Anticancer evaluation of some newly synthesized N-nicotinonitrile derivative

Article abstract of DOI:10.1016/j.ejmech.2013.09.005

Some novel N-nicotinonitrile derivatives 3-14 have been synthesized starting with compound 1. The key step of this work is the coupling between compound 1 and activated sugars to afford the corresponding cyclic nucleosides 3-6. Moreover, the cytotoxicity

Full text of DOI:10.1016/j.ejmech.2013.09.005

Accepted Manuscript
Anticancer Evaluation of Some Newly Synthesized N- Nicotinonitrile Derivative
Ahmed H. Shamroukh, Mahmoud El-Shahat, Józef Drabowicz, Mamdouh M. Ali,
Aymn E. Rashad
PII:
S0223-5234(13)00576-X
10.1016/j.ejmech.2013.09.005
EJMECH 6406
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 April 2013
Revised Date: 2 September 2013
Accepted Date: 6 September 2013
Please cite this article as: A.H. Shamroukh, M. El-Shahat, J. Drabowicz, M.M. Ali, A.E. Rashad,
Anticancer Evaluation of Some Newly Synthesized N- Nicotinonitrile Derivative, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.09.005.
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ACCEPTED MANUSCRIPT
Graphical Abstract
O
O
COPh
Ar
CH2Ph
Ar
NC
Ar
NC
Ar
N
N
11
12
Ar = p- ClC6H4-
Pyridine-3-carbonitrile derivatives possess significant antitumor activity comparable
to the activity of commonly used anticancer drug, Doxorubicin

ACCEPTED MANUSCRIPT
Anticancer Evaluation of Some Newly Synthesized N- Nicotinonitrile
Derivative
Ahmed H. Shamroukh* a, Mahmoud El-Shahat a, Józef Drabowicz b, Mamdouh M. Ali c,
Aymn E. Rashad a,d
a Photochemistry Department, National Research Center, Dokki,12311 Cairo, Egypt.
b Center of Molecular and Macromolecular Studies, Polish Academy of Science, Łódź, Poland.
cBiochemistry Departments, Division of Genetic Engeneering and Biotechnology, National Research Center,
Dokki,Cairo,Egypt.
d Chemistry Department, Faculty of Science and Human Studies, Huryaimla, Shaqra Univerisity, KSA
* Corresponding author email: ahshamroukh@yahoo.com, Tel number: +20(2)7617590, Fax: +20 20337 0931.
ABSTRACT
Some novel N-nicotinonitrile derivatives 3-14 have been synthesized starting with compound 1.
The key step of this work is the coupling between compound 1 and activated sugars to afford the
corresponding cyclic nucleosides 3-6. Moreover, the cytotoxicity and in vitro anticancer
evaluation of the prepared compounds have also been assessed against breast MCF-7 cancer,
liver HepG2 cancer and lung A549 carcinoma cell lines with investigation the effect of the
synthesized compounds on the expression of urokinase plasminogen activator (uPA). The results
revealed that, although all the compounds showed no anticancer activity against A549 cells
without showing any effect on the expression of uPA, the tested compounds exhibited
remarkable cytotoxicity activity against MCF-7 and HepG2 cell lines. Among the tested
compounds, compounds 11 and 12 revealed promising anticancer activity compared to the
activity of the commonly used anticancer drug, doxorubicin with inhibiting the expression of
uPA.
Keywords: Nicotinonitrile/ nucleosides/ Anticancer / Urokinase.
1. Introduction
Targeted therapies have a high specificity toward tumor cells, providing a broader therapeutic
window with less toxicity. They are also often useful in combination with cytotoxic
chemotherapy or radiation to produce additive or synergistic anticancer activity because their
toxicity profiles often do not overlap with traditional cytotoxic chemotherapy. Thus, targeted
therapies represent a new and promising approach to cancer therapy, one that is already leading
to beneficial clinical effects. There are multiple types of targeted therapies available, including
monoclonal antibodies, inhibitors of tyrosine kinases and antisense inhibitors of growth factor
receptors [1].
Urokinase plasminogen activator (uPA) is a serine protease that functions in the conversion of
the circulating plasminogen to the active, broad-spectrum, serine protease plasmin. uPA is
secreted as an inactive single-chain proenzyme by many different cell types and exists in a
soluble or cell-associated form by binding to a specific membrane uPA receptor (uPAR) [2,3].
The uPA is involved in many physiological functions and, along with members of the matrix
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metalloproteinases (MMPs) family; it has been implicated in cancer invasion and metastatization
[4-6]. Besides the proteolytic function, upon binding to uPAR, uPA is involved in initiating
versatile intracellular signal pathways that regulate cell proliferation, adhesion, and migration
through its interaction with various integrins and vitronectin [7]. Urokinase is implicated in a
large number of malignancies, e.g. cancers of breast, lung, bladder, cervix, kidney, stomach and
brain [8,9]. Also, the expression of urokinase is associated with tumor growth, invasion and may
be a useful prognostic factor for hepatocellular carcinoma [10]. The role of uPA in human cancer
progression is further supported by clinical evidences demonstrating that high tissue levels of its
components correlate with a poor prognosis in different types of cancer as breast, gastrointestinal
cancers [11].
