New sorafenib derivatives: Synthesis, antiproliferative activity against tumour cell lines and antimetabolic evaluation

Article abstract of DOI:10.3390/molecules17011124

Sorafenib is a relatively new cytostatic drug approved for the treatment of renal cell and hepatocellular carcinoma. In this report we describe the synthesis of sorafenib derivatives 4a-e which differ from sorafenib in their amide part. A 4-step synthetic

Full text of DOI:10.3390/molecules17011124

Molecules 2012, 17, 1124-1137; doi:10.3390/molecules17011124
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New Sorafenib Derivatives: Synthesis, Antiproliferative Activity
Against Tumour Cell Lines and Antimetabolic Evaluation
Željka Babić 1, Maja Crkvenčić 1, Zrinka Rajić 1, Ana-Matea Mikecin 2, Marijeta Kralj 2,
Jan Balzarini 3, Mariya Petrova 4, Jos Vanderleyden 4 and Branka Zorc 1,*
1
Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, HR-10000 Zagreb,
Croatia; E-Mails: zeljka.babic@sk.t-com.hr (Ž.B.); maja.crkvencic@kr.t-com.hr (M.C.);
zrajic@pharma.hr (Z.R.)
2
Division of Molecular Medicine, Rudjer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb,
Croatia; E-Mails: amikecin@irb.hr (A.M.M.); marijeta.kralj@irb.hr (M.K.)
3
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10,
B-3000 Leuven, Belgium; E-Mail: Jan.Balzarini@rega.kuleuven.be
4
Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20,
Bus 2300, B-3001 Heverlee, Belgium; E-Mails: Mariya.Petrova@biw.kuleuven.be (M.P.);
Jozef.Vanderleyden@biw.kuleuven.be (J.V.)
* Author to whom correspondence should be addressed; E-Mail: bzbz@pharma.hr;
Tel.: +385-148-562-02; Fax: +385-148-562-01.
Received: 26 November 2011; in revised form: 1 January 2012 / Accepted: 5 January 2012 /
Published: 23 January 2012
Abstract: Sorafenib is a relatively new cytostatic drug approved for the treatment of renal
cell and hepatocellular carcinoma. In this report we describe the synthesis of sorafenib
derivatives 4a–e which differ from sorafenib in their amide part. A 4-step synthetic pathway
includes preparation of 4-chloropyridine-2-carbonyl chloride hydrochloride (1), 4-chloro-
pyridine-2-carboxamides 2a–e, 4-(4-aminophenoxy)-pyridine-2-carboxamides 3a–e and the
target compounds 4-4-4-chloro-3-(trifluoromethyl)phenylcarbamoylamino-phenoxy-
pyridine-2-carboxamides 4a–e. All compounds were fully chemically characterized and
evaluated for their cytostatic activity against a panel of carcinoma, lymphoma and leukemia
tumour cell lines. In addition, their antimetabolic potential was investigated as well. The most
prominent antiproliferative activity was obtained for compounds 4a–e (IC50 = 14.3 μmol·L−1).
Their potency was comparable to the potency of sorafenib, or even better. The compounds
inhibited DNA, RNA and protein synthesis to a similar extent and did not discriminate
between tumour cell lines and primary fibroblasts in terms of their anti-proliferative activity.

Molecules 2012, 17
Keywords: sorafenib; amides; cytostatic activity; antimetabolic activity; Caco-2 cells
1125
permeability
1. Introduction
Sorafenib, 4-4-4-chloro-3-(trifluoromethyl)phenylcarbamoylaminophenoxy-N-methylpyridine-
2-carboxamide, is an oral multikinase inhibitor that inhibits cell surface tyrosine kinase receptors
(e.g., vascular endothelial growth factor receptors and platelet-derived growth factor receptor-beta) and
downstream intracellular serine/threonine kinases (e.g., Raf-1, wild-type B-Raf and mutant B-Raf);
these kinases are involved in tumour cell proliferation and tumour angiogenesis 1–3. Sorafenib is
approved for the treatment of advanced renal cell carcinoma and hepatocellular carcinoma 4,5.
Clinical trials to use sorafenib for non-responsive thyroid cancer and glioblastoma are in progress as
well. However, both median survival and time to progression in sorafenib therapy showed only a
3-month improvement in patients who received sorafenib compared to placebo 6,7. These facts point
to sorafenib as an interesting lead compound for further derivatization in order to find a more effective
drug. The present study is focused on the synthesis of new sorafenib derivatives bearing the same
diarylurea moiety as sorafenib and differing from the parent drug in the amide part. The newly
prepared compounds are more lipophilic in an attempt to increase uptake and/or accumulation of the
drug in tissues 8. Here we report their synthesis and in vitro evaluation of their cytostatic activity
against several carcinoma cell lines.
2. Results and Discussion
2.1. Chemistry
A four step synthesis of sorafenib amide analogues 4a–e is described. Scheme 1 outlines the general
preparative route. The starting material, picolinic acid, was first converted to the acid chloride using
thionyl chloride 9. Under the reaction conditions employed (72 °C, 16 h, nitrogen atmosphere),
chlorination of the pyridine ring at the para-position occurred as well and the final product was
4-chloropyridine-2-carbonyl chloride hydrochloride (1). Amidation of the acid chloride 1 with five
different amines (cyclopentylamine, cyclohexylamine, cyclohexylmethylamine, benzylamine and
phenylethylamine) gave amides 2a–e. The amidation step was performed at room temperature in the
presence of triethylamine as HCl acceptor. The selected amines were chosen on the basis of their
lipophilicity and limited number of possible conformations, providing more lipohilic and rigid final
compounds 4. The reactions of acid chloride 1 with the title amines gave the amides in good yields.
Amide 2c was also prepared from picolinic acid methyl ester (methyl 4-chloropyridine-2-carboxylate)
and cyclohexylmethylamine. However, analogous reactions with cyclopentylamine and cyclohexyl-
amine afforded the corresponding amides in poor yields.

