Tri- and tetrasubstituted pyrazole derivates: Regioisomerism switches activity from p38MAP kinase to important cancer kinases

Article abstract of DOI:10.1021/jm201391u

In the course of searching for new p38α MAP kinase inhibitors, we found that the regioisomeric switch from 3-(4-fluorophenyl)-4-(pyridin-4-yl)-1- (aryl)-1H-pyrazol-5-amine to 4-(4-fluorophenyl)-3-(pyridin-4-yl)-1-(aryl)-1H- pyrazol-5-amine led to an almos

Full text of DOI:10.1021/jm201391u

Brief Article
pubs.acs.org/jmc
Tri- and Tetrasubstituted Pyrazole Derivates: Regioisomerism
Switches Activity from p38MAP Kinase to Important Cancer Kinases
Bassam Abu Thaher,† Martina Arnsmann,‡ Frank Totzke,§ Jan E. Ehlert,§ Michael H. G. Kubbutat,§
Christoph Schachtele,§ Markus O. Zimmermann,∥ Pierre Koch,‡ Frank M. Boeckler,∥ and
̈
Stefan A. Laufer*,‡
†Faculty of Science, Chemistry Department, Islamic University of Gaza, Gaza Strip, Palestine
‡Chair Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls University of Tuebingen, Auf der Morgenstelle 8,
72076 Tuebingen, Germany
§ProQinase GmbH, Breisacher Strasse 117, 79106 Freiburg, Germany
∥Laboratory for Molecular Design and Pharmaceutical Biophysics, Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy,
Eberhard Karls University of Tuebingen, 72076 Tubingen, Germany
̈
S
* Supporting Information
ABSTRACT: In the course of searching for new p38α MAP kinase inhibitors, we found that the regioisomeric switch from
3-(4-fluorophenyl)-4-(pyridin-4-yl)-1-(aryl)-1H-pyrazol-5-amine to 4-(4-fluorophenyl)-3-(pyridin-4-yl)-1-(aryl)-1H-pyrazol-
5-amine led to an almost complete loss of p38α inhibition, but they showed activity against important cancer kinases.
Among the tested derivatives, 4-(4-fluorophenyl)-3-(pyridin-4-yl)-1-(2,4,6-trichlorophenyl)-1H-pyrazol-5-amine (6a) exhibited
the best activity, with IC50 in the nanomolar range against Src, B-Raf wt, B-Raf V600E, EGFRs, and VEGFR-2, making it a good
lead for novel anticancer programs.
based inhibitors.7 Both findings motivated us to aim for 1,3,4-
trisubstituted 5-aminopyrazoles.
INTRODUCTION
Pyrazoles represent a class of heterocyclic compounds of
■
Aminopyrazoles A (Figure 1) were reported by Minami et al.
significant importance and are considered as extremely versatile
as inhibitors of p38α MAP kinase;8 however, no biological data
on the regioisomer of A (switch of substituents in position 3
and 4) are known.
building blocks in organic chemistry.1,2 They are also an impor-
tant class of compounds in the pharmaceutical industry and
medicinal chemistry.3 However, only a few examples of 1,3,4-
trisubstituted pyrazoles are reported in the literature.4
Generally, the regioisomer A showed highly efficient inhi-
bition of p38α MAP kinase. Examination of pyrazole A showed
that it has the same skeleton as B of p38α MAP kinase,6 making
a binding mode of A similar to SB-like compounds very likely
(Figure 2a). In contrast, the regioisomeric switch of substi-
tuents in position 3 and 4 should cause issues in the B-like
interaction pattern (Figure 2b,c). To verify this hypothesis, a
series of B pyrazole analogues was designed. The interactions of
these new compounds with the hinge region and the hydro-
phobic region (Figure 2b) or the interactions of the central
aminopyrazole should be affected (Figure 2c) by the topology
of compounds 6. To prove this, we prepared compounds 6
(regioisomer of A) (Figure 1). As expected, the tested deriva-
tives of 6 were inactive against p38α MAP kinase (data not
shown). Likewise, docking of the compounds into different
structures of p38 yielded only low-scoring poses without hinge
binding.
