cl
R ¼ 3-Cl
O
H
N
4
R
O
N
H
4a
4b
4c
4d
R ¼ 4-CH3
>100
>100
>100
>100
52
N.T.
N.T.
N.T.
N.T.
R ¼ 3,4-di-OCH3
R ¼ 4-Cl
>100
>100
>100
R ¼ 3-Cl
O
O
H
N
N
7a-k
H
O
N
H
R1
R2
7a
7b
7c
7d
7e
7f
R1, R2 ¼ eH
>100
95
96
36
49
48
14
>100
> 100
82
54
4.4 (ꢁ0.5)
4.1 (ꢁ2.7)
16
N.T.
N.T.
N.T.
R1 ¼ H, R2 ¼ eCH3
R1 ¼ H, R2 ¼ eOCH3
R1 ¼ H, R2 ¼ eC(CH3)3
R1 ¼ H, R2 ¼ eCF3
R1 ¼ H, R2 ¼ eCl
R1 ¼ H, R2 ¼ eI
5.9 (ꢁ2.5)
1.3
N.T.
7g
7h
4.0 (ꢁ0.2)
4.5 (ꢁ1.0)
R1 ¼ F, R2 ¼ eH
91
96
CH3
R1 ¼ H, R2
¼
16
4.9 (ꢁ0.6)
3.6 (ꢁ0.6)
N
7i
H3C
N
O
CH3
7j
R1 ¼ H, R2
¼
¼
95
12
31
22
40
N
N
7k
R1 ¼ H, R2
>100
(continued on next page)
O
N
CH3

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S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
Table 1 (continued )
Entry
Structure
IC50 c-MET [
m
M]
IC50 IGF-1R [
m
M]
IC50 SRC [mM]
O
O
H
N
R1
N
H
7l-q
N
H
R2
O
O
7l
R2 ¼ eH, R1
R2 ¼ eH, R1
R1 ¼ eH, R2
¼
13
4.9 (ꢁ1.1)
1.9 (ꢁ0.4)
F
CF3
O
O
7m
7n
¼
¼
5.2
7.6
0.81 (ꢁ0.06)
0.99 (ꢁ0.31)
0.62 (ꢁ0.24)
0.72 (ꢁ0.21)
CF3
CH3
O
7o
7p
R1 ¼ eH, R2
¼
¼
4.1
8.8
0.67 (ꢁ0.02)
0.68 (ꢁ0.07)
0.52 (ꢁ0.12)
0.38 (ꢁ0.06)
C(CH3)3
O
R1 ¼ eH, R2
CH3
O
SO2CH3
7q
R1 ¼ eH, R2
¼
27
3.3 (ꢁ0.2)
2.8 (ꢁ0.2)
N
H
N.T.: not tested.
a
Compounds 2, 3 and 4 have been described before. See experimental part for references.
Values with range given in brackets represent the mean of two independent determinations.
Values without range indicate a single measurement.
b
c
the side chain of Ser1009 in IGF-1R and the backbone amide of
Phe278 in SRC.
considered as working hypotheses guiding the further structure
modification and optimisation process.
According to these docking results, in both IGF-1R and SRC the
inhibitor 7o occupies not only the ATP binding pocket but also
binds with its tert-butyl phenyl moiety to a large pocket in the
neighbourhood. This binding mode may account for the selectivity
for IGF-1R and SRC and needs consideration during future studies
in which detailed kinetic investigations will be necessary to
determine the inhibition modes (ATP-competitive or allosteric or
mixed). As long as such results are not available and no crystal
structure is solved for a complex between the ligands 7 and the
target kinases, the binding modes presented here should be
4. Conclusion
The N0-aroyl-2-(1H-indol-3-yl)-2-oxoacetohydrazide structure
was discovered as a promising template for the development of
novel protein kinase inhibitors, namely of IGF-1R and/or SRC.
Although a distinct compound belonging to this structure class has
already demonstrated antiproliferative activity, a broad synthesis
program will be necessary to increase protein kinase inhibitory
potency and selectivity and to tailor ADME properties. The simple
synthetic procedures and the plausible docking mode of 7o in IGF-
1R and SRC are good arguments to consider such a development
campaign.
Table 2
Inhibition of cancer cell proliferation caused by compound 7oa
Cancer cell line
Cell growth (%) in presence of 7o
(10
M) related to untreated controlsb
m
5. Experimental protocols
A549/ATCC
NCI-H522
OVCAR-8
Average of all cell lines
7.38
ꢂ7.70
ꢂ9.59
81.05
5.1. Synthetic chemistry
5.1.1. General
Negative values indicate net cell kill instead of proliferation.
Monomode microwave device: CEM Discover focused micro-
wave synthesis system with ChemDriver software. Melting points
(mp) were determined on an electric variable heater (Barnstead
a
Data from the IVCLSP of the NCI. Cells were incubated for 48 h with the test
compound. Read out by a sulforhodamine assay.
b
Controls ¼ 100%.

S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
2763
Fig. 1. Docking poses generated for inhibitor 7o in the protein structures IGF-1R [28] (upper row) and SRC [29] (lower row). On the left side the inhibitor (H-atoms depicted, carbon
atoms orange-red) is shown with the amino acids (H-atoms omitted) of the glycine-rich loop (Leu1005-Phe1010 for IGF-1R, Leu273-Phe278 for SRC) and the hinge sequence
(Met1079(gatekeeper)-Met1082 for IGF-1R, Thr338(gatekeeper)-Met341 for SRC). On the pictures on the right side the surface of the protein structure is given showing the general
accommodation of the inhibitor in the cleft between the N-terminal lobe (on top of the inhibitor) and the C-terminal lobe (below the inhibitor). Illustration by UCSF Chimera [36].
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Electrothermal IA 9100) and were not corrected. IR spectra were
recorded as KBr discs on a Thermo Nicolet FT-IR 200.1H NMR spectra
and 13C NMR spectra were recorded on the following instruments:
Bruker Avance DRX-400 and Bruker Avance II-600, solvent DMSO-d6
if not stated otherwise, internal standard trimethylsilane, signals in
chloride (5; 521 mg, 2.51 mmol) in anhydrous tert-butyl methyl
ether (30 mL) was slowly added. The reaction mixture was stirred
at 0 ꢀC for 15 min and then for 1.5 h at room temperature, moni-
toring the reaction by TLC analysis. After completion of the reaction
the mixture was washed with a saturated NaHCO3 aqueous solution
and water (3 ꢃ 20 mL in each case). The organic layer was dried
over anhydrous Na2SO4 and evaporated. The crude product was
purified and isolated by silica gel chromatography (ethyl acetate/
petroleum ether). Subsequent recrystallization from toluene and
ethanol 70% yielded yellow crystals (5%).
ppm (d scale). Elemental analyses were determined on a CE Instru-
ments FlashEA 1112 elemental analyzer (Thermo Quest).
Mass spectra were recorded on a double-focused sector field
mass spectrometer Finnigan-MAT 95. Accurate measurements
were conducted according to the peakmatch method using per-
fluorokerosene (PFK) as an internal mass reference; (EI)-MS: ioni-
zation energy 70 eV. TLC: Polygram Sil G/UV254, Macherey-Nagel,
40 mm ꢃ 80 mm, visualization by UV illumination (254 nm).
Column chromatography: silica gel 60 (Merck), column width
2.5e3 cm, column height 15e30 cm. Purity was determined by
HPLC using the following devices and settings [30]: LaChrom Elite
(Merck/Hitachi), pump L-2130, autosampler L-2200, diode array
detector L-2450, organizer box L-2000; column Merck LiChroCART
Mp.: 220e223 ꢀC (dec.); IR (KBr): 3352 cmꢂ1 and 3283 cmꢂ1
(NH), 1677 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 6.72e6.74 (m, 2H, ar-H), 7.27e7.31 (m, 2H, ar-H),
7.32e7.34 (m, 2H, ar-H), 7.55e7.57 (m, 1H, ar-H), 8.17 (d, 1H,
J ¼ 2.7 Hz, NH), 8.23e8.25 (m, 1H, ar-H), 8.57 (d, 1H, J ¼ 2.5 Hz, ar-
H), 10.65 (d, 1H, J ¼ 2.7 Hz, NH), 12.29 (s, 1H, indole-NH); 13C NMR
(DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 112.2, 113.8 (2C), 120.7, 122.3,
123.1,130.9 (2C),137.7 (tert. C); 109.1,111.8,125.4, 135.9,147.7,163.4,
181.7 (quat. C); C16H12BrN3O2 (358.19); calcd. C 53.65, H 3.38, N
11.73; found C 53.69, H 3.29, N 11.35; HPLC-purity: 98.9% at 254 nm
and 97.6% at 280 nm, tN ¼ 2.90 min, tM ¼ 1.03 min (ACN/H2O;
50:50), lmax: 245 nm and 328 nm.
