The synthesis, characterization, cytotoxic activity assessment and structure-activity relationship of 4-aryl-6-(2,5-dichlorothiophen-3-yl)-2-methoxypyridine-3-carbonitriles

Article abstract of DOI:10.3390/molecules24224072

A series of 2-methoxypyridine-3-carbonitrile (5a-i)-bearing aryl substituents were successfully synthesized in good yields by the condensation of chalcones (4a-i) with malononitrile in basic medium. The condensation process, in most cases, offers a route to a variety of methoxypyridine derivatives (6a-g) as side products in poor yields. All new compounds were fully characterized using different spectroscopic methods. Mass ESI-HMRS measurements were also performed. Furthermore, these compounds were screened for their in vitro cytotoxicity activities against three cancer cell lines; namely, those of the liver (line HepG2), prostate (line DU145) and breast (line MBA-MB-231). The cytotoxicity assessment revealed that compounds 5d, 5g, 5h and 5i exhibit promising antiproliferative effects (IC50 1-5 μM) against those three cancer cell lines.

Full text of DOI:10.3390/molecules24224072

Article
The Synthesis, Characterization, Cytotoxic Activity
Assessment and Structure–Activity Relationship of 4-
Aryl-6-(2,5-dichlorothiophen-3-yl)-2-
methoxypyridine-3-carbonitriles
1,
1
2
2
Mahmoud Al-Refai *, Mohammad M. Ibrahim , Mohamad Nurul Azmi , Hasnah Osman ,
3
4
Mohamad Hafizi Abu Bakar and Armin Geyer
1
Department of Chemistry, Faculty of Science, Al al-Bayt University, P.O.BOX 130040, Al-Mafraq 25113,
Jordan; mohammadibrahim@aabu.edu.jo
2
3
4
School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia;
mnazmi@usm.my (M.N.A.); ohasnah@usm.my (H.O.)
Bioprocess Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800
Minden, Pulau Pinang, Malaysia; mhafizi88@usm.my
Faculty of Chemistry, Philipps University Marburg, Hans-Meerwein-Straße 4, 35032 Marburg, Germany;
geyer@staff.uni-marburg.de
* Correspondence: Mahmoud_alrefai@aabu.edu.jo
Received: 20 October 2019; Accepted: 8 November 2019; Published: 10 November 2019
Abstract: A series of 2-methoxypyridine-3-carbonitrile (5a–i)-bearing aryl substituents were
successfully synthesized in good yields by the condensation of chalcones (4a–i) with malononitrile
in basic medium. The condensation process, in most cases, offers a route to a variety of
methoxypyridine derivatives (6a–g) as side products in poor yields. All new compounds were fully
characterized using different spectroscopic methods. Mass ESI-HMRS measurements were also
performed. Furthermore, these compounds were screened for their in vitro cytotoxicity activities
against three cancer cell lines; namely, those of the liver (line HepG2), prostate (line DU145) and
breast (line MBA-MB-231). The cytotoxicity assessment revealed that compounds 5d, 5g, 5h and 5i
exhibit promising antiproliferative effects (IC50 1–5 µM) against those three cancer cell lines.
Keywords: carbonitrile; methoxypyridine; thiophene; malononitrile; cytotoxicity; liver cancer;
prostate cancer; breast cancer
1. Introduction
Pyridine is an important skeletal substituent and valuable organic moiety in many biologically-
active and clinically-used compounds [1–3]. Such compounds display a wide spectrum of
pharmacological activities: antimicrobial [4], antioxidant [5], HIV inhibitors [6], antimalarial [7],
anticancer [8], and many others [9]. Pyridine is also used in many clinically-used agents; namely,
rosiglitazone A [1–3], pioglitazone B [1–3], milrinone C [1–3], amrinone D [3] and etoricoxib E [4,10]
(Figure 1). Pyridine-3-carbonitriles, in particular, have been found to possess many biological
activities and many optical and electrical qualities [11]. Furthermore, they are important precursors
for synthetic manipulations and have been commonly used as precursors and key intermediates in
organic synthesis [12,13]. In recent years, we reported the synthesis of different pyridine derivatives
bearing carbonitrile and 2,5-dichlorothiophene substituents [14–19]. In continuation of our ongoing
work, herein, we present the synthesis and characterization of new pyridine-3-carbonitrile
derivatives (Scheme 1). The new derivatives were screened for their cytotoxic activity against three
Molecules 2019, 24, 4072; doi:10.3390/molecules24224072
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cancer cell lines, namely, those of the liver (line HepG2), prostate (line DU145) and breast (line MBA-
MB-231) are discussed.
Figure 1. Clinically-used pyridine-containing compounds.
2. Results and Discussion
2.1. Synthesis and Characterization
The derivative 3-acetyl-2,5-dichlorothiophene, 2, was prepared following a published procedure
[20] by Friedel–Crafts acylation of 2,5-dichlorthiophene, 1, in carbon disulfide (CS2). The
condensation of 3-acetyl-2,5-dichloro-thiophene (2) with different aromatic aldehydes, 3a–i, in the
presence of potassium hydroxide, afforded chalcones 4a–i (Scheme 1). The targeted pyridine-3-
carbonitrile derivatives, 5a–i were obtained by the reaction of chalcones, 4a–i, with malononitrile
using methanolic solution of sodium hydroxide in good yields. In some cases, decyanation of
pyridine-3-carbonitrile derivatives, 5a–i, provided the pyridine compounds 6a–g in poor yields; see
1
13
Scheme 1. Spectroscopic data ( H-NMR, C-NMR, COSY, HSQC and HMBC) and mass ESI-HMRS
measurements support the proposed structures for compounds 5a–i and 6a–g.

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Cl
Cl
O
O
AlCl3
S
S
+
Cl
CS2
Cl
Cl
rt, 24 hr
(1)
(2)
O
Cl
S
KOH
Ar
H
(3a-i)
Cl
N
O
CN
Ar
(5a-i)
Cl
S
CH3OH
+
Cl
+
CN
NC
Ar
Cl
NaOH
S
O
reflux 2 hr
Cl
N
O
(4a-i)
Ar
(6a-g)
Br
Cl
Ar =
O
a
b
d
e
c
NO2
O
O
NO2
Br
g
f
h
i
Scheme 1. Synthesis of pyridine derivatives (5 and 6).
