Microwave-assisted synthesis of KN-93, a potent and selective inhibitor of Ca2+/calmoduline-dependent protein kinase II

Article abstract of DOI:10.1055/s-0030-1258298

Convenient synthetic routes to KN-93, N-(2-{[[(2E)-3-(4-chlorophenyl)prop- 2-enyl](methyl)amino]methyl}phenyl)-N-(2-hydroxyethyl)-4- methoxybenzenesulfonamide, a well-known Ca²⁺/calmoduline-dependent protein kinase II (CaMKII) inhibitor, are de

Full text of DOI:10.1055/s-0030-1258298

PAPER
4193
Microwave-Assisted Synthesis of KN-93, a Potent and Selective Inhibitor of
Ca2+/Calmoduline-Dependent Protein Kinase II
S
ynthesisof
K
N
-
l
93 audio Bruno,a Giovanni Lentini,a Alessia Catalano,a Alessia Carocci,a Angelo Lovece,a Antonia Di Mola,a
Maria Maddalena Cavalluzzi,a Paolo Tortorella,a Fulvio Loiodice,a Guido Iaccarino,b Pietro Campiglia,c
Ettore Novellino,d Carlo Franchini*a
a
Dipartimento Farmaco-Chimico, Università di Bari, Via E. Orabona 4, 70126 Bari, Italy
Fax +39(080)5442724; E-mail: cfranc@farmchim.uniba.it
Dipartimento di Medicina Clinica e Scienze Cardiovascolari e Immunologiche, Università degli Studi di Napoli Federico II,
b
Corso Umberto I, 80138 Napoli, Italy
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
Dipartimento di Chimica Farmaceutica e Tossicologica, Università degli Studi di Napoli Federico II, Via D. Montesano 49,
c
d
80131 Napoli, Italy
Received 1 July 2010; revised 16 September 2010
flammatory pain,14 and that CaMKII is involved in cardi-
Abstract: Convenient synthetic routes to KN-93, N-(2-{[[(2E)-
ac remodelling secondary to pathological stresses such as
3-(4-chlorophenyl)prop-2-enyl](methyl)amino]methyl}phenyl)-N-
hypertension or myocardial infarction,15 as well as endo-
(2-hydroxyethyl)-4-methoxybenzenesulfonamide, a well-known
thelial cell pathophysiology.16 Generally, the studies con-
Ca2+/calmoduline-dependent protein kinase II (CaMKII) inhibitor,
are described. The methods proposed start from easily available cerning CaMKII activity are carried out by using a
selective CaMKII inhibitor, called KN-93 (Figure 1).17
reagents and allow ready preparation of the final compound in
moderate overall yields. Most of the synthetic steps proposed were
microwave assisted.
O
Me
Key words: Heck reaction, sulfonamides, enzymes, multicompo-
O
nent reaction, medicinal chemistry
S
OH
N
O
N
Calmoduline-dependent protein kinase II (CaMKII) be-
longs to the superfamily of multifunctional CaM kinases
(CaMKs),1–3 which are involved in inducing or modulat-
ing cellular responses to Ca2+ signals in cells. CaMKII is
a ubiquitous mediator of Ca2+-linked signalling that phos-
phorylates a wide range of substrates to coordinate and
regulate Ca2+-mediated alterations in cellular function. It
is an oligomeric serine/threonine kinase that is activated
by calcium-bound calmoduline (Ca2+/CaM). Multiple iso-
forms of human CaMKII are encoded by four closely re-
lated yet distinct genes (a, b, c, and d) that are spliced
differentially to produce more than 20 isoforms of the en-
zyme.4,5 CaMKII is expressed throughout the body and it
is particularly abundant in brain tissue4 and in the heart.6
Given that CaMKII phosphorylates a variety of substrates,
this multifunctional serine/threonine kinase has been im-
plicated in regulating many aspects of cellular function in
response to Ca2+ signalling, including the regulation of
carbohydrate, amino acid and lipid metabolism, ion chan-
nel/receptors, neurotransmitter synthesis and release,
transcription and translation, cytoskeletal organization,
and calcium homeostasis.7–12 Moreover, CaMKII can
modulate opioid tolerance via its action on learning and
memory.13 Recently, it has been reported that sufficient
inhibition of CaMKII is capable of reversing chronic in-
Me
Cl
KN-93
Figure 1 Structure of KN-93
This compound is commercially available in the form of
either free amine or its corresponding phosphate salt. In
both forms, KN-93 is sold only in small quantities (1 or 5
mg) and it is quite expensive, thus discouraging in vivo
pharmacological tests. Therefore, a less expensive access
to KN-93 is desirable. To our knowledge, the only syn-
thetic route to KN-93 has been reported in a patent written
in Japanese and it consists of six steps which start from 1-
(2-nitrophenyl)methanamine and 4-chlorocinnamalde-
hyde.18 Here, we present our efforts to develop more con-
venient and facile synthetic routes to KN-93.