125-4, LiChrosphere 100 RP 18, 5
mm, flow rate 1.000 mL/min,
isocratic, volume of injection 10 L; detection (DAD) at 254 and
m
280 nm; AUC % method; time of detection 15 min, net retention
time (tN), dead time (tM) related to DMSO; eluent water/acetonitrile
if not stated otherwise. All compounds employed in biological tests
were used in ꢄ95% purity. The following compounds were
prepared according to literature methods: 2a-b [22], 3a [23], 3b
[25], 3c-d [23], 4a [24], 4b-c [21] and 6g [31]. Compounds 5, 6a-f,
6h, 6i, 8 and 9 were purchased from commercial suppliers and were
used without further purification.
5.1.3. 2-(1H-Indol-3-yl)-N0-(4-iodophenyl)-2-oxoacetohydrazide
(2d)
To
1.00 mmol) in anhydrous tert-butyl methyl ether (8 mL) triethyl-
amine (153
L, 1.10 mmol) was added at 0 ꢀC. Then a suspension of
a stirred solution of 4-iodophenylhydrazine (234 mg,
m
2-(1H-indol-3-yl)-2-oxoacetyl chloride (5; 208 mg, 1.00 mmol) in
anhydrous tert-butyl methyl ether (12 mL) was slowly added. The
reaction mixture was stirred at 0 ꢀC for 15 min and then for 1.5 h at
room temperature, monitoring the reaction by TLC analysis. After
completion of the reaction the mixture was washed with a satu-
rated NaHCO3 aqueous solution and water (3 ꢃ 20 mL in each case).
The organic layer was dried over anhydrous Na2SO4 and evapo-
rated. The crude product was purified and isolated by silica gel
5.1.2. N0-(4-Bromophenyl)-2-(1H-indol-3-yl)-2-oxoacetohydrazide
(2c)
A solution of triethylamine (765 mL, 5.50 mmol) in anhydrous
tert-butyl methyl ether (10 mL) was added dropwise at 0 ꢀC to
a stirred suspension of 4-bromophenylhydrazine hydrochloride
(559 mg, 2.50 mmol) in the same solvent (20 mL). After stirring for
30 min at 0 ꢀC a suspension of 2-(1H-indol-3-yl)-2-oxoacetyl

2764
S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
chromatography (eluent: ethyl acetate/petroleum ether (1:1)).
Subsequent recrystallization from toluene and ethanol 70% yielded
yellow crystals (2%).
Yield: 16%, white powder; mp.: 166e167 ꢀC; 1H NMR (DMSO-d6,
400.1 MHz):
(m, 2H, ar-H), 7.86e7.90 (m, 2H, ar-H), 8.32 (d, 1H, J ¼ 0.5 Hz,
pyrazine-H), 8.34 (d, 1H, J ¼ 0.5 Hz, pyrazine-H), 9.77 (s, 1H, NH).
d
(ppm) ¼ 2.35 (s, 3H, CH3), 4.51 (s, 2H, NH2), 7.23e7.27
Mp.: 222e228 ꢀC (dec.); IR (KBr): 3388 cmꢂ1 and 3279 cmꢂ1
(NH), 1685 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 6.60e6.63 (m, 2H, ar-H), 7.27e7.31 (m, 2H, ar-H),
5.1.7. General procedure A for the preparation of the biphenyl-4-
carbohydrazides 6l-6q
4-Iodobenzohydrazide (6g; 262 mg, 1.00 mmol) was reacted
with the appropriate boronic acid 9 (1.00 mmol), 1,8-diazabicyclo
7.46e7.48 (m, 2H, ar-H), 7.54e7.57 (m, 1H, ar-H), 8.16 (d, 1H,
J ¼ 2.8 Hz, NH), 8.22e8.25 (m, 1H, ar-H), 8.57 (d, 1H, J ¼ 3.2 Hz, ar-
H), 10.63 (d, 1H, J ¼ 2.7 Hz, NH), 12.29 (d, 1H, J ¼ 2.5 Hz, indole-NH);
13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 112.6, 114.7 (2C), 121.1,
[5.4.0]undec-7-ene (149 mL, 1.00 mmol) and 5 mol% tetrakis(-
122.6, 123.5, 137.0 (2C), 138.1 (tert. C); 80.0, 112.1, 125.7, 136.3, 148.5,
163.7, 182.1 (quat. C); C16H12IN3O2 (405.19); calcd. C 47.43, H 2.99, N
10.37; found C 47.37, H 3.00, N 9.75; MS (EI): m/z (%) ¼ 405 [M]þ$
(27); HRMS (EI): m/z [M]þ$ calcd. 404.99747; found 404.99763;
HPLC-purity: 98.6% at 254 nm and 97.0% at 280 nm, tN ¼ 3.54 min,
tM ¼ 1.03 min (ACN/H2O; 50:50), lmax: 248 nm and 328 nm.
triphenylphosphine)palladium(0) (58 mg, 0.050 mmol) in water
(1 mL) and ethanol (1 mL). The reaction was conducted in
a microwave device using a sealed microwave reaction vessel. The
reaction mixture was stirred for 10 min at 150 ꢀC, 60 W, ramp time
5 min, maximum pressure 250 psi. After cooling to room temper-
ature the mixture was poured into a separating funnel. Water was
added and the aqueous phase was extracted with ethyl acetate
(3 ꢃ 20 mL). The collected organic layers were dried over anhy-
drous Na2SO4 and evaporated. Except for 6p, which was purified
and isolated by silica gel chromatography (eluent: ethyl acetate),
the crude products were used in the next step without further
purification according to general procedure B yielding compounds
7l-q.
5.1.4. N-[2-(3-Chlorophenyl)ethyl]-2-(1H-indol-3-yl)-2-
oxoacetamide (4d)
Triethylamine (153
a stirred suspension of 2-(1H-indol-3-yl)-2-oxoacetyl chloride (5;
208 mg, 1.00 mmol) and 2-(3-chlorophenyl)ethylamine (139 L,
m
L,1.10 mmol) was added dropwise at 0 ꢀC to
m
1.00 mmol) in anhydrous tert-butyl methyl ether (20 mL). The
reaction mixture was stirred at 0 ꢀC for 15 min and then for 1 h at
room temperature, monitoring the reaction by TLC analysis. After
completion of the reaction the mixture was washed with a satu-
rated NaHCO3 aqueous solution and water (3 ꢃ 20 mL in each case).
The organic layer was dried over anhydrous Na2SO4 and evapo-
rated. Recrystallization of the crude product from ethanol 70%
yielded colourless crystals (33%).
5.1.7.1. 20-[(4-Fluorobenzyl)oxy][1,10-biphenyl]-4-carbohydrazide
(6l). Preparation according to General Procedure
A yielded
a yellow, viscous product (57%).
1H NMR (DMSO-d6, 400.1 MHz):
d
(ppm) ¼ 4.54 (s, 2H, NH2),
5.13 (s, 2H, OCH2), 7.07 (ddd, 1H, J ¼ 1.0/7.4/7.4 Hz, ar-H), 7.16e7.20
(m, 2H, ar-H), 7.21e7.23 (m, 1H, ar-H), 7.34e7.37 (m, 2H, ar-H),
7.40e7.44 (m, 2H, ar-H), 7.58e7.61 (m, 2H, ar-H), 7.82e7.85 (m,
2H, ar-H), 9.78 (s, 1H, NH).
Mp.: 184e186 ꢀC; IR (KBr): 3347 cmꢂ1 and 3190 cmꢂ1 (NH),
2938 cmꢂ1 (CH aliph.), 1657 cmꢂ1 (C]O); 1H NMR (DMSO-d6,
400.1 MHz):
d
(ppm) ¼ 2.88 (t, 2H, J ¼ 7.0 Hz, NHCH2CH2), 3.50 (dt,
2H, J ¼ 6.4/6.8 Hz, NHCH2CH2), 7.21e7.34 (m, 6H, ar-H), 7.52e7.57
(m, 1H, ar-H), 8.21e8.27 (m, 1H, ar-H), 8.67 (s, 1H, ar-H), 8.81 (t, 1H,
J ¼ 5.8 Hz, NH), 12.24 (s, 1H, indole-NH); 13C NMR (DMSO-d6,
5.1.7.2. 20-{[3-(Trifluoromethyl)phenoxy]methyl}[1,10-biphenyl]-4-
carbohydrazide (6m). Preparation according to General Procedure
A yielded a yellow, viscous product (65%).
