Development of first lead structures for phosphoinositide 3-kinase-c2γ inhibitors

Article abstract of DOI:10.1021/jm5006034

The importance of complete elucidation of the biological functions of phosphoinositide 3-kinases (PI3K) was realized years ago. They generate 3-phosphoinositides, which are known to function as important second messengers in many inter- and intracellular signaling pathways. However, the functional role of class II PI3Ks is still unclear. Herein, we describe the synthesis of a panel of compounds that were tested against all eight mammalian PI3K-isoforms. We found inhibitors with some selectivity for class II PI3K-C2γ and also compounds with preferred inhibition of class II PI3K-C2β, providing structural leads to develop selective tool compounds.

Full text of DOI:10.1021/jm5006034

Article
pubs.acs.org/jmc
Development of First Lead Structures for Phosphoinositide 3‑Kinase-
C2γ Inhibitors
Anne Freitag,† Prajwal Prajwal,‡ Aliaksei Shymanets,‡ Christian Harteneck,‡ Bernd Nurnberg,‡
̈
Christoph Schachtele,§ Michael Kubbutat,§ Frank Totzke,§ and Stefan A. Laufer*,†
̈
†Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls University Tuebingen, Auf der
Morgenstelle 8, 72076 Tuebingen, Germany
‡Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology,
Eberhard Karls University Tuebingen, 72074 Tuebingen, Germany
§ProQinase GmbH, Breisacher Strasse 117, 79106 Freiburg, Germany
S
* Supporting Information
ABSTRACT: The importance of complete elucidation of the biological
functions of phosphoinositide 3-kinases (PI3K) was realized years ago.
They generate 3-phosphoinositides, which are known to function as
important second messengers in many inter- and intracellular signaling
pathways. However, the functional role of class II PI3Ks is still unclear.
Herein, we describe the synthesis of a panel of compounds that were
tested against all eight mammalian PI3K-isoforms. We found inhibitors
with some selectivity for class II PI3K-C2γ and also compounds with
preferred inhibition of class II PI3K-C2β, providing structural leads to
develop selective tool compounds.
molecule inhibitors are already being investigated in clinical
trials.7
INTRODUCTION
■
Phosphoinositide 3-kinases (PI3K) are lipid kinases phosphor-
ylating phosphoinositides at the 3′-OH position of the inositol
ring, giving rise to PtdIns(3)P, PtdIns(3,4)P2, and PtdIns-
(3,4,5)P3.1 These lipids are known to function as second
messengers and contribute to signaling pathways involved in
cell growth, proliferation, survival, metabolism, cytoskeletal
rearrangement, and vesicle trafficking.2 There are eight different
isoforms of these lipid kinases in mammals, which are divided
into three classes depending on their sequence homology and
substrate specifity.3 Of these, class I has been the most
commonly investigated.
Members of class I exist as heterodimers containing a
catalytic and a regulatory subunit. Class I is further subdivided
into class IA (α-, β-, and δ-isoform) based on their catalytic
subunits (p110α, -β and -δ) and class IB (γ-isoform, p110γ).
The catalytic subunits of class IA PI3Ks form heterodimers with
noncatalytic regulatory p85-subunits, and the sole class IB
isoform PI3Kγ associates with one of the two noncatalytic
subunits p87 (also known as p84) or p101.4 Their preferred
substrate in vivo is PtdIns(4,5)P2, generating the second
messenger PtdIns(3,4,5)P3 (also known as PIP3).3 Distribution
of PI3Kα and -β is almost ubiquitous,5 whereas PI3Kγ and -δ
are mainly expressed in leukocytes.5,6 Because PI3Ks are
involved in tumorigenesis and inflammatory processes, their
inhibition has been established as a target of study and small
The only class III member, Vps34 (vacuolar protein sorting),
was originally discovered in Saccharomyces cerevisiae, but it is
conserved from yeast and plants to mammals.8 It exists as a
constitutive heterodimer with Vps15, and the expression of this
enzyme seems to be ubiquitous.9 Its substrate in vitro and in
vivo is PtdIns. The main functions of the kinase known to date
all concern endosomal trafficking.
Class II is the least understood class of PI3Ks. This class
consists of three isoforms named PI3K-C2α, -C2β, and -C2γ,
which were discovered only by PCR and not by cellular
function.10−12 In contrast to the other PI3Ks, these kinases
exist as monomers, with PI3K-C2α and -C2β being almost
ubiquitously expressed13 and PI3K-C2γ having a much more
restrictive expression pattern. High levels have been found in
liver, breast, prostate, and salivary glands.9 They are able to
phosphorylate PtdIns and PtdIns(4)P in vitro, but in vivo there
is a strong preference for PtdIns.2 In the past few years,
researchers have gained some insight into the physiological
roles of PI3K-C2α and -C2β, revealing their involvement in
some disease-related processes. The two isoforms have
responded to cytokines like EGF and PDGF14 as well as
Special Issue: New Frontiers in Kinases
Received: April 17, 2014
© XXXX American Chemical Society
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insulin,15 integrin,16 and chemokine MCP-1.17 PI3K-C2β
overexpression was recently shown to participate to increased
PKB activity in cancer malignancies like acute myeloid leukemia
(AML), glioblastoma multiforme, and small-cell lung cancer
(SCLC).18 Furthermore, a role for PI3K-C2γ in hepatitis C
virus replication has been discovered,19 showing that these
kinases are potential drug targets. Even though the expression
of PI3K-C2γ is restricted to only a few tissues, its biological role
remains unclear. Besides genetic knockout-experiments,
selective inhibitors have proved to be useful pharmacological
tools for the investigation of the biological function of an
enzyme. In contrast to class I PI3Ks, no isoform-specific
inhibitors for class II PI3Ks have been reported.7 The lack of
crystal structures of these kinases represents a handicap in
designing new potential inhibitors. Herein, we describe the
synthesis of a panel of compounds and the results of an initial
testing against all eight mammalian PI3Ks, revealing promising
new approaches for PI3K research.