In our attempt to investigate the role of the central five-
membered heterocyclic core of teardrop-binder based p38α
MAP kinase inhibitors such as SB2035805 (B) (Figure 1), we
Recently, Liu et al. reported moderate antitumor activity for
1-aryl-5-amino-4-pyrazolecarboxylate derivatives.9 These results
encouraged us to screen the synthesized series of compounds
6a−h against various kinases relevant in cancer.
Figure 1. Pyridinylimidazole B and pyridinyl substituted pyrazole A
and 6a−h (regioisomers of A, see Table 2).
switched from imidazole to oxazole, isoxazole, furan, and
pyrrole derivatives, as already described earlier.6 Furthermore,
we investigated the role of exocyclic amino groups in imidazole
Received: October 14, 2011
Published: December 19, 2011
© 2011 American Chemical Society
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Brief Article
Figure 2. (a) Schematic drawing of suggested relevant interactions between compound A and the ATP binding site of p38α. (b,c) Schematic
drawing of suggested interaction issues arising from compounds 6 (regioisomer of A). The schematic depiction is derived from Traxler et al.20 AB,
adenine binding region; SP, sugar pocket; PB, phosphate binding region; HR I, hydrophobic region I (also called selectivity pocket) adjacent to
Thr106; HR II, hydrophobic region II, where the cleft opens to the cytosol.
a
Scheme 1. Synthesis of Pyrazoles 6a−h and 7−9
RESULTS AND DISCUSSIONS
■
Chemistry. There are several reactions reported for the
preparation of pyrazole compounds. Among them, the Knorr
pyrazole synthesis is considered as one of the standard methods
due to its generality. The reaction leads to the pyrazole
derivatives via the condensation of substituted hydrazine with
1,3-dicarbonyl compounds or their derivatives.10−13 The other
method is the 1,3-dipolar cycloaddition of diazoalkanes or
nitrile imines with olefins or alkynes.11 Recently, a novel
regioselective synthesis of 1,3,4-trisubstituted pyrazoles has
been successfully employed.4
Some aminopyrazoles were prepared by reacting aryl hydrazines
with 3-methylthio or 3-methoxy-2-cyanoacrylate in ethanol in
the presence of sodium ethoxide under reflux.9,14 The other
method is via the reaction of hydrazonyl chlorides with
malononitrile in anhydrous ethanol in the presence of sodium
ethoxide at room temperature.14
The synthesis of the pyrazole compounds 6a−h and 7−9 is
outlined in Scheme 1. 4-Pyridine carbaldehyde (1) was reacted
with arylhydrazines 2a−h to furnish hydrazone derivates 3a−h,
followed by chlorination with N-chlorosuccinimide (NCS),
yielding the hydrazonyl chlorides 4a−h. To freshly prepared
LDA, 4-fluorophenylacetonitrile (5) was added, and then sub-
sequently hydrazonyl chlorides 4a−h were added to the
reaction mixture, giving the corresponding aminopyrazole deri-
vatives 6a−h. Aminopyrazole derivatives 7−9 were synthesized
from the reaction of the corresponding hydrazonyl chloride
4a,b with the respective acetonitrile derivatives 5i,j in dry
ethanol in the presence of sodium ethoxide.
The acid derivative of aminopyrazole 10 was obtained by
hydrolysis of its ester compound 8 using an aqueous KOH
solution, and it was decarboxylated under reflux conditions in a
solution of MeOH/conc HCl to give compound 11 (Scheme 2).
The target compounds 13a,h were prepared according to a
protocol of Deng et al. from the corresponding hydrazone
(3a,h) with trans-p-fluoro-ω-nitrostyrene (12) in the presence
of potassium tert-butoxide and TFA in dry THF (Scheme 3).4
Biological Studies. No inhibitory effect of compounds
6a−h, 7−11, and 13a,h was found using an isolated p38α
MAP kinase assay.15 This confirms the notion illustrated in
Figure 2 proposing that the switch of the substituents in
positions 3 and 4 of inhibitor A to regioisomer 6 should lead
to a dramatic loss of affinity for the p38α ATP binding site.