100.6 MHz):
d
(ppm) ¼ 34.2 (CH2), 39.2 (CH2); 112.5, 121.2, 122.5,
IR (KBr): 3310 cmꢂ1 (NH), 3065 cmꢂ1 (CH arom.), 2929 cmꢂ1 (CH
123.4, 126.1, 127.5, 128.5, 130.1, 138.4 (tert. C); 112.1, 132.9, 136.3,
141.9, 163.6, 182.1 (quat. C, one signal is missing due to peak over-
lap); C18H15ClN2O2 (326.78); calcd. C 66.16, H 4.63, N 8.57; found C
65.84, H 4.60, N 8.31; HPLC-purity: 99.3% at 254 nm and 98.9% at
aliph.); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 4.54 (s, 2H,
NH2), 5.05 (s, 2H, OCH2), 7.19e7.20 (m, 2H, ar-H), 7.27e7.29 (m, 1H,
ar-H), 7.37e7.38 (m, 1H, ar-H), 7.47e7.49 (m, 3H, ar-H), 7.50e7.51
(m, 2H, ar-H), 7.64e7.65 (m, 1H, ar-H), 7.86e7.88 (m, 2H, ar-H), 9.83
280 nm, tN ¼ 3.38 min, tM ¼ 1.03 min (ACN/H2O; 55:45), lmax
:
(s, 1H, NH); 13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 68.0
3
3
256 nm and 327 nm.
(OCH2); 111.1 (q, JC,F ¼ 3.5 Hz, CeCeCF3), 117.3 (q, JC,F ¼ 3.9 Hz,
CeCeCF3), 118.9, 126.8 (2C), 128.0, 128.6, 128.8 (2C), 129.9, 130.0,
130.6 (tert. C); 123.9 (q, 1JC,F ¼ 272 Hz, CF3), 130.2 (q, 2JC,F ¼ 32 Hz,
CeCF3), 132.1, 133.2, 140.9, 142.5, 158.2, 165.3 (quat. C).
5.1.5. 4-(5-Methyl-1,2,4-oxadiazol-3-yl)benzohydrazide (6j)
Hydrazine hydrate (485
mL, 9.98 mmol) was added dropwise
to a solution of ethyl 4-(5-methyl-1,2,4-oxadiazol-3-yl)benzoate
(232 mg, 1.00 mmol) in anhydrous ethanol (4 mL). The reaction
mixture was refluxed for 30 h. After cooling to room tempera-
ture the resulting precipitate was collected and washed with
ethanol. The crude product was used in the next step without
further purification according to general procedure B yielding
compound 7j.
5.1.7.3. 30-{[3-(Trifluoromethyl)phenoxy]methyl}[1,10-biphenyl]-4-
carbohydrazide (6n). Preparation according to General Procedure A
yielded a yellow, viscous product (56%).
IR (KBr): 3288 cmꢂ1 (NH), 3057 cmꢂ1 (CH arom.), 2922 cmꢂ1
und 2863 cmꢂ1 (CH aliph.); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 4.56 (s, 2H, NH2), 5.28 (s, 2H, OCH2), 7.31 (ddd,1H, J ¼ 0.7/
Yield: 84%, white powder; mp.: 237e241 ꢀC; 1H NMR (DMSO-d6,
7.6/7.6 Hz, ar-H), 7.36e7.39 (m, 2H, ar-H), 7.51e7.56 (m, 3H, ar-H),
7.72 (ddd, 1H, J ¼ 2.0/6.9/6.9 Hz, ar-H), 7.77e7.79 (m, 2H, ar-H),
7.85 (m, 1H, ar-H), 7.93e7.95 (m, 2H, ar-H), 9.86 (s, 1H, NH); 13C
400.1 MHz):
d
(ppm) ¼ 2.68 (s, 3H, CH3), 4.57 (s, 2H, NH2),
7.97e8.00 (m, 2H, ar-H), 8.05e8.08 (m, 2H, ar-H), 9.94 (s, 1H, NH).
NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 69.4 (OCH2); 111.3 (q,
3
5.1.6. 4-[(6-methylpyrazin-2-yl)oxy]benzohydrazide (6k)
3JC,F ¼ 3.7 Hz, CeCeCF3), 117.2 (q, JC,F ¼ 3.9 Hz, CeCeCF3), 119.0,
Hydrazine hydrate (121
mL, 2.49 mmol) was added dropwise to
126.3, 126.4, 126.5 (2C), 127.4, 127.6 (2C), 129.2, 130.7 (tert. C); 123.9
2
a
solution of ethyl 4-[(6-methylpyrazin-2-yl)oxy]benzoate
(q, 1JC,F ¼ 272 Hz, CF3), 130.2 (q, JC,F ¼ 32 Hz, CeCF3), 132.2, 137.2,
(258 mg, 1.00 mmol) in ethanol (5 mL). The reaction mixture was
refluxed for 32 h. After cooling to room temperature the resulting
precipitate was collected and washed with ethanol. The crude
product was used in the next step without further purification
according to general procedure B yielding compound 7k.
139.3, 142.2, 158.5, 165.4 (quat. C).
5.1.7.4. 30-{[4-tert-butyl-2-methylphenoxy]methyl}[1,10-biphenyl]-4-
carbohydrazide (6o). Preparation according to General Procedure A
yielded a yellow, viscous product (81%).

S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
2765
IR (KBr): 3280 cmꢂ1 (NH), 3053 cmꢂ1 (CH arom.), 2953 cmꢂ1 und
132.3,136.5,163.7,165.5,182.4 (quat. C); C17H13N3O3 (307.30); calcd.
C 66.44, H 4.26, N 13.67; found C 66.25, H 4.19, N 13.45; HPLC-purity:
99.8% at 254 nm and 99.8% at 280 nm, tN ¼ 4.19 min, tM ¼ 1.09 min
(ACN/H2O; 30:70), lmax: 330 nm.
2863 cmꢂ1 (CH aliph.), 1643 cmꢂ1 (C]O); 1H NMR (DMSO-d6,
600.1 MHz):
d
(ppm) ¼ 1.24 (s, 9H, CH3), 2.21 (s, 3H, CH3), 4.55 (s, 2H,
NH2), 5.17 (s, 2H, OCH2), 6.94 (d, 1H, J ¼ 8.6 Hz, ar-H), 7.13 (dd, 1H,
J ¼ 2.2/8.2 Hz, ar-H), 7.19 (dd, 1H, J ¼ 0.6/2.5 Hz, ar-H), 7.49 (ddd, 1H,
J ¼ 1.6/7.7/7.7 Hz, ar-H), 7.51 (dd, 1H, J ¼ 7.6/7.6 Hz, ar-H), 7.68 (ddd,
1H, J ¼ 1.7/7.1/7.1 Hz, ar-H), 7.75e7.77 (m, 2H, ar-H), 7.80 (m, 1H, ar-
H), 7.92e7.95 (m, 2H, ar-H), 9.85 (s, 1H, NH); 13C NMR (DMSO-d6,
5.1.8.2. 2-(1H-Indol-3-yl)-N0-(4-methylbenzoyl)-2-oxoacetohy-
drazide (7b). Preparation according to General Procedure B. Reac-
tion time:
4 h. Recrystallization from ethanol 96% yielded
150.9 MHz):
d
(ppm) ¼ 16.4 (CH3), 31.3 (3xCH3), 68.9 (OCH2); 111.2,
colourless crystals (24%).
123.1, 125.7, 126.1, 126.5 (2C), 126.8, 127.5, 127.6 (2C), 129.1 (tert. C);
33.6, 125.1, 132.1, 138.4, 139.2, 142.4, 154.0, 165.4, 170.3 (quat. C).
Mp.: 301e305 ꢀC (dec.); IR (KBr): 3301 cmꢂ1 and 3147 cmꢂ1
(NH), 3057 cmꢂ1 (CH arom.), 2916 cmꢂ1 (CH aliph.), 1709 cmꢂ1 and
1638 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 2.39
5.1.7.5. 30-{[4-(2-Methoxyethyl)phenoxy]methyl}[1,10-biphenyl]-4-
carbohydrazide (6p). Preparation according to General Procedure A
yielded a yellow, viscous product (81%).