All the synthetic routes herein proposed start from easily
available reagents and most of the reactions were carried
out under microwave (MW) irradiation.
(E)-4-Chlorocinnamaldehyde (1) was submitted to direct
reductive amination with N-methyl-1-(2-nitrophe-
nyl)methanamine (2) to give compound 3 in 56% yield
(method A; see Scheme 1). This reaction was carried out
following a previously reported procedure,19 shortening
the reaction time to 11 minutes by microwave assistance.
Reduction of the nitro group with zinc and ammonium
chloride20 gave aniline 4, which was in turn reacted with
4-methoxybenzenesulfonyl chloride to give KN-92, an
SYNTHESIS 2010, No. 24, pp 4193–4198
x
x.
x
x
.
2
0
1
0
Advanced online publication: 14.10.2010
DOI: 10.1055/s-0030-1258298; Art ID: T14310SS
© Georg Thieme Verlag Stuttgart · New York

4194
C. Bruno et al.
PAPER
Me
N
NO2
H
H
a
O2N
N
O
+
Me
56%
Cl
Cl
1
2
method A
3
>99%
b
O
Me
O
S
R
NH2
N
O
c
82%
N
N
Me
Me
R = H
R = CH2CH2OH
Cl
Cl
KN-92
KN-93
4
d
61%
Scheme 1 Reagents and conditions: (a) NaBH3CN, MeOH, MW, 110 °C, 11 min; the yield increased to 78% when homemade 2·HCl was
used (see text); (b) Zn, NH4Cl, H2O, acetone, reflux, 6 h; (c) 4-MeOC6H4SO2Cl, py, anhyd CH2Cl2, 0 °C, 3 h; (d) 1,3-dioxolan-2-one, NaOH,
DMF, MW, 150 °C, 45 min.
inactive analogue of KN-93.21 KN-92 was converted into (J3 = 16.0 Hz). For comparison, the Z-isomer of 1 (com-
1
KN-93 by modifying a literature procedure.22 The H pound 12) was obtained following a previously reported
NMR aliphatic, cinnamyl, and benzyl signals were unam- procedure (Scheme 3).26 Starting from 4-chlorobenzalde-
biguously assigned by COSY experiments. KN-93 was hyde (8), a mixture of isomeric 1,3-dioxolanes 10 and 11
converted into the corresponding phosphate salt by treat- (Z/E = 1.6) was obtained by a Wittig-type reaction under
ment with phosphoric acid.
phase-transfer conditions using (1,3-dioxolan-2-ylmeth-
yl)triphenylphosphonium bromide (9). Dioxolane 10 was
isolated by silica gel column chromatography and submit-
ted to mild acidic treatment to give 12 (J3 = 11.7 Hz). On
standing, 12 isomerizes to the more stable isomer 1
(DEZ/E = 13.0 kJ·mol–1, gas phase, 3-21G*//3-21G*).
The synthesis of (E)-4-chlorocinnamaldehyde (1) was
achieved from 1-chloro-4-iodobenzene (5) modifying a
procedure reported in the literature23 by using microwave
irradiation, thus shortening the reaction time (from 3.5 h
to 7 min) (Scheme 2).
As an alternative, aldehyde 1 can be obtained in good
yields by Swern oxidation24 of 4-chlorocinnamyl alcohol
(6), in turn obtained from 4-chlorocinnamic acid (7) fol-
lowing a previously reported procedure (Scheme 2).25 In
agreement with the literature,23,25 both procedures gave
O
O
+
O
Br–
+
H
Ph3P
Cl
8
9
1
the E-isomer, as assessed by H NMR spectroscopy
H
68% (Z/E = 62:38)
a
I
O
a
Cl
68%
Cl
O
5
1
O
+
Cl
O
O
Cl
b
>99%
10
11
O
b
61%
OH
OH
c
Cl
83%
Cl
O
7
6
Cl
12
Scheme 2 Reagents and conditions: (a) acrolein diethyl acetal, n-
Bu4NOAc, K2CO3, KCl, Pd(OAc)2, DMF, MW, 140 °C, 7 min; b)
(COCl)2, anhyd DMSO, CH2Cl2, –60 °C, 30 min; c) ClCO2Et, Et3N,
anhyd THF, NaBH4, –10 °C → r.t.