However, this simple regioisomeric exchange gives rise to
significant affinities toward other kinases. Evaluation of lead
compound 6a in 252 different kinases shows inhibition of the
a
Reagents and conditions: (i) EtOH, reflux temperature; (ii) dry
DMF, NCS; (iii) dry THF, LDA, −78 °C; (iv) dry EtOH, EtONa,
0 °C.
important cancer kinases Src, B-Raf, EGFR, and VEGFR-2
(Table 1). Parallel inhibition of different protein kinases is an
approach to increase the effectiveness of protein kinase
inhibitors in cancer therapy. This increase of effectiveness can
be due to the inhibition of two different cancer related
phenotypes such as proliferation (EGFR) and angiogenesis
(VEGFR-2).16 Parallel inhibition of B-Raf and Src can be
especially attractive for the treatment of melanoma, since both
kinases have been found to be constitutively active in this type
of cancer.17 Since activation of B-Raf in melanoma occurs
predominantly by a mutation of valine 600, it is even more
intriguing that 6a also inhibits B-Raf mutant V600E.
Structure−Activity Relationships and Interaction
Modeling. A focused library of 14 analogues of 6a was tested
side by side with 6a against a selected panel of three particularly
interesting cancer kinases, B-Raf V600E, Src, and VEGFR-2, to
elucidate structure−activity relationships (SAR) in this class of
5-aminopyrazoles (Table 2).
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 2. Decarboxylation of Compound 8
a
Reagents and conditions: (i) aqueous KOH; (ii) MeOH/conc HCl, reflux temperature.
a
Scheme 3. General Method for Pyrazole Synthesis (13a,h)
carbamoyl (7, 9), ethoxycarbonyl (8), carboxy (10), or hydro-
gen (11) leads to significant reduction or complete loss of
activity in all three kinases. As the 4-fluorophenyl ring is either
placed in the hydrophobic region I or II in the proposed
binding modes, this effect is easily comprehensible.
Absence of the amino group (R2) in compound 13a as com-
pared to compound 6a leads to a 5−10-fold reduction of
activity. In all kinases this amino function is directed toward an
aspartate of the sugar binding pocket (Asp594 in B-Raf V600E,
Asp404 in Src, or Asp1046 in VEGFR-2). Taking protein
flexibility into account, direct or water mediated hydrogen
bonds might occur. The electrostatic interaction between aro-
matic amine and aspartate is likely to be weakened by the adja-
cent aqueous environment. This can explain the moderate
decrease of activity. The same exchange of amine to hydrogen
in compound 13h shows similar effects in VEGFR-2 but only
small differences in B-Raf and Src when compared to 6h.
Pharmacological Impact of Inhibitory Profile. Given
the variability within and across tumor types, practical medicine
has adopted a multifaceted approach to tumor therapy encom-
passing surgery, radiation, cytotoxics, immune stimulation, and
combined noncytotoxic drug therapy. The rationale is clear:
tumor variability and propensity to resistance demands
variation of selection pressure. The drug discoverer can learn
from this that a drug with one or few targets conferring a tumor
suppressive effect will quickly select for resistance. Too many
targets will lead to limited tolerance, which will limit dose and,
thereby, effect.
Empirically driven cancer polypharmacology suggests that
activity on a suite of key targets may provide the twin virtues of
“cancer spectrum” and “efficacy”. Starting with spectrum, all
cancers rely on growth factors, however, not the same factors,
or their mutants, and to varying degrees. Thus, a substance that
is similarly potent on EGFR, VEGFR, PDGFR, and B-Raf will
exert broad pressure on growth regulatory pathways without
necessarily selecting for any one locus for mutation toward
resistance.19
Resistance prevention by multitarget effects is, of course,
circumvented by generalized resistance via efflux and meta-
bolism. Here, however, fortuitous polypharmacology can also
be beneficial in that binding to specific cell stress-related targets
also hinders the up-regulation of general defense proteins (e.g.,
MAPKs, ERKs). This, in turn leaves the tumor vulnerable to
cytotoxic substances and local immune reactivation if the kinase
inhibitory profile supports rather than inhibits a pro-TH-1 activa-
tion response.
a
Reagents and conditions: (i) dry THF, tert-BuONa, −78 °C, then
TFA.
To illustrate and rationalize this SAR information, we have
docked these ligands into crystal structures of all three kinases
using GOLD v3.2 (CCDC).18 The proposed binding modes
shown for lead compound 6a in Figure 3 represent top scoring
poses for this inhibitor that reflect trends in the measured IC50
values rather well.