(s, 3H, CH3), 7.28e7.32 (m, 2H, ar-H), 7.34e7.35 (m, 2H, ar-H),
7.56e7.59 (m, 1H, ar-H), 7.84e7.85 (m, 2H, ar-H), 8.23e8.25 (m,
1H, ar-H), 8.72 (d,1H, J ¼ 2.9 Hz, ar-H),10.51 (s,1H, NH),10.70 (s,1H,
NH), 12.35 (d, 1H, J ¼ 2.5 Hz, indole-NH); 13C NMR (DMSO-d6,
IR (KBr): 3308 cmꢂ1 (NH), 3031 cmꢂ1 (CH arom.), 2979 cmꢂ1
,
2926 cmꢂ1, 2863 cmꢂ1 und 2824 cmꢂ1 (CH aliph.), 1625 cmꢂ1 (C]
100.6 MHz):
d
(ppm) ¼ 20.1 (CH3); 111.7, 120.2, 121.8, 122.7, 126.5
O); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 2.73 (t, 2H,
(2C), 128.1 (2C), 137.6 (tert. C); 111.6, 124.7, 128.5, 135.5, 141.1, 162.7,
164.4, 181.5 (quat. C); C18H15N3O3 (321.33); calcd. C 67.28, H 4.71, N
13.08; found C 67.64, H 4.78, N 12.98; HPLC-purity: 99.6% at 254 nm
and 99.4% at 280 nm, tN ¼ 2.43 min, tM ¼ 1.02 min (ACN/H2O;
40:60), lmax: 246 nm and 329 nm.
J ¼ 6.9 Hz, CH2), 3.22 (s, 3H, OCH3), 3.47 (t, 2H, J ¼ 7.0 Hz, CH2), 4.58
(s, 2H, NH2), 5.15 (s, 2H, OCH2), 6.94e6.96 (m, 2H, ar-H), 7.13e7.16
(m, 2H, ar-H), 7.48 (m, 1H, ar-H), 7.50 (dd, 1H, J ¼ 7.6/7.6 Hz, ar-H),
7.69 (ddd, 1H, J ¼ 1.5/7.5/7.5 Hz, ar-H), 7.75e7.78 (m, 2H, ar-H), 7.79
(m, 1H, ar-H), 7.92e7.94 (m, 2H, ar-H), 9.86 (s, 1H, NH); 13C NMR
(DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 34.4 (OCH2CH2Ar), 57.7 (OCH3),
5.1.8.3. 2-(1H-Indol-3-yl)-N0-(4-methoxybenzoyl)-2-oxoacetohy-
drazide (7c). Preparation according to General Procedure B. Reac-
tion time: 23 h. Recrystallization from ethanol 96% yielded yellow
powder (24%).
68.9 (OCH2), 72.9 (OCH2); 114.5 (2C), 126.0, 126.2, 126.5 (2C), 127.2,
127.5 (2C), 129.1, 129.7 (2C) (tert. C); 131.1, 132.1, 138.0, 139.2, 142.2,
156.6, 165.4 (quat. C).
Mp.: 300e305 ꢀC (dec.); IR (KBr): 3284 cmꢂ1 (NH), 1679 cmꢂ1
5.1.7.6. N-{[40-(Hydrazinocarbonyl)[1,10-biphenyl]-3-yl]methyl}
methanesulfonamide (6q). Preparation according to General
Procedure A yielded light yellow powder (50%).
and 1644 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 400.1 MHz):
d
(ppm) ¼ 3.84 (s, 3H, OCH3), 7.06e7.08 (m, 2H, ar-H), 7.27e7.32 (m,
2H, ar-H), 7.56e7.58 (m, 1H, ar-H), 7.92e7.94 (m, 2H, ar-H),
8.23e8.26 (m, 1H, ar-H), 8.72 (d, 1H, J ¼ 3.1 Hz, ar-H), 10.41 (s, 1H,
NH), 10.63 (s, 1H, NH), 12.32 (d, 1H, J ¼ 2.5 Hz, indole-NH); 13C NMR
1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 2.89 (s, 3H, CH3),
4.25 (d, 2H, J ¼ 6.3 Hz, CH2), 4.54 (s, 2H, NH2), 7.38e7.39 (m, 1H, ar-
H), 7.47 (dd, 1H, J ¼ 7.7/7.7 Hz, ar-H), 7.62e7.65 (m, 2H, ar-H,
superimposed with sulfonamide-NH), 7.71 (m, 1H, ar-H),
7.75e7.77 (m, 2H, ar-H), 7.9e7.95 (m, 2H, ar-H), 9.85 (s, 1H, NH);
(DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 55.4 (OCH3); 112.7, 113.8 (2C),
121.2, 122.7, 123.6, 129.4 (2C), 138.5 (tert. C); 112.6, 124.4, 125.7,
136.5,162.1,163.8,165.0,182.5 (quat. C); C18H15N3O4 (337.33); calcd.
C 64.09, H 4.48, N 12.46; found C 63.98, H 4.54, N 12.47; HPLC-
purity: 99.8% at 254 nm and 99.9% at 280 nm, tN ¼ 5.12 min,
tM ¼ 1.02 min (ACN/H2O; 30:70), lmax: 258 nm and 329 nm.
13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 39.8 (CH3), 45.9 (CH2);
125.6, 126.1, 126.4 (2C), 127.2, 127.5 (2C), 129.0 (tert. C); 132.1, 139.0,
139.1, 142.3, 165.4 (quat. C).
5.1.8. General procedure B for the preparation of the N0-aroyl-2-
5.1.8.4. N0-(4-tert-Butylbenzoyl)-2-(1H-indol-3-yl)-2-oxoacetohy-
drazide (7d). Preparation according to General Procedure B. Reac-
tion time: 22 h. Recrystallization from ethanol 96% yielded light
yellow crystals (56%).
(1H-indol-3-yl)-2-oxoacetohydrazides 7
Triethylamine (153 m
L,1.10 mmol) was added dropwise at 0 ꢀC to
a stirred suspension of 2-(1H-indol-3-yl)-2-oxoacetyl chloride (5;
208 mg, 1.00 mmol) and the appropriate benzohydrazide 6
(1.00 mmol) in anhydrous tert-butyl methyl ether (20 mL). The
reaction mixture was stirred at 0 ꢀC for 15 min and then for 1e26 h
at room temperature, monitoring the reaction by TLC analysis. After
completion of the reaction the solvent was removed in vacuo and
the residue was washed with a saturated NaHCO3 aqueous solution,
water (3 ꢃ 20 mL in each case) and petroleum ether. The crude
product was purified by recrystallization from the given solvent.
Mp.: 270e271 ꢀC (dec.); IR (KBr): 3340 cmꢂ1, 3277 cmꢂ1 and
3158 cmꢂ1 (NH), 2952 cmꢂ1, 2903 cmꢂ1 and 2867 cmꢂ1 (CH aliph.),
1694 cmꢂ1 and 1653 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 1.32 (s, 9H, CH3), 7.27e7.33 (m, 2H, ar-H), 7.55e7.56 (m,
2H, ar-H), 7.56e7.59 (m, 1H, ar-H), 7.87e7.89 (m, 2H, ar-H),
8.23e8.26 (m, 1H, ar-H), 8.72 (s, 1H, ar-H), 10.51 (s, 1H, NH), 10.71
(s, 1H, NH), 12.35 (s, 1H, indole-NH); 13C NMR (DMSO-d6,
150.9 MHz):
d
(ppm) ¼ 30.8 (3xCH3); 112.6, 121.1, 122.6, 123.6, 125.2
(2C), 127.3 (2C), 138.5 (tert. C); 34.6, 112.5, 125.6, 129.4, 136.4, 154.8,
163.6, 165.3, 182.4 (quat. C); C21H21N3O3 (363.41); calcd. C 69.41, H
5.82, N 11.56; found C 69.17, H 5.70, N 11.39; HPLC-purity: 99.9% at
254 nm and 99.7% at 280 nm, tN ¼ 2.97 min, tM ¼ 1.01 min (ACN/
H2O; 50:50), lmax: 246 nm and 329 nm.
5.1.8.1. N0-Benzoyl-2-(1H-indol-3-yl)-2-oxoacetohydrazide
(7a). Preparation according to General Procedure B. Reaction time:
2.5 h. Recrystallization from ethanol 96% yielded light yellow
crystals (30%).
Mp.: 305e309 ꢀC (dec.); IR (KBr): 3300 cmꢂ1 (NH),1710 cmꢂ1 and
1639 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 400.1 MHz):
5.1.8.5. 2-(1H-Indol-3-yl)-2-oxo-N0-[4-(trifluoromethyl)benzoyl]ace-
tohydrazide (7e). Preparation according to General Procedure B.