Scheme 1. Direct Two-Step Approach to Build the Desired
New Core-Structure Was Not Successful
A general synthetic approach has been established beginning
with commercially available 2,3-dichloropyrazine and the
corresponding aniline derivatives. In Scheme 3, the established
synthetic route is represented by the example of the synthesis
of compound 2. Because pyrazines are quite electron deficient,
nucleophilic substitution of chlorine is possible under basic
conditions. Here, 2-chloro-5-methoxyaniline was activated with
the strong non-nucleophilic base Na-HMDS. Fortunately, no
second substitution of 2a was observed, probably because the
resulting product was not sufficiently electron deficient for a
second attack of the nucleophile. This result is in accordance
with the failure of direct reaction of the aniline derivatives with
2-amino-3-chloro-pyrazine (Scheme 1). In the next step, the
remaining chlorine has to be converted into an amine for the
subsequent reaction with sulfonyl chloride derivatives. Any kind
of direct substitution with an N-nucleophile failed, so we made
a detour via the corresponding azides, which are known to be
susceptible for reductions and form the corresponding amines.
Compound 2a was therefore heated with sodium azide in DMF
to replace the chlorine by an azide function.23 Interestingly,
infrared spectroscopy showed no typical azide absorption and
NMR spectroscopy confirmed the formation of a tetrazolopyr-
azine 2b (Scheme 2). Azidopyrazines isomerize to ring-closed
tetrazolopyrazines depending on substituents, solvent, and
temperature.24
CHEMISTRY
■
Starting from a core structure of a known pan-class I-inhibitor
(1, XL147, displayed IC50 values in the range of 23−383 nM for
class I PI3Ks20), we pursued different strategies (Chart 1).
Chart 1. Three Series of Compounds Were Synthesized
Derived from a Reduced Core-Structure of 1 (XL147)21,22
Scheme 2. Azidopyrazines Exist at Equilibrium with the
Isomeric Ring-Closed Tetrazolopyrazines
Docking studies with known crystal structures of class I PI3Ks
showed no interaction of chinoxaline with the kinase, so the
core was reduced to the pyrazine. We synthesized a series of
compounds with high similarity to the lead compound by
varying the substituent at the sulfonamide (series 1).
Furthermore, we varied the substituents at the 3-methoxyani-
line in different combinations with substituents at the
benzenesulfonamide (series 2), and for series 3, we varied
substituents at the pyrazin-2-ylamine.
Tetrazolopyrazines are very stable, so subsequent reduction
needed more rigorous conditions than expected. We finally
succeeded in formation of 2c by refluxing the reaction mixture
with Sn(II)Cl2 in concentrated HCl. These conditions were
used for all chloro-substituted tetrazolopyrazines because
catalytic reduction with Pd/C and H2 caused the cleavage of
the chlorine as side reaction. For all tetrazolopyrazines not
comprising an aromatic chloro-substituent, the catalytical
reduction with palladium on activated carbon with H2 at
elevated temperature was the method of choice.
Because a direct two-step synthesis starting from 2-amino-3-
chloro-pyrazine was not successful (Scheme 1), we developed a
new synthetic strategy.
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a
Scheme 3. Synthesis of Compound 2 as an Example of the Established Synthetic Route
a
Reactands and conditions: (a) Na-HMDS, THF, 0 °C → room temperature; (b) NaN3, DMF, 100 °C; (c) Sn(II)Cl2 in concd HCl, reflux; (d)
pyridine, room temperature.
Scheme 4. Amidation of Compound 5 with N-Carbobenzyloxy-2-methylalanine with Subsequent Deprotection Led to
a
Compound 7
a
Reactands and conditions: (a) DCC in dry DCM, room temperature; (b) Pd/C, H2 in ethyl acetate, room temperature, 3 h.
Subsequently, construction of the sulfonamide functionality
could be performed by stirring 2c with the desired sulfonyl
chloride in pyridine at room temperature.
The benzenesulfonamides with an amino- or an acetamide
substituent in m-position (5, 11, 16, 21, and 26 or 6, 12, 17, 22,
and 27, respectively) were prepared by reducing the
corresponding nitro-substituted compounds (4, 10, 15, 20,
and 25) and acetylating them with acetic anhydride.