Scheme
3
Reagents and conditions: (a) tris(methoxyethoxy-
ethyl)amine, sat. aq K2CO3, CH2Cl2, 40 °C, 18 h; (b) AcOH, THF,
H2O, r.t., 8 h.
Synthesis 2010, No. 24, 4193–4198 © Thieme Stuttgart · New York

PAPER
Synthesis of KN-93
4195
spectrophotometer; band positions are given in reciprocal centime-
As an alternative for the synthesis of compound 3 (method
B; see Scheme 4), a one-pot, microwave, three-compo-
nent reductive amination of aldehyde 1 and 2-nitrobenzal-
dehyde (13) with methylamine hydrochloride was
studied.
1
ters (cm–1). H and 13C NMR spectra were recorded on a Bruker
300-MHz spectrometer (ASPECT 3000) (or a Bruker AM-500 in-
strument, where indicated), operating at 300 and 75 MHz (500 and
1
125 MHz when the Bruker AM-500 instrument was used) for H
and 13C, respectively, using CDCl3, unless otherwise indicated, as a
solvent. Chemical shifts are reported in parts per million (ppm) rel-
ative to the residual non-deuterated CDCl3 resonance [d 7.26 (1H
NMR) and d 77.3 (13C NMR)]. J values are given in Hz. EIMS data
were recorded on a Hewlett-Packard 6890/5973 MSD gas chro-
matograph–mass spectrometer at low resolution. ESI+/MS/MS
analyses were performed with an Agilent 1100 series LC/MSD trap
system VL Workstation. Elemental analyses were performed on a
EuroVector Euro EA3000 analyzer. Electrospray ionization (ESI)
time-of-flight reflectron experiments were performed on an Agilent
ESI-TOF mass spectrometer. Samples were electrosprayed into the
TOF reflectron analyzer at an ESI voltage of 4000 V and a flow rate
of 200 mL/min. Chromatographic separations were performed on
silica gel columns (Kieselgel 60, 0.040–0.063 mm, Merck, Darm-
stadt, Germany) by flash chromatography using the technique de-
scribed by Still and co-workers.27 TLC analyses were performed on
precoated silica gel on aluminum sheets (Kieselgel 60 F254, Merck).
NO2
O
N
O2N
a
Me
1
+
Cl
32%
method B
13
3
Scheme 4 Reagents and conditions: (a) MeNH2·HCl, NaBH3CN,
MeOH, MW, 90 °C, 8 min.
This route, even though of lower yield, may be preferable
to method A described previously (see Scheme 1) because
it avoids the use of commercial N-methyl-1-(2-nitrophe-
nyl)methanamine (2), the purity of which is not high, as
assessed by 1H NMR analysis, and also for atom-economy
reasons. On the other hand, pure 2·HCl may be obtained
by submitting 2-nitrobenzaldehyde (13) to microwave re-
(E)-4-Chlorocinnamaldehyde (1)
Via a Microwave-Assisted Heck Reaction
A mixture of 1-chloro-4-iodobenzene (5; 0.50 g, 2.10 mmol), ac-
ductive amination with methylamine hydrochloride rolein diethyl acetal (0.96 mL, 6.30 mmol), n-Bu4NOAc (1.27 g,
4.20 mmol), K2CO3 (0.44 g, 3.14 mmol), KCl (0.16 g, 2.10 mmol),
(Scheme 5).
and Pd(OAc)2 (0.014 g, 0.063 mmol) in DMF (10 mL) was stirred
for 7 min at 140 °C in a microwave reactor under continuous stir-
ring. When the reaction was completed, the mixture was cooled, 2
Me
O
N
N HCl (3 mL) was slowly added, and the reaction mixture was
stirred at r.t. for 10 min. Then, the mixture was diluted with Et2O
(50 mL) and washed with H2O (3 × 20 mL). The organic layer was
dried (Na2SO4) and concentrated under reduced pressure. Purifica-
tion of the residue by flash chromatography (EtOAc–petroleum
ether, 1:9) gave 1 as a white solid; yield: 0.24 g (68%); mp 62–63 °C
(Lit.23 59–60 °C, Lit.28 61–63 °C).
