Inhibitors of the RET tyrosine kinase based on a 2-(alkylsulfanyl)-4-(3- thienyl)nicotinonitrile scaffold

Article abstract of DOI:10.1016/j.ejmech.2010.03.017

In an approach to optimize 2-(4-fluorobenzylsulfanyl)-4-(2-thienyl)-5,6,7, 8-tetrahydroquinoline-3-carbonitrile (1a), a weak inhibitor of the cancer-related tyrosine kinase RET originating from a screening campaign, analogues with 3-thienyl substitution were prepared. Among the novel derivatives, 2-amino-6-{[2-(4-chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-thienyl) pyridine-3,5-dicarbonitrile (13g) was identified as a submicromolar RET inhibitor, displaying 3- and 100-fold selectivity versus ALK and ABL kinases, respectively. The novel inhibitor exhibited antiproliferative activity in the micromolar concentration range against both RET-dependent and RET-independent cancer cell lines. Docking experiments suggest a binding mode of the new inhibitors in the ATP binding pocket of the target kinase, explaining the observed structure-activity relationships.

Full text of DOI:10.1016/j.ejmech.2010.03.017

European Journal of Medicinal Chemistry 45 (2010) 2919e2927
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibitors of the RET tyrosine kinase based on a 2-(alkylsulfanyl)-4-(3-thienyl)
nicotinonitrile scaffold
,
,
Wiebke Brandt a 1, Luca Mologni b 1, Lutz Preu a, Thomas Lemcke c, Carlo Gambacorti-Passerini b,
,
Conrad Kunick a
*
a Technische Universität Braunschweig, Institut für Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany
b University of Milano-Bicocca, Department of Clinical Medicine and Biotechnology, via Cadore 48, 20052 Monza, Italy
c Universität Hamburg, Institut für Pharmazie, Bundesstraße 45, D-20146 Hamburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In an approach to optimize 2-(4-fluorobenzylsulfanyl)-4-(2-thienyl)-5,6,7,8-tetrahydroquinoline-3-
carbonitrile (1a), a weak inhibitor of the cancer-related tyrosine kinase RET originating from a screening
campaign, analogues with 3-thienyl substitution were prepared. Among the novel derivatives, 2-amino-
6-{[2-(4-chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-thienyl)pyridine-3,5-dicarbonitrile (13g) was identi-
fied as a submicromolar RET inhibitor, displaying 3- and 100-fold selectivity versus ALK and ABL kinases,
respectively. The novel inhibitor exhibited antiproliferative activity in the micromolar concentration
range against both RET-dependent and RET-independent cancer cell lines. Docking experiments suggest
a binding mode of the new inhibitors in the ATP binding pocket of the target kinase, explaining the
observed structureeactivity relationships.
Received 20 November 2009
Received in revised form
11 March 2010
Accepted 12 March 2010
Available online 19 March 2010
Keywords:
Antiproliferative activity
Enzyme inhibition
RET tyrosine kinase
Cancer cell line
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
mutations is associated with the development of several forms of
endocrine tumors. Somatic chromosomal rearrangements result in
The RET (rearranged during transfection) protooncogene
encodes for a membrane-bound receptor-like tyrosine kinase that
is mainly expressed in the central and peripheral nervous system
and in the developing urogenital tract [1,2]. The RET tyrosine kinase
is essential for the early development of the enteric nervous system
and the kidney [3,4]. The extracellular part of RET contains a cad-
herin-like domain probably responsible for interaction with the
ligand and a cysteine-rich region that is important for the tertiary
structure and the dimerization of two RET proteins [5,6]. The
transmembrane domain is followed by the tyrosine kinase domain
on the cytoplasmic side [7,8]. RET can be activated by the four
members of the glial cell line-derived neurotrophic factor (GDNF)
family of ligands in complex with a ligand-specific coreceptor
chimeric RET/PTC oncogenes that encode for constitutively acti-
vated RET. These chimeric oncogenes have been identified in
5e30% of sporadic and 60e70% of radiation-induced papillary
thyroid carcinomas (PTC) that arise from thyroid follicular cells [9].
Germline point mutations of the RET protooncogene lead to
multiple endocrine neoplasia type 2 (MEN 2) which includes MEN
2A, MEN 2B and familial medullary thyroid carcinoma (FMTC),
which differ in the clinical manifestation [10e12]. FMTC is char-
acterized only by MTC (medullary thyroid carcinoma, a tumor
arising from the C cells), whereas MEN 2A also involves pheo-
chromocytoma and hyperplasia of the parathyroid. MEN 2B
patients have a more complex phenotype including MTC, pheo-
chromocytoma, ganglioneuromatosis of the gastroenteric mucosa
and marfanoid habitus [13]. The constitutively activated RET tyro-
sine kinase has been suggested as a suitable target for therapeutic
intervention for distinct thyroid neoplastic diseases [14]. Along
these lines, both established kinase inhibitors and novel small
molecular entities have been found to block RET kinase activity, e.g.
PP1 [15], PP2 [16], vandetanib (14) [17], sorafenib [18], semaxanib
(SU5416) [19], RPI-1 [14], imatinib [20], the indolocarbazole CEP-
751 [21], and beta-carbolin-1-ones [22]. Several of these
compounds constitute potential agents in treatment of thyroid
carcinomas. It has been pointed out that, similar to other cancer
(GFRa1-4, GDNF receptor a) [6]. Binding of the complex is followed
by dimerization of RET and autophosphorylation of tyrosine resi-
dues in the intracellular kinase domain, which initiates signaling
cascades regulating survival, differentiation, proliferation, migra-
tion and chemotaxis [7]. Constitutively activated RET evolving from
* Corresponding author. Tel.: þ49 531 391 2754; fax: þ49 531 391 2799.
E-mail address: c.kunick@tu-braunschweig.de (C. Kunick).
1
These authors contributed equally.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.017

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W. Brandt et al. / European Journal of Medicinal Chemistry 45 (2010) 2919e2927
S
analogue 7f are depicted in Scheme 1. Reaction of the Knoevenagel
product 3 with either cyclohexanone (4a) or N-methylpiperidone
(4b) in the presence of piperidine yielded thiolactams 5 or 6,
respectively. Without further purification, intermediate 5 or 6 was
then S-alkylated by reaction with an appropriate alkyl halide in
DMF. For the latter reaction, it is important to restrict the amount of
the base potassium hydroxide to one equivalent in order to avoid
a subsequent ZieglereThorpe ring closure reaction [26], which
might lead to thieno[2,3-b]pyridine derivates 8 as undesired
byproducts depicted in Scheme 2.
S
N
N
R1
R2
R3
N
S
N
S
R
Scheme 3 illustrates the preparation of the more polar deriva-
tives 13ael of the general structure 2. For the synthesis of the
intermediate thiolactam 12 [27], the three components thiophene-
3-carbaldehyde (9), malononitrile (10), and cyanothioacetamide
(11) were refluxed in ethanol in the presence of piperidine as basic
catalyst. Without further purification, the resulting 12 was alky-
lated at the sulphur atom by treatment with an appropriate alkyl
halide in DMF in the presence of 1 equiv potassium hydroxide. The
6-(alkylsulfanyl)-2-amino-4-(3-thienyl)pyridine-3,5-dicarboni-
triles 13ael produced by this method are listed in Table 2.
