Novel pyrazolo[3,4-b]pyridine derivatives: Synthesis, characterization, antimicrobial and antiproliferative profile

Article abstract of DOI:10.1248/bpb.b15-00586

Three novel series of pyridine derivatives, namely Schiff's bases, 4-thiazolidinones and azetidin-2-ones bearing pyrazolo[3,4-b]pyridine moiety, have been synthesized. The chemical structures of the synthesized compounds were characterized. The compounds

Full text of DOI:10.1248/bpb.b15-00586

Vol. 39, No. 4
Biol. Pharm. Bull. 39, 473–483 (2016)
473
Regular Article
Novel Pyrazolo[3,4-b]pyridine Derivatives: Synthesis, Characterization,
Antimicrobial and Antiproliferative Profile
,
a
b
Marwa Sayed Salem* and Mohamed Ahmed Mohamed Ali
a
Synthetic Organic Chemistry Laboratory, Department of Chemistry, Faculty of Science, Ain Shams University;
b
Abbasiya, Cairo 11566, Egypt: and Department of Biochemistry, Faculty of Science, Ain Shams University;
Abbassia, Cairo 11566, Egypt.
Received July 28, 2015; accepted November 27, 2015
Three novel series of pyridine derivatives, namely Schiff’s bases, 4-thiazolidinones and azetidin-2-ones
bearing pyrazolo[3,4-b]pyridine moiety, have been synthesized. The chemical structures of the synthesized
compounds were characterized. The compounds were tested for their antimicrobial activity using the agar
well diffusion and broth macrodilution methods. The compounds were also evaluated for their antipro-
liferative activity using the sulforhodamine B (SRB) assay. The majority of the tested compounds exhibited
slight to high antimicrobial activity against the test microorganisms with minimum inhibitory concentra-
tions (MICs) of 0.12–62.5µg/mL when compared to that of 3 standard antimicrobial agents (Ampicillin,
0
.007–0.03µg/mL; Gentamicin; 0.015–0.24µg/mL; and Amphotericin B, 0.03–0.98µg/mL). Compound (7b)
was found to be nearly as active as the standard antimicrobial drug Amphotericin B against Fusarium oxy-
sporum fungal strain with MIC of 0.98µg/mL. Some of the test compounds showed remarkable cytotoxic
activities against Hep G2 (hepatocellular carcinoma) cells (IC ꢀ0.0158–71.3µM) in comparison to the stan-
5
0
dard anticancer drug doxorubicin (IC ꢀ0.008µM). Among the compounds tested, (5), (6a), (6b), (7b), and
5
0
(
10) exhibited antiproliferative potency (IC ꢀ0.0001–0.0211µM) that was found to be better than that of
50
doxorubicin (IC ꢀ0.099µM) against MCF7 (breast adenocarcinoma) cells. In particular, (7b) displayed the
5
0
highest significant antiproliferative efficacy against both Hep G2 and MCF7 cell lines showing IC50 values of
.0158µM and 0.0001µM, respectively. Our findings suggest that the synthesized compounds may be promis-
0
ing candidates as novel antimicrobial and antiproliferative agents.
Key words pyrazolo[3,4-b]pyridine; Schiff’s base; 4-thiazolidinone; azetidin-2-one; antimicrobial activity;
antiproliferative activity
18)
19)
20)
Microbial infections remain a leading cause of mortality leishmanial, anti-inflammatory, and antitumor activities.
and morbidity worldwide. Unfortunately, a number of the cur- Schiff’s bases (imines or azomethines) were reported to
rent clinically efficacious antimicrobial agents are becoming possess a broad spectrum of potent pharmacological activities
1)
less effective. With all of this in mind, there is an urgent with a wide variety of biological properties including antimi-
2
1)
22)
23)
24)
need for the discovery of novel compounds endowed with crobial,
anticancer,
antioxidant,
and anticonvulsant
2)
antimicrobial activity.
Cancer is a serious health problem and the most frighten-
ing disease of mankind. Inherent and acquired resistance to tigated classes of five member heterocycles and forms an im-
activities.
The 4-thiazolidinone is one of the most intensively inves-
3)
4)
25)
treatment and the dose-limiting toxicity are recognized as portant class of clinically used drugs. The 4-thiazolidinone
major obstacles for effective cancer therapy. Therefore, there scaffold is very versatile and possesses various pharmacologi-
is a need for more innovative approaches to design anticancer cal activity profiles, namely antimicrobial, antitubercular,
5
)
2
6)
27)
6
,7)
28)
29)
30)
drugs.
antiviral, anti-inflammatory, and antiproliferative activi-
The synthesis of heterocyclic compounds has drawn the ties.
attention of medicinal chemists mainly because of their
Azetidin-2-ones are four-membered heterocyclic com-
structural diversity coupled with their important therapeutic pounds commonly known as β-lactams. The azetidin-2-one
properties. A number of heterocyclic derivatives containing scaffold is the common structural feature of a number of
nitrogen atom serve as a versatile scaffolds for experimental broad spectrum β-lactam antibiotics, which are the most
8)
31,32)
drug design.
prescribed antibiotics used in medicine.
The biological
Pyridine is the parent ring system of a large number of activity of the β-lactam ring systems is generally believed to
naturally occurring products and important pharmaceuticals. be associated with the chemical reactivity of their β-lactam
Pyridine derivatives exhibit diverse pharmacological activities skeleton and on the substituents especially at nitrogen of the
9
)
10)
11)
33)
such as antimicrobial, antimycobacterial, antimalarial,
2-azetidinone ring.
Azetidinones are of great biological
12)
13)
14)
34)
35)
antitumor, cytotoxic, antidiabetic, antiarrhythmic, and interest, especially as antimicrobial, antiviral, anti-inflam-
15)
29)
36)
antidepressant.
The pyrazole nucleus has attracted great attention due to the
matory, and anticancer agents.
Prompted by the various pharmacological and biologi-
use of this ring system as an important core structure in many cal activities of pyrazolo[3,4-b]pyridine and as a part of our
drug substances. A large number of substituted pyrazolo[3,4- ongoing studies to identify novel candidates that may be of
b]pyridine derivatives have been found to possess diverse value in designing new and potent antimicrobial and anti-
16)
17)
biological properties such as antimicrobial, antiviral, anti- proliferative agents, we envisioned our approach toward the
*
To whom correspondence should be addressed. e-mail: marwa_k@sci.asu.edu.eg
©
2016 The Pharmaceutical Society of Japan

4
74
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
synthesis of some novel heterocyclic pyridine derivatives in- propriate aromatic or heterocyclic aldehyde, namely, p-chloro-
cluding Schiff’s bases, 4-thiazolidinones and azetidin-2-ones benzaldehyde, 1H-indole-3-carbaldehyde, and/or 1,3-diphen-
bearing pyrazolo[3,4-b]pyridine moiety in order to identify yl-1H-pyrazole-4-carbaldehyde (0.005mol) was refluxed in
compounds having better therapeutic activities. With a view to absolute ethanol (20mL) containing few drops of piperidine
further assess the pharmacological profile of pyrazolo[3,4-b]- for 5h. The reaction mixture was concentrated, cooled and the
pyridine derivatives, it was thought worthwhile to investigate formed precipitate was filtered off, dried and recrystallized to
the antimicrobial and antiproliferative profile of the synthe- give compounds 6a–c, respectively.
sized compounds.
N-(4-Chlorobenzylidene)-5-bromo-4,6-dimethyl-1H-
pyrazolo[3,4-b]pyridin-3-amine (6a)
MATERIALS AND METHODS
Orange crystals. Yield: 93%. mp: 211–212°C (EtOH).