Compound 7 was prepared by amidation of compound 5.
Therefore, the amino-function of 2-methylalanine was
protected and subsequently the carboxylic acid had to be
activated. Amidation with N-Boc-protected 2-methylalanine
could not be achieved, neither with DCC nor with CDI or as
acid chloride. So we used N-carbobenzyloxy-2-methylalanine
instead, and amidation was successful after activation with
DCC. Deprotection could be achieved by hydrogenation with
Pd/C and H2 (Scheme 4).
Methyl 3-(chlorosulfonyl)benzoate was needed as precursor
for methyl carboxylate compounds 8, 13, 18, 23, and 28 and
carboxylic acid compounds 9, 14, 19, 24, and 29. Therefore, 3-
(chlorosulfonyl)benzoic acid was activated with oxalyl chloride
to increase electrophilicity of the carbonyl compared to that of
sulfonyl chloride. After a short reaction time with methanol of
15 min the reaction was quenched, so selective esterification of
the carboxylic function was achieved without formation of
methyl sulfonate. The corresponding carboxylic acids were
subsequently prepared by alkaline hydrolysis.
Preparation of compound 30 was achieved by demethylation
of compound 2 with BBr3 in a nearly quantitative manner.
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a25−27
Scheme 5. Curtius Rearrangement and Alcoholysis of 41a Leads to N-Boc Protected Compound 41b
a
Reactands and conditions: triethylamine (TEA) and diphenylphosphoryl azide (DPPA) in toluene, 75 °C, 1 h, tert-butanol, reflux, 2 h.
Some aniline derivatives were not commercially available.
The precursor of compounds 36 and 37 was prepared by
protecting 3-nitroaniline with di-tert-butyldicarbonate and
subsequent reduction of the nitro functionality. With this,
intermediate synthesis was carried out as described in Scheme
3. Cleavage of the N-Boc protection group succeeded
quantitatively using trifluoroacetic acid.
The precursor of compounds 41 and 42 was synthesized
starting with 2-amino-isonicotinic acid. First, the amino
function was protected by acetylation with acetic anhydride.
Then the carboxylic function was converted to a Boc-protected
amino function by Curtius rearrangement (Scheme 5): The
acid was deprotonated with triethylamine and activated with
diphenylphosphoryl azide. By nucleophilic attack of the azide
anion, a carbonyl azide was formed, which separates N2 at
elevated temperatures and rearranges to an isocyanate.
Alcoholysis of isocyanates with tert-butanol leads to N-Boc-
protected amines which can be easily purificated by silica flash
chromatography. The following cleavage of the protection
group was achieved quantitatively by using trifluoroacetic acid.
Compounds 41 and 42 were subsequently prepared according
to Scheme 3.
None of the shown compounds inhibited class III PI3K
Vps34 or class II PI3K-C2α. Only a few compounds showed
slight effects on the activity of class IA PI3K associated with the
noncatalytic p85 subunits and monomeric class IB PI3Kγ.
As mentioned before, class IB PI3Kγ exists as one of the two
heterodimers p101/p110γ or p87/p110γ in vivo. Recent results
indicated that these dimers showed unequal conformational
alterations of several regions within the catalytic p110γ subunit
and/or lipids.28 On the basis of this background, we also tested
the compounds against the activity of the physiologically
relevant heterodimeric PI3Kγ enzymes. With this approach, we
identified several other compounds with inhibitory effects
against the kinase as compared to the data obtained from the
monomeric enzyme. Nevertheless, the inhibitory effects were
not impressive as compared to inhibition of other isoforms.
The results of these tests are available in the Supporting
Information.
Interestingly, the compounds with the new scaffold
preferentially inhibit the nearly unexplored isoforms PI3K-
C2γ and to some extent PI3K-C2β.
With series 1 candidates, we examined the influence of
substitution at the benzenesulfonamide (Table 1). Surprisingly,
the closest derivative to 1 (XL147), compound 7, showed no
inhibition of class I PI3Ks. Rather, 7 slightly inhibited PI3K-
C2γ. Because there is no data available for selectivity of XL147
beyond class I PI3Ks, we do not know whether this reduced
scaffold also improves inhibition of C2γ. Compounds 2 (R1 =
−H) and 6 (R1 = −acetamide) showed an inhibition similar to
7, so the presence of a free amine in the side chain appears not
to favor further interactions with the kinase. Notably, the best
inhibition was achieved by the compounds with amine- (5) and
carboxylic acid residue (9). These compounds are quite
different in nature concerning their interaction; perhaps the
RESULTS AND DISCUSSION
■
Selectivity Screening. The compounds were screened in a
nonradiometric ADP Glo Assay against eight PI3K isoforms by
singlicate measurement at the concentration of 10 μM to obtain
preliminary information about potency and selectivity. Assay
conditions and performance of the screening are specified in
the Supporting Information.
Results of the three series of compounds are shown in Tables
1, 2, and 3.