1a (R = F)
1b (R = H)
2
Fig. 1. Hit structures 1a and 1b and core structure 2 present in the test compounds 7
and 13. Compounds 1a and 1b were identified in a screening campaign for RET kinase
inhibitors (IC50 ¼ 4.8
mM). In the basic structure 2 present in derivatives 7 and 13 the
thiophene substituent is rotated counter clockwise by one position.
therapies based on protein kinase inhibitors, clinical resistance
caused by mutations of the targeted oncogene is conceivable also
for RET inhibitors. For this reason, a broad array of structurally
different inhibitors would be desirable to circumvent such resis-
tances [23]. Consequently, there is still an urgent need for addi-
tional RET inhibitors with proven antiproliferative activity. In this
study we present the development of a novel RET inhibitor che-
motype based on a nicotinonitrile scaffold. The investigations were
started with a screening campaign using a collection of commer-
cially available compounds, which identified the substituted
4,5,6,7-tetrahydroquinoline-3-carbonitrile 1a as a moderate in
3. Biological evaluation and discussion
3.1. Results of in vitro RET kinase assays
All synthesized derivatives 7aef and 13ael were evaluated for
inhibitory activity of recombinant RET kinase by an ELISA assay
described recently [28]. Derivatives 7a and 7b, which are struc-
turally closest related to the hit structures 1a and 1b, showed
a decreased RET inhibitory activity. Interestingly, formal insertion
of a carbonyl group in the side chain attached to the exocyclic
sulphur as realized in compound 7c improved the activity.
However, this structure modification turned out to be rather
sensitive against enlargement of the side chain as demonstrated
by both derivatives 7d and 7e, which are clearly inferior to 7c.
The insertion of an ionisable nitrogen into the parent ring
system, which was undertaken to improve the water solubility
within the series, led to a definite loss of RET inhibitory activity
vitro inhibitor of RET, exhibiting an IC50 of 4.8 mM. Unfortunately, 1a
failed to inhibit the growth of RET-dependent TPC1 thyroid carci-
noma cells. Analogue 1b showed a slightly increased RET inhibition,
but was also not antiproliferative for cultivated cancer cell lines in
vitro (refer to Table 3). In the context of our interest in thiophene
derivatives as antiproliferative compounds, we were interested in
optimizing the kinase inhibitory and antiproliferative activity
within this compound class and in extending the study towards
entities of higher structural novelty. In this regard, we synthesized
and evaluated derivatives of the general structure 2 in which the
thienyl substituent is rotated by one position related to 1. Examples
of published compounds with the 2-(alkylsulfanyl)-4-(3-thienyl)
nicotinonitrile partial structure present in 2 are rare in the litera-
ture [24,25] and have not been mentioned as kinase inhibitors
before (Fig. 1).
(compound 7f, IC50 ¼ 27
mM, refer to Table 1 for kinase inhibition
results).
In compounds 13ael, the hydrophobic cyclohexene fusion as
present in series 7 is exchanged for a pair of polar substituents,
namely, a cyano and an amino group. Testing of these compounds
showed that similar to our findings with compounds 7a and 7b
substituted or unsubstituted S-benzyl side chains (13ae13e) were
less active than the hit structures 1a and 1b. Again, insertion of
a carbonyl group improved the RET kinase inhibitory activity (13f).
In contrast to 7d, compounds 13fel tolerate substituents at the para
position of the phenacyl residue to a certain extent (13h, 13i and
13k). The p-chloro derivative 13g showed the highest RET
2. Chemistry
The syntheses of the 2-(alkylsulfanyl)-4-(3-thienyl)-5,6,7,8-tet-
rahydroquinoline-3-carbonitriles 7aee and the 6-methyl-6-aza-
S
S
S
X
O
N
N
N
R
(ii)
4a,b, (i)
X
X
H2N
S
N
H
S
N
S
3
5 (X = CH2)
6 (X = N-CH3)
7a-e (X = CH2)
7f (X = N-CH3)
Scheme 1. Synthesis of 2-(alkylsulfanyl)-4-(3-thienyl)-5,6,7,8-tetrahydroquinoline-3-carbonitriles 7aee and analogue 7f. Reagents and conditions: (i) dioxane, piperidine, air,
80 ꢀC, 5 h (/5) or EtOH, piperidine, air, reflux, 1 h (/6); (ii) appropriate alkyl halide, 1 equiv. KOH, DMF, room temperature, 30 min. For substituents R, refer to Table 1.

W. Brandt et al. / European Journal of Medicinal Chemistry 45 (2010) 2919e2927
2921
S
S
to the two RET-independent carcinoma cell lines. The p-unsub-
stituted analogue 13f was clearly inferior to the p-chloro derivative
13g regarding its antiproliferative activity for cancer cell lines. Of
note, the fully optimized RET inhibitor vandetanib (14) exhibited
a more than 100-fold higher antiproliferative potency when
compared with 13g in the proliferation assay with TPC1 cells.
NH2
N
KOH
X
X
R'
S
N
N
S
R'
3.3. Docking experiments
7
8
For a rationalization of the observed structureeactivity rela-
tionships in series 7 and 13, docking studies were carried out
applying the docking program FlexX. As template, the X-ray
structure of a complex of RET with the inhibitor vandetanib (14)
(pdb-file 2ivu [29]) was used. Docking of 7c and 13g as represen-
tatives of highest inhibitory activity from each series produced
poses in which the inhibitors occupy a comparable area in the ATP
binding site of the RET kinase (Fig. 2). In the vandetanib/RET X-ray
structure [29], the quinazoline core element of the inhibitor is
located in the center of the pocket where it is kept in position by
a hydrogen bond to Ala807 of the hinge element. Furthermore,
vandetanib is connected via two water molecules to Ser891 at the
bottom of the pocket by hydrogen bonds. While the N-methyl-
piperidine ring protrudes out of the ATP binding pocket towards
the solvent, the 4-bromo-2-fluorophenyl substituent is buried in
a hydrophobic pocket bordered by Glu775, Leu779, Ile788, Leu802
and Val804 (Fig. 2B). The depicted docking solution of 7c shows its
tetrahydroquinoline ring in the plane occupied by the quinazoline
ring of vandetanib (Fig. 2C). Compound 7c addresses the same
amino acids as vandetanib: The nitrile group of 7c is hydrogen
bonded to Ala807, and a direct hydrogen bond is connecting the
carbonyl group of 7c and the hydroxyl function of Ser891.
Compared to vandetanib, the phenyl substituent is located deeper
in the hydrophobic pocket which explains the observed loss of
activity upon enlargement of this moiety (e.g. 7d and 7e). Similar to
the N-methylpiperidine ring of vandetanib, the thiophene ring of 7c
is directed out of the ATP binding pocket in the depicted docking
solution, suggesting that further molecular modifications should be
possible in this part of the molecule without loss of activity [30].
The depicted docking pose of 13g resembles the orientation of 7c
on first sight (Fig. 2D). However, 13g is not located as deeply in the
binding pocket as 7c, and 13g uses its carbonyl oxygen to address
Ala807 by a hydrogen bond. The depicted docking solution illus-
trates why in this series certain phenyl para substituents are
tolerated (refer to 13g,13h,13i and 13k) and why the ketone moiety
is important for the kinase inhibitory activity (compare with
13aee). On the other hand, the poses fail to explain why
Scheme 2. Possible ZieglereThorpe cyclization in the presence of excess KOH as a side
reaction in the synthesis of target compounds 7.
inhibitory potency reported in this study. Of note, a para-cyano
group (13j) or a meta-chloro substituent (13l) are detrimental for
kinase inhibition, suggesting that also in this series the substitution
pattern at the phenyl ring is important for biological activity
(Table 2).
3.2. Kinase selectivity and antiproliferative activity
To obtain a preliminary impression of the kinase selectivity
profile within this new class of inhibitors, the three derivatives with
highest inhibitory potency (7c, 13f and 13g) were tested in an
analogous ELISA assay for inhibition of ALK (anaplastic lymphoma
kinase) and ABL (Abelson leukemia kinase) (Table 3). In contrast to
1b, the new derivatives 7c, 13f and 13g affected RET with highest
potency. However, a modest selectivity for the inhibition of RET
compared to ALK was observed. In contrast, a selectivity index of
one to two orders of magnitude was found for the RET/ABL
comparison.