Molecules 2012, 17
1126
Scheme 1. Synthesis of sorafenib derivatives 4a–e.
Cl
Cl
O
3
c
CONHR
(i)
(ii)
(iii)
N
N
COOH
N
COCl
N
CONHR
H2N
1
2
CF3
Cl
O
CONHR
(iv)
O
a
b
d
e
N
N
N
R
H
H
4
Reagents and conditions: (i) SOCl2, DMF, N2 atmosphere, 72 C, 16 h; (ii) amine, TEA, toluene, rt,
0.5 h; (iii); 4-aminophenol, t-BuOK, DMF, K2CO3, DMF, 10 min, 173 C, microwaves;
(iv) 4-chloro-3-(fluoromethyl)phenyl isocyanate, CH2Cl2, N2 atmosphere, rt, 16 h.
In the next reaction step products 2a–e were coupled with 4-aminophenol to give the ethers 3a–e.
The ether side chain was introduced using potassium tert-butoxide in the presence of potassium
carbonate (2 h, 80 C). Under basic conditions employed, the alkoxide derived from 4-aminophenol
was a stronger nucleophile than the amino group, therefore the main product was the ether, not the
secondary amine. The reaction time was shortened to 10 min when the ether formation reactions were
run in a microwave reactor (P = 150 W, t = 173 °C). Ethers 3a–e readily reacted with isocyanate to
give the final products 4a–e. Urea bond formation was performed at room temperature, in nitrogen
atmosphere, using a slight excess of 4-chloro-3-(fluoromethyl)phenyl isocyanate.
All intermediates and final amides 4a–e were isolated and fully characterized by elemental analysis
1
13
and usual spectroscopic methods (IR, H-, C-NMR and MS). The spectral data are consistent with the
proposed structures (Table 1).
Table 1. 1H and 13C-NMR spectra of compounds 2a–e, 3a–e, and 4a–e.
13
15
H
N
H
N
11
11
21
13
H2N
14
12
20
19
12
10
10
16
17
Cl
N
O
9
9
7
5
O
Cl
O
7
8
18
6
4
3
8
5
5
1´
NH
CF
223
2
4
3
6
2
1
R
4
6
2
1´
NH R
1
1
NH
R
3
N
O
N
O
O
2a-e
3a-e
4a-e
Compd.
2a
R
1H and 13C-NMR spectra/δ ppm, J/Hz
8.62 (d, 1H, 3, J = 5.4), 8.58 (d, 1H, 1', J = 7.6), 8.02 (d, 1H, 6, J = 1.8), 7.75 (dd,
1H, 4, J = 2.1, J = 3.2), 4.31–4.19 (m, 1H, 2'), 1.94–1.53 (m, 8H, 3'–6')
161.26 (1), 151.02 (2), 149.02 (3), 143.61 (5), 125.36 (6), 120.98 (4), 49.82 (2'),
31.12 (3', 6'), 22.62 (4', 5')
3´
5´
2´
6´
4´

Molecules 2012, 17
1127
Table 1. Cont.
1H and 13C-NMR spectra/δ ppm, J/Hz
Compd.
2b
R
8.61 (d, 1H, 3, J = 5.2), 8.48 (d, 1H, 1', J = 8.1), 8.01 (d, 1H, 6, J = 1.9), 7.74 (dd,
1H, 4, J = 1.9, J = 3.2), 3.80–3.74 (m, 1H, 2'), 1.79–1.16 (m, 10H, 3'–7')
161.54 (1), 151.90 (2), 149.91 (3), 144.53 (5), 126.28 (6), 121.90 (4), 48.13 (2'),
32.03 (3', 7'), 25.03 (5'), 24.73 (4', 6')
3´
6´
2´
4´
5´
7´
8.82 (t, 1H, 1', J = 8.8), 8.62 (d, 1H, 3, J = 8.6), 8.02 (d, 1H, 6, J = 1.8), 7.76 (dd,
1H, 4, J = 2.1, J = 3.1), 3.16 (t, 2H, 2', J = 6.6), 1.68–0.97 (m, 11H, 3'–8')
163.08 (1), 152.37 (2), 150.46 (3), 145.00 (5), 126.76 (6), 122.39 (4), 45.52 (2'),
30.86 (4', 8'), 26.50 (6'), 25.81 (5', 7')
5´
8´
6´
7´
4´
3´
2´
2c
2d
2e
9.40 (t, 1H, 1', J = 6.2), 8.62 (d, 1H, 3, J = 5.5), 8.01 (d, 1H, 6, J = 2.0), 7.77 (dd,
1H, 4, J = 2.1, J = 3.3), 7.33–7.21 (m, 5H, arom. 4'–8'), 4.50 (d, 2H, 2', J = 6.1)
163.26 (1), 152.23 (2), 150.57 (3), 145.04 (5), 139.76 (3'), 128.72 (4', 8'), 127.83 (5', 7'),
126.94 (6'), 125.36 (6), 120.98 (4), 42.98 (2')
5´
6´
7´
4´
3´
8´
2´
8.88 (t, 1H, 1', J = 5.6), 8.61 (d, 1H, 3, J = 5.0), 8.01 (d, 1H, 6, J = 1.8), 7.73 (dd,
1H, 4, J = 2.1, J = 3.2), 3.55–3.52 (q, 2H, 2', J = 7.0), 2.86 (t, 2H, 3', J = 7.5)
162.50 (1), 151.73 (2), 149.98 (3), 144.52 (5), 139.24 (4'), 128.54 (5', 9'), 128.31 (6', 8'),
126.33 (6), 126.08 (7'), 121.86 (4), 40.44 (2'), 34.97 (3')
3´
5´
4´
6´
7´
2´
9´
8´
8.46 (d, 1H, 3, J = 2.5), 8.44 (d, 1H, 1', J = 8.5), 7.34 (d, 1H, 6, J = 2.4), 7.08 (dd,
1H, 4, J = 2.6, J = 2.9), 6.85 (d, 2H, 8, 12, J = 8.9), 6.66 (d, 2H, 9, 11, J = 8.9), 5.16
(s, 2H, 13), 4.22–4.15 (m, 1H, 2'), 1.99–1.53 (m, 8H, 3'–6')
3´
2´
6´
4´
3a
3b
5´
167.30 (5), 163.31 (1), 152.74 (2), 150.51 (3), 147.32 (7), 143.37 (10), 121.99 (8, 12),
115.36 (9, 11), 114.23 (6), 108.86 (4), 51.04 (2'), 32.56 (3', 6'), 23.95 (4', 5')
8.45 (d, 1H, 3, J = 5.7), 8.37 (d, 1H, 1', J = 8.5), 7.34 (d, 1H, 6, J = 2.4), 7.08 (dd,
1H, 4, J = 2.6, J = 2.9), 6.85 (d, 2H, 8, 12, J = 8.9), 6.65 (d, 2H, 9, 11, J = 8.9), 5.17
(s, 2H, 13), 3.77–3.68 (m, 1H, 2'), 1.78–1.04 (m, 10H, 3'–7')
3´
6´
2´
4´
5´
7´
167.31 (5), 162.70 (1), 152.77 (2), 150.52 (3), 147.30 (7), 143.38 (10), 121.98 (8, 12),
115.37 (9, 11), 114.26 (6), 108.92 (4), 48.38 (2'), 32.58 (3', 7'), 25.54 (5'), 25.18 (4', 6')
8.65 (t, 1H, 1', J = 6.2), 8.45 (d, 1H, 3, J = 5.6), 7.34 (d, 1H, 6, J = 2.3), 7.07 (dd,
1H, 4, J = 2.6, J = 3.1), 6.86 (d, 2H, 8, 12, J = 8.6), 6.65 (d, 2H, 9, 11, J = 8.9), 5.15
(s, 2H, 13), 3.12 (t, 2H, 2', J = 6.6), 1.66–0.84 (m, 11H, 3'–8')
5´
6´
7´
4´
3c
3´
167.31 (5), 163.76 (1), 152.78 (2), 150.54 (3), 147.32 (7), 143.37 (10), 122.00 (8, 12),
115.36 (9, 11), 114.19 (6), 108.90 (4), 45.39 (2'), 37.91 (3'), 30.86 (4', 8'), 26.50 (6'),
25.81 (5', 7')
8´
2´
9.27 (t, 1H, 1', J = 6.4), 8.47 (d, 1H, 3, J = 5.6), 7.38 (d, 1H, 6, J = 2.4), 7.31–7.21
(m, 5H, 4'–8'), 7.08 (dd, 1H, 4, J = 2.7, J = 2.9), 6.85 (d, 2H, 8, 12, J = 8.8), 6.65 (d,
2H, 9, 11, J = 8.8), 5.16 (s, 2H, 13), 4.46 (d, 2H, 2', J = 6.4)
5´
8´
6´
7´
4´
3´
3d
167.32 (5), 163.96 (1), 152.63 (2), 150.66 (3), 147.33 (7), 143.36 (10), 139.89 (3'),
128.70 (4', 8'), 127.81 (5', 7'), 127.22 (6'), 122.01 (8, 12), 115.38 (9, 11), 114.35 (6),
109.09 (4), 42.91 (2')
2´