Reaction time: 6.5 h. Recrystallization from ethanol 96%/toluene
(1:1) yielded light yellow crystals (39%).
d
(ppm) ¼ 7.27e7.33 (m, 2H, ar-H), 7.52e7.57 (m, 2H, ar-H),
7.57e7.59 (m, 1H, ar-H), 7.62 (tt, 1H, J ¼ 1.3/6.2 Hz, ar-H), 7.94e7.97
(m, 2H, ar-H), 8.23e8.28 (m, 1H, ar-H), 8.72 (d, 1H, J ¼ 3.1 Hz, ar-H),
10.57 (s, 1H, NH), 10.72 (s, 1H, NH), 12.33 (d, 1H, J ¼ 2.8 Hz, indole-
Mp.: 325e330 ꢀC (dec.); IR (KBr): 3359 cmꢂ1 and 3293 cmꢂ1
NH); 13C NMR (DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 112.6, 121.2,
(NH), 1709 cmꢂ1 and 1645 cmꢂ1 (C]O); 1H NMR (DMSO-d6,
122.7, 123.7, 127.5 (2C), 128.5 (2C), 132.0, 138.5 (tert. C); 112.7, 125.7,
600.1 MHz):
d
(ppm) ¼ 7.28e7.33 (m, 2H, ar-H), 7.56e7.59 (m, 1H,

2766
S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
ar-H), 7.94e7.96 (m, 2H, ar-H), 8.12e8.14 (m, 2H, ar-H), 8.23e8.26
(m,1H, ar-H), 8.71 (d,1H, J ¼ 3.2 Hz, ar-H),10.85 (s, 2H, 2xNH), 12.37
5.1.8.9. N0-[4-(2,5-Dimethyl-1H-pyrrol-1-yl)benzoyl]-2-(1H-indol-3-
yl)-2-oxoacetohydrazide (7i). Preparation according to General
Procedure B. Reaction time: 24 h. Recrystallization from ethanol
96% yielded orange powder (53%).
(d, 1H, J ¼ 2.1 Hz, indole-NH); 13C NMR (DMSO-d6, 150.9 MHz):
3
d
(ppm) ¼ 111.5, 119.9, 121.5, 122.4, 124.4 (q, 2C, JC,F ¼ 3.6 Hz,
CeCeCF3), 127.2 (2C), 137.3 (tert. C); 111.2, 122.6 (q, 1JC,F ¼ 273 Hz,
CF3), 124.5, 130.5 (q, 2JC,F ¼ 32 Hz, CeCF3), 134.8, 135.2, 162.2, 163.1,
180.8 (quat. C); C18H12F3N3O3 (375.30); calcd. C 57.61, H 3.22, N
11.20; found C 57.59, H 3.14, N 10.86; HPLC-purity: 99.9% at 254 nm
and 99.8% at 280 nm, tN ¼ 2.55 min, tM ¼ 1.02 min (ACN/H2O;
45:55), lmax: 254 nm and 331 nm.
Mp.: 279e282 ꢀC (dec.); IR (KBr): 3295 cmꢂ1 and 3148 cmꢂ1
(NH), 2921 cmꢂ1 (CH aliph.), 1709 cmꢂ1 and 1636 cmꢂ1 (C]O); 1H
NMR (DMSO-d6, 400.1 MHz):
d
(ppm) ¼ 2.02 (s, 6H, CH3), 5.85 (s,
2H, pyrrole-H), 7.27e7.33 (m, 2H, ar-H), 7.43e7.46 (m, 2H, ar-H),
7.56e7.60 (m, 1H, ar-H), 8.04e8.08 (m, 2H, ar-H), 8.23e8.27 (m,
1H, ar-H), 8.71 (d, 1H, J ¼ 3.1 Hz, ar-H),10.66 (s, 1H, NH),10.76 (s, 1H,
NH), 12.32 (d, 1H, J ¼ 3.0 Hz, indole-NH); 13C NMR (DMSO-d6,
5.1.8.6. N0-(4-Chlorobenzoyl)-2-(1H-indol-3-yl)-2-oxoacetohy-
drazide (7f). Preparation according to General Procedure B. Reac-
tion time: 2 h. Recrystallization from ethanol 70%/toluene (3:1)
yielded colourless crystals (11%).
100.6 MHz):
d
(ppm) ¼ 12.8 (2xCH3); 106.5 (2C), 112.6, 121.2, 122.7,
123.6, 128.0 (2C), 128.5 (2C), 138.4 (tert. C); 112.6, 125.7, 127.5 (2C),
131.3, 136.5, 141.4, 163.6, 164.8, 182.2 (quat. C); C23H20N4O3
(400.43); calcd. C 68.99, H 5.03, N 13.99; found C 68.74, H 4.85, N
13.80; HPLC-purity: 96.1% at 254 nm and 96.1% at 280 nm,
tN ¼ 2.68 min, tM ¼ 1.01 min (ACN/H2O; 50:50), lmax: 267 nm and
328 nm.
Mp.: 320e328 ꢀC (dec.); IR (KBr): 3295 cmꢂ1 and 3147 cmꢂ1
(NH), 3067 cmꢂ1 (CH arom.), 1709 cmꢂ1 and 1641 cmꢂ1 (C]O); 1H
NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 7.28e7.32 (m, 2H, ar-H),
7.56e7.59 (m, 1H, ar-H), 7.62e7.65 (m, 2H, ar-H), 7.94e7.97 (m,
2H, ar-H), 8.23e8.26 (m, 1H, ar-H), 8.70 (d, 1H, J ¼ 3.2 Hz, ar-H),
10.69 (s, 1H, NH), 10.78 (s, 1H, NH), 12.36 (d, 1H, J ¼ 2.2 Hz,
5.1.8.10. 2-(1H-Indol-3-yl)-N0-[4-(5-methyl-1,2,4-oxadiazol-3-yl)
benzoyl]-2-oxoacetohydrazide (7j). Preparation according to
General Procedure B. Reaction time: 26 h. Recrystallization from
ethanol 96%/toluene (1:1) yielded yellow powder (50%).
indole-NH); 13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 112.6,121.1,
122.7,123.6,128.6 (2C),129.3 (2C),138.4 (tert. C); 112.4,125.6,130.9,
136.4, 136.8, 163.4, 164.4, 182.1 (quat. C); C17H12ClN3O3 (341.75);
calcd. C 59.75, H 3.54, N 12.30; found C 59.68, H 3.53, N 12.07;
HPLC-purity: 99.9% at 254 nm and 99.7% at 280 nm, tN ¼ 3.23 min,
tM ¼ 1.02 min (ACN/H2O; 40:60), lmax: 245 nm and 330 nm.
Mp.: 312e317 ꢀC (dec.); IR (KBr): 3302 cmꢂ1 and 3146 cmꢂ1
(NH), 1708 cmꢂ1 and 1647 cmꢂ1 (C]O); 1H NMR (DMSO-d6,
600.1 MHz):
d
(ppm) ¼ 2.71 (s, 3H, CH3), 7.28e7.33 (m, 2H, ar-H),
7.56e7.59 (m, 1H, ar-H), 8.10e8.11 (m, 2H, ar-H), 8.16e8.17 (m,
2H, ar-H), 8.24e8.26 (m, 1H, ar-H), 8.72 (d, 1H, J ¼ 3.0 Hz, ar-H),
10.77 (s, 1H, NH), 10.82 (s, 1H, NH), 12.36 (d, 1H, J ¼ 2.6 Hz,
5.1.8.7. 2-(1H-Indol-3-yl)-N0-(4-iodobenzoyl)-2-oxoacetohydrazide
(7g). Preparation according to General Procedure B. Reaction time:
3.5 h. Recrystallization from ethanol 96%/toluene (5:1) yielded light
yellow powder (40%).
indole-NH); 13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 12.0 (CH3);
112.6, 121.1, 122.7, 123.6, 127.1 (2C), 128.3 (2C), 138.4 (tert. C); 112.4,
125.6, 129.2, 134.6, 136.4, 163.4, 164.7, 166.9, 177.8, 182.1 (quat. C);
C20H15N5O4 (389.36); calcd. C 61.69, H 3.88, N 17.99; found C 59.27,
H 3.60, N 16.70; MS (EI): m/z (%) ¼ 389 [M]þ$ (4); HRMS (EI): m/z
[M]þ$ calcd. 389.11243; found 389.11181; HPLC-purity: 99.9% at
254 nm and 99.9% at 280 nm, tN ¼ 3.50 min, tM ¼ 1.03 min (ACN/
H2O; 35:65), lmax: 256 nm and 330 nm.