H⋅HCl
O2N
a, b
O2N
57%
13
2⋅HCl
Scheme 5 Reagents and conditions: (a) MeNH2·HCl, NaBH3CN,
Spectroscopic and spectrometric data were in agreement with the
literature.23
MeOH, MW, 110 °C, 11 min; (b) aq HCl.
Via Swern Oxidation24
Reaction of 2·HCl and (E)-4-chlorocinnamaldehyde (1),
as described above, gave compound 3 in 78% yield (see
experimental), thus increasing the yield obtained with
commercial 2 (56%, see Scheme 1).
A soln of oxalyl chloride (2.0 mL, 22 mmol) in CH2Cl2 (50 mL) was
stirred under N2 atmosphere for 5 min. Then, DMSO (1.7 mL, 22
mmol) dissolved in CH2Cl2 (5 mL) was added dropwise to the solu-
tion at –60 °C. The reaction mixture was stirred for 3 min and alco-
hol 6 (3.3 g, 20 mmol) in CH2Cl2 (20 mL) was added within 5 min
using a syringe; stirring was continued for an additional 15 min.
Et3N (14.0 mL, 100 mmol) was then added and the reaction mixture
was allowed to warm to r.t. H2O (100 mL) was added and the aque-
ous layer was extracted with additional CH2Cl2 (100 mL). The com-
bined organic layers were washed with sat. NaCl soln and dried
(anhyd MgSO4). Evaporation of the solvent gave an oil, which was
purified on silica gel (EtOAc–hexane, 3:7) to afford 1 as a white sol-
id; yield: 3.28 g (quant).
In summary, a facile synthesis of KN-93 has been devel-
oped. The final product was obtained in moderate yields
and short reaction times. With the one exception of a
patent written in Japanese,18 this simple, sustainable syn-
thesis of KN-93 is the first method for the preparation of
the target compound. Due to the importance of the prod-
uct, our approach may contribute to widening the phar-
maceutical and biomedical applications of CaMKII
inhibitors.
2-[(Z)-2-(4-Chlorophenyl)ethenyl]-1,3-dioxolane (10) and 2-
[(E)-2-(4-Chlorophenyl)ethenyl]-1,3-dioxolane (11)
All chemicals were purchased from Sigma-Aldrich or Lancaster
and used without further purification. 4-Chlorocinnamyl alcohol (6)
was obtained as previously reported.25 The reactions under micro-
wave irradiation were carried out at constant temperature in a CEM
Discover BenchMate microwave reactor, with continuous stirring.
The temperature was measured and controlled by a built-in infrared
detector. Melting points were determined on a Gallenkamp melting
point apparatus in open glass capillary tubes and are uncorrected.
Infrared spectra were recorded on a Perkin-Elmer Spectrum One FT
To a soln of 4-chlorobenzaldehyde (8; 0.14 g, 1.0 mmol) and
tris(methoxyethoxyethyl)amine (0.32 g, 1.0 mmol) in CH2Cl2 (15
mL), sat. aq K2CO3 soln (15 mL) and phosphonium bromide 9 (0.43
g, 1.0 mmol) were added. The mixture was heated at 40 °C for 18 h.
The products were extracted with CH2Cl2 (3 × 20 mL), and the ex-
tracts were washed with H2O (3 × 10 mL) and then dried (Na2SO4).
Dioxolanes 10 and 11 were separated by silica gel chromatography
(EtOAc–petroleum ether, 0.5:9.5) to give 10 (0.090 g) as a colorless
Synthesis 2010, No. 24, 4193–4198 © Thieme Stuttgart · New York

4196
C. Bruno et al.
PAPER
oil and 11 (0.054 g) as a white solid; combined yield: 68%; Z/E =
62:38.
1H NMR (compound 10): d = 3.8–4.2 (m, 4 H, 2 CH2O), 5.45 (d,
J = 7.4 Hz, 1 H, OCHO), 5.73 (dd, J = 11.5, 7.4 Hz, 1 H,
CHCH=CH), 6.75 (d, J = 11.5 Hz, 1 H, ArCH), 7.31 (s, 4 H, Ar).
petroleum ether, 1:9) gave 0.28 g of a yellow solid consisting of a
mixture of amine 3 (56%) and its analogue reduced at the vinylic
bond (8%), as assessed by 1H NMR spectroscopy. Yields increased
to 78% and 11% for the desired product 3 and its reduced analogue,
respectively, when homemade 2·HCl was used.