Molecules 2012, 17
1128
Table 1. Cont.
Compd.
3e
R
1H and 13C-NMR spectra/δ ppm, J/Hz
8.78 (t, 1H, 1', J = 6.0), 8.44 (d, 1H, 3, J = 5.5), 7.35 (d, 1H, 6, J = 2.6), 7.30–7.18
(m, 5H, 5'–9'), 7.08 (dd, 1H, 4, J = 2.6, J = 3.1), 6.86 (d, 2H, 8, 12, J = 8.7), 6.65 (d,
2H, 9, 11, J = 8.7), 5.17 (s, 2H, 13), 3.52 (q, 2H, 2', J = 7.0), 2.84 (t, 2H, 3', J = 7.5)
166.80 (5), 163.23 (1), 152.14 (2), 150.09 (3), 146.84 (7), 142.83 (10), 139.29 (4'),
128.53 (5', 9'), 128.31 (6', 8'), 126.06 (7'), 121.51 (8, 12), 114.86 (9, 11), 113.73 (6),
108.42 (4), 39.55 (2'), 24.06 (3')
3´
5´
8´
4´
6´
7´
2´
9´
9.20 (s, 1H, 15), 8.98 (s, 1H, 13), 8.50 (d, 1H, 3, J = 5.4), 8.47 (d, 1H, 1', J = 6.7),
8.17 (s, 1H, 17), 7.69–7.58 (m, 4H, 20, 21, 8, 12), 7.38 (d, 1H, 6, J = 2.6), 7.18–7.14
(m, 3H, 4, 9, 11), 4.23–4.16 (m, 1H, 2'), 1.99–1.53 (m, 8H, 3'–6')
3´
2´
6´
166.47 (5), 163.21 (1), 152.94 (14), 152.92 (2), 150.75 (3), 148.36 (7), 139.80 (10),
137.52 (16), 132.44 (22), 124.00 (19), 123.58 (20), 122.54 (18), 121.86 (8, 12),
120.99 (9, 11), 117.36 (17), 117.28 (21), 114.59 (6), 109.20 (4), 51.07 (2'), 32.55 (3', 6'),
23.96 (4', 5')
4´
4a
4b
4c
4d
4e
5´
9.45 (s, 1H, 15), 9.19 (s, 1H, 13), 8.50 (d, 1H, 3, J = 5.6), 8.40 (d, 1H, 1', J = 8.6),
8.11 (s, 1H, 17), 7.68–7.58 (m, 4H, 20, 21, 8, 12), 7.39 (d, 1H, 6, J = 2.4), 7.18–7.14
(m, 3H, 4, 9, 11), 3.75–3.72 (m, 1H, 2'), 1.78–1.56 (m, 10H, 3'–7')
3´
6´
2´
4´
5´
166.49 (5), 162.61 (1), 152.99 (14), 152.93 (2), 150.77 (3), 148.31 (7), 139.85 (10),
137.56 (16), 132.47 (22), 123.93 (19), 123.45 (20), 122.46 (18), 121.87 (8, 12),
120.84 (9, 11), 117.21 (17), 117.13 (21), 114.59 (6), 109.31 (4), 48.42 (2'), 32.57 (3', 7'),
25.53 (5'), 25.18 (4',6')
7´
9.20 (s, 1H, 15), 8.97 (s, 1H, 13), 8.69 (t, 1H, 1', J = 6.1), 8.51 (d, 1H, 3, J = 5.5),
8.12 (s, 1H, 17), 7.69–7.58 (m, 4H, 20, 21, 8, 12), 7.38 (d, 1H, 6, J = 2.5), 7.18–7.14
(m, 3H, 4, 9, 11), 3.13 (t, 2H, 2', J = 6.4), 1.66–0.78 (m, 11H, 3'–8')
5´
6´
7´
4´
3´
166.49 (5), 163.66 (1), 152.94 (14), 152.92 (2), 150.78 (3), 148.35 (7), 139.80 (10),
137.52 (16), 132.45 (22), 124.04 (19), 123.58 (20), 122.58 (18), 121.90 (8, 12),
120.99 (9, 11), 117.36 (17), 117.29 (21), 114.57 (6), 109.22 (4), 45.41 (2'), 37.90
(3'), 30.86 (4', 8'), 26.50 (6'), 25.81 (5', 7')
8´
2´
9.49 (s, 1H, 15), 9.31 (t, 1H, 1', J = 6.3), 9.22 (s, 1H, 13), 8.54 (d, 1H, 3, J = 5.6),
8.12 (s, 1H, 17), 7.65–7.58 (m, 4H, 20, 21, 8, 12), 7.42 (d, 1H, 6, J = 2.4), 7.31–7.16
(m, 8H, 4, 9, 11, 4'–8'), 4.46 (d, 2H, 2', J = 6.3)
5´
6´
7´
4´
3´
166.50 (5), 163.85 (1), 153.00 (14), 152.80 (2), 150.91 (3), 148.27 (7), 139.86 (10),
137.60 (16), 132.48 (22), 128.70 (4', 8'), 127.83 (5', 7'), 127.23 (6'), 126.66 (3'),
123.99 (19), 123.43 (20), 122.57 (18), 121.91 (8, 12), 120.83 (9, 11), 117.18 (17),
117.11 (21), 114.67 (6), 109.45 (4), 42.92 (2')
8´
2´
9.20 (s, 1H, 15), 8.98 (s, 1H, 13), 8.81 (t, 1H, 1', J = 5.8), 8.50 (d, 1H, 3, J = 5.6),
8.12 (s, 1H, 17), 7.68–7.58 (m, 4H, 20, 21, 8, 12), 7.39 (d, 1H, 6, J = 2.3), 7.31–7.14
(m, 8H, 4, 9, 11, 4'–8'), 3.51 (q, 2H, 2', J = 6.9), 2.84 (t, 2H, 3', J = 7.4)
166.48 (5), 163.62 (1), 152.94 (14), 152.81 (2), 150.83 (3), 148.33 (7), 139.79 (10),
137.53 (16), 132.46 (22), 129.03 (5', 9'), 128.81 (6', 8'), 126.73 (4'), 126.57 (7'),
125.11 (19), 123.60 (20), 122.58 (18), 121.90 (8, 12), 121.01 (9, 11), 117.60 (17, 21),
114.60 (6), 109.24 (4), 40.83 (2'), 35.50 (3')
3´
5´
8´
4´
6´
7´
2´
9´