To check whether 7c, 13f and 13g are able to penetrate cell
membranes the properties of the three compounds in a cellular
assay were studied (Table 3). When incubated with TPC1, which is
a human papillary thyroid carcinoma cell line growth-dependent
on the constitutively activated RET/PTC1 fusion kinase, both 7c and
13g exhibited clear antiproliferative activity with IC50 values in the
low two-digit micromolar concentration range. Interestingly, 13g
was even more active against the RET-negative carcinoma cell lines
LS174 T (colon) and ARO (thyroid). As a speculative explanation for
this observation, it can be assumed that other cancer-related
kinases besides RET, which are important for the growth of LS174 T
and ARO, are inhibited by 13g as well. Compound 7c showed the
expected selectivity in the cancer cell line assays, since it inhibited
the thyroid carcinoma cell line in lower concentrations compared
S
S
S
9
CHO
N
N
N
N
R
N
(i)
N
(ii)
+
H2N
N
H
S
H2N
N
S
H2N
S
N
11
12
13a-l
10
Scheme 3. Synthesis of 6-(alkylsulfanyl)-2-amino-4-(3-thienyl)pyridine-3,5-dicarbonitriles 13ael. Reagents and conditions: (i) EtOH, piperidine, air, reflux, 3 h; (ii) appropriate
alkyl halide, 1 equiv. KOH, DMF, room temperature, 30 min. For substituents R, refer to Table 2.

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W. Brandt et al. / European Journal of Medicinal Chemistry 45 (2010) 2919e2927
Table 1
Table 2
Structures and in vitro inhibition of RET kinase by 2-(alkylsulfanyl)-4-(3-thienyl)-
Structures and in vitro inhibition of RET kinase by 6-(alkylsulfanyl)-2-amino-4-
5,6,7,8-tetrahydroquinoline-3-carbonitriles 7aee and by the 6-methyl-6-aza-
(3-thienyl)pyridine-3,5-dicarbonitriles 13ael.
analogue 7f.
S
S
N
N
R
N
X
H2N
N
S
R
N
S
13
7a-e (X = CH2)
7f (X = N-CH3)
R
IC50 RET (m
M)a
R
IC50 RET (m
M)a
13a
13b
19 ꢁ 2
7a
7b
8.0 ꢁ 1.1
8.3 ꢁ 2.1
13 ꢁ 1
Cl
10 ꢁ 2
Cl
13c
13d
OCH3
7c
2.3 ꢁ 0.2
O
11 ꢁ 2
CH3
Cl
Cl
7d
7e
8.6 ꢁ 0.5
O
13e
13f
9.2 ꢁ 1.9
3.4 ꢁ 0.2
Cl
H
N
14 ꢁ 4
27 ꢁ 5
O
Cl
O
Cl
7f
Cl
O
a
Results are mean values of at least three experiments ꢁ SEM.
13g
0.69 ꢁ 0.01
O
compounds 13 are superior to analogues of series 7. Presumably,
water molecules which were not considered in the docking routine
are involved in the binding of inhibitors 13, adding favourable
contacts.
OCH3
13h
13i
3.6 ꢁ 0.4
5 ꢁ 0.1
4. Conclusion
O
The molecular modification of the screening hits 1a and 1b
identified two series 7 and 13 as inhibitors of the cancer-related
tyrosine kinase RET. Both novel inhibitor series are structurally
based on a nicotinonitrile partial structure. The most potent
representatives of the series, namely, 7c and 13g, displayed
a modest degree of selectivity against ALK and pronounced
selectivity against ABL. Both compounds inhibited the growth of
TPC1 cancer cells which are dependent on activated RET kinase.
Docking experiments suggested a binding mode comparable to the
structurally unrelated established RET inhibitor vandetanib. The
docking solutions suggest meaningful molecular modifications for
future studies directed to improve RET inhibitors belonging to the
series.
CH3
O
N
13j
39 ꢁ 10
O

W. Brandt et al. / European Journal of Medicinal Chemistry 45 (2010) 2919e2927
2923
Table 2 (continued)
(St. Louis, MO). With the exception of 9, which was purified by
washing with saturated sodium carbonate solution, all starting
materials were used as supplied.
R
IC50 RET (m
M)a
CF3
5.1.2. 2-Cyano-3-(3-thienyl)-2-propenthioamide (3)
Cyanothioacetamide (11) (1.00 mmol, 100 mg) was dissolved in
EtOH (2 mL). Thiophene-3-carbaldehyde (9, 87.7 mL, 1.00 mmol)
13k
3.6 ꢁ 0.6
O
and triethylamine (one drop) were added successively and the
mixture was stirred for 30 min at 40e50 ꢀC. After cooling to room
temperature, the precipitate was collected and crystallized from
EtOH (96%) to yield yellow crystals (59%); mp 151e155 ꢀC (151 ꢀC
[31]); IR (KBr): 3361, 3263 and 3139 cmꢂ1 (NH), 2207 cmꢂ1 (C^N);
13l
26 ꢁ 6
Cl
O
1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 7.80 (d, 2H, J ¼ 2.0 Hz,
a
Results are mean values of at least three experiments ꢁ SEM.
thiophene-H), 8.15 (s, 1H, CH), 8.40 (t, 1H, J ¼ 2.0 Hz, thiophene-H),
9.51 (s, 1H, NH), 10.03 (s, 1H, NH); 13C NMR (DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 131.8, 134.1, 141.0, 146.3 (tert. C); 115.3, 122.0, 139.5, 197.5
(quat. C); Anal (C8H6N2S2) C, H, N.
5. Experimental protocols
5.1. Chemistry
5.1.3. 4-(3-Thienyl)-2-thioxo-1,2,5,6,7,8-hexahydroquinoline-
3-carbonitrile (5)
To a mixture of cyclohexanone (4a) (104 mL, 1.00 mmol), 1,4-
5.1.1. General
dioxane (2 mL) and piperidine (1 drop) 3 (194 mg, 1.00 mmol) was
added in small portions. Before the addition of a new portion of 3,
completeness of the reaction was ensured by tlc monitoring. The
reaction required 5 h overall. After cooling to room temperature,
the precipitate was collected to yield a yellow powder (46%) which
was used for the subsequent syntheses without further purifica-
Melting points (mp) were determined on an electric variable
heater (Electrothermal IA 9100). Infrared spectra were recorded
using KBr pellets on a Thermo Nicolet FT-IR 200 spectrometer.
Nuclear magnetic resonance spectra were recorded on a Bruker
Avance DRX-400 or a Bruker Avance II-600 (Bruker, Billerica, MA,
spectra recorded at NMR laboratories of the Chemical Institutes of
the Technische Universität Braunschweig) using DMSO-d6 as
solvent and tetramethylsilane as internal standard. NMR signals
tion. mp 258e259 ꢀC; 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼
1.56e1.73 (m, 4H, 2ꢃ CH2), 2.20 (t, 2H, J ¼ 6.3 Hz, CH2), 2.77 (t, 2H,
J ¼ 6.3 Hz, CH2), 7.21 (dd, 1H, J ¼ 5.1/1.3 Hz, thiophene-H), 7.74 (dd,
1H, J ¼ 5.1/3.0 Hz, thiophene-H), 7.80 (dd, 1H, J ¼ 3.0/1.3 Hz, thio-
phene-H), 13.91 (br s, 1H, NH).
are reported in ppm on a
d scale. C, H, N analyses were performed
with a CE Instruments FlashEAÒ 1112 Elemental Analyzer (Thermo
Quest, San Jose, CA). Analyses indicated by the symbols of
elements were within ꢁ0.4% of the theoretical values. Mass
spectra were recorded on a Finnigan-MAT 95 instrument (Fin-
nigan-MAT GmbH, Bremen, Germany, performed at department of
mass spectrometry of the Chemical Institutes of the Technische
Universität Braunschweig). The HPLC purity analyses were carried
out using a Merck Hitachi LaChrom Elite system (pump: L-2130,
DAD detector: L-2450; autosampler: L-2200; column: Merck
5.1.4. 6-Methyl-4-(3-thienyl)-2-thioxo-1,2,5,6,7,8-hexahydro[1,6]
naphthyridine-3-carbonitrile (6)
A mixture of 3 (194 mg, 1.00 mmol), 1-methyl-4-piperidone (4b,
116
mL, 1.00 mmol), EtOH (3 mL) and piperidine (1 drop) was
refluxed for 1 h. After cooling to room temperature, the precipitate
was collected and washed with petrol ether to yield a yellow
powder (18%) which was used for the synthesis of 8 without further
purification; mp 235e247 ꢀC (dec.); IR (KBr): 3429 cmꢂ1 (NH),
LiChroCART 125-4, LiChrospher 100 RP-18 (5 mm); eluent:aceto-
nitrile/water mixtures, or acetonitrile/buffer mixtures (prepara-
tion of the buffer: triethylamine (20 mL) and sodium hydroxide
(242 mg) are dissolved in distilled water (1 L). The solution is
adjusted to pH 2.5 by the addition of concentrated sulphuric
acid); elution rate 1.000 mL/min; detection wavelength: 254 and
280 nm; overall run time: 15 min); tM ¼ hold-up time, tN ¼ net
retention time.