1H NMR (compound 11): d = 3.9–4.1 (m, 4 H, 2 CH2O), 5.42 (d,
J = 5.8 Hz, 1 H, OCHO), 6.14 (dd, J = 16.0, 6.0 Hz, 1 H,
CHCH=CH), 6.73 (d, J = 16.0 Hz, 1 H, ArCH), 7.26–7.38 (m, 4 H,
Ar).
Method B (Scheme 4)
A soln of (E)-4-chlorocinnamaldehyde (1; 0.33 g, 1.99 mmol), 2-ni-
trobenzaldehyde (13; 0.68 g, 4.48 mmol), MeNH2·HCl (0.23 g, 3.48
mmol), and NaBH3CN (0.16 g, 2.49 mmol) in MeOH (15 mL) was
stirred for 8 min at 90 °C in a microwave reactor under continuous
stirring. When the reaction was completed, 6 N HCl was added
dropwise to destroy the excess cyanohydride. The aqueous solution
was made alkaline and extracted with Et2O (2 × 20 mL). The com-
bined extracts were dried (Na2SO4) and concentrated under reduced
pressure. Purification of the residue by flash chromatography
(EtOAc–petroleum ether, 1:9) gave 0.26 g of a yellow solid consist-
ing of a mixture of amine 3 (32%) and its analogue reduced at the
vinylic bond (9%), as assessed by 1H NMR spectroscopy. Amine 3
was recrystallized (CHCl3–hexane) to give pale yellow crystals;
yield: 0.18 g (29%); mp 66–67 °C.
(2Z)-3-(4-Chlorophenyl)prop-2-enal (12, see Scheme 3)
To a mixture of THF (15 mL), H2O (15 mL), and AcOH (7.5 mL),
dioxolane 10 (0.29 g, 1.4 mmol) was added. The mixture was stirred
at r.t. for 8 h and then concentrated under reduced pressure. The
product was extracted with CH2Cl2 (3 × 20 mL), and the extracts
were washed with sat. aq NaHCO3 soln (2 × 10 mL) and H2O (10
mL), then dried (Na2SO4) and purified by chromatography (EtOAc–
petroleum ether, 0.5:9.5) to give 12 as a white solid; yield: 0.14 g
(61%); mp 43–44 °C.
IR (KBr): 1678 (CO) cm–1.
IR (KBr): 1522 (NO2) cm–1.
1H NMR: d = 6.21 (dd, J = 11.7, 8.1 Hz, 1 H, CHCHO), 7.3–7.4 (m,
4 H, Ar), 7.55 (d, J = 11.7 Hz, 1 H, ArCH), 9.94 (d, J = 8.1 Hz, 1 H,
CHO).
13C NMR: d = 129.1 (2 C), 131.1 (2 C), 131.3 (1 C), 132.8 (1 C),
136.2 (1 C), 147.2 (1 C), 192.1 (1 C).
1H NMR: d = 2.20 (s, 3 H, CH3), 3.16 (d, J = 6.3 Hz, 2 H, CH2CH),
3.82 (s, 2 H, ArCH2), 6.20 (dt, J = 16.0, 6.5 Hz, 1 H, CHCH2), 6.46
(d, J = 16.0 Hz, 1 H, ArCH), 7.18–7.33 (m, 4 H, Ar), 7.38 (t, J = 7.7
Hz, 1 H, Ar), 7.53 (t, J = 7.4 Hz, 1 H, Ar), 7.63 (d, J = 7.4 Hz, 1 H,
Ar), 7.81 (d, J = 8.0 Hz, 1 H, Ar).
13C NMR: d = 42.7 (1 C), 58.4 (1 C), 60.3 (1 C), 124.6 (1 C), 127.8
(2 C), 128.1 (1 C), 128.3 (1 C), 128.9 (2 C), 131.1 (1 C), 131.5 (1
C), 132.6 (1 C), 133.2 (1 C), 134.9 (1 C), 135.8 (1 C), 150.1 (1 C).
MS (70 eV): m/z (%) = 166 (25) [M+], 131 (100).