−1
FT-IR (KBr, cm ): 3398 ν(NH), 3045 ν(CH aromatic), 2939
1
Chemistry All reagents and solvents were of analytical ν(CH aliphatic), 1622 ν(C=N). H-NMR (300MHz, DMSO-
grade and were purchased from Sigma-Aldrich (St. Louis, d6, δ, ppm): 13.70 (s, 1H, NH pyrazolo, exchangeable), 8.72
MO, U.S.A.). The melting points were measured using a Gal- (s, 1H, N=CH), 7.91–7.57 (2d, 4H, Ar-H), 2.97 and 2.72 (2s,
13
lenkamp electric melting point apparatus and are uncorrected. 6H, 2CH3). C-NMR (125MHz, DMSO-d ): 161.55, 151.54,
6
The IR spectra were recorded on a PyeUnicam SP-3-300 infra- 148.34, 136.21, 132.42, 130.57, 129.95, 121.58, 105.65, 26.95,
·
+
red spectrophotometer using potassium bromide (KBr) disks. 19.97. MS (EI, m/z, %): 362 (M , 00.0), 305.2 (8.5), 215.6
1
13
The H-NMR spectra and C-NMR experiments were run at (4.9), 167.1 (15.9), 127.7 (20.7), 83.8 (100.0). Anal. Calcd for
00 and 400MHz, respectively on a Varian Mercury VX-300 C H BrClN (363.64): C, 49.54; H, 3.33; Br, 21.97; Cl, 9.75;
3
15
12
4
NMR spectrometer (Varian, U.K.) using tetramethylsilane N, 15.41. Found: C, 49.45; H, 3.24; Br, 21.89; Cl, 9.62; N,
TMS) as internal standard (IS) in deuterated chloroform or 15.35.
deuterated dimethyl sulfoxide (DMSO) and the chemical shifts N-((1H-Indol-3-yl)methylene)-5-bromo-4,6-dimethyl-1H-
(
(δ) were reported in parts per million (ppm) and the coupling pyrazolo[3,4-b]pyridin-3-amine (6b)
constants (J) in Hertz. The mass spectra were recorded using
Shimadzu GCMS-QP-1000EX mass spectrometer (Shimadzu, FT-IR (KBr, cm ): 3462, 3143 ν(NH), 3064 ν(CH aromatic),
Japan) at ionization energy of 70eV with the source 200°C 2981 ν(CH aliphatic), 1593 ν(C=N). H-NMR (300MHz,
and an accelerative voltage of 8kV. All the spectral measure- DMSO-d , δ, ppm): 13.12 (s, 1H, NH pyrazolo, exchangeable),
Yellow crystals. Yield: 72%. mp: 272–275°C (dioxane).
−1
1
6
ments as well as the elemental analyses were carried out at 11.92 (s, 1H, NH indol, exchangeable), 9.22 (s, 1H, N=CH),
the Microanalytical Center, Cairo University and the Main 8.43–7.20 (m, 5H, Ar-H), 2.94 and 2.69 (2s, 6H, 2CH ).
3
13
laboratories for chemical warfare. All the newly synthesized
C-NMR (125MHz, DMSO-d ): 155.85, 150.88, 148.29,
6
compounds gave satisfactory elemental analyses. The purity of 142.29, 137.64, 131.21, 128.76, 122.21, 120.12, 115.95, 113.61,
·
+
the synthesized compounds was checked by TLC.
106.24, 26.95, 19.54. MS (EI, m/z, %): 369 (M+2] , 100.0),
·
+
General Procedures for Compounds’ Synthesis
367.2 (M , 75.4), 287.9 (56.1), 226.8 (52.6), 184.3 (35.1), 142.1
5
-Bromo-3-cyano-4,6-dimethyl-2-oxo-N-phenylpyridine- (49.1), 88.4 (31.6). Anal. Calcd for C H BrN (368.23): C,
17 14 5
1
(2H)-carbothioamide (5)
A mixture of compound 1 (1.13g, 0.005mol) and phenyl Br, 21.65; N, 19.10.
isothiocyanate (0.6mL, 0.005mol) was refluxed in dimethyl
5-Bromo-N-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)-4,6-
formamide (DMF; 20mL) containing a catalytic amount of dimethyl-1H-pyrazolo[3,4-b]pyridin-3-amine (6c)
triethylamine (TEA; 4 drops) for 5h and then left to cool to
Pale yellow crystals. Yield: 92%. mp: 289–291°C (dioxane).
55.45; H, 3.83; Br, 21.70; N, 19.02. Found: C, 55.54; H, 3.90;
−1
room temperature. The reaction mixture was poured into cold FT-IR (KBr, cm ): 3133 ν(NH), 3058 ν(CH aromatic), 2983
1
water for complete precipitation, and the formed precipitate ν(CH aliphatic), 1595 ν(C=N). H-NMR (300MHz, DMSO-
was filtered off, dried and recrystallized from ethanol to give d6, δ, ppm): 13.32 (s, 1H, NH pyrazolo, exchangeable), 9.26
compound 5 as orange crystals. Yield: 82%, mp: 202–205°C. and 9.13 (each s, 1H, N=CH, two stereo-isomers), 8.05–7.42
−
1
13
Fourier transform (FT)-IR (KBr, cm ): 3297, 3137 ν(NH), (m, 11H, Ar-H), 2.77 and 2.68 (2s, 6H, 2CH3). C-NMR
003 ν(CH aromatic), 2849 and 2775 ν(CH aliphatic), 2221 (125MHz, DMSO-d ): 156.74, 153.44, 152.98, 151.01, 143.07,
3
6
1
ν(C≡N), 1658 ν(C=O). H-NMR (300MHz, DMSO-d , δ, 139.34, 132.45, 131.19, 130.12, 129.27, 129.10, 127.79, 120.14,
ppm): 8.64 (s, 1H, NH, exchangeable), 7.46–6.90 (m, 5H, 119.57, 116.59, 110.02, 26.97, 19.44. MS (EI, m/z, %): 471.6
Ar-H), 2.30 and 2.22 (2s, 6H, 2CH3). C-NMR (125MHz, (M+2] , 41.6), 390.7 (M , 13.7), 244.8 (15.4), 230.9 (64.0),
DMSO-d ): 160.54, 155.38, 150.37, 141.81, 135.97, 132.56, 119.1 (10.5), 104.1 (14.5), 77.1 (100.0). Anal. Calcd for
6
13
·+
·+
6
1
29.95, 128.98, 111.55, 105.73, 26.75, 19.89. MS (electron C H BrN (471.35): C, 61.16; H, 4.06; Br, 16.95; N, 17.83.
24 19 6
·
+
ionization (EI), m/z, %): 361 (M , 00.0), 225.7 (24.1), 193.8 Found: C, 61.22; H, 4.15; Br, 16.87; N, 17.76.
57.4), 148.0 (38.9), 104.1 (27.8), 92.6 (100.0). Anal. Calcd for 3-(5-Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-
C H BrN OS (362.24): C, 49.73; H, 3.34; Br, 22.06; N, 11.60; yl)-2-(1H-indol-3-yl)thiazolidin-4-one (7a) and/or 3-(5-
(
15
12
3
S, 8.85. Found: C, 49.68; H, 3.29; Br, 22.00; N, 11.51; S, 8.74.
N-(4-Chlorobenzylidene)-5-bromo-4,6-dimethyl-1H- diphenyl-1H-pyrazol-4-yl)thiazolidin-4-one (7b)
pyrazolo[3,4-b]pyridin-3-amine (6a), N-((1H-Indol-3-yl)- In 250mL round-bottom flask, the Schiff’s bases 6b and c
Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-2-(1,3-
methylene)-5-bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]- (0.01mol) in dry benzene (30mL) were taken, Dean–Stark ap-
pyridin-3-amine (6b), and/or 5-Bromo-N-((1,3-diphenyl-1H- paratus was attached and thioglycolic acid (0.92g, 0.01mol) in
pyrazol-4-yl)methylene)-4,6-dimethyl-1H-pyrazolo[3,4-b]- dry benzene (20mL) was added slowly. The reaction mixture
pyridin-3-amine (6c)
was refluxed for 6–8h and water was removed continuously
A mixture of compound 4 (1.2g, 0.005mol) and the ap- during the reaction’s course. The solvent was evaporated and

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
475
the reaction mixture was neutralized with cold diluted sodium 7.97; N, 15.75. Found: C, 51.25; H, 3.38; Br, 17.86; Cl, 7.96; N,
bicarbonate solution. The formed product was filtered off and 15.64.
recrystallized to afford compounds 7a and b, respectively.