2222 cmꢂ1 (C^N); 1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 2.24
(s, 3H, N-CH3), 2.60 (t, 2H, J ¼ 5.7 Hz, CH2), 2.85 (t, 2H, J ¼ 5.8 Hz,
CH2), 3.01 (s, 2H, CH2), 7.24 (dd, 1H, J ¼ 4.9/1.3 Hz, thiophene-H),
7.77 (dd, 1H, J ¼ 4.9/3.0 Hz, thiophene-H), 7.86 (dd, 1H, J ¼ 2.8/
1.3 Hz, thiophene-H), 14.06 (br s, 1H, NH); 13C NMR (DMSO-d6,
150.9 MHz):
d
(ppm) ¼ 27.4 (CH2), 44.9 (N-CH3), 49.5, 53.4 (2ꢃ
CH2); 127.0, 127.4, 127.4 (tert. C); 113.6, 116.4, 118.7, 133.3, 150.0,
151.1, 175.8 (quat. C).
Starting materials 4a, 4b, 9 and 11 were purchased from Acros
Organics (Geel, Belgium); 10 was purchased from Sigma-Aldrich
Table 3
In vitro kinase inhibition and antiproliferative activity of compounds 1a, 1b, 7c, 13f, 13g and the standard vandetanib (14).
Entry
IC50 RET (
m
M)a
IC50 ALK (
m
M)a
IC50 ABL (
m
M)a
IC50 TPC1b
(m
M)a
IC50 LS174 Tc
(m
M)a
IC50 AROd M)a
(m
1a
1b
7c
13f
13g
14
4.8 ꢁ 0.7
1.6 ꢁ 0.4
35 ꢁ 4
>150
>300
>300
>300
>300
92 ꢁ 23
>300
6.2 ꢁ 1.2
NTf
9.0 ꢁ 3.3
>300
1.7 ꢁ 0.3
9.7 ꢁ 1.4
4.0 ꢁ 0.4
2.1 ꢁ 0.7
NTf
0.29 ꢁ 0.02
60 ꢁ 2
74 ꢁ 22
72 ꢁ 8
NTf
2.3 ꢁ 0.2
28 ꢁ 6
101 ꢁ 48
17 ꢁ 1
159 ꢁ 42
3.4 ꢁ 0.2
>300
0.69 ꢁ 0.01
0.097 ꢁ 0.006
8.5 ꢁ 2.0
NTf
0.116 ꢁ 0.037
a
Results are mean values of at least three experiments ꢁ SEM.
b
c
d
f
Human papillary thyroid carcinoma cell line expressing the RET/PCT1 fusion kinase.
RET-negative colon carcinoma cells.
RET-negative thyroid carcinoma cells.
NT: not tested.

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W. Brandt et al. / European Journal of Medicinal Chemistry 45 (2010) 2919e2927
Fig. 2. Inhibitors in the ATP binding pocket of the RET protein kinase. Legend: Upper row: structure of vandetanib 14 (A); vandetanib in the ATP binding site (pdb-file 2ivu [29]) (B);
Lower row: compound 7c (C) and 13g (D) docked to the ATP binding site of RET. A hydrophobic pocket lined by the amino acids Glu775, Leu779, Ile788, Leu802 and Val804 is
depicted in orange colour. Water molecules are depicted as red balls and hydrogen bonds are depicted green.
5.1.5. 6-Amino-4-(3-thienyl)-2-thioxo-1,2-dihydropyridine-3,
5-dicarbonitrile (12)
thiophene-H), 7.23e7.26 (m, 1H, ArH), 7.29e7.33 (m, 2H, ArH),
7.43e7.47 (m, 2H, ArH), 7.74 (dd, 1H, J ¼ 4.8/2.8 Hz, thiophene-H),
7.77 (dd, 1H, J ¼ 2.8/1.3 Hz, thiophene-H); 13C NMR (DMSO-d6,
A mixture of thiophene-3-carbaldehyde (438
malonodinitrile (330 mg, 5.00 mmol), 2-cyanothioacetamide
(501 mg, 5.00 mmol), piperidine (50 L), and EtOH (4 mL) was
mL, 5.00 mmol),
100.6 MHz):
d
(ppm) ¼ 21.6, 21.8, 26.1, 32.8, 33.2 (sec. C); 126.4,
m
127.0 (2 C), 127.8, 128.3 (2 C), 129.1 (2 C) (tert. C); 103.9, 115.2, 127.1,
134.4, 137.8, 149.2, 157.2, 161.1 (quat. C); Anal. (C21H18N2S2) C, H, N;
HPLC: 98.6% at 254 nm and 97.9% at 280 nm, tN ¼ 3.83 min,
tM ¼ 1.08 min (ACN/H2O; 80:20).
refluxed for 3 h. Subsequently, the solvent was evaporated. Acetic
acid (six drops), water (10 mL) and CH2Cl2 (2 mL) were added. After
shaking the mixture for 2 min at room temperature, it was left
standing overnight at 2e5 ꢀC. The precipitate was collected to yield
a yellow powder (47%) which was used for the subsequent
syntheses without further purification. Mp 234e238 ꢀC; IR (KBr):
3477, 3384 and 3324 cmꢂ1 (NH), 2214 cmꢂ1, 2201 cmꢂ1 (C^N); 1H
5.1.6.2. 2-[(4-Chlorobenzyl)sulfanyl]-4-(3-thienyl)-5,6,7,8-tetrahy-
droquinoline-3-carbonitrile (7b). Prepared from 5 and 4-chlor-
obenzylbromide. Light yellow powder (16%); mp 108e109 ꢀC
(EtOH); IR (KBr): 2218 cmꢂ1 (C^N); 1H NMR (DMSO-d6, 600 MHz):
NMR (DMSO-d6, 400 MHz):
600 MHz):
7.45e7.93 (br s, 2H, NH2), 7.74 (dd, 1H, J ¼ 5.1/2.8 Hz, thiophene-H,
superimposed by br s at 7.45e7.93), 8.00 (dd, 1H, J ¼ 2.8/1.3 Hz,
thiophene-H), 12.87 (br s, 1H, NH).
d
(ppm) ¼ 1H NMR (DMSO-d6,
d
(ppm) ¼ 1.64e1.68 (m, 2H, CH2), 1.80e1.84 (m, 2H, CH2), 2.45 (t,
d
(ppm) ¼ 7.34 (dd, 1H, J ¼ 5.1/1.3 Hz, thiophene-H),
2H, J ¼ 6.4 Hz, CH2), 2.97 (t, 2H, J ¼ 6.4 Hz, CH2), 4.49 (s, 2H, CH2),
7.22e7.23 (m, 1H, ArH), 7.36e7.39 (m, 2H, ArH), 7.48e7.50 (m, 2H,
ArH), 7.74e7.75 (m, 1H, ArH), 7.77e7.79 (m, 1H, ArH); 13C NMR
(DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 21.6, 21.8, 26.1, 32.3, 32.8 (sec. C);
5.1.6. General procedure for the synthesis of 7aef 8, and 13ael
Compound 5, 6, or 12 (0.400 mmol), respectively, is dissolved in
DMF (0.5 mL). Ten percent aqueous potassium hydroxide solution
126.6, 127.1, 127.8, 128.2 (2C), 131.0 (2C) (tert. C); 103.9, 115.3, 127.1,
131.6, 134.3, 137.2, 149.2, 156.9, 161.2 (quat. C); Anal. (C21H17ClN2S2)
C, H, N; HPLC: 99.3% at 254 nm and 98.6% at 280 nm, tN ¼ 5.17 min,
tM ¼ 1.06 min (ACN/H2O; 80:20).