The formation of 2-methoxypyridine-3-carbonitrile products 5a–i, Scheme 2, was assumed to
take place via conjugated addition of the malononitrile anion to the β-carbon of the corresponding
chalcone, followed by methanol aꢀack to C≡N group, leading to the imine A which tautomerizes to
amine derivative B. The intermolecular dehydrative cyclization of amine B furnished the
dihydropyridine C which oxidizes (aromatization) to the desired 5a–i product (Scheme 2) [21–24].
The decyanation of 5a–i is believed to proceed through the hydrolysis of the nitrile derivative to the
corresponding carboxylic acid followed by the decarboxylation to form pyridines 6a–g (Scheme 2).

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OCH3
CN
NC
Ar
CN
NC
O
CN
HN
O
O
NaOH
-H2O
CH3OH
R
R
Ar
R
A
R
Ar
NaOH
OCH3
CN
H
N
R
N
OCH3
CN
H3CO
NC
cyclization
-H2O
-2H
H2N
O
Ar
R
Ar
Ar
B
(5a-i)
C
NaOH
R
N
OCH3
O
R
N
OCH3
R =
heat
Cl
Cl
S
-CO2
Ar = listed in scheme 1
Ar ONa
Ar
(6a-g)
Scheme 2. Proposed mechanism for the formation of pyridine derivatives (5 and 6).
1
13
All new compounds were characterized by H and C-NMR, and 2D-NMR (See Supplementary
1
Materials). In the H-NMR spectra of compounds 5a–i, the methoxy protons (OCH3-2) appeared at δ
4.16–4.20. The singlet signals which resonated at 7.58–7.67 and 7.40–7.43 were assigned to pyridine
(H-5) and thiophene (H-4′) protons, respectively. This was supported by HMBC and COSY
1
correlations. In addition, the H-NMR spectra of compounds 6a–g exhibit two singlets at 7.41–7.43
and 4.04–4.08 attributed to H-4′ of thiophene and the methoxy protons (OCH3–2), respectively. The
two mutually-coupled doublet signals at δ 7.66–7.72 and 6.89–6.97, respectively, could be assigned to
H-5 and H-3 protons, respectively, based on their coupling constants (J = 1.2–1.7 Hz), and the chemical
shifts of carbons to which they are attached (C-5, δ 113.9–117.0; C-3, δ 107.3–110.3).
13
The C-NMR spectra of compounds 5a–i and 6a–g are very similar, with one significant
exception that the chemical shifts of C-3 in the series 5a–i, appeared at 95.9 ppm instead of the 107.3
ppm for compounds 6a–g. Finally, all structural assignments are well-matched with the high-
resolution HSESIMS data and further confirmed by a combination of COSY, HSQC and HMBC
experiments (See Supplementary Materials). A complete analysis of all NMR data is listed in the
experimental section.
2.2. Cytotoxicity Properties
All compounds tested, 5a–i and 6a–g, were subjected to different concentrations for cytotoxicity
screening against three human cancer cell lines, namely, HepG2 (liver), DU145 (prostate) and MBA-
MB-231 (breast) along with standard 5-fluorouracil. The cytotoxicity scanning results showed
variable degrees of toxicity on all the cancer cell lines (Table 1).

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Table 1. Cytotoxic activities and selectivity indexes (SIs) of 2-methoxypyridine derivatives.
IC50 Values (µM)
Tested cpd.
MDA-MB-
DU145
>100
SI
HepG2
SI
SI
3.90
HSF1184
231
49.55 ±
0.51
5a
5b
5c
5d
5f
ND
ND
2.18
1.66
21.15 ± 0.37
82.39 ± 0.04
114.54 ±
0.38
>100
9.48 ± 0.63 12.08
>100 ND
>100
ND
42.36 ±
0.21
3.63 ±
0.17
27.45 ±
0.63
4.97 ±
0.35
1.77 ±
0.38
1.82 ±
0.21
53.49 ±
0.02
64.59 ±
0.08
21.65 ±
0.12
29.45 ±
0.88
18.45 ±
0.02
25.42 ±
0.38
28.34 ± 0.42
2.59 ± 0.04
43.49 ± 0.06
1.93 ± 0.03
2.14 ± 0.06
1.38 ± 0.03
15.34 ± 0.41
52.39 ± 0.31
3.26
92.38 ± 0.38
423.45 ±
0.49
116.65 1.62 ± 0.15 261.39
18.34 ±
163.49
2.20
3.48
5.21
95.49 ± 0.29
0.31
135.59 ±
0.55
187.49 ±
0.14
234.45 ±
0.27
5g
5h
5i
27.28 2.11 ± 0.26 64.26
105.93 1.53 ± 0.38 122.54
70.25
87.61
169.89
5.82
128.82 1.72 ± 0.42 136.31
32.31 ±
6a
6b
6c
6d
6f
1.67
1.59
5.19
3.14
5.68
3.33
2.77
2.59
9.96
1.43
2.73
2.14
89.34 ± 0.55
0.23
39.49 ±
0.58
11.28 ±
0.22
64.59 ±
0.33
38.45 ±
0.62
102.39 ±
0.30
112.38 ±
0.47
1.95
10.34 ± 0. 37 10.87
15.43 ± 0.48
64.39 ± 0.28
18.34 ± 0.34
2.21 ± 0.28
6.00
1.63
92.55 ± 0.42
104.83 ±
0.71
39.43 ±
0.09
6g
4.61
84.56 ± 0.40
5-
1.92 ±
0.24
185.48 ±
0.48
96.60 1.65 ± 0.09 112.41
83.93
fluorouracil
In general, the cytotoxicity results, based on IC50 values for the compounds we tested, displayed
considerable effects (<100 µM) against all cancer cell lines, but 5a, 5b and 5c did not (>100 µM).