Molecules 2012, 17
1129
2.2. Biological Evaluation
2.2.1. Cytostatic Activity
The experiments were carried out on six human tumour and one murine cell lines, which are
derived from five solid tumour types and two (one murine and one human) suspension tumour cell
lines. The following cell lines were used: HCT 116 (colon carcinoma), SW 620 (colon carcinoma),
MCF-7 (breast carcinoma), H 460 (lung carcinoma), L1210 (murine leukemia), CEM (human
lymphoma) and HeLa (cervix carcinoma). The results of the cytostatic effect of the tested compounds are
presented in Table 2. Compounds 2a–e and 3a–c showed no, or rather modest antiproliferative effect,
only at the maximal tested concentration (104 M). Compounds 3d and 3e with a benzene ring in the
amide moiety, exerted a somewhat stronger (but still rather poor) cytostatic effect (IC50 ≈ 2090 µM).
In contrast, compounds 4a–e showed the most prominent effect with IC50 values in the lower
micromolar range (IC50 from 1 to 4.3 M, with an average of 2.6 ± 1.6), but without substantial
selectivity between the tumour cell lines. The compounds were also equally cytostatic against primary
human embryonic lung (HEL) fibroblast cells (Table 2). The obtained data clearly point to the
obligatory presence of the [4-chloro-3-(trifluoromethyl)phenyl]carbamoyl group being crucial for high
potency. Also, the 4a–e series of compounds are far more lipophilic than the 2 and 3 series of
compounds, which may be related to their increased cytostatic potential. The cytostatic activities of
compounds 4a–e are comparable with the data obtained for sorafenib 10 or even better (median IC50
value for sorafenib is 4.3 M; twenty of 23 cell lines had values between 1.0 and 10.0 M), which
indicate that the pyridine-2-carboxamide modifications had a minor effect on the antiproliferative activity.
Log P of sorafenib is 3.76, while amides 4a–e have log P values between 4.89 and 5.77 (Table 2) 11.
Table 2. IC50 values of compounds 2a–e, 3a–e, and 4a–e a.
Tumour cell growth/IC50 (µmol·L−1)
log P
Compd.
(Clog P) b
HCT 116 SW 620 MCF-7 H 460
L1210
CEM HeLa HEL
1.95
(2.72)
2.37
2a
2b
2c
2d
2e
3a
3b
3c
>100
>100
>100
>100
≥100
71 ± 28
>100
≥100
≥100
≥100
>100
≥100
≥100
35 ± 12
>100
>100
>100
>100
>100
≥100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
≥100
>100
(3.28)
2.80
≥100
≥100
(3.90)
2.55
≥100
≥100
>100
(3.22)
2.83
≥100
57 ± 46
53 ± 40
≥100
>100
(3.35)
2.13
54 ± 21
37 ± 1
≥100
>100
121 ± 50.5 ≥100 77 ± 3.4 >100
(2.80)
2.54
65 ± 33 234 ± 4.5 >100
>100 163 ± 147 >100
>100
>100
>100
>100
(3.36)
2.97
≥100
(3.98)