On the other hand, the chemistry of cyclic and acyclic nicotinonitrile nucleosides has attracted
attention during the last few decades because of its interesting pharmacological activities as
antiviral [12], antitumor [13], antibacterial [14,15], anticancer [16,17] and other biological
activity [18-25]. In the same direction and in continuation of our previous work for the synthesis
of different nucleosides [26-28], to find more potent and selective anticancer compounds, we
synthesized a series of cyclic nucleosides of pyridine derivatives.
2. Results and Discussion
2.1. Chemistry
Heating of 4,6-Bis-(4-chloro-phenyl)-2-oxo-1,2 -dihydro-pyridine-3-carbonitrile (1) [27] with
hexamethyldisilazane (HMDS) in the presence of ammonium sulfate gave the silyloxypyridine
derivative 2, which was subsequently treated with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-
ribofuranose in the presence of SnCl4 according to the method of Niedball and Vorbrüggen [29]
1
to afford the corresponding N-riboside 3 (Scheme 1). The H NMR spectrum of compound 3
showed a doublet at δ 6.05 ppm (J1’,2’ = 4.30 Hz) assigned to the anomeric proton indicating the
β- configuration [28,30] and the other sugar protons resonate at δ 3.37–3.55(m, 1H, 4’-H); 4.43–
4.76 (m, 4H, 2’-H, 3’-H, 5’-H2). Similarly, when the silyloxypyridine derivative 2 was treated
with β-D-glycopyranose pentaacetate in the presence of SnCl4, it afforded the corresponding N-
glycoside 4. The 1H NMR spectrum of compound 4 showed a doublet at 5.26 ppm (J1’,2’ = 10.40
Hz) assigned to the anomeric proton of the glucose moiety with a diaxial orientation of H-1’ and
H-2’ indicating the β-configuration. The other protons of the glucopyranose ring resonate at
3.91–4.09 (m, 2H, 6’-H2); 4.26– 4.69 (m, 4H, 2’-H; 3’-H, 4’-H, 5’-H), while the four acetoxy
groups appear as four singlets in the 2.01-2.23 ppm region providing further verification of the
structure [31]. Debenzoylation of the blocked riboside 3 was achieved in methanolic ammonia at
0◦C to afford the desired free 1-β-D-ribofuranosyl derivative 5. The IR and IH NMR spectrum of
the latter compound showed the presence of hydroxyl groups and the 13C NMR spectrum
providing further verification of the structure (see Experimental). Also, deprotection of
compound 4 with methanolic ammonia at room temperature afforded the free N-glycoside 6
1
(Scheme 1). The H NMR spectrum of the latter compound showed the anomeric proton as a
doublet at δ 6.02 (J1’,2’ = 9.80 Hz) indicative for the β-D-configuration, and the signals of the
other six carbon-bonded glucose protons appear as multiplets at δ 3.72–4.01 and 4.27–4.59,
while the signals of the four hydroxyl groups of the glucose moiety are observed at δ 5.01, 5.12,
5.20 and 5.34 (exchangeable with D2O). The 13C NMR spectrum of compound 6 is characterized
by the appearance of the C=O signal and the presence of a signal at δ 90.23 corresponding to the
C-1’ atom. Another five signals at δ 59.77, 64.21, 67.01, 70.68, and 74.11 are assigned to C-6’,
C-4’, C-2’, C-3’ and C-5’, respectively (see Experimental). Moreover, the IR and 13C NMR
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spectra of compounds 3-6 indicated that the site of attack was on the nitrogen since it behaves as
a nucleophile which attacks an electrophilic carbon atom of an alkyl halide.
Moreover, the sodium salt of derivative 1 was treated with ethyl iodide, allyl bromide, propagyl
bromide, chloro acetonitrile, benzyl chloride, benzoyl chloride, epichlorohydrine or 1,3-dichloro-
2-prpanol to afford the corresponding N-substituted derivatives 7–14, respectively (Scheme 2).
The IR and 13C NMR spectra of the latter compounds revealed the appearance of the C= O signal
1
and the H NMR spectra indicated the absence of the NH group and the presence of ethyl, allyl,
prop-2ynyl, cyanomethyl, benzyl, benzoyl, oxiran-2ylmethyl and 3-chloro-2-hydroxypropyl
signals, respectively (see Experimental). This due to the fact that the nitrogen atom behaves as a
nucleophile attacks an electrophilic carbon atom of an alkyl halide [27].
Scheme 1
Scheme 2
2.2. Anticancer Evaluation
2.2. 1. In vitro cytotoxicity activity
The cytotoxicity of the synthetic compounds 1, 3-14 were tested using SRB assay as
described by Skehan et al. [32] in breast cancer cell line MCF-7, liver cancer cell line HepG2,
and lung carcinoma cell line A549. For comparison, doxorubicin was also tested. The results
revealed that all the compounds did not exert any activity against lung carcinoma cell line A549.
In case of breast cancer cell line MCF-7 and liver cancer cell line HepG2 although
compounds 1 and 10 showed no activity in the two cell lines, compound 12 (IC50: 2.60±0.27 and
3.75±0.44 µg/ml, respectively) exhibited similar activity to doxorubicin (IC50: 2.80±0.24 and
3.75±0.35µg/ml, respectively) in both MCF-7 and HepG2. The order of cytotoxicity activity of
the tested compounds was 12, 11, 14, 13, 3, 5, 4, 6, 9, 8, and 7 in a descending order.