Among compounds tested, 5i exhibited the most promising anticancer activity with a considerably
broad spectrum of cytotoxic activity against all three human cancer cell lines. In tissue-specific
toxicity, compounds 5d, 5h and 5i showed very strong cytotoxic activity against HepG2 cell lines, all
having IC50 values of 1.53 µM; comparatively, 5-fluorouracil had an IC50 value of 1.65 µM. Both
compounds 5h and 5i demonstrated potent cytotoxic effects on DU145 cell lines with respect to the
cytotoxicity of the 5-fluorouracil. Compound 5i was the most potent cytotoxic agent against MBA-
MB-231 cell lines, with an IC50 value of 1.38 µM for each.
On the other hand, compounds 6a–g displayed mild to moderate cytotoxic activity with IC50
values in the range of 10.34–64.59 µM against all of the cancer cell lines. In addition, the selectivity
indexes of compounds were determined in this study. All compounds were tested on normal human
fibroblast cell lines. The IC50 values of compounds 5d, 5g, 5h and 5i were found to be high in normal
human fibroblast cell lines (Table 1), suggesting their high selectivity for cancer cells over normal
cells. These results may provide some important justification for further development as anticancer
agents.

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2.3. Structure–Activity Relationship (SAR)
The pyridine-3-carbonitrile derivatives (5a–i) showed more potent inhibition between 1 and 5
µM than the pyridine derivatives (6a–g) (Table 1). To explore the structure–activity relationship of
pyridine-3-carbonitriles (5), the aromatic substituents in 5a were initially replaced with 4-
methylbenzene (5b), 4-chlorobenzene (5c), 4-bromobenzene (5d), 2-methoxybenzene (5e), 3-
methoxybenzene (5f), 3-nitrobenzene (5g), 4-nitrobenzene (5h) and 3-bromo-4-methoxybenzene (5i)
groups.
The activity was increased in the 5d derivative compared to the chloro-5c one. This leads to the
conclusion that relatively larger substituents are valuable for good activity. Introducing a methoxy
group at position 2 of the phenyl ring of 5f, increases the activity. Therefore, the presence of a
hydrophilic substituent on the phenyl ring will increase the activity. To explore the influence of
electron withdrawing groups, a nitro group was introduced at positions 3 and 4 for 5g and 5h,
respectively. Surprisingly, these two compounds showed very potent inhibitions against cell lines
between 1 and 5 µM. Consequently, the nitro group can be considered an interesting moiety. In
addition, compound 5i with bromo and methoxy substitutions exhibited a similar activity to 5h.
In conclusion, the structural requirements of pyridine-3-carbonitrile derivatives to enhance their
cytotoxic activity against tested cell lines are as follows: (a) the nitrile substituent at position 3 on the
pyridine ring is essential for activity and as a basic pharmacophore; (b) the nitro, methoxy and chloro
substituents on the benzene ring are useful for the enhancement of activity (Figure 2). Such
substituents were selected to offer variable lipophilic, electronic and steric environments in order to
influence the biological activity being targeted; said specificity is believed to be responsible for the
biological significance of some anticancer agents [11].
Necessary
- to have the pyridine moiety
Cl
S
Cl
N
O
Important
Essential
CN
R1
- to have the nitrile
moiety at C-3 position
- to have the 2,5-dichlorothiophene
moiety
R2
R3
Compulsory
- Introduction of EWD (e.g NO2) at
C-3 and C-4 will increase the activity
- Introduction of hydrophilic group will
give the good activity
- Increase lipophilicity
Figure 2. Chemical structure of the pyridine-3-carbonitriles with important sites for the structure–
activity relationship (SAR).
3. Materials and Methods
3.1. Materials

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The starting material, 3-acetyl-2,5-dichlorothiophene (2) was prepared as described in the
previously-reported procedure [20]. The chalcone compounds, 4a–i, were synthesized according to
literature’s methods [25]. All reagents were purchased from Fluka and used as purchased. Solvents
were dried and distilled according to standard protocols.
3.2. Instrumentation
1
13
H (500 MHz) and C (125 MHz) NMR spectra were recorded in CDCl3 used as an internal
standard at 300 K on Bruker spectrometers (Bruker Biospin Mri GmbH, Ettlingen, Germany).
Chemical shifts (δ) are given in parts per million (ppm) and were determined from the center of the
respective coupling pattern (s: singlet; d: doublet; dd: doublet of d; t: triplet). ESI-HMRS
measurements were performed on an LTQ-FT mass spectrometer (Thermo Fisher Scientific,
Schwerte, Germany). All reactions were monitored using thin layer chromatography (TLC) coated
with silica gel (60 F254, Merck, Darmstadt, Germany). Pure compounds were obtained by preparative
TLC plates using a CHCl3/pentane (30:70) mixture as an eluent.
3.3. Synthesis
Synthesis of chalcones 4a–i, went according to the reported procedure [19]. A solution of 3-
acetyl-2,5-dichlorothiophene, 2, in MeOH (10 mL) was added dropwise to an equimolar mixture of
the corresponding aldehydes 3a–I and KOH in MeOH (50 mL). After the addition was completed,
the reaction mixture was stirred at RT for 10 h. The precipitate formed was filtered off, washed with
MeOH and dried without any further purification.
3.3.1. General procedure for the Synthesis 6-(2,5-dichlorothiophen-3-yl)-2-methoxy-4-(4-
methoxyphenyl)pyridines (5a–i) and (6a–g)
The pyridines (5a–i) and (6a–g) were prepared according to the methods reported in
[19,20,26,27]. A mixture of chalcone, 4, (0.01 mol), malononitrile (0.66g, 0.01 mol) and KOH (0.56 g,
0.01 mol) in MeOH (50 mL) was refluxed for about 2 h. The reaction mixture was cooled, and then
the solid obtained was filtered off, air-dried and purified using preparative TLC (20 × 20 cm) by
CHCl3/pentane (70:30) as a mobile phase to give the desired product, Scheme 1.