Molecules 2012, 17
1130
Table 2. Cont.
Tumour cell growth/IC50 (µmol·L−1)
log P
Compd.
(Clog P) b
HCT 116 SW 620
MCF-7
H 460
L1210
CEM
HeLa
HEL
2.73
3d
29 ± 4
25 ± 2
37 ± 6
20 ± 7
29 ± 13
42 ± 9
66 ± 3.1 94 ± 2.2 94 ± 1.9
>100
>100
(3.30)
3.01
3e
34 ± 17
20 ± 0.0 63 ± 8.9 42 ± 3.0 39 ± 3.0
(3.43)
4.89
4a
3.0 ± 0.5
3.0 ± 0.8
3.0 ± 1.0
2.0 ± 0.4 4.0 ± 1.0 1.0 ± 0.0 4.2 ± 0.8 4.2 ± 0.6 3.1 ± 0.6 2.8 ± 0.0
3.0 ± 0.2 2.0 ± 0.6 2.0 ± 0.0 3.2 ± 1.7 3.6 ± 0.9 3.0 ± 0.2 3.2 ± 0.2
3.0 ± 0.4 2.0 ± 0.1 2.0 ± 0.2 1.8 ± 0.3 2.0 ± 0.5 2.7 ± 1.8 2.9 ± 0.0
(6.93)
5.31
4b
(7.49)
5.73
4c
(8.11)
5.49
4d
2.0 ± 0.8 2.0 ± 0.08 2.0 ± 0.2 2.0 ± 0.2 2.0 ± 0.4 4.3 ± 0.4 2.6 ± 0.6 3.1 ± 0.2
3.0 ± 0.7 2.0 ± 0.4 2.0 ± 0.1 2.0 ± 0.2 2.2 ± 0.2 2.5 ± 0.9 1.7 ± 0.2 4.6 ± 0.36
(7.42)
5.77
4e
(7.55)
3.76
Sorafenib
3.0 ± 0.8 6.0 ± 1.0 3.0 ± 0.7 3.0 ± 0.8 4.1 ± 0.0 3.4 ± 0.5 3.7 ± 0.6 7.5 ± 0.7
(5.46)
a
b
IC50, the concentration that causes 50% growth inhibition; Lipophilicity was theoretically calculated by the
CLOGP Programme of Biobyte Corp. 11.
2.2.2. Antimetabolic Activity
A variety of compounds (2a, 3a, 4a with a cycloalkyl substituent directly attached to nitrogen atom,
2c, 3c, 4c with a cycloalkyl bound to nitrogen through a short spacer and 2e, 3e, 4e with an aromatic
substituent) have been investigated on their inhibitory activity against DNA, RNA and protein
synthesis in CEM cell cultures 12. The compounds were equally inhibitory to dThd (DNA synthesis),
Urd (RNA synthesis) and Leu (protein synthesis) incorporation into TCA-insoluble cell material.
The 2 series of compounds were least inhibitory whereas the 4 series were most inhibitory (IC50 in the
lower micromolar range). Their antimetabolic IC50 values closely corresponded to their antiproliferative
IC50 values. They also behaved as sorafenib in terms of antimetabolic potential (Table 3).
Table 3. Antimetabolic activity of tested compounds in CEM cell cultures.
IC50 (µmol·L−1)
CEM
[CH3-3H]dThd
>100
[5-3H]Urd
>100
[4,5-3H]Leu
>100
2a
2c
>100
>100
>100
2e
>100
>100
>100
3a
>100
>100
>100
3c
>100
92.1 ± 43.0 70.6 ± 27.6
3e
4a
4c
>100
49.9 ± 17.9
7.7 ± 2.1
12.2 ± 8.4
6.6 ± 1.8
7.1 ± 0.2
>100
12.5 ± 6.7
20.1 ± 10.9
12.7 ± 9.0
8.6 ± 1.1
6.1 ± 0.7
27.4 ± 12.7
11.8 ± 7.7
12.0 ± 7.0
4e
Sorafenib
a
50% Inhibitory concentration or compound concentration required to inhibit
tritated dThd, Urd or Leu into TCA-insoluble cell materials.

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2.2.3. Uptake and Efflux of Tested Compounds in CaCo-2 Cell Cultures
Derivatives 2a, 3a and 4a have been examined for their efficiency of uptake at the apical site and
subsequent delivery at the basolateral site of colon carcinoma CaCo-2 cell cultures 13. This model
system can be used to predict the potential of a drug to show oral bioavailability. Sorafenib was
included as the reference compound. Whereas 2a and 3a showed a higher drug delivery at the
basolateral site than sorafenib in function of exposure time, 4a performed less efficient than the
reference compound (Figure 1). The molecular basis of this phenomenon is yet unclear.
Figure 1. Release of the tested compounds at the basolateral side by CaCo-2 cell cultures.
Hundred micromolar concentrations of the tested compounds were added at the apical side
of confluent CaCo-2 cell cultures, and the appearance of the intact tested compound at the
basolateral side was measured by HPLC analysis.
3. Experimental Section
3.1. Chemistry
3.1.1. General
Melting points were determined on a Stuart Melting Point Apparatus SMP3 and were uncorrected.
1
IR spectra were recorded on a FTIR Perkin Elmer Paragon 500 spectrometer. H- and 13C-NMR
spectra were recorded on a Bruker AV-600 spectrometer operating at 300.13 and 75.47 MHz for the
1H and 13C nuclei, respectively. Samples were measured in DMSO-d6 solutions at 20 °C in 5 mm
NMR tubes. Chemical shifts () were referred to TMS. Mass spectra were taken on a HPLC-MS/MS
(HPLC, Agilent Technologies 1200 Series; MS, Agilent Technologies 6410 Triple Quad). CEM
Discover microwave reactor was used for microwave reactions. Precoated Merck silica gel 60 F254
plates (Merck, Germany) and solvent systems petrolether/ethyl acetate/methanol (3:1:0.5; 3:1:0.25
or 3:1:0.15), cyclohexane/ethyl acetate (1:1), petrolether/ethyl acetate (1:1), ethyl acetate and
dichloromethane/methanol (9:1 or 9.5:0.5), were used for thin-layer chromatography. Spots were
visualized by short-wave UV light and iodine vapour. Column chromatography was performed on
silica gel (0.063–0.200 mm), with petroleum ether/ethyl acetate/methanol (3:1:0.5, 3:1:0.25 or
3:1:0.15) or dichloromethane/methanol (9:1). Preparative chromatography was performed on silica gel