When comparing inhibitory activities of compounds 6a−h
that comprise variations in substituent R1, remarkable differ-
ences become obvious between the three kinases. While 2,4,6-
trichlorophenyl (6a) is clearly superior to the unsubstituted
(6b) or 4-substituted phenyl (6c−h) moieties in VEGFR-2 and
Src showing activity differences of >35-fold and >17-fold,
respectively, this ratio is less pronounced in B-Raf V600E
(>1.4-fold). In VEGFR-2 this can be rationalized by the
orientation of the ligand placing the R1 substituent in the
hydrophobic region I, which is located between Thr916 and
Lys868 (see Figure 3c). This hydrophobic area is just large
enough to accommodate both chlorines in the ortho position,
as well as substituents in the para position of the phenyl ring.
Thus, loss of the chlorines in positions 2 and 6 of the phenyl
ring seems detrimental for the inhibitory activity, whereas
modifications of position 4 only have minor influence. In
contrast, in B-Raf V600E (Figure 3a) R1 seems to be placed
within the hydrophobic region II (HR II), which is located at
the entrance of the ATP binding site. Chlorine atoms in
positions 2 and 6 are not tightly bound within the rather
spacious HR II environment. Substituents in position 4 are
oriented toward the water environment of the cytoplasm. This
might explain why the inhibitory activity of compounds 6a−h
on B-Raf V600E is barely affected by modifications in the
substitution pattern of the phenyl ring. For Src the measured
SAR is difficult to explain from the predicted binding mode.
Exchange of the 4-fluorophenyl substitution (R3) by smaller,
nonaromatic, and more hydrophilic substituents such as
Table 1. Inhibitory Profile of 6a against Important Cancer Kinases and Their Mutants
IC50 (μM)
a
a
b
c
a
b
VEGFR-2
0.034 0.003
Src
B-Raf wt
0.27
B-Raf V600E
0.592 0.154
EGFR wt
0.113 0.052
EGFR L858R
0.031
0.399 0.082
a
b
c
Mean SEM of three experiments. Value of one experiment. Mean SEM of five experiments.
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Journal of Medicinal Chemistry
Brief Article
Table 2. Biological Data of Compounds 6a−h, 7−11, and 13a,h
IC50 (μM)
compd
R1
R2
R3
B-Raf V600E
Src
VEGFR-2
6a
6b
6c
6d
6e
6f
2,4,6-trichloro
4-H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
CONH2
CO2C2H5
CONH2
COOH
H
0.59
0.90
0.80
0.86
1.4
0.40
52
0.034
2.4
4-Cl
23
1.2
4-OCH3
4-NO2
23
1.9
82
1.4
4-CH3
0.89
1.7
53
2.6
6g
6h
7
4-CN
>100
6.7
1.3
4-CF3
1.5
2.4
2,4,6-trichloro
2,4,6-trichloro
4-H
>100
>100
64
6.4
31
8
>100
19
>100
48
9
10
11
13a
13h
2,4,6-trichloro
2,4,6-trichloro
2,4,6-trichloro
4-CF3
>100
>100
2.5
55
43
>100
2.0
87
4-FC6H4
4-FC6H4
0.21
27
H
2.7
12
Figure 3. Proposed models of interaction between 6a and the ATP binding site of B-Raf (a), Src (b), and VEGFR-2 (c). Binding modes are
suggested on the basis of docking results using GOLD v3.2 (CCDC). The best scoring poses were carefully checked for consistency with the
observed SAR data.
employing a gradient of 0.01 M KH2PO4 (pH 2.3) and methanol as
the solvent system with a flow rate of 1.5 mL/min. All final co-
mpounds have a purity of >95%.
Taking this line, 6a represents an excellent starting point for
a phenomenological lead kinase inhibitor optimization built
upon a desirable balance of effects rather than excessive faith in
a specific screenable target.
General Procedure for Synthesis of Amino Pyrazole
Derivatives (6a−h). Twenty mmol of LDA was added to dry THF
(30 mL) in a three neck flask and cooled to −78 °C. Fourteen mmol
of 4-fluorophenyl acetonitrile (5) dissolved in THF (10 mL) was
added dropwise, and the reaction mixture was stirred for 45 min. Five
mmol of the appropriate hydrazonyl chloride 4a−h (neat or dissolved
in THF) was added slowly to the reaction. After about 1.0 h, the
reaction was completed, and the reaction mixture was warmed to room
temperature. Water (50 mL), followed by ethyl acetate (50 mL), was
added to the reaction mixture, and the organic phase was separated.