1-(5-Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-
3
-(5-Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3- yl)-3-chloro-4-(1,3-diphenyl-1H-pyrazol-4-yl)azetidin-2-one
yl)-2-(1H-indol-3-yl)thiazolidin-4-one (7a)
(8b)
Yellow crystals. Yield: 91.2%. mp: 312–314°C (toluene/etha-
Red crystals. Yield: 95%. mp: over 300°C (EtOH). FT-IR
−1
−1
(
KBr, cm ): 3236, 3142 ν(NH), 3064 ν(CH aromatic), 2985 nol). FT-IR (KBr, cm ): 3238 ν(NH), 3137 ν(CH aromatic),
1
1
ν(CH aliphatic), 1657 ν(C=O), 1583 ν(C=N). H-NMR 2985 ν(CH aliphatic), 1667 ν(C=O), 1585 ν(C=N). H-NMR
300MHz, DMSO-d , δ, ppm): 13.45 (s, 1H, NH pyrazolo, ex- (300MHz, DMSO-d , δ, ppm): 13.49 (s, 1H, NH pyrazolo,
(
6
6
changeable), 10.39 (s, 1H, NH indol, exchangeable), 8.10–7.11 exchangeable), 8.32–7.45 (m, 11H, Ar-H), 5.34 (s, 1H, Cl–CH),
1
3
(
m, 5H, Ar-H), 5.09 (s, 1H, N–CH–S), 3.86 (s, 2H, CH of 4.35 (s, 1H, N–CH), 2.68 and 2.54 (2s, 6H, 2CH3). C-NMR
2
13
thiazolidinone ring), 2.68 and 2.54 (2s, 6H, 2CH3). C-NMR (125MHz, DMSO-d ): 167.70, 157.56, 150.32, 148.75, 141.72,
6
(
1
3
125MHz, DMSO-d ): 170.21, 161.23, 155.66, 150.88, 149.56, 131.21, 128.76, 127.89, 122.21, 121.65, 120.12, 116.78, 110.00,
6
·
+
37.45, 127.65, 122.98, 120.23, 119.54, 112.03, 105.45, 64.12, 74.23, 67.57, 27.09, 18.78. MS (EI, m/z, %): 546.7 (M+1] ,
·
+
4.54, 26.76, 19.75. MS (EI, m/z, %): 441 (M , 00.0), 348.6 8.1), 320.6 (4.1), 282.3 (8.1), 280.8 (16.2), 266.5 (14.9), 244.9
(54.5), 167.1 (63.6), 141.2 (63.6), 121.6 (54.5), 95.2 (45.5), 56.0 (5.9), 239.8 (100.0), 239.5 (77.0), 162.4 (16.2). Anal. Calcd for
(
100.0). Anal. Calcd for C H BrN OS (442.33): C, 51.59; H, C H BrClN O (547.83): C, 57.00; H, 3.68; Br, 14.59; Cl,
19
16
5
26 20
6
3
1
.65; Br, 18.06; N, 15.83; S, 7.25. Found: C, 51.46; H, 3.59; Br, 6.47; N, 15.34. Found: C, 57.07; H, 3.76; Br, 14.48; Cl, 6.37;
8.00; N, 15.78; S, 7.17. N, 15.22.
-(5-Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3- 2-(2-(5-Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-
3
yl)-2-(1,3-diphenyl-1H-pyrazol-4-yl)thiazolidin-4-one (7b)
yl)hydrazono)malononitrile (10)
White crystals. Yield: 59%. mp: 240–242°C (EtOH). FT-IR
Compound 4 (0.241g, 0.001mol) was dissolved in con-
−1
(
KBr, cm ): 3198 ν(NH), 3068 ν(CH aromatic), 2983 ν(CH centrated hydrochloric acid (5mL) and the solution was then
1
aliphatic), 1682 ν(C=O), 1595 ν(C=N). H-NMR (300MHz, cooled to 0–5°C. Sodium nitrite (0.069g, 0.001mol) in water
DMSO-d , δ, ppm): 12.03 (s, 1H, NH pyrazolo, exchangeable), (3mL) was then added to this solution dropwise with vigor-
6
8.80–7.34 (m, 11H, Ar-H), 5.88 (s, 1H, N–C–S), 3.75 (s, 2H, ous stirring for 1h, while cooling at 0–5°C. The clear diazo-
CH of thiazolidinone ring), 2.68 and 2.54 (2s, 6H, 2CH ). nium salt solution (compound 9) was then added dropwise to
2
3
13
C-NMR (125MHz, DMSO-d6): 170.45, 160.57, 153.75, a well-cooled (0–5°C) and stirred solution of malononitrile
50.21, 149.89, 139.51, 133.23, 129.27, 129.10, 127.79, 123.14, (0.001mol) in sodium acetate (1g, dissolved in 5mL of 25%
21.57, 116.59, 105.31, 56.75, 33.56, 26.97, 19.44. MS (EI, aqueous ethanol). The pH of the coupling mixture, in each
1
1
·
+
m/z, %): 545.8 (M+2] , 15.0), 468.4 (9.7), 320.0 (21.2), 218.8 case, was maintained at 5–6 through the coupling process
8.8), 143.7 (3.5), 76.9 (100.0). Anal. Calcd for C H BrN OS by adding sodium acetate. Stirring was continued for 4h at
(
2
6
21
6
(
545.45): C, 57.25; H, 3.88; Br, 14.65; N, 15.41; S, 5.88. Found: 0–5°C and the precipitated product separated upon dilution
C, 57.14; H, 3.79; Br, 14.57; N, 15.35; S, 5.76.
with cold water (25mL) was filtered, washed with water sev-
-(5-Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3- eral times, dried, and recrystallized from benzene–ethanol
yl)-3-chloro-4-(1H-indol-3-yl)azetidin-2-one (8a) and/or 1-(5- to give compound 10 as orange crystals. Yield: 40%. mp:
1
−1
Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-yl)-3-chlo- 100–102°C. FT-IR (KBr, cm ): 3125 ν(NH), 3061 ν(CH
ro-4-(1,3-diphenyl-1H-pyrazol-4-yl)azetidin-2-one (8b)
To a well-stirred solution (0.01mol) of the Schiff’s bases 6b
aromatic), 2924 ν(CH aliphatic), 2229 ν(C≡N), 1597 ν(C=N).
1
H-NMR (300MHz, DMSO-d , δ, ppm): 13.01 (s, 1H, NH
6
and c and 0.02mol of TEA in 100mL of dry dioxane, 0.02mol pyrazolo, exchangeable), 7.23 (s, 1H, NH–N=, exchangeable),
13
of monochloroacetyl chloride was added dropwise at room 2.68 and 2.56 (2s, 6H, 2CH3). C-NMR (125MHz, DMSO-
temperature. The mixture is stirred for extra 9h, and left at d ): 161.32, 154.43, 150.94, 149.96, 122.05, 111.55, 105.73,
6
·
+
room temperature for 3d. The reaction mixture was poured 85.75, 26.98, 19.74. MS (EI, m/z, %): 319 (M+2] , 86.4), 317.0
onto crushed ice and the formed precipitate was filtered off, (M , 100.0), 238.6 (18.2), 238.5 (36.4), 226.2 (36.4), 210.0
washed with 10% sodium bicarbonate solution. The residue (31.8), 147.7 (22.7), 117.2 (68.2). Anal. Calcd for C H BrN
·
+
11
8
7
was collected and purified to yield compounds 8a and b.