(224 mL) is added. After stirring the mixture for 1 min, the appro-
priate alkyl halide is added and stirring is continued for 0.5 h. Water
(5 mL) is added. The precipitate is filtered off and washed succes-
sively with water and petrol ether.
5.1.6.3. 2-[(2-Oxo-2-phenylethyl)sulfanyl]-4-(3-thienyl)-5,6,7,8-tet-
rahydroquinoline-3-carbonitrile (7c). Prepared from
5 and 2-
bromo-1-phenylethanone. Greyegreen powder (88%); mp
5.1.6.1. 2-(Benzylsulfanyl)-4-(3-thienyl)-5,6,7,8-tetrahydroquinoline-
3-carbonitrile (7a). Prepared from 5 and benzylbromide. Greye-
green crystals (48%); mp 132e133 ꢀC (EtOH); IR (KBr): 2218 cmꢂ1
123e124 ꢀC; IR (KBr): 2218 cmꢂ1 (C^N), 1685 cmꢂ1 (C ¼ O); 1H
NMR (CDCl3, 400 MHz):
d
(ppm) ¼ 1.61e1.68 (m, 2H, CH2),
1.71e1.78 (m, 2H, CH2), 2.45 (t, 2H, J ¼ 6.3 Hz, CH2), 2.64 (t, 2H,
J ¼ 6.6 Hz, CH2), 4.64 (s, 2H, CH2), 7.06 (dd, 1H, J ¼ 5.1/1.3 Hz, thio-
phene-H), 7.36 (dd, 1H, J ¼ 3.0/1.3 Hz, thiophene-H), 7.45 (dd, 1H,
J ¼ 5.1/3.0 Hz, thiophene-H), 7.49e7.54 (m, 2H, ArH), 7.60e7.64 (m,
(C^N); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 1.63e1.69 (m, 2H,
CH2),1.79e1.85 (m, 2H, CH2), 2.45 (t, 2H, J ¼ 6.3 Hz, CH2), 2.98 (t, 2H,
J ¼ 6.3 Hz, CH2), 4.51 (s, 2H, CH2), 7.22 (dd, 1H, J ¼ 4.8/1.3 Hz,

W. Brandt et al. / European Journal of Medicinal Chemistry 45 (2010) 2919e2927
2925
1H, ArH), 8.07e8.10 (m, 2H, ArH); 13C NMR (DMSO-d6, 150.9 MHz):
(quat. C); Anal. (C18H12N4S2) C, H, N; HPLC: 99.6% at 254 nm and
d
(ppm) ¼ 20.7, 20.9, 25.3, 31.7, 36.2 (sec. C); 125.8, 126.4, 127.1,
99.4% at 280 nm, tN ¼ 7.35 min, tM ¼ 1.03 min (ACN/H2O; 50:50).
127.4 (2C), 127.9 (2C), 132.6 (tert. C); 102.9, 114.7, 126.3, 133.6, 135.7,
148.3, 156.0, 160.2, 192.9 (quat. C); Anal. (C22H18N2OS2) C, H, N;
HPLC: 99.1% at 254 nm and 98.4% at 280 nm, tN ¼ 4.71 min,
tM ¼ 1.04 min (ACN/H2O; 70:30).
5.1.6.8. 2-Amino-6-[(4-chlorobenzyl)sulfanyl]-4-(3-thienyl)pyridine-
3,5-dicarbonitrile (13b). Prepared from 12 and 4-chlorobenzyl-
bromide. Beige crystals (48%); mp 217e219 ꢀC (EtOH); IR (KBr):
3468, 3352 and 3214 cmꢂ1 (NH), 2210 cmꢂ1 (C^N); 1H NMR
5.1.6.4. 2-{[2-(4-Chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-thienyl)-
(DMSO-d6, 400 MHz):
d
(ppm) ¼ 4.48 (s, 2H, CH2), 7.35e7.38 (m,
5,6,7,8-tetrahydroquinoline-3-carbonitrile (7d). Prepared from
5
3H, ArH, superimposed by thiophene-H), 7.54e7.58 (m, 2H, ArH),
7.76 (dd, 1H, J ¼ 5.1/3.0 Hz, thiophene-H), 8.03 (dd, 1H, J ¼ 3.0/
1.3 Hz, thiophene-H), 8.10 (br s, 2H, NH2, superimposed by dd at
and 2-bromo-1-(4-chlorphenyl)ethanone. Light yellow powder
(66%); mp 179e180 ꢀC (EtOH); IR (KBr): 2216 cmꢂ1 (C^N),
1702 cmꢂ1 (C ¼ O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 1.55-
8.03 ppm); 13C NMR (DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 32.3 (CH2);
1.61 (m, 2H, CH2), 1.65e1.70 (m, 2H, CH2), 2.41 (t, 2H, J ¼ 6.3 Hz,
CH2), 4.80 (s, 2H, CH2), 7.23 (dd, 1H, J ¼ 5.1/1.3 Hz, thiophene-H),
7.66 (m, 2H, ArH), 7.75 (dd, 1H, J ¼ 5.1/2.8 Hz, thiophene-H), 7.80
(dd, 1H, J ¼ 2.8/1.3 Hz, thiophene-H), 8.11 (m, 2H, ArH), 2 H (5,6,7,8-
tetrahydroquinoline) are overlapped by DMSO-Peak; 13C NMR
127.3, 127.7, 128.2 (2C), 128.7, 131.2 (2C) (tert. C); 85.5, 92.9, 115.3,
115.4, 131.8, 133.4, 136.9, 153.1, 159.6, 166.1 (quat. C); Anal.
(C18H11ClN4S2) C, H, N; HPLC: 99.6% at 254 nm and 99.8% at 280 nm,
tN ¼ 4.01 min, tM ¼ 1.03 min (ACN/H2O; 60:40).
(DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 21.4, 21.7, 26.0, 32.4, 36.8 (sec. C);
5.1.6.9. 2-Amino-6-[(4-methoxybenzyl)sulfanyl]-4-(3-thienyl)pyri-
dine-3,5-dicarbonitrile (13c). Prepared from 12 and 4-methox-
ybenzylbromide. Light grey powder (74%); mp 208e209 ꢀC; IR
(KBr): 3439 3348 and 3238 cmꢂ1 (NH), 2212 cmꢂ1 (C^N); 1H NMR
126.6, 127.2, 127.8, 128.8 (2C), 130.1 (2C) (tert. C); 103.6, 115.4, 127.0,
134.3, 135.1, 138.2, 149.1, 156.6, 160.9, 192.9 (quat. C); Anal.
(C22H17ClN2OS2) C, H, N; HPLC: 99.3% at 254 nm and 98.7% at
280 nm, tN ¼ 3.51 min, tM ¼ 1.07 min (ACN/H2O; 75:25).