3.3.2. Characterization Data for Products
(6-(2,5-dichlorothiophen-3-yl)-2-methoxy-4-phenylpyridine-3-carbonitrile (5a). White solid (1.30 g, 72%
−1
yield). M.p.: 156–157 °C. IR (cm ) ν = 2219 (-CN, nitrile), 1689, 1535, 1451, 1440, 1355, 1224, 1112, 1014,
1
839, 763. H-NMR (CDCl3, 500 MHz): δ = 7.67 (m, 2H, H-2”, 6”), 7.65 (s, 1H, H-5), 7.56 (m, 3H, H-3”,
13
4”, 5”), 7.42 (s, 1H, H-4′), 4.17 (s, 3H, OCH3-2). C-NMR (CDCl3, 125 MHz)  = 164.8 (C-2), 156.6 (C-
4), 151.6 (C-6), 135.9 (C-1”), 135.6 (C-3′), 130.2 (C-4”), 129.1 (C-3”,5”), 128.4 (C-2”,6”), 127.5 (C-4′), 126.8
+
(C-5′), 126.4 (C-2′), 115.8 (C-5), 115.2 (CN-3), 93.7 (C-3), 54.9 (OCH3-2). (+)-ESIMS m/z 361([M + H] ,
+
+
+
+
74), 363 ([M + H + 2] , 56), 356 ([M + H + 4] , 14), 383 ([M + Na] , 100), 385 ([M + Na + 2] , 62), 387 ([M
+
+
+
+ Na + 4] , 17). (+)-HRESIMS m/z 382.9781 [M + Na] , 384.9752 [M + Na + 2] (calculated for
C17H10Cl2N2OSNa, 382.9783).
6-(2,5-dichlorothiophen-3-yl)-2-methoxy-4-p-tolylpyridine-3-carbonitrile (5b). White solid (1.46 g, 78%
1
1
yield). M.p.: 178–179 °C. IR (cm- ) ν = 2223 (-CN, nitrile), 1584, 1551, 1540, 1457, 1352, 1014, 807. H-
NMR (CDCl3, 500 MHz): δ = 7.64 (s,1H, H-5), 7.58 (d, J. = 8.3 Hz, 2H, H-2”,6”), 7.41 (s, 1H, H-4′), 7.36
13
(d, J. = 8.3 Hz, 2H, H-3”, 5”), 4.16 (s, 3H, OCH3-2), 2.47 (s, 3H, CH3-4”). C-NMR (CDCl3, 125 MHz) 
= 164.8 (C-2), 156.6 (C-4), 151.5 (C-6), 140.6 (C-4”), 135.7 (C-3′), 133.1 (C-1”), 129.8 (C-3”,5”), 128.3 (C-
2”,6”), 127.5 (C-4′), 126.8 (C-5′), 126.2 (C-2′), 115.7 (C-5), 115.4 (CN-3), 93.5 (C-3), 54.8 (OCH3-2), 21.4
+
+
+
(CH3-4”). (+)-ESIMS m/z 397 ([M + Na] , 100), 399 ([M + Na + 2] , 58), 401 ([M + Na + 4] , 14). (+)-
+
+
+
HRESIMS m/z 396.9936 [M + Na] , 398.9907 [M + Na + 2] , 400.9879 [M + Na + 4] (calculated for
C18H12Cl2N2OSNa, 396.9940).

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4-(4-chlorophenyl)-6-(2,5-dichlorothiophen-3-yl)-2-methoxypyridine-3-carbonitrile (5c). White solid (1.28 g,
−1
65% yield). M.p.: 195–196 °C. IR (cm ) ν = 2228 (-CN, nitrile), 1582, 1547, 1454, 1357, 1299, 1091, 1025,
1
1011, 826. H-NMR (CDCl3, 500 MHz): δ = 7.61 (d, J. = 8.9 Hz, 2H, H-2”,6”), 7.62 (s,1H, H-5), 7.54 (d, J.
13
= 8.9 Hz, 2H, H-3”, 5”), 7.42 (s, 1H, H-4′), 4.18 (s, 3H, OCH3-2). C-NMR (CDCl3, 125 MHz)  = 164.8
(C-2), 155.3 (C-4), 151.9 (C-6), 136.6 (C-4”), 135.5 (C-3′), 134.3 (C-1”), 129.8 (C-2”,6”), 129.4 (C-3”,5”),
127.4 (C-4′), 127.0 (C-5′), 126.5 (C-2′), 115.5 (C-5), 115.0 (CN-3), 93.5 (C-3), 55.0 (OCH3-2). (+)-ESIMS
+
+
+
+
m/z 395([M + H] , 16), 397 ([M + H+2] , 15), 417 ([M + Na] , 20), 419 ([M + Na + 2] , 20). (+)-HRESIMS
+
+
+
+
m/z 394.9578 [M + H] , 396.9548 [M + H+2] , 398.9519 [M + H + 4] , 400.9487 [M + H + 6] (calculated
for C17H10Cl3N2OS, 394.9574).
4-(4-bromophenyl)-6-(2,5-dichlorothiophen-3-yl)-2-methoxypyridine-3-carbonitrile (5d). White solid (1.47 g,
1
67% yield). M.p.: 214–215 °C. H-NMR (CDCl3, 500 MHz): δ = 7.71 (d, J. = 8.6 Hz, 2H, H-3”, 5”), 7.62
13
(s,1H, H-5), 7.54 (d, J. = 8.6 Hz, 2H, H-2”,6”), 7.41 (s, 1H, H-4′), 4.18 (s, 3H, OCH3-2). C-NMR (CDCl3,
125 MHz)  =164.8 (C-2), 155.4 (C-4), 151.9 (C-6), 135.5 (C-3′), 134.8 (C-1”), 132.4 (C-3”,5”), 130.0 (C-
2”,6”), 127.4 (C-4′), 127.0 (C-5′), 126.5 (C-2′), 125.1 (C-4”), 115.5 (C-5), 115.0 (CN-3), 93.5 (C-3), 55.0
+
+
+
(OCH3-2). (+)-ESIMS m/z 439 ([M + H] , 63), 441 ([M + H+2] , 100), 443 ([M + H + 4] , 48). (+)-HRESIMS
+
+
+
m/z 460.8890 [M + Na] , 462.8864 [M + Na + 2] , 464.8839 [M + Na + 4] (calculated for
C17H9BrCl2N2OSNa, 460.8888).