Mp.: 345e348 ꢀC (dec.); IR (KBr): 3338 cmꢂ1, 3294 cmꢂ1 and
3147 cmꢂ1 (NH), 1709 cmꢂ1 and 1635 cmꢂ1 (C]O); 1H NMR
(DMSO-d6, 400.1 MHz):
d
(ppm) ¼ 7.27e7.32 (m, 2H, ar-H),
7.55e7.59 (m, 1H, ar-H), 7.70e7.72 (m, 2H, ar-H), 7.92e7.95 (m,
2H, ar-H), 8.22e8.25 (m, 1H, ar-H), 8.68 (d, 1H, J ¼ 3.2 Hz, ar-H),
10.62 (s, 1H, NH), 10.71 (s, 1H, NH), 12.30 (d, 1H, J ¼ 2.7 Hz,
indole-NH); 13C NMR (DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 112.6,
5.1.8.11. 2-(1H-Indol-3-yl)-N0-{4-[(6-methylpyrazin-2-yl)oxy]
benzoyl}-2-oxoacetohydrazide (7k). Preparation according to
General Procedure B. Reaction time: 3 h. Recrystallization from
ethanol 96% yielded yellow powder (8%).
121.1, 122.6, 123.5, 129.3 (2C), 137.4 (2C), 138.3 (tert. C); 99.6, 112.5,
125.6, 131.7, 136.4, 163.4, 164.8, 182.1 (quat. C); C17H12IN3O3
(433.20); calcd. C 47.13, H 2.79, N 9.70; found C 47.20, H 2.66, N
9.41; HPLC-purity: 99.9% at 254 nm and 99.7% at 280 nm,
tN ¼ 4.15 min, tM ¼ 1.04 min (ACN/H2O; 40:60), lmax: 257 nm and
330 nm.
Mp.: 273e275 ꢀC (dec.); IR (KBr): 3288 cmꢂ1 (NH), 1707 cmꢂ1
and 1637 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 2.37 (s, 3H, CH3), 7.28e7.32 (m, 2H, ar-H), 7.34e7.36 (m,
2H, ar-H), 7.56e7.59 (m, 1H, ar-H), 7.99e8.02 (m, 2H, ar-H),
8.23e8.25 (m, 1H, ar-H), 8.35 (s, 1H, pyrazine-H), 8.40 (s, 1H,
pyrazine-H), 8.71 (d, 1H, J ¼ 3.1 Hz, ar-H), 10.60 (s, 1H, NH), 10.75 (s,
1H, NH), 12.35 (d, 1H, J ¼ 2.9 Hz, indole-NH); 13C NMR (DMSO-d6,
5.1.8.8. N0-(2-Fluorobenzoyl)-2-(1H-indol-3-yl)-2-oxoacetohy-
drazide (7h). Preparation according to General Procedure B. Reac-
tion time: 5 h. Recrystallization from ethanol 96% yielded yellow-
green crystals (32%).
150.9 MHz):
d
(ppm) ¼ 20.5 (CH3); 112.6, 120.7 (2C), 121.1, 122.6,
Mp.: 271e275 ꢀC (dec.); IR (KBr): 3376 cmꢂ1, 3298 cmꢂ1 and
123.6, 129.3 (2C), 132.3, 138.4, 138.9 (tert. C); 112.5, 125.6, 128.6,
136.4, 151.1, 156.1, 158.1, 163.6, 164.7, 182.2 (quat. C); C22H17N5O4
(415.40); MS (EI): m/z (%) ¼ 415 [M]þ$ (6); HRMS (EI): m/z [M]þ$
calcd. 415.12805; found 415.12737; HPLC-purity: 97.3% at 254 nm
and 97.8% at 280 nm, tN ¼ 3.60 min, tM ¼ 1.03 min (ACN/H2O;
35:65), lmax: 252 nm, 303 nm and 329 nm.
3145 cmꢂ1 (NH), 1709 cmꢂ1 and 1639 cmꢂ1 (C]O); 1H NMR
(DMSO-d6, 400.1 MHz):
d
(ppm) ¼ 7.27e7.32 (m, 2H, ar-H),
7.34e7.38 (m, 2H, ar-H), 7.55e7.64 (m, 2H, ar-H), 7.70 (ddd, 1H,
J ¼ 2.7/7.1/7.1 Hz, ar-H), 8.22e8.26 (m, 1H, ar-H), 8.70 (d, 1H,
J ¼ 3.2 Hz, ar-H), 10.39 (s, 1H, NH), 10.76 (s, 1H, NH), 12.33 (d, 1H,
J
d
¼
3.0 Hz, indole-NH); 13C NMR (DMSO-d6, 100.6 MHz):
2
(ppm) ¼ 112.6, 116.3 (d, JC,F ¼ 22 Hz, CeCF), 121.2, 122.6, 123.6,
5.1.8.12. N0-({20-[(4-Fluorobenzyl)oxy][1,10-biphenyl]-4-yl}carbonyl)-
2-(1H-indol-3-yl)-2-oxoacetohydrazide (7l). Preparation according
to General Procedure B. Reaction time: 24 h. Recrystallization from
ethanol 96%/toluene (1:3) yielded yellow powder (24%).
3
4
124.5 (d, JC,F ¼ 3.4 Hz, CeCeCF), 130.1 (d, JC,F ¼ 2.8 Hz,
CeCeCeCF), 133.1 (d, 3JC,F ¼ 9.0 Hz, CeCeCF), 138.4 (tert. C); 112.5,
122.0 (d, JC,F ¼ 14 Hz, CeCF), 125.7, 136.4, 159.2 (d, 1JC,F ¼ 250 Hz,
2
CF), 163.0, 163.2, 182.1 (quat. C); C17H12FN3O3 (325.29); calcd. C
62.77, H 3.72, N 12.92; found C 62.71, H 3.70, N 12.77; HPLC-purity:
99.9% at 254 nm and 99.8% at 280 nm, tN ¼ 2.95 min, tM ¼ 1.03 min
(ACN/H2O; 35:65), lmax: 254 nm, 266 nm and 330 nm.
Mp.: 227e231 ꢀC (dec.); IR (KBr): 3247 cmꢂ1 (NH), 1701 cmꢂ1
and 1659 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 5.15 (s, 2H, OCH2), 7.10 (ddd, 1H, J ¼ 1.0/7.5/7.5 Hz, ar-H),
7.19e7.23 (m, 2H, ar-H), 7.25 (dd,1H, J ¼ 0.9/8.7 Hz, ar-H), 7.28e7.33

S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
2767
(m, 2H, ar-H), 7.38e7.41 (m, 2H, ar-H), 7.44e7.47 (m, 2H, ar-H),
7.56e7.59 (m, 1H, ar-H), 7.69e7.70 (m, 2H, ar-H), 7.95e7.96 (m,
2H, ar-H), 8.23e8.26 (m, 1H, ar-H), 8.72 (d, 1H, J ¼ 2.3 Hz, ar-H),
10.59 (s, 1H, NH), 10.74 (s, 1H, NH), 12.35 (s, 1H, indole-NH); 13C
Mp.: 256e261 ꢀC (dec.); IR (KBr): 3308 cmꢂ1 (NH), 3150 cmꢂ1
(CH arom.), 2956 cmꢂ1 and 2866 cmꢂ1 (CH aliph.), 1709 cmꢂ1 and
1638 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 400.1 MHz):
d
(ppm) ¼ 1.25
(s, 9H, CH3), 2.23 (s, 3H, CH3), 5.19 (s, 2H, OCH2), 6.95 (d, 1H,
J ¼ 8.5 Hz, ar-H), 7.14 (dd, 1H, J ¼ 2.5/8.5 Hz, ar-H), 7.19 (d, 1H,
J ¼ 2.2 Hz, ar-H), 7.27e7.33 (m, 2H, ar-H), 7.51e7.54 (m, 2H, ar-H),
7.56e7.60 (m, 1H, ar-H), 7.73 (ddd, 1H, J ¼ 2.2/6.6/6.6 Hz, ar-H),
7.85e7.87 (m, 3H, ar-H), 8.04e8.06 (m, 2H, ar-H), 8.23e8.27 (m,
1H, ar-H), 8.73 (d,1H, J ¼ 3.3 Hz, ar-H),10.63 (s,1H, NH),10.73 (s,1H,
NH), 12.34 (d, 1H, J ¼ 3.3 Hz, indole-NH); 13C NMR (DMSO-d6,
NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 68.9 (OCH2); 112.6, 113.2,
115.1 (d, 2C, 2JC,F ¼ 21 Hz, CeCF), 121.1, 121.2, 122.6 (2C), 123.6, 127.1
3
(2C), 129.3 (2C), 129.4 (d, 2C, JC,F ¼ 8.0 Hz, CeCeCF), 130.5, 138.4
4
(tert. C); 112.5, 125.6, 129.0, 130.5, 133.1 (d, JC,F ¼ 3.0 Hz,
CeCeCeCF), 136.4, 141.6, 154.9, 161.5 (d, 1JC,F ¼ 244 Hz, CF), 163.6,
165.2, 182.3 (quat. C); C30H22FN3O4 (507.51); calcd. C 71.00, H 4.37,
N 8.28; found C 71.05, H 4.36, N 8.32; HPLC-purity: 98.4% at 254 nm
and 98.2% at 280 nm, tN ¼ 2.91 min, tM ¼ 1.08 min (ACN/H2O;
60:40), lmax: 267 nm and 303 nm.