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a
Table 1. Series 1, Varying Substituents at the m-Position of the Benzensulfonamide
a
Residual activity of class II PI3K-C2γ and -C2β is given in %; 25−45% is indicated in yellow, 45−70% in red. Testing was performed at a
concentration of 10 μM by singlicate measurement.
residue is exposed to the solvent and hence improves inhibition
potency to some extent. These two compounds also showed
some slight effects on class IA PI3Ks α and δ.
residues at pyrazine-2-ylamine seemed to have a greater effect
on potency of the inhibitors.
We therefore prepared a panel of compounds with broader
variation at pyrazine-2-ylamine (series 3), and the residual
kinase activity after treatment with 10 μM of the inhibitors is
shown in Table 3.
Here, the importance of an H-bond-acceptor in the
appropriate position is evident. All compounds lacking an H-
bond-acceptor showed no inhibition of any tested kinase. To
our surprise, demethylation of 2 greatly improved inhibition of
C2β, while inhibition of C2γ remained unaffected. The
compound had no effects on any other isoform. Compounds
38 and 40 also showed a preference for C2β over C2γ.
Compound 38 does not affect any other kinase, while
compound 40 slightly inhibits class IA PI3Kβ. Compound 41
contained a typical hinge-binder motif, and indeed it was the
most nonselective compound in our screening, as it displayed
equal potency against C2β and C2γ and furthermore inhibited
class IB PI3Kγ (displaying remaining kinase activity of
approximately 40%).
In series 2, several substituents at the benzenesulfonamide
were combined with different 3-methoxyphenyl-substituents at
the pyrazine-2-ylamine. The results of inhibition studies at 10
μM (Table 2) showed a significant difference in inhibition
potency against PI3K-C2γ between the distinct 3-methox-
yphenyl-derivatives. The mean inhibition of the 3-methox-
yphenyl-compounds (10−14) of series 2 and the 2-chloro-5-
methoxyphenyl-derivatives of series 1 were comparable, while
5-methoxy-2-methylphenyl-substituted compounds (20−24)
showed no inhibition in any of the tested kinases. This
indicates that the substituents in the o-position strongly affected
binding to the kinase, probably due to the influence on rotation
of the residue. Interestingly, 3,5-dimethoxyphenyl-substitutents
(25−29) were the most potent inhibitors of PI3K-C2γ in this
series; probably a further interaction can be formed.
Compounds 27 and 28 also inhibit class IA PI3Ks to some
extent, but compounds 25 and 26 seem to inhibit PI3K-C2γ in
a selective way.
Eleven compounds were further tested against a panel of 60
protein kinases. Only 30, 38, and 40 showed slight inhibition of
p38α, but no additional protein kinases were inhibited.
Complete testing results are available in the Supporting
Information.
Methyl carboxylate substitution at the benzenesulfonamide
was not tolerated, suggesting that binding of these compounds
is sterically hindered. Here also no concrete correlation of the
properties of substituents and inhibition effect can be seen;
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a
Table 2. Testing Results of Series 2, Combination of Residues at Position 2 of Pyrazine (R2) and Benzenesulfonamide (R1)
a
Residual activity of the kinases up to 25% is marked in green, 25−45% in yellow, 45−70% in red. Testing was performed at an inhibitor
concentration of 10 μM by singlicate measurement.
Dose-Dependent Inhibition of PI3K-C2β and -C2γ.
Four compounds of this first set were additionally tested as
dose-dependent on the inhibition of the two isoforms PI3K-
C2β and -C2γ. The IC50 values were determined in a
nonradiometric ADP Glo Assay at 10 concentrations from
100 μM to 3 nM by singlicate measurements (Table 4). Assay
conditions and performance of the screening as well as the raw
data are specified in the Supporting Information.
Compounds 17 and 19 showed more than 10-fold selectivity
for PI3K-C2γ over -C2β (Table 4). The IC50 values against
PI3K-C2γ are in the range of 3.5 μM, whereas PI3K-C2β was
inhibited with an IC50 of 44.8 or 61.2 μM, respectively. The
inhibition profile of compound 26 revealed an IC50 of 0.34 μM
against PI3K-C2γ with about 80-fold selectivity over PI3K-C2β
(IC50 28.8 μM, Scheme 6).
Compound 30 showed preferred inhibition the PI3K-C2β
isoform in the selectivity screening. Indeed determination of
the IC50 confirmed 10-fold selectivity over PI3K-C2γ (IC50 of
2.71 or 23.2 μM, respectively).
CONCLUSION
■
We established a general synthetic route to obtain a panel of
compounds. Three series of compounds were synthesized and
screened against all eight mammalian isoforms of PI3Ks. We
found several compounds which show 60−80% inhibition of
PI3K-C2γ activity at 10 μM, whereas they did not affect any of
the other tested protein kinases. The most promising
compound 26 showed an IC50 of 0.34 μM with over 80-fold
selectivity over PI3K-C2β.