1
(318.13): C, 41.53; H, 2.53; Br, 25.12; N, 30.82. Found: C,
-(5-Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3- 41.45; H, 2.47; Br, 25.01; N, 30.75.
yl)-3-chloro-4-(1H-indol-3-yl)azetidin-2-one (8a)
5-((5-Bromo-4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-
Yellow crystals. Yield: 47.9%. mp: 212–214°C (EtOH). yl)diazenyl)quinolin-8-ol (11)
−
1
FT-IR (KBr, cm ): 3216 ν(NH), 3057 ν(CH aromatic),
2
The clear diazonium salt solution (9) was then added
1
927 ν(CH aliphatic), 1667 ν(C=O), 1619 ν(C=N). H-NMR dropwise to a cold solution (0–5°C) of 8-hydroxyquinoline
300MHz, DMSO-d , δ, ppm): 13.56 (s, 1H, NH pyrazolo, ex- (0.001mol) in 10% sodium hydroxide (25mL). The reaction
(
6
changeable), 10.03 (s, 1H, NH indol, exchangeable), 7.98–6.87 mixture was stirred at 0–5°C for 2h, and then neutralized
(
m, 5H, Ar-H), 5.52 (s, 1H, Cl–CH), 5.21 (s, 1H, N–CH), 2.55 with diluted HCl. The solid product was collected by filtera-
13
and 2.46 (2s, 6H, 2CH3). C-NMR (125MHz, DMSO-d6): tion, washed with water several times, dried, and recrystal-
1
60.40, 155.52, 150.71, 148.62, 137.63, 125.16, 123.05, 122.54, lized from benzene–ethanol to afford compound 11 as orange
−1
120.95, 112.52, 112.37, 104.56, 72.39, 71.71, 26.97, 19.56. MS crystals. Yield: 45%. mp: 258–259°C. FT-IR (KBr, cm ):
·
+
(
EI, m/z, %): 387.6 (M−(CH ) ] , 17.8), 316.0 (44.0), 266.8 3191 ν(NH), 3061, 3019 ν(CH aromatic), 2929, 2887 ν(CH
2 4
13
(
28.9), 240.0 (82.2), 239.3 (35.6), 211.0 (15.6), 184.6 (11.1), aliphatic), 1598 ν(C=N). C-NMR (125MHz, DMSO-d6):
61.4 (13.3), 105.1 (33.3), 90.8 (26.7), 77.0 (100.0). Anal. Calcd 160.65, 153.42, 152.21, 150.32, 149.79, 137.45, 136.02, 133.42,
for C H BrClN O (444.71): C, 51.31; H, 3.40; Br, 17.97; Cl, 130.75, 128.32, 122.54, 120.11, 112.68, 105.01, 26.89, 19.74. MS
1
19
15
5

4
76
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
·
·+
(
(
(
EI, m/z, %): 371.4 (M−CN, +2H ] , 12.5), 175.7 (15.0), 134.0 ducted in triplicate and the results were reported as the mean
10.0), 128.0 (22.5), 122.2 (27.5), 104.2 (30.0), 99.0 (40.0), 55.1 of inhibition zone diameter in mm± standard deviation (S.D.).
100.0). Anal. Calcd for C H BrN O (397.23): C, 51.40; H,
The results were compared with those of the reference an-
17
13
6
3.30; Br, 20.12; N, 21.16. Found: C, 51.35; H, 3.24; Br, 20.00; timicrobial drugs. The diameter of inhibition zone is directly
N, 21.02.
proportional to the degree of sensitivity of the microbial strain
Antimicrobial Activity Testing All the test compounds and the concentration of the compound under test. Results
were evaluated in vitro for their antimicrobial activity against were interpreted according to the standards of the Clinical and
®
3
Gram-positive strains (Bacillus subtilis ATCC 6633™, Laboratory Standards Institute (CLSI, formerly the National
®
38–40)
Staphylococcus aureus ATCC 25923™ and Streptococ- Committee for Clinical Laboratory Standards or NCCLS)
®
cus pneumoniae ATCC 49619™), 3 Gram-negative strains where CLSI tables were used to convert the mm zone diam-
®
(
Escherichia coli ATCC 25922™, Pseudomonas aerugi- eters into interpretive breakpoints (inactive, mildly active,
®
®
®
nosa ATCC 27853™ and Salmonella Typhimurium ATCC
moderately active, or highly active).
1
6
3311™) and 3 fungal strains (Aspergillus niger ATCC
Determination of Minimum Inhibitory Concentration
®
275™, Candida albicans ATCC 10231™ and Fusarium (MIC) The MIC was defined as the lowest concentration
®
oxysporum ATCC 7601™). The strains under study were of the assayed compound that inhibited the visible growth of
purchased from the American type culture collection (ATCC; the microorganism being tested. Test compounds that showed
Manassas, VA, U.S.A.). Nutrient broth/agar and Sabouraud significant antimicrobial activity in the preliminary screening
dextrose broth/agar (Oxoid™, Basingstoke, Hampshire, U.K.) were selected for MIC determinations using the broth macro-
41)
were used as cultivation media for bacterial and fungal dilution method. In brief, cultures of each microorganism on
strains, respectively.
The antimicrobial activity of the test compounds was tested ile broth medium which were then incubated at 37°C (bacte-
agar media were prepared and used to inoculate 5mL of ster-
37)
using the agar well diffusion method. Briefly, the microbial ria) or at 25°C (fungi) until an optical density of 0.1 at 600nm
strains under test were initially grown as cultures on agar was achieved to obtain an inoculum containing approximately
8
media. The microbial inoculum used was prepared using the 1–2×10 CFU/mL.
bacteria/fungi from a fresh culture on agar medium where
A stock solution (1000µg/mL) of each test compound was
the top of the microbial colonies in the Petri dish was touched prepared in DMSO (0.25% (v/v)) and a serial two-fold dilution
with a sterile wire loop and the growth was transferred into of each compound was carried out to obtain the concentrations
a tube containing 5mL of broth medium. The broth culture of 1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81, 3.90, 1.95,
was incubated at 37°C (bacteria) or at 25°C (fungi) until 0.98, 0.49, 0.24, 0.12, 0.06, 0.03, 0.015, 0.007, and 0.0035µg/
it achieved or exceeded the turbidity of the 0.5 McFarland mL. The various concentrations of the test compounds were
standard. The turbidity of the actively growing broth culture introduced into culture tubes containing 2mL of the standard-
was adjusted to have an optical density of 0.1 at 600nm with ized microbial suspensions. The culture tubes were incubated
sterile broth in order to obtain a turbidity comparable to that at 37°C for 24h (bacterial strains) or at 25°C for 4–10d (fun-
of the 0.5 McFarland standard. This resulted in a microbial gal strains) and then examined for the presence or absence of
8
suspension containing approximately 1–2×10 colony forming visible microbial growth as evidenced by turbidity. A set of
units per milliliter (CFU/mL).
culture tubes containing only culture broth and culture broth
Nutrient/Sabouraud dextrose agar was melted, cooled to with an equivalent volume of the solvent (DMSO; 0.25% (v/v))
5°C, poured into Petri dishes to get a uniform thickness of were used as negative controls. Ampicillin and Gentamicin
4
6
mm, and allowed to solidify on a leveled surface. Once the were used as standard reference antibacterial drugs for Gram-
medium had solidified, the entire surface of the agar was positive and Gram-negative bacteria, respectively. Ampho-
uniformly inoculated by streaking a sterile cotton swab that tericin B was used as a standard reference antifungal drug for
was previously dipped into the microorganism suspension. fungal strains.
Six wells, each 6mm in diameter, were then carefully cut out
of the agar using a sterile cork borer allowing at least 30mm under test where the test tube remained clear without turbidity
between the adjacent wells.
was recorded to represent the MIC expressed in µg/mL, indi-
The last culture tube with no growth of the microorganism
The test compounds were dissolved in 0.25% (v/v) (DMSO; cating that the bacterial or fungal growth was completely in-
Sigma-Aldrich) at a concentration of 1000µg/mL and 100µL hibited at this concentration. All experiments were performed
of each compound solution was introduced into the corre- in triplicate and the results were reported as the mean of MIC
sponding well using a sterile micro-pipette. An additional well in µg/mL± S.D. The results were interpreted according to the
42,43)
containing an equivalent volume of the solvent (DMSO; 0.25% NCCLS.
v/v)) was used as a negative control in each Petri dish. Ampi-
Assessment of Antiproliferative Activity The cytotoxic
(
cillin and Gentamicin (30µg/mL) were used as standard refer- activity of the test compounds was determined by the sulfo-
ence antibacterial drugs for Gram-positive and Gram-negative rhodamine B (SRB) assay which is used for measuring the
bacteria, respectively. Amphotericin B (30µg/mL) was used as cellular protein content of cultures as previously described
4
4)
a standard reference antifungal drug for fungal strains.
by Skehan et al. The growth inhibitory effect was tested in
The Petri dishes were subsequently incubated at 37°C for vitro against two human tumor cell lines that were purchased
2
4h (bacteria) or at 25°C for 4–10d (fungi). Following incu- from The American Type Culture Collection (ATCC) (Manas-
®
bation of the Petri dishes, the antimicrobial activity of each sas): 1) Hep G2 (ATCC HB8065™, hepatocellular carci-
®
test compound was determined by measuring the diameter of noma); 2) MCF7 (ATCC HTB22™, breast adenocarcinoma).
inhibition zone in mm, if any, surrounding the well containing The cell lines were cultured in Eagle’s Minimum Essential
®
that compound. In all determinations, experiments were con- Medium (EMEM; ATCC 302003™, Manassas, VA, U.S.A.)