100.6 MHz):
d
(ppm) ¼ 16.4 (CH3), 31.3 (3xCH3), 69.0 (OCH2); 111.3,
112.6, 121.2, 122.7, 123.2, 123.6, 125.7, 125.8, 126.7 (2C), 127.0, 127.5,
128.2 (2C), 129.2, 138.5 (tert. C); 33.6, 112.7, 125.2, 126.2, 131.2,
136.5, 138.6, 139.1, 142.6, 143.2, 154.1, 163.6, 165.1, 182.3 (quat. C);
C35H33N3O4 (559.65); calcd. C 75.11, H 5.94, N 7.51; found C 74.85, H
5.75, N 7.35; HPLC-purity: 99.1% at 254 nm and 99.3% at 280 nm,
tN ¼ 3.00 min, tM ¼ 1.08 min (ACN/H2O; 72:25), lmax: 272 nm and
327 nm.
5.1.8.13. 2-(1H-Indol-3-yl)-2-oxo-N0-[(20-{[3-(trifluoromethyl)phe-
noxy]methyl}[1,10-biphenyl]-4-yl)carbonyl]acetohydrazide
(7m). Preparation according to General Procedure B. Reaction
time: 3 h. Recrystallization from ethanol 96% yielded light yellow
powder (37%).
Mp.: 221e228 ꢀC (dec.); IR (KBr): 3355 cmꢂ1 and 3310 cmꢂ1
(NH), 1707 cmꢂ1 and 1639 cmꢂ1 (C]O); 1H NMR (DMSO-d6,
5.1.8.16. 2-(1H-Indol-3-yl)-2-oxo-N0-[(30-{[4-(2-methoxyethyl)phe-
noxy]methyl}[1,10-biphenyl]-4-yl)carbonyl]acetohydrazide
(7p). Preparation according to General Procedure B. Reaction time:
5 h. Recrystallization from ethanol 96%/toluene (2:1) yielded
yellow powder (16%).
600.1 MHz):
d
(ppm) ¼ 5.08 (s, 2H, OCH2), 7.21e7.23 (m, 2H, ar-H),
7.28e7.32 (m, 3H, ar-H), 7.41e7.42 (m, 1H, ar-H), 7.48e7.53 (m, 3H,
ar-H), 7.57e7.58 (m, 1H, ar-H), 7.59e7.60 (m, 2H, ar-H), 7.66e7.68
(m, 1H, ar-H), 7.98e8.00 (m, 2H, ar-H), 8.23e8.26 (m, 1H, ar-H), 8.71
(d, 1H, J ¼ 3.1 Hz, ar-H), 10.63 (s, 1H, NH), 10.75 (s, 1H, NH), 12.35 (d,
Mp.: 251e255 ꢀC; IR (KBr): 3305 cmꢂ1 (NH), 2925 cmꢂ1 and
2860 cmꢂ1 (CH aliph.), 1708 cmꢂ1 and 1639 cmꢂ1 (C]O); 1H NMR
1H, J ¼ 2.7 Hz, indole-NH); 13C NMR (DMSO-d6, 150.9 MHz):
(DMSO-d6, 600.1 MHz):
d
(ppm) ¼ 2.73 (t, 2H, J ¼ 7.0 Hz, OCH2
-
3
d
(ppm) ¼ 68.0 (OCH2); 111.2 (q, JC,F ¼ 3.8 Hz, CeCeCF3), 112.6,
CH2Ar), 3.23 (s, 3H, OCH3), 3.48 (t, 2H, J ¼ 7.0 Hz, OCH2CH2Ar), 5.17
(s, 2H, OCH2), 6.95e6.98 (m, 2H, ar-H), 7.14e7.17 (m, 2H, ar-H),
7.29e7.33 (m, 2H, ar-H), 7.50 (ddd, 1H, J ¼ 1.4/7.5/7.5 Hz, ar-H),
7.53 (dd, 1H, J ¼ 7.7/7.7 Hz, ar-H), 7.57e7.60 (m, 1H, ar-H), 7.74
(ddd, 1H, J ¼ 1.5/7.3/7.3 Hz, ar-H), 7.85 (m, 1H, ar-H), 7.86e7.88 (m,
2H, ar-H), 8.04e8.06 (m, 2H, ar-H), 8.24e8.27 (m, 1H, ar-H), 8.73 (d,
1H, J ¼ 3.2 Hz, ar-H),10.65 (s,1H, NH),10.76 (s, 1H, NH),12.36 (d,1H,
3
117.3 (q, JC,F ¼ 3.9 Hz, CeCeCF3), 118.8, 121.1, 122.7, 123.6, 127.4
(2C), 128.1, 128.7, 129.1 (2C), 129.9, 130.1, 130.7, 138.4 (tert. C); 112.5,
123.7 (q, 1JC,F ¼ 272 Hz, CF3), 125.6, 130.3 (q, 2JC,F ¼ 32 Hz, CeCF3),
131.0, 133.2, 136.4, 140.8, 143.4, 158.2, 163.6, 165.0, 182.2 (quat. C);
C31H22F3N3O4 (557.52); calcd. C 66.78, H 3.98, N 7.54; found C 66.85,
H 3.85, N 7.26; HPLC-purity: 99.1% at 254 nm and 98.8% at 280 nm,
tN ¼ 4.96 min, tM ¼ 1.09 min (ACN/H2O þ 0.05% HCOOH; 60:40),
lmax: 260 nm and 328 nm.
J
d
¼
2.9 Hz, indole-NH); 13C NMR (DMSO-d6, 150.9 MHz):
(ppm) ¼ 34.4 (OCH2CH2Ar), 57.7 (OCH3), 68.9 (OCH2), 72.9
(OCH2CH2Ar); 112.6, 114.5 (2C),121.1, 122.7, 123.6, 126.1, 126.3, 126.7
(2C), 127.4, 128.1 (2C), 129.1, 129.7 (2C), 138.4 (tert. C); 112.5, 125.6,
131.0, 131.1, 136.4, 138.1, 139.0, 143.1, 156.6, 163.6, 165.1, 182.3 (quat.
C); C33H29N3O5 (547.60); calcd. C 72.38, H 5.34, N 7.67; found C
72.11, H 5.26, N 7.76; HPLC-purity: 98.4% at 254 nm and 98.3% at
280 nm, tN ¼ 4.06 min, tM ¼ 1.09 min (ACN/H2O þ 0.05% HCOOH;
60:40), lmax: 272 nm and 327 nm.
5.1.8.14. 2-(1H-Indol-3-yl)-2-oxo-N0-[(30-{[3-(trifluoromethyl)phe-
noxy]methyl}[1,10-biphenyl]-4-yl)carbonyl]acetohydrazide
(7n). Preparation according to General Procedure B. Reaction time:
4 h. Recrystallization from ethanol 96%/toluene (2:1) yielded light
yellow powder (43%).