2.2. 2. The level of uPA protein expression
To identify the mechanism of action responsible for the cytotoxicity of prepared
compounds 1, 3-14 the level of uPA protein expressed in the two cell lines (breast cancer cell
line MCF-7 and liver cancer cell line HepG2) were estimated quantitatively. The result revealed
that the data of uPA expression were in consistent with the cytotoxicity activity.
In case of breast cancer cell line MCF-7 and liver cancer cell line HepG2, compounds 1 and 10
have no effect on the expression of uPA, the level of uPA decreased in compounds by the
following percent in MCF-7 and HepG2 respectively 3 (66, 60%), 4 (54, 48%), 5 (60, 56%), 6
(9, 6%), 7 (3, 2%), 8 (3, 5%), 9 (7, 5%), 11 (86, 83%), 12 (90, 87%), 13 (77, 63%) and 14 (82,
67%). From the results, compound 12 exhibited a good activity in MCF-7 (90%) and HepG2
(87%) similar to doxorubicin (91% and 88%, respectively) (Table 1). in both MCF-7 and
HepG2. The order of uPA activity inhibition of the tested compounds was 12, 11, 14, 13, 3, 5, 4,
6, 9, 8, and 7 in a descending order which are in accordance with the cytotoxicity activity.
Taken together, these findings suggested that there are correlation between the cytotoxicity of
the tested compounds and inhibition of the urokinase activity. The tested compounds exert anti-
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carcinogenic activity in MCF-7 breast and HepG2 liver cancer cells through inhibiting the
activity of urokinase enzyme which may reduce the cell proliferation and resulted in significant
growth inhibitory.
Table 1.
2.3. Conclusion
In conclusion, the present results suggested that there are correlation between the cytotoxicity of
the synthesized compounds 1, 3-14 and inhibition of the urokinase activity. The tested
compounds exert anti-carcinogenic activity in hepatic HepG2 and breast MCF-7 cancer cell lines
through down-regulation the activity of urokinase enzyme which may reduce the cell
proliferation and resulted in significant growth inhibitory, especially, compounds 11 and 12
which revealed promising activity compared to the activity of the commonly used anticancer
drug, doxorubicin.
3. Experimental
3.1. Chemistry
All melting points are uncorrected and measured using Electro-Thermal IA 9100 apparatus
(Shimadzu, Japan). Infrared spectra were recorded as potassium bromide pellets on a Perkin-
Elmer 1650 spectrophotometer (Perkin-Elmer, Norwalk, CT, USA), National Research Center,
1
Cairo, Egypt. H NMR and 13C NMR spectra were determined on a Jeol-Ex-400 NMR
spectrometer (JEOL, Tokyo, Japan) and chemical shifts were expressed as part per million; (δ
values, ppm) against TMS as internal reference (Organic Chemistry Department , Center of
Molecular and Macromolecular Studies, Polish Academy of Science, Łódź, Poland). Mass
spectra were recorded on VG 2AM-3F mass spectrometer (Thermo electron corporation, USA),
National Research Center, Cairo, Egypt. Microanalyses were operated using Mario El Mentar
apparatus, Organic Microanalysis Unit, and the results were within the accepted range (± 0.20)
of the calculated values. Follow up of the reactions and checking the purity of the compounds
was made by TLC on silica gel-precoated aluminum sheets (Type 60 F254; Merck, Darmstadt,
Germany). Column Chromatography was performed on (Merck) Silica gel 60 (particle size 0.06–
0.20 mm). Compound 1 was prepared according to a reported method [27].
3.1.1. Synthesis of 4,6-bis(4-Chlorophenyl)- 2-oxo-1-(2’,3’,5’-tri-O-benzoyl-β-D-
ribofuranosyl)-1,2dihydro pyridine-3-carbonitrile (3)
Compound 1 (0.340 g, 1 mmol) was refluxed by stirring under anhydrous condition for 12 hours
with hexamethyldisilazane (60 mL) and ammonium sulfate (0.02 g). The obtained clear solution
was evaporated under reduced pressure and the resulting trimethylsilylated pyridine 2 was
dissolved in dry 1,2-dichloromethane (40 mL) and a solution of 1-O-acetyl- 2,3,5-tri-O-benzoyl-
β-D-ribofuranose (1 mmol) and SnCl4 (1.6 mL) in dry 1,2-dichloromethane (10 mL) was then
added dropwise with stirring under inert atmosphere (nitrogen gas) until the reaction was judged
complete by thin layer chromatography. The reaction mixture was poured into saturated
NaHCO3 solution and extracted with CHCl3 (2 × 30 mL). The extracts were collected and dried
over anhydrous Na2SO4, filtered and concentrated producing the crude nucleoside that was
purified on silica gel column using n-hexane: ethyl acetate (4:1) as an eluent to give 3. Yield
1
73%; oil; IR (KBr, ν, cm−1): 2214 (CN), 1716, 1682 (C=O); H NMR (CDCl3, δ ppm): 3.37-
4

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3.55(m, 1H, 4’-H), 4.43–4.76 (m, 4H, 5’-H21,32’-H, 3’-H), 6.05 (d, 1H, J1’,2’ = 4.30 Hz, 1’-H),
7.49–8.15 (m, 24H, 23Ar-H + pyridine-H5); C NMR (CDCl3, δ ppm): 60.77 (C-5’), 67.28 (C-
3’), 68.93 (C-2’), 73.01 (C-4’), 93.25 (C-1’), 114.37 (CN), 129.33–136.16 (Ar-C), 164.37,
174.34, 179.84, 185.12 (4C=O); Anal. Calcd. For C44H30Cl2N2O8 (785.64): C 67.27, H 3.85, Cl
9.03, N 3.57. Found: C 66.97, H 3.61, Cl 8.74, N 3.21.