6-(2,5-dichlorothiophen-3-yl)-2-methoxy-4-(2-methoxyphenyl)pyridine-3-carbonitrile (5e). White solid (1.56
1
g, 80% yield). M.p.: 149–150 °C. H-NMR (CDCl3, 500 MHz): δ = 7.61 (s,1H, H-5), 7.50 (m, 1H, H-4”),
7.41 (s, 1H, H-4′), 7.35 (dd, J. = 8.3, 1.7 Hz, 1H, H-6”), 7.11 (m, 1H, H-5”), 7.09 (m, 1H, H-3”), 4.16 (s,
13
3H, OCH3-2), 3.91 (s, 3H, OCH3-2”). C-NMR (CDCl3, 125 MHz)  = 164.1 (C-2), 156.3 (C-2”), 154.4
(C-4), 151.3 (C-6), 135.8 (C-3′), 131.5 (C-4”), 130.3 (C-6”), 127.5 (C-4′), 126.7 (C-5′), 126.1 (C-2′), 125.1
(C-1”), 121.0 (C-5”), 117.1 (C-5), 115.1 (CN-3), 111.6 (C-3”), 95.9 (C-3), 55.6 (OCH3-2”), 54.7 (OCH3-2).
+
+
+
+
(+)-ESIMS m/z 391([M + H] , 48), 393 ([M + H + 2] , 17), 413 ([M + Na] , 100), 415 ([M + Na + 2] , 58),
+
+
+
417 ([M + Na + 4] , 13). (+)-HRESIMS m/z 412.9896 [M + Na] , 414.9867 [M + Na + 2] , 416.9837 [M + Na
+ 4] (calculated for C18H12Cl2N2O2SNa, 412.9889).
+
6-(2,5-dichlorothiophen-3-yl)-2-methoxy-4-(3-methoxyphenyl)pyridine-3-carbonitrile (5f). White solid (1.54
−1
g, 79% yield). M.p.: 179–180 °C. IR (cm ) ν = 2223 (-CN, nitrile), 1601, 1540, 1511, 1376, 1350, 1257,
1
1169, 1022, 820. H-NMR (CDCl3, 500 MHz): δ = 7.64 (s,1H, H-5), 7.45 (t, J. = 7.8 Hz, 1H, H-5”), 7.41 (s,
1H, H-4′), 7.22 (ddd, J. = 7.6, 1.7, 0.9 Hz, 1H, H-6”), 7.18 (t, J. = 2.4 Hz, 1H, H-2”), 7.07 (ddd, J. = 8.3,
13
2.6, 0.9 Hz, 1H, H-4”), 4.16 (s, 3H, OCH3-2), 3.90 (s, 3H, OCH3-3”). C-NMR (CDCl3, 125 MHz)  =
164.7 (C-2), 159.9 (C-3”), 156.5 (C-4), 151.6 (C-6), 137.2 (C-1”), 135.5 (C-3′), 130.2 (C-5”), 127.4 (C-4′),
126.8 (C-5′), 126.4 (C-2′), 120.7 (C-6”), 115.9 (C-4”),115.7 (C-5), 115.2 (CN-3), 113.9 (C-2”), 93.7 (C-3),
+
+
+
54.9 (OCH3-3”) 55.5 (OCH3-2). (+)-ESIMS m/z 391([M + H] , 48), 393 ([M + H+2] , 17), 413 ([M + Na] ,
+
+
+
100), 415 ([M + Na + 2] , 58), 417 ([M + Na + 4] , 13). (+)-HRESIMS m/z 412.9896 [M + Na] , 414.9867 [M
+ Na + 2] , 416.9837 [M + Na + 4] (calculated for C18H12Cl2N2O2SNa, 412.9889).
+
+
6-(2,5-dichlorothiophen-3-yl)-2-methoxy-4-(3-nitrophenyl)pyridine-3-carbonitrile (5g). Yellow solid (1.26 g,
1
62% yield). M.p.: 213–214 °C. IR (cm- ) ν = 2225 (-CN, nitrile), 1582, 1553, 1537, 1453, 1356, 1282, 1018,
1
837, 770. H-NMR (CDCl3, 500 MHz): δ = 8.48 (t, J. = 2.02Hz, 1H, H-2”), 8.42 (ddd, J. = 8.2, 2.3, 1.1Hz,
1H, H-4”), 8.03 (ddd, J. = 7.7, 1.8, 1.0 Hz, 1H, H-6”), 7.78 (t, J. = 8.1Hz, 1H, H-5”), 7.67 (s,1H, H-5), 7.43
13
(s, 1H, H-4′), 4.20 (s, 3H, OCH3-2). C-NMR (CDCl3, 125 MHz)  =164.8 (C-2), 154.0 (C-4), 152.4 (C-6),
148.6 (C-3”), 137.5 (C-1”), 135.1 (C-3′), 134.3 (C-6”), 130.3 (C-5”), 127.3 (C-4′), 127.2 (C-5′), 127.0 (C-2′),
124.8 (C-4”),123.5 (C-2”), 115.4 (C-5), 114.5 (CN-3), 93.7 (C-3), 55.1 (OCH3-2). (+)-ESIMS m/z 428 ([M +
+
+
+
+
Na] , 100), 430 ([M + Na + 2] , 66), 432 ([M + Na + 4] , 14). (+)-HRESIMS m/z 427.9629 [M + Na] , 429.9601
+
+
[M + Na + 2] , 431.9574 [M + Na + 4] (calculated for C17H9Cl2N3O3SNa, 427.9634).