Molecules 2012, 17
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preparative plates using dichloromethane/methanol (9:1 or 9.5:0.5) as eluents. Picolinic acid,
4-amino-phenol and 4-chloro-3-(fluoromethyl)phenyl isocyanate were purchased from Acros Organics,
cyclopentylamine, cyclohexylamine, cyclohexylmethylamine, benzylamine, phenylethylamine and
potassium tert-butoxyde from Aldrich, and potassium carbonate from Kemika. Anhydrous solvents
(DMF, CH2Cl2, toluene) were prepared prior to use. Human cell lines for cytostatic activity were
obtained from American Type Culture Collection.
4-Chloropyridine-2-carbonyl chloride hydrochloride (1). Compound 1 was prepared from picolinic
acid (1.231 g, 10 mmol) and thionyl chloride (5 mL) following procedure previously described 9.
The crude product 1 was used in the next step without further purification.
3.1.2. General Method for the Synthesis of 4-chloropyridine-2-carboxamides 2a–e
To a cold solution of chloride 1 (1.760 g, 10 mmol) in anhydrous toluene (10 mL) a solution of
appropriate amine (10 mmol) and triethylamine (2.02 g, 20 mmol) in toluene (5 mL) was added
dropwise. The reaction mixture was stirred at room temperature for 30 min and then extracted 3 times
with saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4, filtered and
evaporated under reduced pressure. The crude products 2a–e were purified by column chromatography.
4-Chloro-N-cyclopentylpyridine-2-carboxamide (2a). From the reaction of chloride 1 (1.760 g, 10 mmol)
and cyclopentylamine (0.086 g, 10 mmol) at room temperature for 0.5 h and column chromatography
(mobile phase petroleum ether/ethyl acetate/methanol 3:1:0.5 and petroleum ether/ethyl acetate 1:1)
1.621 g (72%) of 2a was obtained; mp 7980 C; IR (KBr): max 3328, 3064, 2961, 2870, 1666, 1575,
1556, 1519, 732 cm–1. MS/MS m/z 225.1 (M+H)+. Anal. (C11H13ClN2O) C, H, N.
4-Chloro-N-cyclohexylpyridine-2-carboxamide (2b). From the reaction of chloride 1 (1.760 g, 10 mmol)
and cyclohexylamine (0.099 g, 10 mmol) at room temperature for 0.5 h and column chromatography
(mobile phase petroleum ether/ethyl acetate/methanol 3:1:0.15) 1.243 g (52%) of 2b was obtained; mp
116119 C; IR (KBr): max 3330, 2930, 2853, 1650, 1578, 1554, 1534, 736 cm–1. MS/MS m/z 239.1
(M+H)+. Anal. (C12H15ClN2O) C, H, N.
4-Chloro-N-cyclohexylmethylpyridine-2-carboxamide (2c). From the reaction of chloride 1 (1.760 g,
10 mmol) and cyclohexylmethylamine (1.132 g, 10 mmol) at room temperature for 0.5 h and column
chromatography (mobile phase petroleum ether/ethyl acetate/methanol 3:1:0.15) 0.999 g (40%) of 2c
was obtained; mp 107108 C; IR (KBr): max 3341, 3089, 2926, 2855, 1649, 1537, 736 cm–1. MS/MS
m/z 253.1 (M+H)+. Anal. (C13H17ClN2O) C, H, N.
4-Chloro-N-benzylpyridine-2-carboxamide (2d). From the reaction of chloride 1 (1.760 g, 10 mmol)
and benzylamine (1.072 g, 10 mmol) at room temperature for 0.5 h and column chromatography
(mobile phase petroleum ether/ethyl acetate/methanol 3:1:0.25) 1.577 g (64%) of 2d was obtained; mp
7072 C; IR (KBr): max 3326, 1649, 1578, 1552, 1536, 736, 725 cm–1. MS/MS m/z 269.1 (M+Na)+.
Anal. (C13H11ClN2O) C, H, N.