3.1.2. Synthesis of 4,6-bis(4-Chlorophenyl)- 2-oxo-1-(2’,3’,4’5’-tetra-O-acetyl-β-D-
glucopyranosyl)-1,2-dihydro pyridine-3-carbonitrile (4)
The same procedure as in preparation of compound 3 was performed, but 1,2,3,4,6-penta-O-
acetyl-β-D-glucopyranose (1 mmol) was used to give product 4. Yield 59%; m.p. 183–185◦C; IR
(KBr, ν, cm−1): 2222 (CN), 1713, 1688 (C=O). 1H NMR (CDCl3, δ ppm): 2.01, 2.09, 2.13, 2.23
(4s, 12H, 4CH3CO), 3.91–4.09 (m, 2H, 6’-H2), 4.26–4.69(m, 4H, 5’-H, 4’-H, 3’-H, 2’-H), 5.26
(d, 1H, J1’,2’ = 10.40 Hz, 1’-H), 7.36–8.01(m,9H, 8Ar-H+ pyridine-H5); 13C NMR (DMSO, δ
ppm): 20.30–20.5 (4CH3CO), 61.00 (C-6’), 66.49 (C-2’), 68.22 (C-3’), 71.69 (C-4’), 75.90 (C-
5’), 81.80 (C-1’), 115.22 (CN), 125.11 – 139.02 (Ar-C), 167.90, 169.44, 169.72, 170.02,170.60
(5C=O); Anal. Calcd. For C32H28Cl2N2O10 (671.49): C 57.24, H 4.20, Cl 10.56, N 4.17. Found:
C 56.98, H 3.89, Cl 10.31, N 3.82.
3.1.3. Synthesis of 4,6-bis(4-Chlorophenyl)- 1-(1-β-D-ribofuranosyl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile (5)
Dry ammonia gas was passed into a solution of protected nucleoside 3 (1 mmol) in dry methanol
(20 mL) at 0◦C for 30 minute, then the reaction mixture was stirred at room temperature until the
reaction was judged complete by TLC. The resulting mixture was then concentrated under
reduced pressure at 40◦C to afford a solid residue that was purified on silica gel column using
chloroform: methanol (4:1) as an eluent to give 5.Yield 53%; oil; IR (KBr, ν, cm−1): 3444–3232
1
(OH), 2220 (CN), 1670 (C=O); H NMR (DMSO-d6, δ ppm): 3.53–3.57 (m, 3H, 4’-H, 5’-H2),
3.88–4.89 (m, 2H, 2’-H, 3’-H), 4.60–4.62 (m,1H, OH, D2O exchangeable), 4.72–4.74 (m, 1H,
OH, D2O exchangeable), 4.83– 4.87(m, 1H, OH, D2O exchangeable), 6.53 (d, 1H, J1’,2’ = 4.60
Hz, 1’-H),7.61–8.31 (m, 9H, 8Ar-H+ pyridine-H5); 13CNMR (DMSO, δ ppm): 62.01(C-5’),
71.22(C-3’), 76.21(C-2’), 81.55 (C-4’), 88.33 (C-l’), 115.51 (CN), 124.46 – 133.65 (Ar-C),
167.22 (C=O); Anal. Calcd. For C23H18Cl2N2O5 (473.32): C 58.37,H 3.83, Cl 14.98, N 5.92.
Found: C 58.09, H 3.63, Cl 14.71, N 5.60.
3.1.4. Synthesis of 4,6-bis(4-Chlorophenyl)- 1-(1-β-D-glucopyranosyl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile (6)
The same procedure as in preparation of compound 5 was used to give product 6 from 4.Yield
1
42%; 284–286◦C; IR (KBr, ν, cm−1): 3460–3220 (OH), 2217 (CN), 1680 (C=O); H NMR
(DMSO-d6, δ ppm): 3.72–4.01(m, 3H, 6’-H2, H-5’), 4.27–4.59 (m, 3H, H-4’, H-3’, H-2’), 5.01
(m, 1H, OH,D2O exchangeable), 5.12 (m, 1H, OH, D2O exchangeable), 5.20 (m, 1H, OH, D2O
exchangeable), 5.34 (m, 1H, OH, D2O exchangeable), 6.02 (d, 1H, J1’,2’ = 9.80 Hz, H-1’), 7.21
(s, 1H, pyridine-H5), 7.45–8.39 (m, 8H, Ar-H); 13C NMR (DMSO, δ ppm): 59.77 (C-6’), 64.21
(C-2’), 67.01 (C-3’), 70.68 (C-4’), 74.11 (C-5’), 90.23 (C-1’), 115.28 (CN), 129.22–138.69 (Ar-
C), 169.68 (C=O); Anal. Calcd. forC24H20Cl2N2O6 (503.34): C 57.27, H 4.01, Cl 14.09, N 5.57.