6-(2,5-dichlorothiophen-3-yl)-2-methoxy-4-(4-nitrophenyl)pyridine-3-carbonitrile (5h). Brown solid (1.15 g,
1
57% yield). M.p.: 204–205 °C. H-NMR (CDCl3, 500 MHz): δ = 8.43 (d, J. = 8.3 Hz, 2H, H-3”, 5”), 7.84
13
(d, J. = 8.3 Hz, 2H, H-2”,6”), 7.66 (s,1H, H-5), 7.44 (s, 1H, H-4′), 4.20 (s, 3H, OCH3-2). C-NMR (CDCl3,
125 MHz)  = 164.8 (C-2), 154.1 (C-4), 152.4 (C-6), 148.8 (C-4”), 142.0 (C-1”), 135.2 (C-3′), 129.6 (C-
2”,6”), 127.3 (C-4′), 127.2 (C-5′), 126.9 (C-2′), 124.3 (C-3”,5”), 115.3 (C-5), 114.5 (CN-3), 93.7 (C-3), 55.1

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+
+
+
(OCH3-2). (+)-ESIMS m/z 428 ([M + Na] , 100), 430 ([M + Na + 2] , 66), 432 ([M + Na + 4] , 14). (+)-
+
+
+
HRESIMS m/z 427.9629 [M + Na] , 429.9601 [M + Na + 2] , 431.9574 [M + Na + 4] (calculated for
C17H9Cl2N3O3SNa, 427.9634).
4-(3-bromo-4-methoxyphenyl)-6-(2,5-dichlorothiophen-3-yl)-2-methoxypyridine-3-carbonitrile (5i). Yellow
1
solid (1.76 g, 75% yield). M.p.: 191–192 °C. H-NMR (CDCl3, 500 MHz): δ = 7.82 (d, J. = 2.5 Hz, 1H, H-
2”), 7.67 (dd, J. = 8.6, 2.4 Hz, 1H, H-6”), 7.58 (s,1H, H-5), 7.40 (s, 1H, H-4′), 7.06 (d, J. = 8.6 Hz, 1H, H-
13
5”), 4.16 (s, 3H, OCH3-2), 3.99 (s, 3H, OCH3-4”). C-NMR (CDCl3, 125 MHz)  =164.9 (C-2), 157.5 (C-
4”), 154.7 (C-4), 151.8 (C-6), 135.5 (C-3′), 133.2 (C-2”), 129.4 (C-1”),129.0 (C-6”), 127.4 (C-4′), 126.9 (C-
5′), 126.5 (C-2′), 115.4 (C-5), 115.2 (CN-3), 112.4 (C-3”),112.0 (C-5”), 93.3 (C-3), 56.5 (OCH3-4”), 54.9
+
+
+
(OCH3-2). (+)-ESIMS m/z 491 ([M + Na] , 66), 493 ([M + Na + 2] , 100), 495 ([M + Na + 4] , 46). (+)-
+
+
+
HRESIMS m/z 490.8995 [M + Na] , 492.8971 [M + Na + 2] , 494.8945 [M + Na + 4] (calculated for
C18H11BrCl2N2O2SNa, 490.8994).
2-(2,5-dichlorothiophen-3-yl)-6-methoxy-4-phenylpyridine (6a). Pale yellow solid (0.25 g, 15% yield). M.p.:
1
127–128 °C. H-NMR (CDCl3, 500 MHz): δ = 7.72 (d, J. = 1.5 Hz, 1H, H-5), 7.68 (m, 2H, H-2”, 6”), 7.50
13
(m, 3H, H-3”,4”, 5”), 7.43 (s, 1H, H-4′), 6.95 (d, J. = 1.5 Hz, 1H, H-3), 4.06 (s, 3H, OCH3-2). C-NMR
(CDCl3, 125 MHz)  =164.3 (C-2), 151.9 (C-4), 149.0 (C-6), 138.4 (C-1”), 137.2 (C-3′), 129.1 (C-4”), 129.1
(C-3”, 5”), 127.9 (C-4′),127.1 (C-2”,6”), 126.1 (C-5′), 123.7 (C-2′), 114.4 (C-5), 107.7 (C-3), 53.7 (OCH3-2).
+
+
+
(+)-ESIMS m/z 336 ([M + H] , 100), 338 ([M + H + 2] , 64), 340 ([M + H + 4] , 13). (+)-HRESIMS m/z
+
+
336.0009 [M + H] , 337.9980 [M + Na + 2] (calculated for C16H12Cl2NOS, 336.0011).
2-(2,5-dichlorothiophen-3-yl)-6-methoxy-4-p-tolylpyridine (6b). Pale yellow solid (0.32 g, 18% yield). M.p.:
1
150–151 °C. H-NMR (CDCl3, 500 MHz): δ = 7.69 (d, J. = 1.2 Hz,1H, H-5), 7.57 (d, J. = 7.9 Hz, 2H, H-
2”,6”), 7.42 (s, 1H, H-4′), 7.31 (d, J. = 7.9 Hz, 2H, H-3”,5”), 6.92 (d, J. = 1.2 Hz, 1H, H-3), 4.04 (s, 3H,
13
OCH3-2), 2.44 (s, 3H, CH3-4”). C-NMR (CDCl3, 125 MHz)  =164.3 (C-2), 151.8 (C-4), 148.8 (C-6), 139.3
(C-4”), 137.2 (C-3′), 135.4 (C-1”), 129.8 (C-3”,5”), 127.9 (C-4′), 126.9 (C-2”,6”), 126.0 (C-5′), 123.6 (C-2′),
+
114.3 (C-5), 107.3 (C-3), 53.7 (OCH3-2), 21.3 (CH3-4”). (+)-ESIMS m/z 350 ([M + H] , 100), 352 ([M +
+
+
+
+
H+2] , 70), 354 ([M + H+4] , 15). (+)-HRESIMS m/z 350.0166 [M + H] , 352.0137 [M + H+2] (calculated
for C17H14Cl2NOS, 350.0168).