N-Methyl-1-(2-nitrophenyl)methanamine Hydrochloride
(2·HCl)
MS (70 eV): m/z (%) = 316 (3) [M+], 151 (100).
A
soln of 2-nitrobenzaldehyde (13; 0.30 g, 1.99 mmol),
MeNH2·HCl (0.37 g, 5.49 mmol), and NaBH3CN (0.088 g, 1.39
mmol) in MeOH (4 mL) was stirred for 11 min at 110 °C in a mi-
crowave reactor under continuous stirring. When the reaction was
completed, 6 N HCl was added dropwise to destroy the excess cy-
anohydride. The aqueous solution was made alkaline and extracted
with EtOAc (2 × 10 mL). The combined extracts were dried
(Na2SO4) and concentrated under reduced pressure to give 0.33 g of
a slightly yellowish oil, which was treated with 2 N HCl, with azeo-
tropic removal of H2O (abs EtOH–toluene), to give a yellow solid
(2·HCl). 2·HCl was recrystallized (EtOH–Et2O) to give yellow
crystals; yield: 0.23 g (57%); mp 141–142 °C.
2-{[[(2E)-3-(4-Chlorophenyl)prop-2-enyl](methyl)amino]meth-
yl}aniline (4)
To a soln of 3 (0.40 g, 1.26 mmol) in acetone (4 mL), a soln of
NH4Cl (0.16 g, 3.02 mmol) in H2O (0.6 mL) was added. The mix-
ture was brought to reflux; then, Zn dust (0.41 g) was added in small
portions to maintain a moderate reaction. After 6 h, the solution was
filtered while hot and the solid was washed with CH2Cl2 (2 ×). The
combined washings and filtrate were concentrated and the residue
was taken up with CH2Cl2. The solution was dried (Na2SO4) and the
solvent was removed under reduced pressure to obtain amine 4 as a
yellow solid; yield: 0.36 g (>99%); mp 69–70 °C.
+
IR (KBr): 3392 (NH2 ), 1530 (NO2) cm–1.
IR (KBr): 3361 (NH2) cm–1.
1H NMR [500 MHz, DMSO-d6 (d 2.50)]: d = 2.63 (t, J = 5.0 Hz, 3
H, CH3), 4.41 (t, J = 5.5 Hz, 2 H, CH2), 7.70–7.75 (m, 1 H, Ar),
7.85–7.90 (m, 2 H, Ar), 8.20 (d, J = 8.3 Hz, 1 H, Ar), 9.50 (br s, 2
1H NMR: d = 2.23 (s, 3 H, CH3), 3.16 (d, J = 6.6 Hz, 2 H, CH2CH),
3.57 (s, 2 H, ArCH2), 4.74 (br s, 2 H, NH2), 6.25 (dt, J = 15.7, 6.6
Hz, 1 H, CHCH2), 6.48 (d, J = 16.0 Hz, 1 H, ArCH), 6.70 (dd,
J = 12.6, 7.4 Hz, 2 H, Ar), 7.03 (d, J = 6.9 Hz, 1 H, Ar), 7.13 (t,
J = 7.7 Hz, 1 H, Ar), 7.30 (s, 4 H, Ar).
13C NMR: d = 42.1 (1 C), 59.5 (1 C), 61.6 (1 C), 115.7 (1 C), 117.8
(1 C), 123.2 (1 C), 127.7 (2 C), 128.3 (1 C), 128.6 (1 C), 128.9 (2
C), 130.7 (1 C), 131.7 (1 C), 133.3 (1 C), 135.8 (1 C), 147.3 (1 C).
+
H, NH2 ).
13C NMR [125 MHz, DMSO-d6 (d 39.5)]: d = 32.6 (1 C), 48.3 (1
C), 125.2 (1 C), 127.2 (1 C), 130.6 (1 C), 133.0 (1 C), 134.2 (1 C),
148.2 (1 C).
ESI-MS: m/z = 167 [M + H]+; ESI+/MS/MS: m/z (%) = 136 (100).
MS (70 eV): m/z (%) = 286 (<1) [M+], 180 (100).