Furthermore, we found three compounds with preferred
inhibition for PI3K-C2β (30, 38, and 40). Here, 10-fold
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a
Table 3. Testing Results of Compounds of Series 3, Varying Substituents at Position 2 of the Pyrazine
a
Residual activity of the kinases up to 25% is marked in green, 25−45% in yellow, and 45−70% in red. Testing was performed at an inhibitor
concentration of 10 μM by singlicate measurement.
selectivity of PI3K-C2β over -C2γ could be confirmed with IC50
values of 2.71 and 23.2 μM, respectively. Because these three
compounds also showed side effects on a protein kinase
(p38α), we can assume that the binding pockets of these two
different types of kinases show similarities.
pyrazin-2-yl-residue have an influence on inhibitor potency,
probably due to rotation effects.
Even with limited test data available, the results may provide
a lead to the development of the first selective compounds for
further investigation of the biological roles of class II PI3K-C2γ.
No crystal structure of class II PI3Ks is available to date, so
interactions of the compounds with the kinase can only be
assumed. Reducing the scaffold from quinoxaline to pyrazine
reduced potency of class I inhibitors. Polar substituents at the
m-position of the benzenesulfonamide moiety slightly improved
inhibition potency for PI3K-C2γ, but no direct interaction
seems to be formed. An appropriate H-bond acceptor at the
phenyl residue at pyrazine-2-ylamine is essential for inhibition.
For PI3K-C2γ selectivity, the 3-methoxyphenyl substitution is
crucial, and substituents in position 2 of the phenylamino-
EXPERIMENTAL SECTION
■
All reagents and (anhydrous) solvents are commercially available and
were used without further purification. Purification was performed on
normal phase silica using Davisil LC60A 20−45 μm silica from Grace
for the column, and separation was conducted by an automated flash
chromatography system (LaFlash). 1H spectra were obtained with
Bruker Avance 200 at 200 MHz or with Bruker 400 Avance at 400
MHz, respectively. The spectra were obtained in the indicated solvent
and calibrated against the residual proton peak of the deuterated
solvent. Chemical shifts (δ) are reported in parts per million relative to
G
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a
Table 4. IC50 Values of Four Compounds against PI3K-C2β and -C2γ
a
Inhibitions curves were determined at 10 concentrations in semilog steps from 100 μM to 3 nM by singlicate measurement. IC50 values up to 5 μM
are marked in green, from 5 to 25 μM in yellow, and from 25 to 50 μM in red.
1H), 7.50 (s, 1H), 7.84 (s, 1H), 8.47 (s, 1H). GC-MS (m/z): 269
[M+].
Scheme 6. Dose-Dependent Inhibition Curves of Compound
26
N-(2-Chloro-5-methoxyphenyl-)tetrazolo[1,5-a]pyrazin-8-
amine (intermediate 2b). Intermediate 2a (1 equiv) and sodium
azide (2.5 equiv) were dissolved in DMF and heated to 100 °C
overnight. After cooling to room temperature, the reaction mixture
was poured into ice−water, resulting in precipitation of the product,
which could be filtered and dried. Without further purification, 93% of
1
compound 2b were obtained. H NMR (400 MHz, DMSO-d6): δ =
3.78 (s, 3H), 6.91 (d, 1H, J = 8.8 Hz), 7.44 (s, 1H), 7.48 (d, 1H, J =
8.8 Hz), 7.76 (d, 1H), 8.69 (d, 1H), 10.05 (s, 1H). EI-MS (m/z): 276
[M+].
N-(2-Chloro-5-methoxyphenyl-)pyrazin-2,3-diamine (inter-
mediate 2c). Intermediate 2b (1 equiv) was dissolved in
concentrated HCl and refluxed after addition of 10 equiv Sn(II)Cl2
for 3 h. The mixture had to be cooled to room temperature, diluted
with water, and then neutralized with K2CO3. Extraction with DCM
(until there was no further product extracted) and evaporation of the
1
solvent yielded 87% of compound 2c with sufficient purity. H NMR
(400 MHz, CDCl3): δ = 3.72 (s, 3H), 4.51 (s, 2H), 6.43 (d, 1H, J =
8.4 Hz), 6.65 (s, 1H), 7.19 (d, 1H, J = 8.4 Hz), 7.58 (s, 1H), 7.62 (s,
1H), 7.69 (s, 1H). GC-MS (m/z): 250 [M+].
tetramethylsilane. GC-MS analyses were carried out on a Hewlett-
Packard HP 6890 series GC-system equipped with a HP-5MS capillary
column (0.25 μm film thickness, 30 m × 250 μm) and a HP 5973 mass
selective detector (electron ionization). Helium was used as carrier gas.
EI-MS data were obtained using a Finnigan Sector Field mass
spectrometer. FAB-MS, Finnigan MAT TSQ 70; matrix, 3-nitrobenzyl
alcohol.
Purity of the tested compounds was confirmed by reverse phase
HPLC on Merck Hitachi. Separation was conducted with a
Lichrospher RP18 column. Detection: Merck Hitachi L-4250 UV−
vis detector at 254 nm. The method used had a flow of 1 mL/min and
a gradient of 0.01 M KH2PO4, pH 2.3, and methanol from 60/40 to
15/85. All shown compounds have a purity >95% determined by
HPLC.