Found: C 56.89, H 3.74, Cl 13.81, N 5.22.
5

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General for synthesis compounds 7-14
To a solution of compound 1 (0.340 g, 1 mmol) in dry dimethylformamide (50 ml), 50% oil-
immersed sodium hydride (0.20 g) was added. Thereafter the reaction mixture was stirred at
room temperature for 1 hour, then ethyl iodide, allyl bromide, propagyl bromide, chloro
acetonitrile, benzyl chloride, benzoyl chloride, epichloro hydrine, or 1,3-dichloro-2-propanol (1
mmol) was added. The reaction mixtures were stirred at 70◦C for 3, 6, 4, 3, 5, 6, 2 and 2 hours,
respectively. After evaporation under reduced pressure, the residues were purified on silica gel
column using chloroform: methanol (9:1) as an eluent to give compounds 7, 8, 9, 10, 11, 12, 13
or 14, respectively.
3.1.5. 4,6-bis(4-Chlorophenyl)-1-ethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (7)
1
Yield 64%; m.p. 149–151◦C; IR (KBr, ν, cm−1): 2218 (CN), 1678 (C=O); H NMR (CDCl3, δ
ppm): 1.33 (t, 3H, J = 6.71Hz, NCH2CH3), 3.33 (q, 2H, J = 7.20 Hz, NCH2CH3), 6.97(s, 1H,
pyridine-H5), 7.36–8.01(m, 8H, 8Ar-H). MS m/z (%): 372 (M++4, 2), 370 (M++2, 11), 368 (M+,
21). Anal. Calcd. For C20H14Cl2N2O2 (369.25): C 65.06, H 3.82, Cl 19.20, N 7.59 Found: C
64.84, H 3.49, Cl 18.92, N 7.33.
3.1.6. 1-Allyl-4,6-bis(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (8)
1
59% yield; m.p. 197–199◦C; IR (KBr, ν, cm−1) 2220 (CN), 1680(C=O); H NMR (CDCl3, δ
ppm): 4.36 (d, J=5.1 Hz, 2H, NCH2), 3.95 (d, J=9.8 Hz, 1H, CHa), 4.03 (d, J=16.2 Hz, 1H, CHb),
5.60 (q, J=16.2 Hz, 1H, CH), 7.67 (s, H, pyridine-H5), 7.76–8.31 (m, 8H, Ar-H); 13C NMR
(CDCl3, δ ppm): 68.0 (NCH2), 112.6 (CH2), 115.51 (CN), 119.11 (CH ), 128.31–138.15 (13Ar-
C). MS m/z (%): 339 (M+- 41, 100). Anal. Calcd for C21H14Cl2N2O (381.26): C 66.16, H 3.70,
Cl 18.60, N, 7.35. Found: C 65.77, H 3.39, Cl 18.41, N 7.09.
3.1.7. 4,6-bis(4-Chlorophenyl)-2-oxo-1(prop-2ynyl)-1,2-dihydropyridine-3-carbonitrile (9)
1
51% yield; m.p. 215–217◦C; IR (KBr, ν, cm−1): 2218 (CN), 1685 (C=O); H NMR (300 MHz;
DMSO-d6, δ ppm): 2.81 (t, J=2.1 Hz, 1H, CH), 4.32 (d, J=2.2 Hz, 2H, NCH2), 7.23 (s, 1H,
pyridine-H5), 7.67– 8.11(m, 8H, Ar-H). MS m/z (%): 339 (M+- 39, 100). Anal. Calcd for
C21H12Cl2N2O (379.25): C 66.51, H 3.19, Cl 18.70, N 7.39. Found: C 66.25, H 2.81, Cl 18.25, N
3.12.
3.1.8. 4,6-bis(4-Chlorophenyl)-1-(cyanomethyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (10)
47% yield; m.p. 165–167◦C; IR (KBr, ν, cm−1) 2222, 2216 (CN), 1677 (C=O); 1H NMR (CDCl3,
δ ppm): 3.90(s, 2H, CH2), 7.77– 8.29(m, 9H, 8Ar-H+pyridine-H5).MS m/z (%): 383 (M++4, 6),
381 (M++2, 31), 379 (M+, 67). Anal. Calcd for C20H11Cl2N3O (380.24): C 63.18, H 2.92, Cl
18.65, N 11.05. Found: C 62.89, H 2.66, Cl 18.41, N 10.91.
3.1.9. 1-Benzyl-4,6-bis(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (11)
42% yield; m.p. 209–211◦C; IR (KBr, ν, cm−1) 2217 (CN), 1680 (C=O); 1H NMR (DMSO-d6, δ
ppm): 4.11 (s, 2H, CH2), 7.36– 8.20(m, 14H, 13Ar-H+pyridine-H5).MS m/z (%): 434(M++4, 3),
6

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432 (M++2, 15), 430 (M+, 34). Anal. Calcd for C25H16Cl2N2O (431.33): C 69.62, H 3.74, Cl
16.44, N 6.49. Found: C 69.49, H 3.52, Cl 16.16, N 6.22.