(2E)-3-(4-Chlorophenyl)-N-methyl-N-(2-nitrobenzyl)prop-2-
en-1-amine (3)
Method A (Scheme 1)
N-(2-{[[(2E)-3-(4-Chlorophenyl)prop-2-enyl](methyl)ami-
no]methyl}phenyl)-4-methoxybenzenesulfonamide (KN-92)
To a stirred soln of amine 4 (0.20 g, 0.70 mmol) in anhyd CH2Cl2 (2
mL) under N2 atmosphere, pyridine (0.17 mL, 2.10 mmol) was add-
ed at 0 °C. Then, a soln of 4-MeOC6H4SO2Cl (0.17 g, 0.84 mmol)
in anhyd CH2Cl2 (2 mL) was added. After 3 h, the reaction mixture
was concentrated and the residue was taken up with H2O, made al-
kaline with NaHCO3, and extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were dried (Na2SO4) and the solvent was
removed under reduced pressure. Purification of the residue by
A soln of (E)-4-chlorocinnamaldehyde (1; 0.23 g, 1.38 mmol), N-
methyl-1-(2-nitrophenyl)methanamine (2; 0.69 g, 4.14 mmol), and
NaBH3CN (0.061 g, 0.97 mmol) in MeOH (5 mL) was stirred for 11
min at 110 °C in a microwave reactor under continuous stirring.
When the reaction was completed, 6 N HCl was added dropwise to
destroy the excess cyanohydride. The aqueous solution was made
alkaline and extracted with Et2O (2 × 10 mL). The combined ex-
tracts were dried (Na2SO4) and concentrated under reduced pres-
sure. Purification of the residue by flash chromatography (EtOAc–
Synthesis 2010, No. 24, 4193–4198 © Thieme Stuttgart · New York

PAPER
Synthesis of KN-93
4197
flash chromatography (EtOAc–petroleum ether, 2:8) gave KN-92
as a colorless oil; yield: 0.26 g (82%).
IR (neat): 1337 (SO2) cm–1.
13C NMR [CD3OD (d 47.8)]: d = 39.5 (1 C), 54.2 (1 C), 55.2 (1 C),
56.4 (1 C), 58.7 (1 C), 59.2 (1 C), 114.3 (2 C), 118.7 (1 C), 126.8 (1
C), 127.9 (1 C), 128.6 (4 C), 129.2 (1 C), 130.8 (2 C), 131.0 (1 C),
133.0 (1 C), 133.7 (1 C), 134.3 (1 C), 134.5 (1 C), 138.7 (1 C), 140.1
(1 C), 164.3 (1 C).
1H NMR: d = 2.16 (s, 3 H, NCH3), 3.14 (d, J = 6.9 Hz, 2 H,
CH2CH), 3.27 (s, 2 H, ArCH2), 3.74 (s, 3 H, OCH3), 6.31 (dt,
J = 15.7, 6.6 Hz, 1 H, CHCH2), 6.50 (d, J = 16.0 Hz, 1 H, ArCH),
6.63–6.72 (m, 2 H, Ar), 6.92–7.02 (m, 2 H, Ar), 7.16–7.27 (m, 2 H,
Ar + NH), 7.28–7.42 (m, 4 H, Ar), 7.53 (d, J = 7.7 Hz, 1 H, Ar),
7.58–7.66 (m, 2 H, Ar).
13C NMR: d = 41.9 (1 C), 55.7 (1 C), 59.7 (1 C), 60.6 (1 C), 114.1
(2 C), 121.1 (2 C), 124.2 (1 C), 126.5 (1 C), 127.5 (1 C), 128.0 (1
C), 128.8 (2 C), 129.0 (1 C), 129.1 (2 C), 130.0 (1 C), 132.5 (1 C),
133.0 (1 C), 133.7 (1 C), 135.3 (1 C), 138.1 (1 C), 163.0 (1 C).
HRMS (ESI-TOF): m/z calcd for C26H29ClN2O4S: 501.1609 ([M +
H]+); found: 501.1610.
Acknowledgment
This work was accomplished thanks to financial support from Pro-
getto Strategico Regione Puglia (PS131) entitled ‘Realizzazione di
molecole a basso peso molecolare attive sulle CAM chinasi da uti-
lizzare nelle procedure diagnostiche riguardanti le strategie di tera-
pia innovativa nelle patologie umane associate ad iperproliferazione
cellulare’.
ESI-MS: m/z = 479 [M + Na]+; ESI+/MS/MS: m/z (%) = 151 (100).
HRMS (ESI-TOF): m/z calcd for C24H25ClN2O3S: 457.1347 ([M +
H]+); found: 457.1345.
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