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
477
supplemented with 10% fetal bovine serum, 2mM L-glutamine atmosphere to allow attachment of cell to the wall of the
and 100µg/mL penicillin–streptomycin at 37°C and in 5% plate. After 24h, the test compounds were dissolved in DMSO
CO atmosphere.
(0.25% (v/v); Sigma-Aldrich) as stock solutions and diluted
2
In brief, cells were seeded in sterile 96-well flat-bottom with culture media to furnish the concentrations of 6.25, 12.5,
4
microtiter plates at a density of 2.5×10 cells/mL (100µL cell 25, 50 and 100µM that were added to the cell monolayer. For
suspension/well) and grown for 24h at 37°C and in 5% CO2 the untreated cells, equivalent volume of the vehicle (DMSO,
Chart 1
Chart 2

4
78
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
0.25% (v/v)) was added to the negative control wells instead of phenylpyridine-1(2H)-carbothioamide 5 (Chart 1).
the test compounds. For comparison purposes, the cytotoxic-
The Schiff’s bases 6a–c were formed when the amine
ity of Adriamycin (doxorubicin; Sigma-Aldrich) as a standard derivative 4 was condensed with the appropriate aromatic
anticancer drug was evaluated under the same conditions as or heterocyclic aldehyde, namely, p-chlorobenzaldehyde,
a positive control. Monolayer cells were incubated with the 1H-indole-3-carbaldehyde and/or 1,3-diphenyl-1H-pyrazole-
compounds for 72h at 37°C and in atmosphere of 5% CO2. 4-carbaldehyde in refluxing ethanol containing few drops of
After 72h, the cells were fixed by gentle layering of cold piperidine as a catalytic base. The IR spectra of the Schiff’s
5
0% trichloroacetic acid (TCA) (Sigma-Aldrich) (50µL/well, bases 6a–c revealed the disappearance of absorption bands
1
10% final concentration) on the top of the culture medium in of the amino group frequencies. Furthermore, H-NMR spec-
.
each well. The plates were incubated at 4°C for 1h and then tra of the Schiff’s bases 6a and b displayed singlet at δ 8.72
washed five times with distilled water. The TCA-fixed cells and 9.22ppm, respectively, due to the azomethine proton. On
1
were stained for 30min with 0.4% (w/v) SRB (Sigma-Aldrich) the other hand, H-NMR spectrum of the Schiff’s base 6c
dissolved in 1% acetic acid (50µL/well). Excess unbound dye displayed two singlet of the azomethine proton at δ 9.26 and
was removed by four washes with 1% acetic acid and the 9.13ppm which were recorded in an integration of 48.86 and
protein-bound dye was extracted with 10mM unbuffered Tris 51.14%, respectively, indicating the existence of the Schiff’s
base [tris(hydroxymethyl)aminomethane] (Sigma-Aldrich) for base 6c in syn- and anti-stereoisomers (Fig. 1).
determination of the optical density (OD) at 540nm in a com-
puter-interfaced, 96-well microtiter plate reader (Benchmark acid in dry benzene using Dean–Stark apparatus afforded
Plus; Bio-Rad, Hercules, CA, U.S.A.). tetrahydrocarbazolyl thiazolidinones 7a and b. On the other
Cyclization of Schiff’s bases 6b and c with thioglycolic
The experiment was performed at least three times for hand, cyclization of Schiff’s bases 6b and c with chloroacetyl
each cell line and each compound in a given concentration chloride in dioxane containing triethylamine afforded tetra-
was tested in triplicates in each experiment. Cell viabil- hydrocarbazolyl azetidinones 8a and b. The IR spectra of the
ity was calculated using the following formula: cell viability 2-azetidinones 8a and b showed the absorption band of the
−
1
(
percentage)=(ODtreated/ODuntreated)×100. The in vitro growth carbonyl group at 1667cm , a reduced value due to hydrogen
inhibition effect of each compound was expressed as inhibi- bonding between the carbonyl group and the hydrogen in the
tory concentration 50% (IC ) and defined as the concentration pyrazole ring.
5
0
of the compound (µM) producing a 50% growth inhibition of
Treatment of 4 with sodium nitrite in the presence
treated cells as compared to the untreated control cells. The of concentrated HCl afforded 5-bromo-4,6-dimethyl-1H-
IC50 was determined by the SigmaPlot 10.0 software (Systat pyrazolo[3,4-b]pyridine-3-diazoniumchloride 9, which upon
Software, Inc., San Jose, CA, U.S.A.) using a four-parameter coupling with malononitrile and/ or 8-hydroxyquinoline af-
logistic function for the sigmoid dose–response curves.
forded hydrazonopyrimidine 10 and the corresponding azo
derivatives 11, respectively (Chart 2).
RESULTS
Antimicrobial Activity The results of the in vitro anti-
microbial testing against a panel of selected Gram-positive
As a part of our ongoing studies in developing new antimi- bacteria, Gram-negative bacteria and fungi are reported in
crobial and antiproliferative agents, we synthesized a series of Table 1 in comparison to those of the reference drugs Am-
pyridine derivatives to evaluate their antimicrobial and anti- picillin, Gentamicin, and Amphotericin B, respectively. The
proliferative profile and to correlate the biological results with antimicrobial effect of DMSO against the test organisms
their molecular properties.
was investigated and no significant inhibitory effect was re-
Chemistry The synthetic procedures adopted to obtain ported so that the concentration of DMSO in all the prepared
the test compounds are depicted in Charts 1 and 2. Chlori- tested compounds with different concentrations did not exceed
nation of 5-bromo-4,6-dimethyl-2-oxo-1,2-dihydropyridine- 0.25% (v/v) to avoid its inhibitory effect. Data presented in
3
-carbonitrile 1 with PCl in POCl afforded 2-chloropyridine Table 1 demonstrate that all the tested compounds exhib-
5 3
45)
derivative 2, which was cyclized under the effect of hydra- ited slight to moderate antibacterial activity against the tested
zine hydrate in boiling ethanol yielded the amine derivative 4 Gram-positive bacterial strains (Bacillus subtilis, Staphylococ-
instead of 5-bromo-2-hydrazinyl-4,6-dimethylnicotinonitrile cus aureus, and Streptococcus pneumoniae). Moreover, all the
4
6,47)
3
.
Reaction of 1 with phenyl isothiocyanate in dry dimeth- tested compounds were found to be highly active, moderately
ylformamide (DMF) containing a catalytic amount of trieth- active, or slightly active against the tested Gram-negative bac-
ylamine (TEA) gave 5-bromo-3-cyano-4,6-dimethyl-2-oxo-N- terial strains (Escherichia coli and Salmonella Typhimurium).