3.1.10. 1-Benzoyl-4,6-bis(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (12)
1
Yield 62%; m.p. 235–237◦C; IR (KBr, ν, cm−1): 2220 (CN), 1691, 1710 (2C=O); H NMR
(CDCl3, δ ppm): 7.37–8.42(m, 14H, 13Ar-H, pyridine-H5); 13C NMR (CDCl3, δ ppm): 115.26
(CN), 125.21–142.40 (Ar-C), 169.67, 163.01 (C=O). MS m/z (%):448 (M++4, 1), 446 (M++2, 6),
444 (M+, 13).Anal. calcd. For C25H14Cl2N2O2 (445.31): C 67.19, H 3.17, Cl 15.92, N 6.29.
Found: C 67.19, H 2.93, Cl 15.77, N 6.01.
3.1.11.
4,6-bis(4-Chlorophenyl)-1-(oxiran-2ylmethyl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile (13)
51% yield; m.p. 171–173◦C; IR (KBr, ν, cm−1): 2216 (CN), 1680; 1H NMR (DMSO-d6, δ ppm)
δ 2.92 (dd, 1H, J = 1.50 Hz; J = 4.50 Hz, H-a’), 2.97 (dd, 1H, J = 4.20 Hz; J =4.55 Hz, H-a),
4.29–4.41 (m, 1H, H-b), 4.77 (dd, 1H, J = 2.17 Hz; J = 5.22 Hz, H-c’), 4.82 (dd, 1H, J = 2.27
Hz; J = 4.99 Hz, H-c), 7.24 (s, 1H, pyridine-H5), 7.66-8.33 (m, 8H, Ar-H). Anal. Calcd for
C21H14Cl2N2O2(397.26): C 63.49, H 3.55, Cl 17.85, N 7.05. Found: C 63.21, H 3.39, Cl 17.81,
N 6.79.
3.1.12.
1-(3-Chloro-2-hydroxypropyl)-4,6-bis(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile (14)
47% yield; m.p. 113–115◦C; (KBr, ν, cm−1): 3424–3230 (OH), 2217 (CN), 1680(C=O). 1H NMR
(DMSO-d6,δ ppm): 3.10 (dd, 1H, J = 5.81 Hz, J = 10.23 Hz, H-c`), 3.37 (dd, 1H, J = 5.15 Hz, J
= 10.23 Hz, H-c ), 3.83 (dd, 1H, J = 5.43 Hz, J = 11.13 Hz, H-a`), 4.01 (dd, 1H, J = 5.64 Hz, J =
11.17 Hz, H-a), 5.66 (m, 1H, H-b), 5.87 (d, 1H, J = 5.23 Hz, OH), 7.38 (s, 1H, pyridine- H-5),
7.39–7.77 (m, 8H, Ar-H); 13C NMR (DMSO): 43.01 (CH2Cl), 67.39(CHOH), 69.34 (NCH2),
115.41 (CN), 129.31–136.00(13Ar-C), 164.54(C=O). Anal. Calcd for C21H15Cl3N2O2 (433.72):
C 58.16, H 3.49, Cl 24.52, N 6.46. Found: C 57.81, H 3.15, Cl 24.35, N 6.20.
3.2. Anticancer
3.2.1. Materials and methods
3.2.1.1. Chemicals
Fetal bovine serum (FBS) and L-glutamine, were obtained from Gibco Invitrogen
Company (Scotland, UK). Dulbecco’s modified Eagle’s (DMEM) medium was provided from
Cambrex (New Jersey, USA). Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, and
streptomycin were obtained from Sigma Chemical Company (Saint Louis, MO, USA). The level
of uPA protein was determined using Assay Max human urokinase (uPA) ELISA kit (Assaypro,
USA).
3.2.1. 2. Cell lines and culturing
Anticancer activity screening for the tested compounds utilizing 3 different human tumor
cell lines including breast cancer cell line MCF-7, liver cancer cell line HepG2, and lung
carcinoma cell line A549 were obtained from the American Type Culture Collection (Rockville,
MD, USA). The tumor cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
7

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supplemented with 10% heat inactivated fetal calf serum (GIBCO), penicillin (100 U/ml) and
o
streptomycin (100 µg/ml) at 37 C in humidified atmosphere containing 5% CO2. Cells at a
concentration of 0.50 x 106 were grown in a 25 cm2 flask in 5 ml of complete culture medium.
3.2.2. In Vitro cytotoxicity assay
The cytotoxicity activity was measured in vitro using the Sulfo-Rhodamine-B stain
(SRB) assay according to the previous reported standard procedure [32]. Cells were inoculated in
96-well microtiter plate (104 cells/ well) for 24 h before treatment with the tested compounds to
allow attachment of cell to the wall of the plate. Test compounds were dissolved in DMSO at 1
mg/ml immediately before use and diluted to the appropriate volume just before addition to the
cell culture. Different concentration of tested compounds and doxorubicin were added to the
cells. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated
with the compounds for 48 h. at 37°C and in atmosphere of 5% CO2. After 48 h cells were fixed,
washed, and stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye
was removed by four washes with 1% acetic acid, and attached stain was recovered with Tris-
EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving
fraction and drug concentration is plotted to get the survival curve for each cell line after the
specified time. The concentration required for 50% inhibition of cell viability (IC50) was
calculated and the results are given in Table 1. The results were compared to the antiproliferative
effects of the reference control doxorubicin.