Mp.: 275e277 ꢀC (dec.); IR (KBr): 3346 cmꢂ1, 3287 cmꢂ1 and
3227 cmꢂ1 (NH), 2961 cmꢂ1 and 2905 cmꢂ1 (CH aliph.), 1699 cmꢂ1
and 1676 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600.1 MHz):
5.1.8.17. N-{[40-({2-[2-(1H-Indol-3-yl)-2-oxoacetyl]hydrazino}
carbonyl)[1,10-biphenyl]-3-yl]methyl}methanesulfonamide
(7q). Preparation according to General Procedure B. Reaction time:
3 h. Recrystallization from ethanol 96% yielded yellow powder
(14%).
d
(ppm) ¼ 5.30 (s, 2H, OCH2), 7.30e7.33 (m, 3H, ar-H), 7.37e7.40 (m,
2H, ar-H), 7.53e7.59 (m, 4H, ar-H), 7.77 (ddd, 1H, J ¼ 2.0/6.7/6.7 Hz,
ar-H), 7.87e7.89 (m, 2H, ar-H), 7.91 (m, 1H, ar-H), 8.05e8.06 (m, 2H,
ar-H), 8.24e8.27 (m, 1H, ar-H), 8.73 (s, 1H, ar-H), 10.65 (s, 1H, NH),
10.76 (s, 1H, NH), 12.35 (s, 1H, indole-NH); 13C NMR (DMSO-d6,
Mp.: 274e280 ꢀC (dec.); IR (KBr): 3305 cmꢂ1 (NH), 1709 cmꢂ1
3
150.9 MHz):
d
(ppm) ¼ 69.4 (OCH2); 111.3 (q, JC,F ¼ 3.8 Hz,
and 1641 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600.1 MHz):
3
CeCeCF3), 112.6, 117.2 (q, JC,F ¼ 3.6 Hz, CeCeCF3), 119.0, 121.1,
122.7, 123.6, 126.4, 126.5, 126.7 (2C), 127.6, 128.1 (2C), 129.2, 130.7,
138.4 (tert. C); 112.5, 123.9 (q, 1JC,F ¼ 272 Hz, CF3), 125.6, 130.2 (q,
2JC,F ¼ 32 Hz, CeCF3), 131.1, 136.4, 137.3, 139.1, 143.0, 158.5, 163.6,
165.0, 182.3 (quat. C); C31H22F3N3O4 (557.52); calcd. C 66.78, H 3.98,
N 7.54; found C 66.62, H 3.94, N 7.42; HPLC-purity: 99.4% at 254 nm
and 99.4% at 280 nm, tN ¼ 5.24 min, tM ¼ 1.09 min (ACN/H2Oþ0.05%
HCOOH; 60:40), lmax: 272 nm and 327 nm.
d
(ppm) ¼ 2.90 (s, 3H, CH3), 4.27 (d, 2H, J ¼ 6.1 Hz, CH2), 7.29e7.32
(m, 2H, ar-H), 7.41 (d, 1H, J ¼ 7.5 Hz, ar-H), 7.50 (dd, 1H, J ¼ 7.5/
7.5 Hz, ar-H), 7.57e7.59 (m, 1H, ar-H), 7.65 (t, 1H, J ¼ 6.2 Hz, NH),
7.70 (d, 1H, J ¼ 7.8 Hz, ar-H), 7.76 (m, 1H, ar-H), 7.85e7.87 (m, 2H, ar-
H), 8.05e8.06 (m, 2H, ar-H), 8.24e8.26 (m, 1H, ar-H), 8.73 (d, 1H,
J ¼ 3.2 Hz, ar-H), 10.65 (s, 1H, NH), 10.76 (s, 1H, NH), 12.35 (d, 1H,
J
d
¼
2.9 Hz, indole-NH); 13C NMR (DMSO-d6, 150.9 MHz):
(ppm) ¼ 39.8 (CH3), 45.9 (CH2); 112.6, 121.1, 122.7, 123.6, 125.7,
126.1, 126.6 (2C), 127.4, 128.1 (2C), 129.1, 138.4 (tert. C); 112.5, 125.6,
131.0, 136.4, 138.9, 139.0, 143.2, 163.6, 165.0, 182.3 (quat. C);
C25H22N4O5S (490.53); calcd. C 61.21, H 4.52, N 11.42; found C 60.96,
H 4.56, N 11.47; HPLC-purity: 96.8% at 254 nm and 98.3% at 280 nm,
tN ¼ 3.81 min, tM ¼ 1.03 min (ACN/H2O þ 0.05% HCOOH; 40:60),
lmax: 272 nm and 327 nm.
5.1.8.15. N0-({30-[(4-tert-Butyl-2-methylphenoxy)methyl][1,100-
biphenyl]-4-yl}carbonyl)-2-(1H-indol-3-yl)-2-oxoacetohydrazide
(7o). Preparation according to General Procedure B. Reaction time:
3 h. Double recrystallization from ethanol 96% yielded yellow
powder (7%).

2768
S. Schmidt et al. / European Journal of Medicinal Chemistry 46 (2011) 2759e2769
5.2. Biological assays
absorbance at 515 nm is measured on a plate reader. The
percentage of growth is calculated using the different absorbance
measurements [time zero (Tz), control growth (C), and growth in
the presence of drug (Ti)]. The growth inhibition 50% (GI50) is the
concentration for a Ti value satisfying the equation [(TieTz)/
(CeTz)] ꢃ 100 ¼ 50.
5.2.1. In vitro kinase assays
A radiometric protein kinase assay (33PanQinaseÒ Activity
Assay) was used for measuring the activity of the protein kinases.
All assays were performed with a BeckmanCoulter/Sagian robotic
system and 96-well FlashPlatesTM from Perkin Elmer (Boston, MA,
Further details of the test method have been published
[26,34,35].
USA) in a 50
pipetted in 4 steps in the following order: 20
L of ATP solution (in H2O); 5 L of test compound (in 10%
DMSO); 10 L of substrate/10 L of enzyme solution (premixed).
The assay for all enzymes contained 60 mM HEPES-NaOH, pH 7.5,
m
L reaction volume. The reaction cocktail was
m
L of assay buffer;
5
m
m
5.3. Docking
m
m
Ligands were prepared for the docking using HyperChem 8.0.4
(Hypercube Inc., Gainesville, Florida) by energy minimization
(MMþ force field, RMS gradient 0.1 kcal/Å mol). Docking was
carried out using the docking engines ArgusDock (parameters: grid
resolution 0.4; ligand: flexible; docking precision: normal;
augmented root node: extend root node by 1 extra torsion) and
GADock (parameters: grid resolution 0.4; ligand: flexible, pop-
ulation size: 100, max. generations: 1000, mutation rate: 0.8;
crossover rate: 0.8; elitism: 5), as implemented in ArgusLab
(ArgusLab 4.0.1, Mark A. Thompson, Planaria Software LLC, Seattle,
WA, http://www.arguslab.com). ArgusLab requires a standard pdb-
file for both receptor and ligand as input. Atomic charges are
assigned internally by the program without involvement of the
user. The resulting docking poses were scored using AScore as
implemented in ArgusLab. Validation of the docking methods was
carried out by redocking the cocrystallized ligand. Visualization
was accomplished with Chimera 1.4 [36].
3 mM MgCl2, 3 mM MnCl2, 3
50 g/mL PEG20000, 1 M [
mM Na-orthovanadate, 1.2 mM DTT,
m
m
g-
33P]-ATP (approx. 5 ꢃ 105 cpm per
well). The following substrates were used [substrate amount in
square brackets given in ng/50 mL]: GSK3(14e27) [2000] for AKT1;
Poly(Glu,Tyr)4:1 [125] for FAK, IGF-1R; SRC, VEGF-R2; Poly(-
Glu,Tyr)4:1 [125] or Poly(Ala,Glu,Lys,Tyr)6:2.5:1 [125] for INS-R;
Poly(Glu,Tyr)4:1 [250] for AXL; RBER-CHKtide [2000] for ARK5
and PRK1; RBER-CHKtide [2000] or Casein [1000] for PLK1; pol-
y(Ala,Glu,Lys,Tyr)6:2:5:1 [125] for MET; p38-alpha-KA [2000] for
SAK. The reaction cocktails were incubated at 30 ꢀC for 80 min.
The reaction was stopped by adding 50
acid. Plates were aspirated and washed two times with 200
m
L of 2% (v/v) phosphoric
L of
m
0.9% (w/v) NaCl. Incorporation of 33P was determined with
a microplate scintillation counter (Microbeta Trilux, Wallac). For
calculation of IC50 values, data were generated for comparison
without enzyme in the presence of substrate (“low control”).
Moreover, data with enzyme but without inhibitor were generated
(“high control”). The difference between high and low control was
taken as 100% enzyme activity. The residual activities for each
concentration and the compound IC50 values were calculated
using Quattro Workflow V2.x (Quattro Research GmbH, Munich,
Germany; www.quattroresearch.com/). The fitting model for the
IC50 determinations was “Sigmoidal response (variable slope)”
with parameters “top” fixed at 100% and “bottom” at 0%. The
fitting method used was a least squares fit. In case of solubility
problems, outliers in the higher concentration range were
removed to generate more realistic sigmoidal curves for IC50
calculation. As parameter for assay quality, the Z0-factor [32] was
calculated for the low and high controls of each plate. Assay plates
were repeated in case the Z0-factor dropped below 0.4.
Acknowledgements
Funding by a grant of the European Commission (Contract No
LSHB-CT-2004-503467; to S.S., F.T., C.S., M.H.G.K., and C.K.) is
gratefully acknowledged. The authors thank the National Cancer
Institute for evaluating compound 7o in the NCI’s in vitro cell line
screening project (IVCLSP).
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