Fig. 1. Stereoisomers of the Schiff’s Base 6c

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
479
Table 1. In Vitro Antimicrobial Activity of the Synthesized Compounds against Different Microbial Strains
Diameter of the inhibition zone (mm)
Bacterial strains
Fungal strains
Test compounds
Gram-positive bacteria
Gram-negative bacteria
(1000µg/mL)
Aspergillus
niger
Candida
albicans
Fusarium
oxysporum
Bacillus Staphylococcs Streptococcus Escherichia Pseudomonas Salmonella
subtilis aureus pneumoniae coli
aeruginosa Typhimurium ATCC 6275 ATCC 10231 ATCC 7601
ATCC 6633 ATCC 25923 ATCC 49619 ATCC 25922 ATCC 27853 ATCC 13311
1
2
4
5
14.3± 0.63
+)
17.8± 0.44
++)
17.6± 0.44
++)
19.6± 0.53
++)
13.3± 0.63
+)
18.9± 0.42
++)
14.3± 0.44
+)
18.2± 0.25
++)
20.3± 0.58
++)
21.2± 0.44
++)
10.2± 0.67
+)
11.7± 0.44
+)
18.4± 0.44
++)
13.4± 0.44
(+)
15.8± 0.53
(+)
15.9± 0.37
(+)
17.9± 0.53
(++)
12.9± 0.44
(+)
17.4± 0.42
(++)
13.5± 0.42
(+)
17.6± 0.63
(++)
18.3± 0.67
(++)
20.6± 0.58
(++)
10.0± 0.67
(+)
10.6± 0.58
(+)
17.8± 0.58
(++)
22.4± 0.25
(++)
18.0± 0.63
(++)
22.4± 0.43
(++)
14.2± 0.25
(+)
20.2± 0.15
(++)
15.2± 0.63
(+)
20.3± 0.44
(++)
22.9± 0.44
(++)
22.9± 0.44
(++)
11.6± 0.44
(+)
16.5± 0.37
(+)
16.3± 0.37
(++)
18.9± 0.44
(+++)
13.4± 0.25
(+)
18.6± 0.25
(+++)
14.3± 0.44
(+)
16.8± 0.53
(++)
13.6± 0.37
(+)
20.3± 0.25
(+++)
17.9± 0.63
(+++)
20.2± 0.25
(+++)
12.3 ± 0.46
(+)
15.2± 0.63
(++)
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
18.2± 0.44 15.3± 0.25
(+++) (++)
23.6± 0.58 18.9± 0.63
(+++) (++)
15.2± 0.44 11.9± 0.58
(++) (+)
21.8± 0.53 20.3± 0.58
(+++) (+++)
15.8± 0.25 12.3± 0.58
(++) (+)
18.8± 0.42 19.8± 0.58
(+++) (++)
15.1± 0.42 16.3± 0.44
(++)
(++)
21.4± 0.58 16.3± 0.58
(+++) (++)
20.6± 0.67 20.9± 0.44
(+++) (+++)
24.4± 0.44 23.8± 0.58
(+++) (+++)
13.6± 0.67 10.6± 0.25
(+) (+)
16.4± 0.58 17.6± 0.44
(++) (++)
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
16.2± 0.44
(++)
20.6± 0.37
(+++)
13.2± 0.34
(+)
21.6± 0.25
(+++)
13.4± 0.37
(+)
20.6± 0.25
(+++)
17.2± 0.58
(++)
18.6± 0.58
(++)
22.0± 0.58
(+++)
20.1± 0.63
(+++)
11.7± 0.34
(+)
18.2± 0.25
(++)
(
(
(
(
6
a
(
6
b
(
6
c
(
7
a
(
7
b
(
8
a
(
8
b
(
1
0
1
(
1
16.3± 0.58
(+)
19.1± 0.63
(++)
16.2± 0.37
(++)
19.8± 0.25 10.6± 0.58
11.7± 0.44
(+)
(
(+++)
(+)
Standard antimicrobial drug (30µg/mL)
Ampicillin
32.4± 0.3
26.3± 0.3
(+++)
23.8± 0.2
(++)
—
—
—
—
—
—
—
—
—
(
+++)
Gentamicin
—
—
—
19.9± 0.3
+++)
17.3± 0.1
(++)
28.8± 0.3
(+++)
(
Amphotericin B
—
—
—
—
—
—
23.7± 0.1
25.4± 0.1
(+++)
19.7± 0.2
(++)
(
+++)
0
.25% (v/v) DMSO
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Gram-positive bacteria: (+++) Highly active=inhibition zone diameter >25mm; (++) Moderately active=inhibition zone diameter 17–25mm; (+) Slightly active=
inhibition zone diameter 9–17mm. Gram-negative bacteria: (+++) Highly active=inhibition zone diameter >18mm; (++) Moderately active=inhibition zone diameter
1
5–18mm; (+) Slightly active=inhibition zone diameter 12–15mm. Fungi: (+++) Highly active=inhibition zone diameter >20mm; (++) Moderately active=inhibition zone
diameter 15–20mm; (+) Slightly active=inhibition zone diameter 10–15mm.
In contrast, none of the compounds were found to be active and MCF7 (breast adenocarcinoma) cells expressed as in-
against Pseudomonas aeruginosa. Furthermore, all the tested hibitory concentration 50% (IC ) are summarized in Table 3
5
0
compounds revealed high, moderate or slight antifungal ac- compared to that of the standard anticancer drug Doxorubicin.
tivity against the tested fungal strains (Aspergillus niger and As illustrated in Table 3, the majority of the test compounds
Fusarium oxysporum). On the other hand, none of the com- possess potential cytotoxic activities against the tested human
pounds were found to be active against Candida albicans. cancer cell lines in submicromolar range.
Based on the in vitro antimicrobial testing data, the majority
of the tested compounds possess promising antimicrobial ac- DISCUSSION
tivity against the test microorganisms, when compared to the
standard antimicrobial drugs. Therefore, the compounds that
Antimicrobial Activity In the pyridine derivatives series
showed significant antimicrobial activity in the preliminary (compounds 1, 2, 5), compound 2 showed improved antimi-
screening were selected for MIC determinations. MIC values crobial activity compared to compound 1 which might be ex-
of the tested compounds for antibacterial and antifungal ac- plained by the introduction of the electron-withdrawing chlo-
tivities are listed in Table 2.
rine atom at position 2 of the pyridine ring. In line with the
Antiproliferative Activity Results of the in vitro antipro- structure–activity relationship (SAR) exploration, the presence
liferative activity against Hep G2 (hepatocellular carcinoma) of electron-withdrawing on the pyridine ring dramatically

4
80
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
Table 2. Minimum Inhibitory Concentration of the Synthesized Compounds against Different Microbial Strains
MIC (µg/mL)
Bacterial strains
Fungal strains
Test compounds
Gram-positive bacteria
Gram-negative bacteria
Fusarium
oxysporum
ATCC 7601
Aspergillus niger
ATCC 6275
Staphylococcus
aureus
Streptococcus
pneumoniae
ATCC 49619
Salmonella
Typhimurium
ATCC 13311
Bacillus subtilis
Escherichia coli
ATCC 6633
ATCC 25922
ATCC 25923
1
2
4
5
125
15.63
31.25
3.9
125
62.5
62.5
15.63
125
31.25
0.98
15.63
0.98
125
62.5
7.81
125
15.63
0.49
125
125
7.81
500
62.5
3.9
125
15.63
125
1.95
62.5
7.81
125
3.9
1.95
125
6
a
125
500
6
b
15.63
125
31.25
125
3.9
31.25
125
3.9
1.95
31.25
7.81
0.98
1.95
500
6
c
125
62.5
31.25
1.95
0.24
500
7
a
7.81
3.9
15.63
15.63
1.95
500
1.95
0.49
0.49
500
1.95
15.63
1.95
500
0.98
3.9
7
b
8
a
0.98
500
0.12
125
8
b
1
1
0
1
500
500
31.25
3.9
125
31.25
1.95
15.63
500
7.81
500
7.81
125
31.25
Standard antimicrobial drug
Ampicillin
0.007
—
0.015
—
0.03
—
—
0.24
—
—
0.015
—
—
—
—
—
Gentamicin
Amphotericin B
—
—
—
0.03
0.98
MIC values were expressed as µg/mL.
Table 3. Antiproliferative Activity of the Test Compounds against HepG2 and MCF7 Cell Lines
®
®
HepG2 (ATCC HB8065™, hepatocellular carcinoma) MCF7 (ATCC HTB22™, breast adenocarcinoma)
Test compound
IC (µM)
IC (µM)
5
0
50
1
2
4
5
168.54± 0.379 (−)
71.3± 0.098 (+)
351.9± 2.647 (−)
1.65± 0.011 (++)
425.280± 4.031 (−)
3.627± 0.014 (++)
680.89± 5.357 (−)
8.31± 0.025 (++)
0.0158± 0.00013 (+++)
38.69± 0.174 (+)
41.02± 0.356 (+)
2.291± 0.018 (++)
1.389± 0.007 (++)
0.0211± 0.00003 (+++)
0.0169± 0.0001 (+++)
0.0198± 0.00015 (+++)
2.421± 0.012 (++)
54.13± 0.29 (+)
6
a
6
b
6
c
7
a
7
b
0.0001± 0.00000036 (+++)
92.96± 0.18 (+)
8
a
8
b
5.47± 0.032 (++)
110.131± 0.76 (−)
127.468± 0.53 (−)
0.008± 0.00005 (+++)
103.22± 0.49 (−)
0.0091± 0.000016 (+++)
147.23± 0.865 (−)
1
1
0
1
Doxorubicin (standard anticancer drug)
0.099± 0.00048 (+++)
IC50: concentration of the compound (µM) producing 50% cell growth inhibition after 72h of compound exposure, as determined by the SRB assay. Each experiment was run
at least three times, and the results are presented as the mean± standard deviation (S.D.). (+++) Highly active=IC <1µM; (++) Moderately active=1µM<IC <10µM; (+)
50
50
Slightly active=10µM<IC <100µM; (−) Inactive=IC >100µM.