N-{3-[(2-Chloro-5-methoxyphenyl)amino]pyrazin-2-yl}-
benzenesulfonamide (2). Intermediate 2c (1 equiv) and
benzenesulfonyl chloride (3 equiv) were dissolved in dry pyridine,
and the reaction mixture was stirred overnight at room temperature.
The reaction was quenched by adding water and acidifying the
solution with HCl. The product was extracted with ethyl acetate and
dried over Na2SO4, and the solvent was evaporated. Purification of the
desired compound was achieved by flash chromatography using
DCM/methanol 95/5 as eluent, affording 65% of the desired
1
compound. H NMR (400 MHz, DMSO-d6): δ = 3.75 (s, 3H), 6.67
(d, 1H, J = 8.8 Hz), 7.20 (s, 1H), 7.40 (d, 1H, J = 8.8 Hz), 7.47 (s,
1H), 7.61 (m, 3H), 7.98 (d, 2H), 8.22 (s, 1H), 8.87 (s, 1H), 12.63 (bs,
1H). FT-ICR-MSFT-ICR-MS (m/z): 413 [M + Na]+.
For detailed syntheses and characterization of all shown compounds
and their precursors see Supporting Information.
N-{3-[(2-Chloro-5-methoxyphenyl)amino]pyrazin-2-yl}-3-ni-
trobenzenesulfonamide (4). Intermediate 2c (1 equiv) and 3-
nitrobenzenesulfonyl chloride (3 equiv) were dissolved in dry pyridine,
and the reaction mixture was stirred overnight at room temperature.
The reaction was quenched by adding water and acidifying the
solution with HCl. The product was extracted with ethyl acetate and
dried over Na2SO4, and the solvent was evaporated. Purification of the
desired compound was achieved by flash chromatography using DCM
3-Chloro-N-(2-chloro-5-methoxyphenyl)pyrazin-2-amine
(intermediate 2a). 2-Chloro-5-methoxyaniline (1.2 equiv) was
dissolved in dry THF under argon and cooled to 0 °C. Then 1.2
equiv Na-HMDS was added dropwise and the reaction mixture was
stirred for 1 h. After addition of 2,3-dichloropyrazine (1 equiv) in dry
THF, the solution was allowed to warm to room temperature and
stirred for 2 h. The mixture was quenched with a saturated solution of
ammonium chloride, and the product was extracted with ethyl acetate.
The combined organic layers were dried over Na2SO4, and solvent was
evaporated. Purification of the desired compound was achieved by
flash chromatography using ethyl acetate/petroleum ether 5/95 as
1
as eluent, affording 57% of the desired compound. H NMR (400
MHz, DMSO-d6): δ = 3.76 (s, 3H), 6.69 (dd, 1H, J = 8.8 Hz), 7.22 (s,
1H), 7.42 (d, 1H, J = 8.8 Hz), 7.52 (s, 1H), 7.89 (t, 1H, J = 8.0 Hz),
8.20 (s, 1H), 8.39 (d, 1H, J = 8.0 Hz), 8.46 (d, 1H, J = 8.0 Hz), 8.70
(s, 1H), 8.86 (s, 1H), 12.84 (bs, 1H). FAB-MS (m/z): 434 [M − H]−.
3-Amino-N-{3-[(2-chloro-5-methoxyphenyl)amino]pyrazin-
2-yl}benzenesulfonamide (5). At room temperature, compound 4
1
eluent, affording 64% of the desired compound. H NMR (400 MHz,
DMSO-d6): δ = 3.77 (s, 3H), 6.81 (m, 1H), 7.43 (m, 1H), 7.44 (s,
H
dx.doi.org/10.1021/jm5006034 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
was dissolved in ethyl acetate, then Pd/C (10% (m/m)) was added
and H2 was led through the reaction mixture. After 3 h of stirring, Pd/
C was filtered and the solvent was removed under reduced pressure.
The crude product was purified by silica flash chromatography using
ethyl acetate/n-hexane as eluent, affording 47% of the desired
= 8.8 Hz), 7.40 (d, 1H, J = 4.2 Hz), 7.61 (t, 1H), 8.22 (m, 2H), 8.38
(m, 1H), 8.69 (m, 1H), 8.93 (s, 1H); −NHSO2− was not detected.
FAB-MS (m/z): 447 [M − H]−.
3-[({3-[(2-Chloro-5-methoxyphenyl)amino]pyrazin-2-yl}-
amino)sulfonyl]benzoic Acid (9). Compound 8 was dissolved in
1,4-dioxane, then a 2 M solution of sodiumhydroxide in water (10
equiv of NaOH) was added and the solution was stirred at room
temperature for 2.5 h. The reaction was quenched by adding water and
acidified with 2 M HCl. The product was extracted with ethyl acetate
and the organic layers were dried over Na2SO4, and the solvent was
removed under reduced pressure. The desired product was obtained to
1
compound. H NMR (200 MHz, CDCl3): δ = 3.82 (s, 3H), 6.56
(dd, 1H), 6.84 (m, 2H), 7.30 (m, 5H), 8.38 (d, 1H), 8.96 (s, 1H);
−NHSO2− was not detected. FAB-MS (m/z): 404 [M − H]−.
