Robust synthesis of ortho-cyanoheteroarylamines, building blocks of ATP like
kinase inhibitors
Frigyes Wáczek1, 2*, Jenő Marosfalvi2, Zoltán Varga1, 2, Zsolt Székelyhidi1, 2, Péter Bánhegyi1, 2, Gyula Bencze1,
Richárd Schwáb1, Edit Szabó1, Beatrix Németh1, György Kéri1, 2,, László Őrfi1, 2, 3
1 Cooperative Research Center, Semmelweis University, Budapest 5, Pf. 131, H-1367 Hungary,
Tel/Fax: +36-1-3010613, E-mail: fwaczek@kkk.sote.hu
2 VICHEM Ltd., Herman O. u. 15, Budapest, H-1022 Hungary,
3 Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes E. u. 9, Budapest, H- 1092
Hungary
*Corresponding author
http://www.vichem.hu
Introduction:
The ATP binding sites of various enzymes are potential targets of enzyme inhibitory drugs,
there is an increasing demand for ATP-like compounds. The structures of ATP analog
inhibitors are mostly contain a condensed bicyclic heteroaryl moiety, within a pyrimidine ring
part seems to be crucial. For the construction of pyrimidine ring in condensed bicyclic
heteroaryl cores, the ortho-cyanoarylamines are very useful building blocks.1 In the present
paper we show an effective and cheap method for the preparation of hundreds of orthocyanoarylamines. We produced more than 150 commercially unavailable molecules as
proprietary intermediates which can serve as good starting points for the development of
potential drug candidates. Biological activity of several derivates are also described.
Cooperative Research Center
Semmelweis University
Budapest, Hungary
http://www.webio.hu/kkk
Published synthesis of the pyrazole building block and a simplified
method of it’s preparation via enoyl chloride intermedier
Synthesis of bioisosteric isoxazoles from common enoyl chloride
intermediers
Traxler et al.1 have published the synthesis of 4-arylamino-3-phenyl-1H-pyrazolo[3,4-d]pyrimidines, as ATP-like
kinase inhibitors (e.g. compound 2), via 5-amino–4-cyano–3-phenyl-1H-pyrazole key-intermediate 1.
(Scheme1.)
We have found enoyl chloride intermediers of general formula A are usful for the synthesis of two
aminoisoxazole carbonitrile isomers E and F (Scheme 3). 5-Aminoisoxazole-4-carbonitriles E, were obtained in
the reaction of A with hydroxylamine in aqueous solution at neutral pH. The pH was adjusted by addition of
adequate amount of NaOH, resulting deliberation of the free base, which is crucial in the first condensation step.
The amino group as nucleophylic functional group reacts with the enoyl chloride in the first step followed by a
fast ring-closure reaction yielding E without the chance of the isolation of intermediate B. Excess amount of
base causes deprotonation of the hydroxyl function in the hydroxylamine, which has stronger nucleophylic
property and reacts better with the enoyl chloride moiety of A, finally resulting the other isomer F. However the
equilibrium may be pushed towards the formation of isomer F by addition of two equivalent NaOH, it is not
suitable method for synthesis of pure product F, because about 20-30% of E isomer always contaminates the
final product.
Cl
N
HN
a, b, c
N
N
NH2
N
H
EGF-R PTK
inhibitor
IC50=30nm
N
N
H
N
R
2
1
N
N
N
N
N
R
Cl
An US patent
from 1974 reported the
application of hydroxyurea as protected
hydroxylamine in the reaction with enoyl
chlorides
resulting
3-aminoisoxazole-4carbonitriles of general formula F.6 The
amino moiety is blocked in the hydroxyurea,
preventing it’s reaction with enoyl chloride
in the first step and directed the reaction via
intermediers of general formula C. The
protecting group leaves easily so it does not
prevent the reaction of the amino moiety
with the nitrile in the ring-closure, resulting
F. The intermediate molecules of general
formula C have not been separated in this
case either. We produced more than 150
commercially unavailable molecules of
general formula D, E and F by the reported
method,
these molecules are unique
intermediates
for
drug
development
purposes. The antiproliferative effect of
some derivates were proven in vitro on A431
cells and the apoptotic effect of some
compounds were studied by flow cytometry
(FACS).
N
D
A
Scheme 1
(a) Triethyl orthoformate/100 oC (b) 3-Chloroaniline/ethanol/reflux (c) H2O/dioxane/reflux
N
The preparation of compound 1 was reported starting from 2-(methylsulfanyl-phenyl-methylene)malononitrile 9 and hydrazine hydrate with high yield.2 Although this reaction seems to be very practical for
preparation of 1, the intermedier 9 is accessible through many reaction steps and complicated intermediates
and it is finally derived from two simple starting materials: malononitrile and benzoyl chloride. (Scheme 2.)
During the preparation of 1 the overall yields calculated for both starting materials individually are: 27,7%
for 3 and 49.8% for 7 according to the literature 3-5
N
N
Br
a
Br
N
N
N
N
N
3
R
O
F
Scheme 3
N
S
d
Cl
O
S
h
N
N
7
8
N
e
N
i
NH2
N
Searching for new antiproliferative agents we have modified compound 2 using our building block set described
above. We have found taht the ring opened urea 12 and the isoxazolo-pyrimidine derivates of general formula
13 had antiproliferative effect in micromolar range on A431 cells.