5
0
50
improved the antimicrobial activity. Furthermore, compound derivative 4 resulted in the introduction of the bulky aromatic
exhibited the best antimicrobial activity among this series of pyrazolo[3,4-b]pyridine group which might be resulted in the
compounds. One explanation is that, the introduction of carbo- loss of antimicrobial activity. These results suggest that the
thioamide moiety might favor the inhibitory potency.
addition of bulky substituents might deteriorate the antimi-
5
In regards to the pyrazolo[3,4-b]pyridines series (com- crobial activity of this derivative in this series of compounds,
pounds 4, 10, 11), compound 4 was recorded to be less active suggesting that this bicyclic system plays a negative role on
than compound 2. It has been known that the introduction of the antimicrobial effectiveness. In contrast, compound 10 ex-
bulky moieties at different positions of the pyridine ring could hibited better antimicrobial activity than 4 against Salmonella
affect the antimicrobial activity. Accordingly, the cyclization Typhimurium, Aspergillus niger and Fusarium oxysporum, an
of 2 under the effect of hydrazine hydrate to yield the amine observation that could be explained by the introduction of the

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
481
aliphatic malononitrile moiety, suggesting that pyrazolo[3,4- is nearly as active as the standard antimicrobial drug Ampho-
b]pyridine system bearing malononitrile moiety might tericin B against Fusarium oxysporum. Notably, the inhibitory
enhance the antimicrobial efficacy against these microbial potency of the tested compounds against Gram-positive bacte-
strains. Similarly, compound 11 was proved to be more active rial strains was somewhat superior when compared to Gram-
than 4 against Bacillus subtilis, Streptococcus pneumonia, negative bacterial strains and fungal strains.
Escherichia coli, and Salmonella Typhimurium, possibly due
Antiproliferative Activity Among the pyridine deriva-
to the introduction of the aromatic hydroxyquinoline moi- tives (compounds 1, 2, 5), compound 5 was proved to be the
eties, indicating that pyrazolo[3,4-b]pyridine system bearing most active among this series against HepG2 and MCF7 cells,
hydroxyquinoline moiety might enhance the antimicrobial confirming that the introduction of carbothioamide moiety
efficacy against these microbial strains. Moreover, compound might favor the cytotoxic potency. The cytotoxic activity
11 appeared to be more active than compound 10 against four against both HepG2 and MCF7 cells was enhanced in com-
of the test microorganisms. It is evident that the presence pound 2 when compared to compound 1, possibly due to the
of a hydroxyl group endowed with an electron-donating and introduction of an electron-withdrawing chlorine atom at posi-
hydrophilic character in 11 could potentially contribute to the tion 2 of the pyridine ring, suggesting that the electronic influ-
enhanced antimicrobial activity with respect to introducing a ence of the substituents in the pyridine ring appears to play an
malononitrile moiety in 10.
important role in the antiproliferative activity.
Concerning the Schiff’s bases series (compounds 6a–c),
In the pyrazolo[3,4-b]pyridines series (compounds 4, 10,
compound 6a was found to be less active than 4. Additionally, 11), the cyclization of 2 under the effect of hydrazine hydrate
compounds 6b and c were proved to be more active than 6a. to yield the amine derivative 4 resulted in the introduction of
Furthermore, compound 6b appeared to be more active than the bulky aromatic pyrazolo[3,4-b]pyridine group, causing a
compound 6c. The varying degree of antimicrobial activity of deterioration of cytotoxic potency against HepG2 cells. Ac-
these derivatives seems to be governed in part by the physico- cordingly, compounds 10 and 11 were found to be inactive
chemical properties of the aromatic or heterocyclic aldehyde against the HepG2 cells. Unexpectedly, the introduction of
that has been condensed with 4 to afford the different Schiff’s the pyrazolo[3,4-b]pyridine group resulted in improved anti-
bases.
proliferative potency of compounds 4 and 10 against MCF7
In the 4-thiazolidinones series (compounds 7a, b), com- cells, demonstrating that compound 10 is more potent than
pound 7a was proved to be more active as antibacterial agent compound 4 against the MCF7 cells, an observation that
than compound 6b, whereas compound 6b was found to be might be attributed to the introduction of the small aliphatic
more active than compound 7a as antifungal agent, suggesting malononitrile moiety. In contrast, the introduction of the
that the cyclization of the Schiff’s base 6b with thioglycolic bulky aromatic hydroxyquinoline moiety to yield 11 resulted
acid to yield the 4-thiazolidinone 7a resulted in improved in the loss of the antiproliferative activity against both HepG2
antibacterial activity and diminished antifungal activity. On and MCF7 cells. The real reason for this discrepancy is not
the other hand, compound 7b was found to be more active clear. It seemed quite probable that this behavior depends on
than compound 6c against most of the test microorganisms, the cancer cells to which the compounds were subjected to.
indicating that the cyclization of the Schiff’s base 6c with
In regards to the Schiff’s bases series (compounds 6a–c), it
thioglycolic acid to yield the 4-thiazolidinone 7b encourages has been observed that compound 6b was found to be the only
the antimicrobial activity. Interestingly, compounds 7a and active among this series of compounds against HepG2 cells.
b were observed to have comparable antimicrobial activity One possibility is that the condensation of 4 with the hetero-
against most of the test microorganisms, suggesting that their cyclic aldehyde, 1H-indole-3-carbaldehyde to afford 6b was
antimicrobial activities were not significantly affected by the favorable for increasing antiproliferative potency. In contrast,
position or physicochemical properties of the different sub- the condensation of 4 with p-chlorobenzaldehyde or 1,3-di-
stituents and functional groups.
phenyl-1H-pyrazole-4-carbaldehyde to yield 6a and c, respec-
Noteworthy, the azetidin-2-one 8a was found to be more tively, led to diminished cytotoxicity against the HepG2 cells.
active than compound 6b against most of the test microor- In contrast, the condensation of 4 with p-chlorobenzaldehyde
ganisms, indicating that the cyclization of the Schiff’s base or 1H-indole-3-carbaldehyde to afford the corresponding
6
b with chloroacetyl chloride to afford the azetidin-2-one 8a Schiff’s bases 6a and b, respectively, might be favorable for
was in favor of enhanced antimicrobial activity. In contrast, improving cytotoxic activity against MCF7 cells. However,
the Schiff’s base 6c was proved to be more active than the the condensation of 4 with 1,3-diphenyl-1H-pyrazole-4-carb-
azetidin-2-one 8b particularly at the antifungal activity level, aldehyde to yield 6c was unfavorable for antiproliferative ef-
suggesting that the cyclization of the Schiff’s base 6c with ficacy against the MCF7 cells.
chloroacetyl chloride to afford the azetidin-2-one 8b resulted
Concerning the 4-thiazolidinones series (compounds 7a,
in a diminished antimicrobial activity. Unexpectedly, although b), compound 7a was found to be less potent than compound
both 8a and b belong to the azetidin-2-one series, 8a was 6b, indicating that the cyclization of the Schiff’s base 6b with
proved to be the most active, whereas 8b was found to be thioglycolic acid to yield the 4-thiazolidinone 7a resulted in
the least active among the tested compounds against most of decreased antiproliferative activity against both HepG2 and
the test microorganisms. Perhaps their antimicrobial activities MCF7 cells. In contrast, compound 7b was found to be more
were significantly affected by the physicochemical properties potent than compound 6c against both HepG2 and MCF7
of the different substituents on the azetidin-2-one nucleus.
cells, suggesting that the cyclization of the Schiff’s base 6c
In most cases, the inhibitory potency exhibited by the test- with thioglycolic acid to yield the 4-thiazolidinone 7b resulted
ed compounds is lower than that of the standard antimicrobial in enhanced cytotoxic potency. It has been shown that com-
agents. Noteworthy exception arises with compound 7b which pound 7b was proved to be more active than compound 7a

4
82
Biol. Pharm. Bull.