N-{3-[({3-[(2-Chloro-5-methoxyphenyl)amino]pyrazin-2-yl}-
amino)sulfonyl]phenyl}acetamide (6). Compound 5 (1 equiv)
was dissolved in water with an excess of acetic anhydride (5000 equiv)
and refluxed for 1 h. Subsequently, the reaction mixture was brought
to pH 5 with K2CO3 and extracted with ethyl acetate. The organic
layers were combined and dried over Na2SO4, and the solvent was
evaporated under reduced pressure. Remaining acetic acid was
codestilled with ethyl acetate to yield 100% of the desired compound
1
100% in sufficient purity. H NMR (200 MHz, DMSO-d6): δ = 3.74
(s, 3H), 6.66 (dd, 1H, J = 8.9 Hz), 7.20 (d, 1H, J = 3.6 Hz), 7.39 (d,
1H, J = 8.9 Hz), 7.48 (d, 1H, J = 3.6 Hz), 7.71 (t, 1H, J = 7.8 Hz), 8.17
(m, 3H), 8.48 (m, 1H), 8.84 (s, 1H), 12.74 (bs, 1H), 13.43 (bs, 1H).
FAB-MS (m/z): 433 [M − H]−.
2-(Acetylamino)isonicotinic Acid (Intermediate 41a). 4-
Aminoisonicotinic acid was suspended in acetic anhydride, a few
drops of sulfuric acid were added, and the reaction mixture was heated
overnight at 90 °C. Acetic acid and excess acetic anhydride were
evaporated under reduced pressure as completely as possible, water
was added to the residue, and the mixture was stirred and cooled for
15 min. The desired product could be filtered and dried to afford 60%
of sufficient purity. 1H NMR (200 MHz, DMSO-d6): δ = 2.11 (s, 3H),
7.49 (dd, 1H, J = 5.0 Hz), 8.45 (d, 1H, J = 5.0 Hz), 8.55 (s, 1H), 10.71
(s, 1H). FAB-MS (m/z): 179 [M + H]+.
1
with sufficient purity. H NMR (200 MHz, DMSO-d6): δ = 2.04 (s,
3H), 3.74 (s, 3H), 6.66 (dd, 1H), 7.20 (m, 1H), 7.43 (m, 3H), 7.62 (d,
1H, J = 7.9 Hz), 7.73 (d, 1H, J = 7.9 Hz), 8.24 (m, 2H), 8.85 (s, 1H),
10.21 (s, 1H), 12.55 (bs, 1H). FAB-MS (m/z): 446 [M − H]−.
Cbz-N1-{3-[({3-[(2-chloro-5-methoxyphenyl)amino]pyrazin-
2-yl}amino)sulfonyl]phenyl}-2-methylalaninamide (7a). Com-
pound 5 (1 equiv) and N-carbobenzyloxy-2-methylalanine (1.5 equiv)
were dissolved in dry DCM, and dicyclohexylcarbodiimide (DCC) in
DCM (1.5 equiv) was added dropwise. The solution was stirred at
room temperature, and further N-Carbobenzyloxy-2-methylalanine
and DCC were added until all of compound 5 was converted (HPLC).
The resulting precipitate was filtered, and the filtrate was concentrated
under reduced pressure. The residue was purified by silica flash
chromatography using ethyl acetate/petroleum ether with a gradient of
35/65 for 1 h, then 65/35 as eluent, affording 37% of the desired
tert-Butyl-2-(acetylamino)pyridin-4-ylcarbamate (Intermedi-
ate 41b). At room temperature under argon, intermediate 41a (1
equiv) was suspended in dry toluene. Diphenylphosphorylazide (1
equiv) and triethylamine (1.2 equiv) were added dropwise, and then
the reaction mixture was warmed to 75 °C for 1 h. Afterward, an
excess of dried tert-butanol (ca. 7 equiv) was added and the mixture
was refluxed for 3 h. Then water was added and brought to pH 8−9
with a 10% solution of sodium hydroxide in water. The layers were
separated, and the water was extracted 3 times with ethyl acetate. The
organic layers were combined and dried over Na2SO4, and the solvent
was removed under reduced pressure. The crude product was purified
by silica flash chromatography using ethyl acetate/petroleum ether 67/
1
compound. H NMR (200 MHz, DMSO-d6): δ = 1.41 (s, 6H), 3.75
(s, 3H), 5.00 (s, 2H), 6.65 (d, 1H), 7.34 (m, 10H), 7.64 (d, 1H, J = 7.4
Hz), 7.87 (d, 1H, J = 7.4 Hz), 8.29 (m, 2H), 8.91 (s, 1H), 9.83 (s,
1H), 12.67 (bs, 1H). FAB-MS (m/z): 255 [M − H]−.