Molecules 2012, 17
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4-Chloro-N-phenylethylpyridine-2-carboxamide (2e). From the reaction of chloride 1 (1.760 g, 10 mmol)
and phenylethylamine (1.212 g, 10 mmol) at room temperature for 0.5 h and column chromatography
(mobile phase petroleum ether/ethyl acetate/methanol 3:1:0.15) 1.404 g (54%) of 2e was obtained; mp
6062 C; IR (KBr): max 3346, 1657, 1556, 1531, 733, 700 cm–1. MS/MS m/z 261.1 (M+H)+. Anal.
(C14H13ClN2O) C, H, N.
3.1.3. General Method for the Synthesis of 4-(4-aminophenoxy)-pyridine-2-carboxamides 3a–e
A suspension of t-BuOK (0.056 g, 0.5 mmol) and 4-aminophenol (0.054 g, 0.5 mmol) in anhydrous
DMF (5 mL) was stirred at room temperature for 30 min. The corresponding 4-chloropicolinamide 2a–e
(0.5 mmol) and potassium carbonate (0.034 g, 0.25 mmol) were added. The reaction mixture was
stirred in microwave reactor for 10 min (P = 150 W, t = 173 °C). DMF was evaporated under reduced
pressure. The residue was dissolved in ethyl acetate end extracted with saturated NaCl solution.
The organic phase was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure.
The crude products 3a–e were purified by column chromatography or by recrystallization.
4-(4-Aminophenoxy)-N-cyclopentylpyridine-2-carboxamide (3a). From the reaction of amide 2a (0.112 g,
0.5 mmol) and purification by column chromatography [mobile phase dichloromethane/methanol
(9:1)] 0.069 g (46%) of 3a was obtained; viscous oil; IR (KBr): max 3353, 2958, 2870, 1666, 1608,
1590, 1521, 1502, 1470, 1284, 1198, 836 cm–1. MS/MS m/z 298.2 (M+H)+. Anal. (C17H19N3O2) C, H, N.
4-(4-Aminophenoxy)-N-cyclohexylpyridine-2-carboxamide (3b). From the reaction of amide 2b (0.119 g,
0.5 mmol) and recrystallization from methanol/water 0.075 g (48%) of 3b was obtained; mp 150153 C.
IR (KBr): max 3357, 3316, 2928, 2855, 1660, 1566, 1523, 1506, 839 cm–1. MS/MS m/z 312.2 (M+H)+.
Anal. (C18H21N3O2) C, H, N.
4-(4-Aminophenoxy)-N-cyclohexylmethylpyridine-2-carboxamide (3c). From the reaction of amide 2c
(0.126 g, 0.5 mmol) and purification by column chromatography (mobile phase petroleum ether/ethyl
acetate/methanol 3:1:0.15) 0.075 g (46%) of 3c was obtained; mp 135137 C; IR (KBr): max 3390,
3364, 3194, 2924, 2838, 1680, 1526, 1504, 1299, 1195, 844 cm–1. MS/MS m/z 326.2 (M+H)+. Anal.
(C19H23N3O2) C, H, N.
4-(4-Aminophenoxy)-N-benzylpyridine-2-carboxamide (3d). From the reaction of amide 2d (0.123 g,
0.5 mmol) and purification by column chromatography (mobile phase petroleum ether/ethyl
acetate/methanol 3:1:0.5) 0.121 g (76%) of 3d was obtained; viscous oil; IR (KBr): max 3348, 3062,
2925, 1665, 1590, 1566, 1526, 1501, 1468, 1261, 1197, 834, 699 cm–1. Anal. (C19H17N3O2) C, H, N.
4-(4-Aminophenoxy)-N-phenylethylpyridine-2-carboxamide (3e). From the reaction of amide 2e (0.130 g,
0.5 mmol) and purification by column chromatography (mobile phase petroleum ether/ethyl
acetate/methanol 3:1:0.5) 0.115 g (69%) of 3e was obtained; viscous oil; IR (KBr): max 3354, 3229,
3061, 3027, 2928, 2862, 1665, 1607, 1590, 1526, 1504, 1469, 1285, 1198, 838, 670 cm–1. MS/MS m/z
334.2 (M+H)+. Anal. (C20H19N3O2) C, H, N.

Molecules 2012, 17
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3.1.4. General Procedure for the Synthesis of 4-4-4-Chloro-3-(trifluoromethyl)phenylcarbamoyl-
aminophenoxy-pyridine-2-carboxamides 4a–e
A solution of 4-(4-aminophenoxy)-picolinamide 3a–e (0.1 mmol) and 4-chloro-3-(fluoromethyl)-
phenyl isocyanate (0.12 mmol) in anhydrous dichloromethane (3 mL) was stirred for 16 h at room
temperature under nitrogen atmosphere. Solvent was evaporated under reduced pressure and the
residue was dissolved in ethyl acetate and extracted three times with brine. The organic phase was
dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. The crude products
4a–e were purified by column chromatography, preparative chromatography or by recrystallization.
4-4-4-Chloro-3-(trifluoromethyl)phenylcarbamoylaminophenoxy-N-cyclopentyl-pyridine-
2-carboxamide (4a). From the reaction of compound 3a (0.030 g, 0.1 mmol) and purification by
column chromatography (mobile phase petroleum ether/ethyl acetate/methanol 3:1:0.5) 0.051 g (98%)
of 4a was obtained; viscous oil; IR (KBr): max 3353, 2962, 2872, 1718, 1654, 1595, 1546, 1506, 1420,
1306, 1199, 1176, 1140, 838, 663 cm–1. MS/MS m/z 519.1 (M+H)+. Anal. (C25H22ClF3N4O3) C, H, N.
4-4-4-Chloro-3-(trifluoromethyl)phenylcarbamoylaminophenoxy-N-cyclohexyl-pyridine-
2-carboxamide (4b). From the reaction of compound 3b (0.031 g, 0.1 mmol) and purification by
column chromatography (mobile phase petroleum ether/ethyl acetate/methanol 3:1:0.5) and
preparative chromatography (mobile phase dichloromethane/methanol 9:1) 0.023 g (43%) of 4b was
obtained; mp 110114 C; IR (KBr): max 3348, 3072, 2932, 2855, 1716, 1650, 1596, 1548, 1505,
1484, 1419, 1293, 1197, 1175, 1137, 840, 663 cm–1. MS/MS m/z 533.2 (M+H)+. Anal.
(C26H24ClF3N4O3) C, H, N.
4-4-4-Chloro-3-(trifluoromethyl)phenylcarbamoylaminophenoxy-N-cyclohexylmethyl-pyridine-2-
carboxamide (4c). From the reaction of compound 3c (0.033 g, 0.1 mmol) and purification by column
chromatography (mobile phase petroleum ether/ethyl acetate 1:1) and recrystallization from
methanol/water 0.030 g (55%) of 4c was obtained. mp 156160 C; IR (KBr): max 3353, 2962, 2872,
1718, 1654, 1595, 1546, 1506, 1420, 1306, 1199, 1176, 1140, 838, 663 cm–1. MS/MS m/z 547.2
(M+H)+. Anal. (C27H26ClF3N4O3) C, H, N.
4-4-4-Chloro-3-(trifluoromethyl)phenylcarbamoylaminophenoxy-N-benzyl-pyridine-2-
carbox-amide (4d). From the reaction of compound 3d (0.032 g, 0.1 mmol) and recrystallization from
methanol/water 0.020 g (37%) of 4d was obtained. mp 160163 C; IR (KBr): max 3351, 3132, 2926,
2854, 1718, 1654, 1595, 1543, 1506, 1420, 1303, 1199, 1176, 1140, 837, 668 cm–1. MS/MS m/z 541.9
(M+H)+. Anal. (C27H20ClF3N4O3) C, H, N.
4-4-4-Chloro-3-(trifluoromethyl)phenylcarbamoylaminophenoxy-N-phenylethyl-pyridine-
2-carboxamide (4e). From the reaction of compound 3e (0.033 g, 0.1 mmol) and purification by
column chromatography (mobile phase petroleum ether/ethyl acetate/methanol 3:1:0.15) 0.041 g
(74%) of 4e was obtained. viscous oil; IR (KBr): max 3345, 3066, 2928, 1716, 1654, 1596, 1545,
1505, 1483, 1419, 1299, 1198, 1175, 1134, 840, 699 cm–1. Anal. (C28H22ClF3N4O3) C, H, N.