(DMSO-d6, 400 MHz):
d
(ppm) ¼ 3.73 (s, 3H, OCH3), 4.45 (s, 2H,
CH2), 6.85e6.89 (m, 2H, ArH), 7.37 (dd, 1H, J ¼ 5.1/1.3 Hz, thio-
phene-H), 7.41e7.45 (m, 2H, ArH), 7.76 (dd, 1H, J ¼ 5.1/3.0 Hz,
thiophene-H), 8.03 (dd, 1H, J ¼ 3.0/1.3 Hz, thiophene-H), 8.08 (br s,
2H, NH2, superimposed by dd at 8.03 ppm); 13C NMR (DMSO-d6,
5.1.6.5. N-(4-Chlorophenyl)-2-{[3-cyano-4-(3-thienyl)-5,6,7,8-tet-
rahydroquinolin-2-yl]-sulfanyl}acetamide (7e). Prepared from 5 and
2-chloro-N-(4-chlorphenyl)acetamide. Yellow needles (61%); mp
204e205 ꢀC (EtOH); IR (KBr): 3284 and 3251 cmꢂ1 (NH), 2215 cmꢂ1
(C^N), 1673 cmꢂ1 (C ¼ O); 1H NMR (DMSO-d6, 600 MHz):
100.6 MHz):
d
(ppm) ¼ 32.7 (CH2); 54.9 (OCH3); 113.7 (2 C), 127.3,
127.6, 128.7, 130.5 (2 C) (tert. C); 85.2, 92.7, 115.4, 115.4, 129.2, 133.3,
152.9, 158.4, 159.6, 166.5 (quat. C); Anal. (C19H14N4OS2) C, H, N;
HPLC: 99.2% at 254 nm and 98.7% at 280 nm, tN ¼ 2.50 min,
tM ¼ 1.03 min (ACN/H2O; 60:40).
d
(ppm) ¼ 1.60e1.64 (m, 2H, CH2), 1.72e1.76 (m, 2H, CH2), 2.44 (t,
2H, J ¼ 6.4 Hz, CH2), 2.80 (t, 2H, J ¼ 6.4 Hz, CH2), 4.16 (s, 2H, CH2),
7.25 (dd, 1 H, J ¼ 5.1/1.3 Hz, thiophene-H), 7.38e7.40 (m, 2H, ArH),
7.62-7.64 (m, 2H, ArH), 7.77 (dd, 1H, J ¼ 5.1/2.8 Hz, thiophene-H),
7.81 (dd, 1H, J ¼ 2.8/1.3 Hz, thiophene-H), 10.49 (s, 1H, NH); 13C
5.1.6.10. 2-Amino-6-[(4-methylbenzyl)sulfanyl]-4-(3-thienyl)pyridine-
3,5-dicarbonitrile (13d). Prepared from 12 and 4-methylbenzyl-
bromide. Beige needles (46%); mp 204e206 ꢀC (EtOH); IR (KBr):
3459, 3339 and 3227 cmꢂ1 (NH), 2218 cmꢂ1 and 2208 cmꢂ1 (C^N);
NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 21.5, 21.7, 26.0, 32.8, 34.8
(sec. C); 120.4 (2C), 126.6, 127.1, 127.8, 128.6 (2C) (tert. C); 103.6,
115.3, 126.7, 127.0, 134.3, 137.9, 149.0, 157.1, 161.0, 166.2 (quat. C);
Anal. (C22H18ClN3OS2) C, H, N; HPLC: 98.9% at 254 nm and 98.5% at
280 nm, tN ¼ 3.71 min, tM ¼ 1.03 min (ACN/H2O; 70:30).
1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 2.27 (s, 3H, CH3), 4.45 (s,
2H, CH2), 7.12 (d, 2H, J ¼ 7.8 Hz, ArH), 7.36 (dd, 1H, J ¼ 5.1/1.3 Hz,
thiophene-H), 7.38 (d, 2 H, J ¼ 7.8 Hz, ArH, superimposed by dd at
7.36 ppm), 7.76 (dd, 1H, J ¼ 5.1/3.0 Hz, thiophene-H), 8.03 (dd, 1H,
J ¼ 3.0/1.3 Hz, thiophene-H), 8.06 (br s, 2H, NH2, superimposed by
5.1.6.6. 2-{[2-(4-Chlorophenyl)-2-oxoethyl]sulfanyl}-6-methyl-4-(3-
thienyl)-5,6,7,8-tetrahydro[1,6]naphthyridine-3-carbonitrile
(7f).
Prepared from
6
and 2-bromo-1-(4-chlorophenyl)ethanone.
dd at 8.03 ppm); 13C NMR (DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 20.7
Orange powder (60%); mp 194e195 ꢀC (EtOH); IR (KBr): 2212 cmꢂ1
(C^N), 1671 cmꢂ1 (C ¼ O); 1H NMR (DMSO-d6, 600 MHz):
(CH3); 33.0 (CH2); 127.3,127.7,128.7,128.9 (2 C),129.2 (2 C) (tert. C);
85.4, 92.9, 115.4, 115.4, 133.4, 134.3, 136.5, 153.0, 159.6, 166.5 (quat.
C); Anal. (C19H14N4S2) C, H, N; HPLC: 98.7% at 254 nm and 99.0% at
280 nm, tN ¼ 3.77 min, tM ¼ 1.04 min (ACN/H2O; 60:40).
d
(ppm) ¼ 2.25 (s, 3H, CH3), 2.60 (s, 4H, 2ꢃ CH2), 3.23 (s, 2H, CH2),
4.83 (s, 2H, CH2), 7.28 (dd, 1H, J ¼ 4.9/1.3 Hz, thiophene-H),
7.65e7.67 (m, 2H, ArH), 7.79 (dd, 1H, J ¼ 4.9/2.8 Hz, thiophene-H),
7.88 (dd, 1H, J ¼ 2.8/1.3 Hz, thiophene-H), 8.10e8.12 (m, 2H, ArH);
5.1.6.11. 2-Amino-6-[(3,4-dichlorobenzyl)sulfanyl]-4-(3-thienyl)pyri-
dine-3,5-dicarbonitrile (13e). Prepared from 12 and 3,4-dichlor-
obenzylbromide. Grey powder (52%); mp 215e218 ꢀC (EtOH); IR
(KBr): 3469, 3348 and 3223 cmꢂ1 (NH), 2214 cmꢂ1 (C^N); 1H NMR
13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 32.1, 36.9, 50.9, 54.2
(sec. C); 45.1 (CH3); 127.0,127.5,127.7,128.8 (2C),130.1 (2C) (tert. C);
103.5, 115.2, 124.7, 133.2, 135.0, 138.2, 147.2, 157.8, 158.4, 192.8 (quat.
C); Anal. (C22H18ClN3OS2) C, H, N; HPLC: 99.4% at 254 nm and 99.4%
at 280 nm, tN ¼ 2.06 min, tM ¼ 1.03 min (ACN/buffer; 40:60).
(DMSO-d6, 400 MHz):
d
(ppm) ¼ 4.48 (s, 2H, CH2), 7.37 (dd, 1H,
J ¼ 5.1/1.5 Hz, thiophene-H), 7.53e7.58 (m, 2H, ArH), 7.76 (dd, 1H,
J ¼ 5.1/3.0 Hz, thiophene-H), 7.86 (d, 1H, J ¼ 1.3 Hz, ArH), 8.03 (dd,
1H, J ¼ 3.0/1.5 Hz, thiophene-H), 8.11 (br s, 2H, NH2, superimposed
by dd at 7.76 ppm, d at 7.86 ppm and dd at 8.03 ppm); 13C NMR
5.1.6.7. 2-Amino-6-(benzylsulfanyl)-4-(3-thienyl)pyridine-3,5-dicar-
bonitrile (13a). Prepared from 12 and benzylbromide. Light beige
powder (40%); mp 207e208 ꢀC (EtOH); IR (KBr): 3440, 3326 and
3216 cmꢂ1 (NH), 2219 cmꢂ1, 2207 cmꢂ1 (C^N); 1H NMR (DMSO-d6,
(DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 31.6 (CH2); 127.3, 127.6, 128.7,
129.8, 130.3, 131.3 (tert. C); 85.5, 92.7, 115.3, 115.3, 129.7, 130.6,
133.3, 139.3, 153.0, 159.5, 165.7 (quat. C); Anal. (C18H10Cl2N4S2) C, H,
N; HPLC: 98.2% at 254 nm and 98.4% at 280 nm, tN ¼ 5.78 min,
tM ¼ 1.04 min (ACN/H2O; 60:40).