6
S
9
Scheme 2
(a) Br2, KBr/water/5-10oC, (b) Cu/Benzene/reflux, (c) Iodosylbenzene /dichloromethane, (d) 2,4Bis(methylthio)-2,4-dithioxo-1,3-dithia-λ5,λ5-diphosphetan/ benzene/80oC, (e) Benzene/r.t.,
(f)N2H4*H2O/MeOH/reflux, (g) Pyridine/0oC, (h) POCl3/reflux (i) N2H4*H2O/MeOH/reflux.
Starting from the same simple molecules 3 and 7 we managed to prepare 1 through another reactive
intermediate -chlorobenzylidene malononitrile 11, which can be obtained in two reaction steps. Although 11
is referred as precursor of other heterocyclic molecules, it has not been published as intermediate for synthesis
of 5-amino–4-cyano–3-phenyl-1H-pyrazole 1 yet. This paper subjects our efforts for replacing intermedier 9
with the corresponding chlorobenzylidene intermedier 11 in the preparation of 1. This substitution of 2methylsulfanyl moiety with 2-chloro function resulted the increase of reactivity while we obtained compound
10 with comparable yield in a significantly shorter reaction time in the ring-closure reaction. Our method is
capable for synthesis of the molecule 1 via 11 from simple compounds 3 and 7 with an overall yield of 60% in 3
reaction steps. This method is independent from the processes and complicated intermediates of the reaction
pathway referred in the literature: bromination of 3, coupling of 4 by copper powder forming 5, epoxidation of
5 and thiation of 7 resulting 8.
N
O
N
N
H
N
F
N
H
F F
F
F
F
A431
cell
12
IC50=1.08 microM
R
HN
N
H
R
N
N
O
N
13 A431 cell
IC50=1-10 microM
Analysis of apoptosis induction based on propidium iodide staining
Flow cytometry was used with propidium iodide staining, which is simple and cheap method for
determination of membrane integrity and DNA content. A431 cells were treated with inhibitors for 24 hours
prior to analysis. The ratio of Sub G1 area provides information about apoptotic events.
DNA content
G1
Sub G1
Conclusion:
In this paper we reviewed the chemistry of enoyl chloride intermediates which make them
applicable as building blocks in the synthesis of biologically active compounds. We have
shown that the applied method is capable for the synthesis of three different types of
heteroaryl building blocks via common enoyl chloride intermediates. The process resulted the
products from moderate to good yields. Commercially available, cheap chemicals were used in
the synthesis like acyl chlorides, malononitrile and simple reagents. The elaborated synthesis
also suffers from some limitations. It cannot be applied in the synthesis of unsaturated (e.g.:
acryl methacryl) derivates. Although acryloyl and methacryloyl chlorides are commercially
available, polymerization during the first acylation reaction diminished the yield of these
derivates. The method is only limited by the feasibility of the acyl chlorides as startingmaterials. We found these ortho-cyanoarylamines useful as starting materials in the
synthesis of condensed heterocycles in the antiproliferative drug development field.
Cl
A431
epidermoid
carcinoma
cell
(overexpressing EGFR) were harvested in
standard cell-culture environment (37 °C, 5%
CO2, 10% FCS). Proliferation assays were
based on the Methylene Blue method.7 Cell
viability was assessed after 6 and 48 hours.
Efficacy (=low, non-specific toxicity + high
rate of apoptosis induction) was judged on
the basis of high early viability connected to
the maximum viability-loss at 48h.
f
N
1
Antiproliferative activity of the derivatives
N
N
NH
11
N
E
4
O
Cl
N
c
10
N
N
N
O
5 N
OH
N
C
N
R
O
N
B
N
g
O
R
O
N
b
N
N
R
N
N
N
G2
S
Most of the cells are healthy and
there is no sign of apoptotic events in
SubG1 in DMSO control after 24h.
51% of the events in positive control
(cycloheximide) are in SubG1 area
after 24h. Both compounds 12 and
13a show very strong apoptotic effect
reducing G1, S and G2 events below
20%.
Cycloheximide
Compound 12
Compound 13a
(10 microM/24h)
(10 microM/24h)
(10 microM/24h)
Marker Events % Ga ted
Al l
37 71 10 0.00
SubG1
M1
32 44
86 .02
Marke r Events % Gated
All
586 7 100 .00
SubG1
M1
469 1
79.96
Marke r Events % Gated
All
457 2 100 .00
SubG1
M1
239 6
52.41
References:
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Traxler, P. M.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.; Mett, H.; Buchdunger, E.; Meyer, T.; Mueller, M. and Furet, P.; J.
Med. Chem. 1997, 40, 3601-3616
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US 2794824
[4]
Pryor, W., A., Govindan, C., K.; J.; Amer. Chem. Soc. 1981, 103, 25, 7681-7682
[5]
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[6]
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[7]
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[1]
[2]
The authors gratefully acknowledge the contributions of Dr. Ildikó Szilágyi and Mr. István Varga in the structure verification and the chemical analysis of the compounds.