Molecules 2012, 17
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3.2. Biological Evaluation
3.2.1. Cytostatic Activity
HCT 116, SW 620, MCF-7, and H 460 cells were cultured as monolayers and maintained in
Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS),
2 mmol·L–1 L-glutamine, 100 U·mL–1 penicillin and 100 μg·mL–1 streptomycin in a humidified
atmosphere with 5% CO2 at 37 C. The growth inhibition activity was assessed as described
previously, according to the slightly modified procedure of the National Cancer Institute,
Developmental Therapeutics Program 14,15. Briefly, the cells were seeded onto standard 96-well
microtiter plates on day 0. The cell concentrations were adjusted according to the cell population
doubling time (PDT): 3000 cells/well for HCT116 and SW 620 cell lines (PDT = 20–24 h) and 4,000
cells/well for MCF-7 cell lines (PDT = 33 h). Tested agents were then added in five consecutive 10-fold
dilutions (10–8 to 10–4 mol·L–1) and incubated for further 72 h. Working dilutions were freshly
prepared on the day of testing. The solvent (DMSO) was also tested for eventual inhibitory activity by
adjusting its concentration to be the same as in working concentrations (maximal concentration of
DMSO was 0.25%). After 72 h of incubation, the cell growth rate was evaluated by performing the
MTT assay (Promega) which detects dehydrogenase activity in viable cells 16. The absorbency (OD,
optical density) was measured on a microplate reader at 570 nm. Each test point was performed in
quadruplicate in at least three individual experiments. The results are expressed as IC50, which is the
concentration necessary for 50% of inhibition. The IC50 values for each compound are calculated from
dose-response curves using linear regression analysis, using FORCAST function in Excel, by fitting
the test concentrations that give PG (percentage of growth) values above and below the reference value
(i.e., 50%). Each result is a mean value from three separate experiments.
The cytostatic activity of the tested compounds against murine leukemia L1210, human cervix
carcinoma HeLa, human embrionic lung (HEL) and T-lymphocyte CEM cells was determined with
cells grown in suspension (L1210, CEM) or monolayers (HeLa, HEL) in 200 µL-wells of 96-well
microtiter plates (initial cell number: 5–7.5 × 104 cells/well). After 48 (L1210) or 72 (CEM, HeLa, HEL) h,
the tumour cell number was determined by the use of a Coulter counter.
3.2.2. Antimetabolic Activity and Drug Influx/Efflux Assay in CaCo-2 Cell Cultures
The procedures have been modified from previously described methods 12,13. Human CEM cells
were seeded in 96-well microtiter plates at 10 × 104 cells/200 µL-well in the presence of serial
dilutions of the tested compounds. Then, 0.25 µCi of [CH3-3H]dThd (radiospecificity: 52 Ci/mmol) or
1 µCi of [5-3H]Urd (radiospecificity: 26 Ci/mmol) or [4,5-3H]Leu (radiospecificity: 160 Ci/mmol)
were added and the drug-exposed tumour cell cultures further incubated at 37 °C for ~20 h. Then the
content of each well was transfered to glass fiber discs (Wattman, GE Healthcare, Maidstone, UK),
mounted on a manifold (Millipore, Billerica, MA, USA) and subsequently washed with PBS
(to remove the radiolabel material that has not been incorporated in the cellular macromolecules),
10% and 5% trichoroacetic acid (TCA) (to precipitate DNA, RNA and proteins) and ethanol.
Then, the radioactivity (TCA precipitate) associated with each disc was determined in a liquid
scintillation counter (Packard, Palo Alto, CA, USA).

Molecules 2012, 17
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Human colon carcinoma CaCo-2 cells were seeded in the inner wells of a double-chamber-well-tray
at 150,000 cells (0.5 mL) per 1 mL-well. The outer wells also contain 0.5 mL medium (without cells).
After the cells were grown to confluency (~2 days) at 37 °C in a humidified CO2-controlled
atmosphere, 100 µmol·L–1 tested compound (final concentration) was added to the inner well, and the
medium from the outer wells was replaced by 800 µL PBS (containing 10% foetal bovine serum).
At different time points (i.e., 0, 2, 4, 6, 8 and 24 h), 50 µL was removed from the outer wells and
mixed with 100 µL cold methanol. The mixture was left on ice for 10 min, centrifuged to remove the
precipitate, and the supernatant was analysed by HPLC (reversed phase) to determine the drug
concentration that appeared in the outer wells (reflecting basolateral drug efflux) in function of time.
4. Conclusions
The synthetic pathway leading to five new sorafenib amide derivatives is described. The final
compounds, as well as intermediates, are fully chemically characterized. Their cytostatic activity was
tested on a panel of seven tumour cell lines, on which they exerted pronounced, but nonselective
antiproliferative activity at the lower micromolar range, comparable to the activity of sorafenib itself.
Detailed pharmacological and pharmacokinetic evaluation still remain to be done to compare the
sorafenib derivatives described herein with the parent compound.
Acknowledgments
Support for this study was provided by Ministry of Science, Education and Sports of the Republic
of Croatia (Project 006-0000000-3216, 098-0982464-2514) and the K.U.Leuven (GOA No. 10/014).
The authors would like to thank Lizette van Berckelaer, Ria Van Berwaer, Leen Ingels, Leentje Persoons
and Frieda De Meyer for the antiviral, cytostatic and antimetabolic assays.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of all compounds are available from the authors.
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