4-(4-chlorophenyl)-2-(2,5-dichlorothiophen-3-yl)-6-methoxypyridine (6c). Pale yellow solid (0.24 g, 13%
1
1
yield). M.p.: 142–143 °C. IR (cm- ) ν = 1608, 1550, 1452, 1361, 1205, 1092, 1024, 1012, 814. H-NMR
(CDCl3, 500 MHz): δ = 7.65 (d, J. = 1.3 Hz, 1H, H-5), 7.59 (d, J. = 8.6 Hz, 2H, H-2”,6”), 7.48 (d, J. = 8.6
13
Hz, 2H, H-3”, 5”), 7.41 (s, 1H, H-4′), 6.89 (d, J. = 1.3 Hz,1H, H-3), 4.04 (s, 3H, OCH3-2). C-NMR
(CDCl3, 125 MHz)  =164.3 (C-2), 150.6 (C-4), 149.1 (C-6), 137.0 (C-3′), 136.7 (C-1”), 135.3 (C-4”), 129.3
(C-3”,5”), 128.4 (C-2”,6”), 127.8 (C-4′), 126.1 (C-5′), 123.8 (C-2′), 114.0 (C-5), 107.5 (C-3), 53.7 (OCH3-2).
+
+
+
(+)-ESIMS m/z 370 ([M + H] , 100), 372 ([M + H + 2] , 98), 374 ([M + H + 4] , 55). (+)-HRESIMS m/z
+
+
+
369.9620 [M + H] , 371.9591 [M + H + 2] , 373.9562 [M + H + 4] (calculated for C16H11Cl3NOS, 369.9621).
4-(4-bromophenyl)-2-(2,5-dichlorothiophen-3-yl)-6-methoxypyridine (6d). Pale yellow solid (0.31 g, 15%
1
1
yield). M.p.: 148–149 °C. IR (cm- ) ν = 1606, 1550, 1448, 1357, 1207, 1019, 1007, 809. H-NMR (CDCl3,
500 MHz): δ = 7.66 (d, J. = 1.4 Hz, 1H, H-5), 7.64 (d, J. = 8.5 Hz, 2H, H-3”, 5”), 7.53 (d, J. = 8.5 Hz, 2H,
13
H-2”,6”), 7.42 (s, 1H, H-4′), 6.89 (d, J. = 1.4 Hz,1H,H-3), 4.05 (s, 3H, OCH3-2). C-NMR (CDCl3, 125
MHz)  = 164.3 (C-2), 150.6 (C-4), 149.2 (C-6), 137.3 (C-1”), 137.0 (C-3′), 132.3 (C-3”,5”), 128.6 (C-2”,6”),
127.8 (C-4′), 126.2 (C-5′), 123.8 (C-2′), 123.5 (C-4”), 114.0 (C-5), 107.5 (C-3), 53.7 (OCH3-2). (+)-ESIMS
+
+
+
+
m/z 414 ([M + H] , 60), 416 ([M + H+2] , 100), 418 ([M + H+4] , 45). (+)-HRESIMS m/z 413.9117 [M + H] ,
+
+
+
415.9094 [M + H+2] , 417.9067 [M + H+4] , 419.9042 [M + H+6] (calculated for C16H11BrCl2NOS,
413.9116).
2-(2,5-dichlorothiophen-3-yl)-6-methoxy-4-(2-methoxyphenyl)pyridine (6e). Pale yellow solid (0.39 g, 21%
1
yield). M.p.: 135–136 °C. H-NMR (CDCl3, 500 MHz): δ = 7.40 (m, J. = 8.3, 1.7 Hz, 1H, H-6”), 7.68 (d, J.
= 1.3 Hz, 1H, H-5), 7.44 (m, 1H, H-4”), 7.41 (s, 1H, H-4′), 7.09 (m, 1H, H-5”), 7.05 (dd, J. = 8.3, 1.1 Hz,
13
1H, H-3”), 6.93 (d, J. = 1.3 Hz,1H, H-3), 4.05 (s, 3H, OCH3-2), 3.88 (s, 3H, OCH3-2”). C-NMR (CDCl3,
125 MHz)  = 163.7 (C-2), 156.6 (C-2”), 149.5 (C-4), 148.0 (C-6), 137.5 (C-3′), 130.4 (C-4”),130.1 (C-6”),
128.0 (C-4′), 127.8 (C-1”), 125.9 (C-5′), 123.3 (C-2′), 121.0 (C-5”), 117.0 (C-5), 111.5 (C-3”), 110.3 (C-3),

Molecules 2019, 24, 4072
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+
+
+
55.6 (OCH3-2”), 53.5 (OCH3-2). (+)-ESIMS m/z 366 ([M + H] , 100), 368 ([M + H+2] , 65), 370 ([M + H+4] ,
+
+
+
15). (+)-HRESIMS m/z 366.0117 [M + H] , 368.0085 [M + H+2] , 370.0057 [M + H+4] (calculated for
C17H14Cl2NO2S, 366.0117).
2-(2,5-dichlorothiophen-3-yl)-6-methoxy-4-(3-methoxyphenyl)pyridine (6f). Pale yellow solid (0.33 g, 18%
1
yield). M.p.: 156–157 °C. H-NMR (CDCl3, 500 MHz): δ = 7.70 (d, J. = 1.7 Hz, 1H, H-5), 7.43 (t, J. = 8.3
Hz, 1H, H-5”), 7.42 (s, 1H, H-4′), 7.26 (ddd, J. = 7.7, 1.4, 0.9 Hz, 1H, H-6”), 7.19 (t, J. = 1.9 Hz, 1H, H-
2”), 7.02 (ddd, J. = 8.3, 2.4, 0.8 Hz, 1H, H-4”), 6.94 (d, J. = 1.7 Hz, 1H, H-3), 4.06 (s, 3H, OCH3-2), 3.91
13
(s, 3H, OCH3-3”). C-NMR (CDCl3, 125 MHz)  = 164.2 (C-2), 160.1 (C-3”), 151.8 (C-4), 149.0 (C-6),
139.8 (C-1”), 137.2 (C-3′), 130.1 (C-5”), 127.9 (C-4′), 126.1 (C-5′), 123.7 (C-2′), 119.5 (C-6”), 114.4 (C-
+
4”),114.4 (C-5),112.9 (C-2”), 107.7 (C-3), 55.4 (OCH3-3”) 53.7 (OCH3-2). (+)-ESIMS m/z 366 ([M + H] ,
+
+
+
+
100), 368 ([M + H+2] , 65), 370 ([M + H+4] , 15). (+)-RESIMS m/z 366.0117 [M + H] , 368.0085 [M + H+2] ,
+
370.0057 [M + H+4] (calculated for C17H14Cl2NO2S, 366.0117).