N1-{3-[({3-[(2-Chloro-5-methoxyphenyl)amino]pyrazin-2-yl}-
amino)sulfonyl]phenyl}-2-methylalaninamide (7). At room
temperature, compound 7a was dissolved in ethyl acetate, then Pd/
C (10% (m/m)) was added and H2 was led through the reaction
mixture. After 3 h of stirring, Pd/C was filtered and the solvent was
removed under reduced pressure. The crude product was purified by
silica flash chromatography using first ethyl acetate/n-hexane 70/30 as
eluent then ethyl acetate/methanol 90/10 to afford 60% of the desired
compound. 1H NMR (200 MHz, MeOD-d3): δ = 1.70 (s, 6H), 3.63 (s,
3H), 6.61 (d, 1H, J = 8.4 Hz), 7.08 (s, 1H), 7.28 (d, 1H, J = 8.4 Hz),
7.46 (s, 1H), 7.72 (m, 4H), 8.02 (d, 1H, J = 7.2 Hz), 8.10 (s, 1H), 8.37
(s, 1H); −NHSO2− and −NH2 were not detected. EI-MS (m/z): 490
[M+].
1
33 as eluent to yield 62% of the desired compound. H NMR (200
MHz, CDCl3): δ = 1.51 (s, 9H), 2.23 (s, 3H), 7.71 (d, 1H), 8.13 (m,
2H), 8.66 (s, 1H), 10.15 (s, 1H). GC-MS (m/z): 251 [M+].
N-(4-Aminopyridin-2-yl)acetamide (Intermediate 41c). At
room temperature, intermediate 41b (1 equiv) was dissolved in 1,2-
dichloroethane, trifluoroacetic acid (3 equiv) was added dropwise, and
the mixture was refluxed for 48 h. After reducing the volume of the
reaction mixture, the precipitate could be filtered. The precipitate was
solved in water and basified with K2CO3. The water was extracted 3
times with ethyl acetate, the organic layers were combined and dried
over Na2SO4, and the solvent was removed under reduced pressure to
Methyl 3-Chlorosulfonylbenzoate. Under argon, 3-chlorosulfo-
nylbenzoic acid (1 equiv) was dissolved in dry DCM, 3 drops of DMF
were added, and then oxalyl chloride (1.5 equiv) was added dropwise
under vigorous stirring. The reaction was stirred at room temperature
for 1.5 h, then dry methanol (5 equiv) was added. After 15 min, the
solvent was removed under reduced pressure and the residue was
purified immediately by silica flash chromatography using ethyl
acetate/petroleum ether 20/80 as eluent. We obtained 58% of the
1
yield 82% of the desired compound with sufficient purity. H NMR
(200 MHz, DMSO-d6): δ = 2.00 (s, 3H), 6.01 (s, 2H), 6.18 (dd, 1H, J
= 5.6 Hz), 7.29 (m, 1H), 7.69 (d, 1H, J = 5.6 Hz), 9.93 (s, 1H). FAB-
MS (m/z): 152 [M + H]+.
ASSOCIATED CONTENT
■
1
desired compound. H NMR (200 MHz, CDCl3): δ = 3.99 (s, 3H),
S
* Supporting Information
7.74 (t, 1H, J = 7.8 Hz), 8.21 (d, 1H, J = 7.8 Hz), 8.40 (d, 1H, J = 7.8
Hz), 8.68 (s, 1H).
Synthesis and characterization of compounds 2−42 and their
intermediates. This material is available free of charge via the
Internet at http://pubs.acs.org.
Methyl 3-[({3-[(2-Chloro-5-methoxyphenyl)amino]pyrazin-
2-yl}amino)sulfonyl]benzoate (8). Compound 2c (1 equiv) and
methyl 3-chlorosulfonylbenzoate (3 equiv) were dissolved in dry
pyridine, and the reaction mixture was stirred overnight at room
temperature. The reaction was quenched by adding water and
acidifying the solution with HCl. The product was extracted with
ethyl acetate and dried over Na2SO4, and the solvent was evaporated.
The crude product was purified by silica flash chromatography using
ethyl acetate/n-hexane 35/65 as eluent, affording 38% of the desired
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49 7071 2972459. E-mail: stefan.laufer@uni-
tuebingen.de.
1
Notes
compound. H NMR (200 MHz, CDCl3): δ = 3.82 (s, 3H), 3.95 (s,
3H), 6.56 (dd, 1H, J = 8.8 Hz), 6.93 (d, 1H, J = 4.2 Hz), 7.27 (d, 1H, J
The authors declare no competing financial interest.
I
dx.doi.org/10.1021/jm5006034 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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ACKNOWLEDGMENTS
■
We thank Peter Druckes (Assays), Jurgen Koppler (Compound
̈
̈
̈
Logistic), and Jorg Trappe (Coordination) from Novartis
̈
Pharma AG, Basel, Switzerland, for the protein kinase screen.
We also thank Peter Keck for the help with the processing of
the raw data.
ABBREVIATIONS USED
■
CDI, 1,1′-carbonyldiimidazole; cpd, compound; MCP-1,
monocyte chemotactic protein 1; Na-HMDS, sodium hexam-
ethyldisilazane/sodium-bis(trimethylsilyl)amide; Pd/C, palladi-
um on activated carbon; PDGF, platelet derived growth factor;
PtdIns, phosphatidyl inositide; Vps, vacuolar protein sorting
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