The aqueous phase was extracted with ethyl acetate (50 mL), and the
combined organic layer was dried over Na2SO4. The solvent was
removed under reduced pressure to about 5 mL and left overnight, and
the product precipitated from the solution. The respective product was
filtered off, washed with diethyl ether and/or petroleum ether, and
dried. In case the product did not precipitate, the residue was purified
by flash chromatography (petroleum ether/ethyl acetate) to yield a
pure solid.
CONCLUSION
■
In summary, regioisomeric switch completely changed the
inhibitory profile of 4-(4-fluorophenyl)-3-(pyridin-4-yl)-1-
(aryl)-1H-pyrazol-5-amines from p38α MAPK to important
cancer kinases, opening a new avenue for structurally novel
leads in cancer research. Further studies in cellular systems have
to prove the potential benefits of this kinase profile.
EXPERIMENTAL SECTION
■
General. All commercially available reagents and solvents were
used without further purification. NMR data were recorded on a
Bruker Spectrospin AC200 or on a Bruker Avance 400 at room
temperature. Chemical shifts are reported in ppm relative to the
solvent resonance. The purity of the final compounds was determined
by HPLC on a Hewlett-Packard HP 1090 series II liquid chro-
matography using a Betasil C8 column (150 mm × 4.6 mm i.d., dp =
5 μm, Thermo Fisher Scientific, Waltham, MA) at 230 and 254 nm,
4-(4-Fluorophenyl)-3-(pyridin-4-yl)-1-(2,4,6-trichlorophen-
yl)-1H-pyrazol-5-amine (6a). Yield: 35%; pale brown solid;
1
mp 215 °C; H NMR (200 MHz, DMSO-d6) δ 5.53 (s, br, 2H, NH2),
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7.12−7.67 (m, 6H), 7.93 (s, 2H), 8.45 (d, J = 6 Hz, 2H); 13C NMR (50
MHz, DMSO-d6) δ 99.7, 116.1, 122.0, 129.2, 129.3, 132.0, 133.1, 135.9,
136.2, 141.2, 147.2, 147.5, 149.7, 161.5; IR (ATR) 3451, 3293, 3164
(NH2), 1639 (CN), 1604, 1573, 1552, 1519 (aromatic rings), 1466,
1212, 972,833 cm−1; EI-HRMS: calcd for C20H12Cl3FN4 434.0112, obsd
434.0058.
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ASSOCIATED CONTENT
* Supporting Information
■
(13) Peruncheralathan, S.; Ehan, T. A.; Ila, H.; Junjappa, H.
Regioselective synthesis of 1-aryl-3,4-substituted/annulated-5-
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nonradioactive immunosorbent assay for p38 alpha mitogen-activated
protein kinase activity. Anal. Biochem. 2010, 406, 233−234.
(16) Herbst, R. S.; Johnson, D. H.; Mininberg, E.; Carbone, D. P.;
Henderson, T.; Kim, E. S.; Blumenschein, G.; Lee, J. J.; Liu, D. D.;
Truong, M. T.; Hong, W. K.; Tran, H.; Tsao, A.; Xie, D.; Ramies, D.
A.; Mass, R.; Seshagiri, S.; Eberhard, D. A.; Kelley, S. K.; Sandler, A.
Phase I/II trial evaluating the anti-vascular endothelial growth factor
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S
Computational methods, biochemical protein kinase assays, as
well as experimental procedures and analytical data for all novel
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +497071-2972459. Fax: +497071-295037. E-mail:
stefan.laufer@uni-tuebingen.de.
■
ACKNOWLEDGMENTS
B.A.T. thanks the Alexander von Humboldt-Foundation (AvH)
for funding.
■
ABBREVIATIONS USED
■
MAP, mitogen-activated protein; Src, sarcoma; B-Raf, serine/
threonine-protein kinase; EGFR, epidermal growth factor
receptor; VEGFR, vascular endothelial growth factor receptor;
NCS, N-chlorosuccinimide; LDA, lithium diisopropylamide;
TFA, trifluoroacetic acid; ATP, adenosine triphosphate; SAR,
structure−activity relationship; HR, hydrophobic region; ERKs,
extracellular-signal-regulated kinases; AB, adenine binding
region; SP, sugar pocket; PB, phosphate binding region
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