2-(2,5-dichlorothiophen-3-yl)-6-methoxy-4-(3-nitrophenyl)pyridine (6g). Pale yellow solid (0.21 g, 11%
1
1
yield). M.p.: 182–183 °C. IR (cm- ) ν = 1604, 1556, 1521, 1454, 1349, 1211, 1025, 834, 737. H-NMR
(CDCl3, 500 MHz): δ = 8.45 (t, J. = 1.8 Hz, 1H, H-2”), 8.34 (ddd, J. = 8.1, 2.0, 1.3 Hz, 1H, H-4”), 7.99
(ddd, J. = 7.3,1.8, 1.8 Hz, 1H, H-6”), 7.73 (t, J. = 8.3 Hz, 1H, H-5”), 7.72 (d, J. = 1.3 Hz, 1H, H-5), 7.44 (s,
13
1H, H-4′), 6.97 (d, J. = 1.3 Hz, 1H, H-3), 4.08 (s, 3H, OCH3-2). C-NMR (CDCl3, 125 MHz)  =164.5 (C-
2), 149.7 (C-6), 149.3 (C-4), 148.9 (C-3”), 140.2 (C-1”), 136.8 (C-3′), 133.0 (C-6”), 130.2 (C-5”), 127.7 (C-
4′), 126.4 (C-5′), 124.1 (C-2′), 123.8 (C-4”),122.1 (C-2”), 113.9 (C-5), 107.9 (C-3), 53.8 (OCH3-2). (+)-ESIMS
+
+
+
+
m/z 381 ([M + H] , 16), 383 ([M + H + 2] , 10). (+)-HRESIMS m/z 380.9860 [M + H] , 382.9831 [M + H+2]
(calculated for C16H11Cl2N2O3S, 380.9862).
3.4. Cell Culture and Maintenance
Three different cancer cell lines; namely, DU145 derived from prostate cancer cells (American
Type Culture Collection No. HTB-81), HepG2 derived from human liver cancer cells (American Type
Culture Collection No. HB-8065), MBA-MB-231 derived from breast cancer cells (American Type
Culture Collection No. HTB-26) and normal human fibroblasts (HSF1184) were grown and cultivated
according to our previously described method [18]. Briefly, cells were seeded in Dulbecco′s modified
Eagle’s media (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS),
100ꢁIU/mL penicillin, 100ꢁmg/mL streptomycin and 2ꢁmM glutamine. The cells were maintained in a
humidified atmosphere with 5% CO2 at 37 °C for 8 days, with subsequent media renewal. Then, cells
were plated in a 96-well flat bottom plate at a concentration of 104 cells/well in fresh complete growth
medium for 24 h and maintained in 37 °C in a 5% CO2-humidified incubator.
3.5. Cytotoxicity Assay
Cells in 96-well flat bottom plates were first serum-starved for 4 h by replacing cultured media
with fresh medium (without serum). All synthesized compounds were properly dissolved in
dimethyl sulfoxide (DMSO) to produce a stock concentration of 0.1 M. Cells were incubated with a
range of different concentrations of the compounds we were testing (0–1000 µM) to give a final
volume of 300 µL per well. In comparison, cells were incubated either alone with DMSO (negative
control) or with 5-fluorouracil as a positive control. DMSO was used as a vehicle for the dissolution
of the tested compounds and its final concentration in cultured media was less than 0.1%. Triplicate
wells were designed for each individual dose. The compounds we synthesized were evaluated for
in-vitro cytotoxicity properties via the standard 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay accordingly to our previously-reported work [2]. The normal human fibroblast
line (HSF1184) was used as a standard reference for the measurement of selectivity indexes (SIs)
relative to cancer cell lines. The IC50 values and SIs were calculated accordingly.
4. Conclusions
In conclusion, a series of 2-methoxypyridine-3-carbonitrile bearing aryl substituents were
synthesized, in most cases in good yields, by the condensation of chalcones with malononitrile in

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basic medium. Although decarbonylation byproducts were observed in poor yields, they offer a route
to a variety of methoxypyridine derivatives. All new compounds were fully characterized using
different spectroscopic methods. Furthermore, these compounds were screened for their in-vitro
cytotoxicity activities against three cancer cell lines; namely, those of the liver (line HepG2), prostate
(line DU145) and breast (line MBA-MB-231). Although the IC50 values are not very impressive, their
selectivity with respect to normal cells is, in some cases, very high. Remarkably, the decarbonylated
substrates show less cytotoxicity on normal cells than in cancer cells. The cytotoxicity assessments
revealed that compounds 5d, 5g, 5h and 5i exhibit promising antiproliferative effects (IC50 1–5 µM)
against tested cancer cell lines.
Supplementary Materials: NMR and Mass spectra for the compounds we prepared are available online.
Author Contributions: M.A.-R. proposed the subject and designed the study. M.A.-R. and Mohammad M.M.I.
carried out the chemical experiments and wrote the article. M.N.A., H.O. and M.H.A.B. carried out the cytotoxic
activity and performed the SAR analysis. A.G. provided the chemicals, performed instrumental measurements
and helped in the characterization of all compounds.
Funding: We are grateful to the deanship of scientific research, Al al-Bayt University, Mafraq, Jordan, for the
financial support of this project (research grant number 8810/2017).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 5a-i are available from the authors.
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