400 MHz):
d
(ppm) ¼ 4.50 (s, 2H, CH2), 7.23e7.28 (m, 1H, ArH),
7.30e7.34 (m, 2H, ArH), 7.37 (dd, 1H, J ¼ 5.1/1.5 Hz, thiophene-H),
7.49e7.52 (m, 2H, ArH), 7.76 (dd, 1H, J ¼ 5.1/2.8 Hz, thiophene-H),
8.03 (dd, 1H, J ¼ 2.8/1.5 Hz, thiophene-H), 8.10 (br s, 2H, NH2,
superimposed by dd at 8.03 ppm); 13C NMR (DMSO-d6,150.9 MHz):
5.1.6.12. 2-Amino-6-[(2-oxo-2-phenylethyl)sulfanyl]-4-(3-thienyl)pyri-
dine-3,5-dicarbonitrile (13f). Prepared from 12 and 2-bromo-1-
phenylethanone. Light grey powder (65%); mp 265e268 ꢀC (EtOH);
d
(ppm) ¼ 33.0 (CH2); 127.2,127.3,127.6,128.3 (2C),128.7,129.3 (2C)
(tert. C); 85.3, 92.7, 115.3, 115.4, 133.3, 137.5, 153.0, 159.6, 166.3

2926
W. Brandt et al. / European Journal of Medicinal Chemistry 45 (2010) 2919e2927
IR (KBr): 3471, 3340 and 3212 cmꢂ1 (NH), 2210 cmꢂ1 (C^N),
superimposed by dd at 7.79 and m at 8.06e8.08), 8.06e8.08 (m, 3H,
ArH), 8.20e8.22 (m, 2H, ArH); 13C NMR (DMSO-d6, 150.9 MHz):
1676 cmꢂ1 (C ¼ O); 1H NMR (DMSO-d6, 400 MHz):
d
(ppm) ¼ 5.01
(s, 2H, CH2), 7.41 (dd,1H, J ¼ 5.1/1.3 Hz, thiophene-H), 7.57e7.61 (m,
2H, ArH), 7.69e7.73 (m, 1H, ArH), 7.79 (dd, 1H, J ¼ 5.1/3.0 Hz, thio-
phene-H), 7.92 (br s, 2H, NH2), 8.07e8.09 (m, 3H, ArH); 13C NMR
d
(ppm) ¼ 38.1 (CH2); 127.4, 127.6, 128.8, 128.9 (2C), 132.7 (2C) (tert.
C); 85.5, 92.8, 115.2, 115.4, 115.4, 118.1, 133.2, 138.8, 152.9, 159.5,
165.7, 192.1 (quat. C); Anal. (C20H11N5OS2) C, H, N; HPLC: 99.3 at
254 nm and 99.3 at 280 nm, tN ¼ 3.99, tM ¼ 1.02 min (ACN/H2O;
50:50).
(DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 37.5 (CH2); 126.9, 127.2, 127.9
(2C), 128.3 (2C), 128.4, 133.2 (tert. C); 84.9, 92.4, 114.8, 115.0, 132.8,
135.0, 152.4, 159.1, 165.5, 192.2 (quat. C); Anal. (C19H12N4OS2) C, H,
N; HPLC: 99.4% at 254 nm and 98.9% at 280 nm, tN ¼ 2.87 min,
tM ¼ 1.03 min (ACN/H2O; 55:45).
5.1.6.17. 2-Amino-6-({2-oxo-2-[4-(trifluoromethyl)phenyl]ethyl}sul-
fanyl)-4-(3-thienyl)pyridine-3,5-dicarbonitrile (13k). Prepared from
12 and 2-bromo-1-[4-(trifluoromethyl)phenyl]ethanone. Light
yellow crystals (50%); mp 236e237 ꢀC (EtOH); IR (KBr): 3409, 3323
and 3231 cmꢂ1 (NH), 2217 cmꢂ1 (C^N), 1697 cmꢂ1 (C ¼ O); 1H
5.1.6.13. 2-Amino-6-{[2-(4-chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-
thienyl)pyridine-3,5-dicarbonitrile (13g). Prepared from 12 and 2-
bromo-1-(4-chlorophenyl)ethanone. Light grey powder (57%); mp
243e244 ꢀC (EtOH); IR (KBr): 3438, 3334 and 3224 cmꢂ1 (NH),
2208 cmꢂ1 (C^N), 1687 cmꢂ1 (C ¼ O); 1H NMR (DMSO-d6,
NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 5.02 (s, 2H, CH2), 7.41 (dd,
1H, J ¼ 5.0/1.3 Hz, thiophene-H), 7.79 (dd, 1H, J ¼ 5.0/3.0 Hz, thio-
phene-H), 7.90 (br s, 2H, NH2, superimposed by dd at 7.79, d at 7.96
and dd at 8.08), 7.96 (d, 2H, J ¼ 8.1 Hz, ArH), 8.08 (dd, 1H, J ¼ 3.0/
400 MHz):
d
(ppm) ¼ 4.97 (s, 2H, CH2), 7.41 (dd, 1H, J ¼ 5.1/1.3 Hz,
thiophene-H), 7.64e7.67 (m, 2H, ArH), 7.78 (dd, 1H, J ¼ 5.1/3.0 Hz,
1.3 Hz, thiophene-H), 8.27 (d, 2H, J ¼ 8.1 Hz, ArH); 13C NMR (DMSO-
thiophene-H), 7.90 (br s, 2H, NH2), 8.07e8.10 (m, 3H, ArH); 13C NMR
3
d6, 150.9 MHz):
d
(ppm) ¼ 38.1 (CH2); 125.7 (q, 2 C, JC,F ¼ 3.6 Hz),
(DMSO-d6, 100.6 MHz):
d
(ppm) ¼ 37.9 (CH2); 127.4, 127.7, 128.9,
127.4, 127.6, 128.8, 129.2 (2 C) (tert. C); 85.5, 92.8, 115.2, 115.4, 123.7
(q, 1JC,F ¼ 273 Hz), 132.7 (q, 2JC,F ¼ 31.8 Hz), 133.3, 138.8, 152.9, 159.5,
165.7, 192.1 (quat. C); Anal. (C20H11F3N4OS2); HPLC: 99.9 at 254 nm
and 99.9 at 280 nm, tN ¼ 5.37, tM ¼ 1.01 min (ACN/H2O; 55:45).
128.9 (2C), 130.3 (2C) (tert. C); 85.6, 92.9, 115.3, 115.5, 133.3, 134.3,
138.6, 153.0, 159.6, 165.9, 191.8 (quat. C); Anal (C19H11ClN4OS2) C, H,
N; HPLC: 99.2% at 254 nm and 98.6% at 280 nm, tN ¼ 4.66 min,
tM ¼ 1.03 min (ACN/H2O; 55:45).