3.2.3. Determination the level of uPA protein expression
The level of uPA protein expression was determined using AssayMax human urokinase
(uPA) ELISA kit (Assaypro, USA) according to manufacturer’s instructions. The prepared
compounds as well as standard drug, doxorubicin were incubated for 48 h with MCF7, HepG2
and A549 cells at concentration of 1/10 of the IC50 values of each compound which shown in
Table 1.
After 48 h from compounds treatment, medium was collected and centrifuged at 2000 xg
for 10 min to remove cellular debris. Add 50 µl of the cell extract per well and incubate for 2 h.
Wells were washed with 200 µl of wash buffer then add 50 ꢀl of biotinylated uPA antibody to
each well and incubate for 1 h at 25ºC. After washing, plates were incubated with 50 ꢀl of
streptavidin-peroxidase conjugate per well and incubate for 30 minutes then wash the microplate
as described above. Add 50 ꢀl of chromogen substrate per well and incubate for about 10 min or
till the optimal blue color density develops. Add 50 ꢀl of stop solution to each well. The color
will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of
450 nm immediately and the concentrations of uPA in the samples were determined and the
percentage of uPA inhibition for each compound was calculated as compared with control cancer
cells (DMSO treated).
3.2.4. Statistical analysis
The results are reported as Mean ± Standard error (S.E.) for at least three times
experiments. Statistical differences were analyzed according to followed by one way ANOVA
test followed by student’s t test wherein the differences were considered to be significant at p <
0.05.
8

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References:
[1]
[2]
A. Arora, E.M. Scholar, J. Pharm. Exp. Ther. 315 (2005) 971–979.
L. Yuli, P. Nana, L. Jianping, Z. Hongqin, H. Zi-Chun, Molecular Cancer 12 (2013)
54–65.
[3]
[4]
T. Syrovets, T. Simmet, Cell Mol. Life Sci. 61 (2004) 873–885.
T. Tamura, E. Morita, S. Kawai, R. Okada, M. Naito, K. Wakai, Y. Hori, T. Kondo,
N. Hamajima, Gene 524 (2013) 363–367.
[5]
[6]
P. Mignatti, D.B. Rifkin, Physiol. Rev. 73 (1993) 161–195.
K. Bdeir, A. Kuo, B.S. Sachais, Blood 102 (2003) 3600–3608.
F. Blasi, P. Carmeliet, P. Nat. Rev. Mol. Cell Biol. 3 (2002) 932–943.
P.A. Andreasen, L. Kjoller, L. Christensen, M.J. Duffy, Int. J. Cancer 72 (1997) 1–22.
T. Meyer, I.R. Hart, Eur. J. Cancer 34 (1998) 214–221.
[7]
[8]
[9]
[10]
N.N. Al-Hassan, A. Behzadian, R. Caldwell, V. S. Ivanova, V. Syed, K. Motamed,
N. A. Said, Neoplasia 14 (2012) 259–270.
[11]
[12]
M.J. Duffy, C. Duggan, Clin. Biochem. 37 (2004) 541–548.
I. Verheggen, A.V. Aerschot, S. Toppet, , R. Snoeck; G. Janssen, J. Balzarini, E. De
Clercq, P. Herdewijn, J. Med. Chem. 36 (1993) 2033–2040.
[13]
[14]
[15]
[16]
S.A.F. Rostom, G.S. Hassan, H.I. El-Subbagh, Arch. Pharm. Chem. Life Sci. 342
(2009) 584 – 590.
V. Berg, P. Das, E. Chorell, M. Hedenström, J.S. Pinkner, S.J. Hultgren, F. Almqvist,
Bioorg. Med. Chem. Lett. 18 (2008) 3536–3540.
T. Suresh, T. Dhanabal, R.N. Kumar, P.S. Mohan, Indian J. Chem. 44B (2005) 2375–
2379.
A.H. Abadi, T.M. Ibrahim, K.M. Abouzid, J. Lehmann, H.N. Tinsley, B.D. Gary, G.
A. Piazza; Bioorg. Med. Chem. 17 (2009) 5974–5982.
[17]
[18]
H.S. Abbas, W.A. El Sayed, N.M. Fathy, Eur. J. Med. Chem., 45 (2010) 973–982.
M.S. Al-Said, M.S. Bashandy, S.I. Al-qasoumi, M.M. Ghorab, Eur. J. Med. Chem. 46
(2011) 137–141.
[19]
[20]
J.J. Starrett, T.A. Montzka , A.R. Crosswell, R.L. Cavanagh ; J Med Chem., 32 (1989)
2204–2210.
C.J. Blankely, L.R. Bennett, R.W. Fleming, R.D. Smith, D.K. Tessman, H.R. Kaplan,
J. Med. Chem. 26 (1983) 403–411.
9

ACCEPTED MANUSCRIPT
[21]
[22]
A.E. Amr, H.H.Sayed,d M.M. Abdulla , Arch. der Pharm. 338 (2005) 433–440.
H.A. Parish, R.D. Gilliom, W.P. Purcell, R.K. Browne, R.F. Spirk, H.D. White, J.
Med. Chem. 25 (1982) 98–102 .
[23]
[24]
[25]
G. Singh, G. Singh,, A.K. Vadav, A.K. Mishra , Phosphorus Sulfur Silicon and
Related Elements, 165 (2000) 107–116.
G. Singh, A.K. Yadav, A.K. Mishre, Phosphorus Sulfur Silicon and Related Elements,
165 (2007) 107– 116.
K.L. Van, C. Cauvin, S. de Walque, B. Georges, S. Boland, V. Martinelli, D. Demont,
F. Durant, L. Hevesi, C. Van Lint , J. Med. Chem. 52 (2009) 3636–3643.
[26]
[27]
[28]
A.E. Rashad, A.H. Shamroukh, M.A. Ali and F.M. Abdel-Motti, Heteroatom Chem.,
18 (2007) 274–282.
A.E. Rashad , A.H. Shamroukh , M.A. El-Hashash , A.F. El-Farargy , N.M. Yousif , M.
A. Salama , A. Mostafa M. El-Shahat , J. Heterocycl.Chem. 49 (2012) 1130–1135.
A.E. Rashad; A.H. Shamroukh; R.E. Abdel-Megeid; A. Mostafa; M.A. Ali, K. Banert;
Nucleosides, Nucleotides 29 (2010) 809–920.
[29]
[30]
U. Niedballa , H. Vorbrüggen, J. Org. Chem. 39 (1974) 3654–3660.
V.D. Patil, D.S. Wise, L.L. Wotring, L.C. Bloomer, L.B. Townsend, J. Med. Chem.
28 (1985) 423–427.
[31]
[32]
A. E. Rashad; M. A. Ali and K. Banert, Nucleosides, Nucleotides. 25 (2006) 17–28.
P. Skehan, R. Storeng,D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T. Warren,
J. Natl. Cancer Inst. 82 (1990) 1107–1112
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Highlights
► Novel N-nicotinonitrile derivatives 3-14 have been synthesized. ► The anticancer
evaluation of all prepared compounds has been assessed against breast MCF-7 cancer, liver
HepG2 cancer and lung A549 carcinoma cell lines. ► Compounds 11 and 12 caused down-
regulation of urokinase activity and revealed promising activity compared to doxorubicin.

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Table 1. In vitro cytotoxicity activity and the percent inhibition of uPA of the synthesized compounds on
different cell lines.
IC50 (µg/ml)
% inhibition of uPA a
Compound
MCF-7
2.80±0.24
N.A.
HepG2
3.90±0.37
N.A.
A549
4.11±0.40
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
MCF-7
HepG2
88±6.36
N.A.
A549
82±6.35
91±3.70
N.A.
Doxorubicin
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
DMSO
N.A.
N.A.
N.A.
N.A.
1
5.90±0.56
6.30±0.60
5.70±0.72
14.18±1.36
19.70±1.90
19.00±1.85
16.70±1.50
N.A.
8.00±0.92
7.25±0.35
6.90±0.65
16.10±1.40
22.30±2.32
20.00±1.92
18.30±1.75
N.A.
66±5.44
54±4.93
60±2.84
9±0.47
3±0.45
3±0.45
7±0.94
N.A.
60±5.09
48±4.82
56±1.98
6±0.45
2±0.27
5±046
3
4
5
6
7
8
5±0.46
N.A.
9
10
11
12
13
14
3.65±0.29
2.60±0.27
5.00±0.63
4.60±0.48
4.20±0.44
3.75±0.44
6.20±0.80
4.80±0.47
86±3.09
90±5.76
77±4.28
82±6.95
83±4.65
87±6.45
63±4.65
67±4.78
Data were expressed as Mean ± Standard error (S.E.) of three independent experiments.
aThe percentage changes as compared with control untreated cancer cells (DMSO treated).
N.A. is no activity
1

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O
NC
NH
Ar
Ar
1
(Me3Si)2NH, (NH4)2SO4 ,
heat
OSiMe3
N
NC
Ar
Ar
2
N gas, SnCl ,
dry CH2Cl2
2
4
OAc
O
BzO
AcO
OAc
O
OAc
OAc
OBz
OBz
OAc
O
O
AcGlucose
Ar
NC
Ar
BzRibose
NC
Ar
N
N
Ar
3
4
O
6
MeOH/NH3 gas
MeOH/NH3 gas
O
NC
Ribose
NC
Ar
Glucose
N
N
Ar
Ar
Ar
5
HO
HO
O
O
OH
OH
Ar = p-ClC6H4-
Ribose
Glucose
=
=
OH
OH
OH
Scheme 1. Synthesis of compounds 2-6
1

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O
CH2CH3
Ar
NC
NaH/DMF,
N
I
heat
,
Ar
7
O
CH2-CH=CH2
Ar
NC
Ar
N
N
N
NaH/DMF,
,
Br
Br
heat
8
O
CH2-C
Ar
CH
NC
Ar
NaH/DMF,
,
heat
heat
9
O
CH2CN
Ar
NC
Ar
NaH/DMF,
1
Cl
CN ,
10
O
CH2Ph
NC
Ar
N
NaH/DMF,
Ar
Ar = p-ClC6H4-
heat
,
Cl
11
O
COPh
Ar
NaH/DMF,
NC
N
O
Ar
12
Cl
heat
,
Hc'
Hc
O
O
Ha
Ha'
NC
N
NaH/DMF,
O
Hb
Ar
Ar
heat
,
13
Ha'
Cl
Ha
Cl
Hb
O Hc' Hc
N
NC
NaH/DMF,
heat
OH
,
Cl
Ar
Cl
Ar
OH
14
Scheme 2. Synthesis of compounds 7-14
1