5.1.6.18. 2-Amino-6-{[2-(3-chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-
thienyl)pyridine-3,5-dicarbonitrile (13l). Prepared from 12 and 2-
bromo-1-(3-chlorphenyl)ethanone. Light yellow crystals (60%); mp
224e225 ꢀC (EtOH); IR (KBr): 3471 and 3353 cmꢂ1 (NH), 2210 cmꢂ1
(C^N), 1685 cmꢂ1 (C]O); 1H NMR (DMSO-d6, 600 MHz):
5.1.6.14. 2-Amino-6-{[2-(4-methoxyphenyl)-2-oxoethyl]sulfanyl}-4-
(3-thienyl)pyridine-3,5-dicarbonitrile (13h). Prepared from 12 and
2-bromo-1-(4-methoxyphenyl)ethanone. Light grey powder (71%);
mp 250e252 ꢀC (EtOH); IR (KBr): 3390, 3321 and 3217 cmꢂ1 (NH),
2210 cmꢂ1 (C^N), 1672 cmꢂ1 (C ¼ O); 1H NMR (DMSO-d6,
d
(ppm) ¼ 4.99 (s, 2H, CH2), 7.41 (dd, 1H, J ¼ 5.1/1.3 Hz, thiophene-
600 MHz):
d
(ppm) ¼ 3.87 (s, 3H, OCH3), 4.95 (s, 2H, CH2), 7.09e7.10
H), 7.63 (t, 1H, J ¼ 7.9 Hz, ArH), 7.77e7.79 (m, 1H, ArH), 7.79 (dd, 1H,
J ¼ 5.1/3.0 Hz, thiophene-H, superimposed by m at 7.77e7.79), 7.93
(brs, 2H, NH2), 8.02e8.04 (m, 1H, ArH), 8.08 (t, 1H, J ¼ 1.7 Hz, ArH),
8.09 (dd, 1H, J ¼ 3.0/1.3 Hz, thiophene-H); 13C NMR (DMSO-d6,
(m, 2H, ArH), 7.41 (dd, 1H, J ¼ 5.1/1.3 Hz, thiophene-H), 7.79 (dd, 1H,
J ¼ 5.1/3.0 Hz, thiophene-H), 7.93 (br s, 2H, NH2, superimposed by
dd at 7.79, m at 8.05e8.06 and dd at 8.08), 8.05e8.06 (m, 2H, ArH),
8.08 (dd, 1H, J ¼ 3.0/1.3 Hz, thiophene-H); 13C NMR (DMSO-d6,
150.9 MHz):
d
(ppm) ¼ 38.0 (CH2); 127.0, 127.4, 127.7, 128.0, 128.8,
150.9 MHz):
d
(ppm) ¼ 37.5 (CH2); 55.5 (OCH3); 113.9 (2 C), 127.3,
130.7, 133.3 (tert. C); 85.4, 92.8, 115.3, 115.5, 133.3, 133.6, 137.3,
152.9, 159.5, 165.8, 191.6 (quat. C); Anal. (C19H11ClN4OS2) C, H, N;
HPLC: 99.7 at 254 nm and 99.7 at 280 nm, tN ¼ 4.48, tM ¼ 1.01 min
(ACN/H2O; 55:45).
127.6, 128.8, 130.8 (2C) (tert. C); 85.4, 92.9, 115.3, 115.5, 128.3, 133.3,
152.9, 159.5, 163.5, 166.1, 191.0 (quat. C); Anal. (C20H14N4O2S2) C, H,
N; HPLC: 99.7% at 254 nm and 99.7% at 280 nm, tN ¼ 4.83 min,
tM ¼ 1.01 min (ACN/H2O; 50:50).
5.1.6.15. 2-Amino-6-{[2-(4-methylphenyl)-2-oxoethyl]sulfanyl}-4-
(3-thienyl)pyridine-3,5-dicarbonitrile (13i). Prepared from 12 and
2-bromo-1-(4-methylphenyl)ethanone. Light yellow needlꢂes1
(70%); mp 234e235 ꢀC (EtOH); IR (KBr): 3423, 3325 and 3221 cm
(NH), 2210 cmꢂ1 (C^N), 1681 cmꢂ1 (C ¼ O); 1H NMR (DMSO-d6,
5.2. Biological assays
5.2.1. In vitro kinase assays
Recombinant kinase domains of human RET (aa 700e1020,
NP_066124), ABL (aa 230e517, isoform a, NP_005148) and ALK (aa
1103e1459, NP_004295) were expressed in Sf9 cells with an amino
terminal histidine tag and purified as described [28].
The ELISA kinase assay was performed as described [28] using
approximately 1 pmol recombinant enzyme and 1 nmol peptide
substrate. In brief, the enzyme was incubated in reaction buffer
(25 mM Hepes pH 7, 1 mM MnCl2, 5 mM MgCl2) with inhibitors and
600 MHz):
d
(ppm) ¼ 2.41 (s, 3H, CH3), 4.97 (s, 2H, CH2), 7.38e7.40
(m, 2H, ArH), 7.41 (dd, 1H, J ¼ 5.1/1.5 Hz, thiophene-H, super-
imposed by m at 7.38e7.40), 7.79 (dd, 1H, J ¼ 5.1/3.0 Hz, thiophene-
H), 7.91 (br s, 2H, NH2, superimposed by dd at 7.79, m at 7.97e7.99
and dd at 8.08), 7.97e7.99 (m, 2H, ArH), 8.08 (dd, 1H, J ¼ 3.0/1.5 Hz,
thiophene-H); 13C NMR (DMSO-d6, 150.9 MHz):
d
(ppm) ¼ 21.1
(CH3); 37.7 (CH2); 127.3, 127.6, 128.5 (2 C), 128.8, 129.3 (2 C) (tert.
C); 85.4, 92.9, 115.3, 115.5, 133.0, 133.3, 144.1, 152.9, 159.5, 166.0,
192.2 (quat. C); Anal. (C20H14N4OS2) C, H, N; HPLC: 99.8% at 254 nm
and 99.8% at 280 nm, tN ¼ 3.77 min, tM ¼ 1.02 min (ACN/H2O;
55:45).
300 mM ATP, in a 96-well plate pre-coated with peptide substrate,
for 15 min at 30 ꢀC. After five washes with wash buffer (0.05%
Tween in PBS), the plate was incubated with anti-phosphotyrosine
primary antibody (clone 4G10, Millipore) and HRP-conjugated anti-
mouse secondary antibody. A colorimetric signal was developed by
addition of TMB substrate (Pierce) and stopped with 0.1 M H2SO4.
Absorbance was read at 450 nm with a 96-well microplate reader
(Bio-Rad). As a substrate, an ALK activation loop-derived peptide
was used [32]. Each data point was done in duplicate and experi-
ments were repeated at least three times.
5.1.6.16. 2-Amino-6-{[2-(4-cyanophenyl)-2-oxoethyl]sulfanyl}-4-(3-
thienyl)pyridine-3,5-dicarbonitrile (13j). Prepared from 12 and 4-
(bromoacetyl)benzonitrile. Light yellow powder (65%); mp 264 ꢀC
(dec.; EtOH); IR (KBr): 3434, 3330 and 3213 cmꢂ1 (NH), 2213 cmꢂ1
(C^N), 1695 cmꢂ1 (C ¼ O); 1H NMR (DMSO-d6, 600 MHz):
d
(ppm) ¼ 5.00 (s, 2H, CH2), 7.41 (dd, 1H, J ¼ 4.9/1.3 Hz, thiophene-
The IC50 values were calculated as the inhibitor concentrations
that caused 50% inhibition of the kinase activity.
H), 7.79 (dd, 1H, J ¼ 4.9/2.8 Hz, thiophene-H), 7.89 (br s, 2H, NH2,

W. Brandt et al. / European Journal of Medicinal Chemistry 45 (2010) 2919e2927
2927
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5.2.2. Cell growth assay
TPC1 is a human papillary thyroid carcinoma cell line expressing
the RET/PTC1 fusion kinase. These cells were grown in DMEM
medium supplemented with 10% fetal calf serum, 100 U/mL peni-
cillin, 100 mg/mL gentamicin, and 2 mM glutamine. RET-negative
colon carcinoma cells LS174 T were cultured in RPMI-1640 with 10%
serum and antibiotics.
Cell proliferation was measured using the tritiated thymidine
(3H-T) incorporation assay. Serial dilutions of the inhibitors were
prepared directly in 96-well plates in DMSO as 100ꢃ concentrated.
The cells (104/well) were then added to the wells and incubated for
72 h. The cells were pulsed with 3H-T (1
mCi/well) during the last
8 h of culture and then harvested onto glass fiber filter paper. Cell-
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5.3. Docking experiments
Compounds 7c and 13g were docked into the ATP binding site of
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Augustin, Germany). The active site was defined as all residues
surrounding the original ligand to about 6.5 Å. The ligands and all
water molecules were deleted from the experimental protein
structure prior to docking. Compounds 7c and 13g were con-
structed in SYBYL (SYBYL 8, Tripos International, 1699 South Hanley
Rd., St. Louis, MO, 63144, USA), AM1 charges were calculated and
forcefield minimisations were done using the Tripos forcefield.
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except the usage of the SIS mode of FlexX for base fragment
placement. Fifty poses were saved for each docking run and clus-
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Funding of the project by the European Commission (Contract
No LSHB-CT-2004-503467, Protein Kinase Research, to L. M., C. G.-P.
and C. K.) is gratefully acknowledged.
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