Vol. 39, No. 4 (2016)
4
)
Kaelin WG Jr. The concept of synthetic lethality in the context of
anticancer therapy. Nat. Rev. Cancer, 5, 689–698 (2005).
Kamb A, Wee S, Lengauer C. Why is cancer drug discovery so dif-
ficult? Nat. Rev. Drug Discov., 6, 115–120 (2007).
Cozzi P, Mongelli N, Suarato A. Recent anticancer cytotoxic agents.
Curr. Med. Chem. Anticancer Agents, 4, 93–121 (2004).
Chabner BA, Roberts TG Jr. Timeline: Chemotherapy and the war
on cancer. Nat. Rev. Cancer, 5, 65–72 (2005).
against both HepG2 and MCF7 cells. One explanation might
be that the presence of the 1,3-diphenyl-1H-pyrazol-4-yl moi-
ety in 7b could enhance its cytotoxicity.
5
6
7
)
)
)
Regarding the azetidin-2-ones series (compounds 8a, b),
compound 8a was found to be less active than the Schiff’s
base 6b against both HepG2 and MCF7 cells, suggesting that
the cyclization of 6b with chloroacetyl chloride to yield the
azetidin-2-one 8a was not in favor of enhanced antiprolifera-
tive activity. In contrast, the azetidin-2-one 8b was proved to
be more active than the Schiff’s base 6c against HepG2 cells,
indicating that the cyclization of 6c with chloroacetyl chloride
to afford 8b resulted in improved cytotoxic efficacy. Surpris-
ingly, this cyclization led to a substantial decrease in antipro-
liferative potency against the MCF7 cells.
8) Horton DA, Bourne GT, Smythe ML. The combinatorial synthesis
of bicyclic privileged structures or privileged substructures. Chem.
Rev., 103, 893–930 (2003).
9)
Patel NB, Agravat SN, Shaikh FM. Synthesis and antimicrobial
activity of new pyridine derivatives-I. Med. Chem. Res., 20, 1033–
1041 (2011).
10) Gezginci MH, Martin AR, Franzblau SG. Antimycobacterial ac-
tivity of substituted isosteres of pyridine- and pyrazinecarboxylic
acids. 2. J. Med. Chem., 44, 1560–1563 (2001).
The test compounds showed comparatively much weaker
cytotoxic profiles when compared to the standard anticancer
drug against HepG2 cells. On the other hand, the cytotoxic
effect revealed by the test compounds (5, 6a, b, 7b, 10) was
1
1) Acharya BN, Thavaselvam D, Kaushik MP. Synthesis and antima-
larial evaluation of novel pyridine quinoline hybrids. Med. Chem.
Res., 17, 487–494 (2008).
found to be better than that of Doxorubicin against MCF7 12) Cocco MT, Congiu C, Lilliu V, Onnis V. Synthesis and in vitro
cells. In particular, compound 7b displayed the highest anti-
proliferative efficacy against both HepG2 and MCF7 cells,
demonstrating that this compound could be an interesting lead
antiproliferative candidate. Noteworthy, the antiproliferative
efficacy of the tested compounds against MCF7 cells was
somewhat superior when compared to HepG2 cells.
antitumoral activity of new 3,5-dicyanopyridine derivatives. Bioorg.
Med. Chem., 15, 1859–1867 (2007).
1
3) Willemann C, Grunert R, Bednarski PJ, Troschutz R. Synthesis and
cytotoxic activity of 5,6-heteroaromatically annulated pyridine-2,4-
diamines. Bioorg. Med. Chem., 17, 4406–4419 (2009).
4) Bahekar RH, Jain MR, Jadav PA, Prajapati VM, Patel DN, Gupta
AA, Sharma A, Tom R, Bandyopadhya D, Modi H, Patel PR. Syn-
thesis and antidiabetic activity of 2,5-disubstituted-3-imidazol-2-yl-
pyrrolo[2,3-b]pyridines and thieno[2,3-b]pyridines. Bioorg. Med.
Chem., 15, 6782–6795 (2007).
1
CONCLUSION
We report herein the synthesis of three series of com- 15) Abele E, Abele R, Lukevics E. Pyridine oximes: Synthesis, reac-
pounds, including Schiff’s bases, 4-thiazolidinones and
azetidin-2-ones. The synthesized compounds were evaluated
for their antimicrobial and antiproliferative efficacy. Our re-
sults revealed that some of the tested compounds exhibited a
significant antimicrobial potency against a wide spectrum of
bacteria and fungi. Moreover, some of the tested compounds
have been found to possess a promising antiproliferative ac-
tivity against HepG2 and MCF7 cell lines. The synthesized
compounds certainly hold great promise towards pursuit to
discover novel antimicrobial and antiproliferative agents. Our
study is a preliminary step toward developing newly synthe-
tions and biological activity. Chem. Heterocycl. Compd., 39, 825–
865 (2003).
6) El-Sayed Ali T. Synthesis of some novel pyrazolo[3,4-b]pyridine
1
and pyrazolo[3,4-d]pyrimidine derivatives bearing 5,6-diphenyl-
1,2,4-triazine moiety as potential antimicrobial agents. Eur. J. Med.
Chem., 44, 4385–4392 (2009).
1
7) Bernardino AMR, de Azevedo AR, Pinheiro LCS, Borges JC, Car-
valho VL, Miranda MD, de Meneses MDF, Nascimento M, Ferreira
D, Rebello MA, Da Silva VAGG, de Frugulhetti ICPP. Syntehsis
and antiviral activity of new 4-(phenylamino)/4-[(methylpidin-2-
yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic
derivatives. Med. Chem. Res., 16, 352–369 (2007).
acid
sized antimicrobial and antiproliferative agents. Further stud- 18) de Mello H, Echevarria A, Bernardino AM, Canto-Cavalheiro M,
ies are being conducted to acquire more information about
SAR of these novel synthesized compounds in order to clarify
their mode of action as antimicrobial and antiproliferative sub-
stances. Modifications to improve the potency of the synthe-
sized derivatives by structural diversification are currently
under progress in our laboratory.
Leon LL. Antileishmanial pyrazolopyridine derivatives: Synthesis
and structure–activity relationship analysis. J. Med. Chem., 47,
5
427–5432 (2004).
1
9) Hamdy NA, Gamal-Eldeen AM. New pyridone, thioxopyridine,
pyrazolopyridine and pyridine derivatives that modulate inflamma-
tory mediators in stimulated RAW264.7 murine macrophage. Eur. J.
Med. Chem., 44, 4547–4556 (2009).
2
0) Lin R, Connolly PJ, Lu Y, Chiu G, Li S, Yu Y, Huang S, Li X,
Emanuel SL, Middleton SA, Gruninger RH, Adams M, Fuentes-
Pesquera AR, Greenberger LM. Synthesis and evaluation of
pyrazolo[3,4-b]pyridine CDK1 inhibitors as anti-tumor agents.
Bioorg. Med. Chem. Lett., 17, 4297–4302 (2007).
Conflict of Interest The authors declare no conflict of
interest.
REFERENCES
21) Panneerselvam P, Rather BA, Ravi Sankar Reddy D, Ramesh
1)
Heinemann JA, Ankenbauer RG, Amabile-Cuevas CF. Do antibiot-
ics maintain antibiotic resistance? Drug Discov. Today, 5, 195–204
Kumar N. Synthesis and anti-microbial screening of some Schiff
bases of 3-amino-6,8-dibromo-2-phenylquinazolin-4(3H)-ones. Eur.
J. Med. Chem., 44, 2328–2333 (2009).
(2000).
2
)
Muroi H, Nihei K, Tsujimoto K, Kubo I. Synergistic effects of
anacardic acids and methicillin against methicillin resistant Staphy-
lococcus aureus. Bioorg. Med. Chem., 12, 583–587 (2004).
22) Barve V, Ahmed F, Adsule S, Banerjee S, Kulkarni S, Katiyar P,
Anson CE, Powell AK, Padhye S, Sarkar FH. Synthesis, molecular
characterization, and biological activity of novel synthetic deriva-
tives of chromen-4-one in human cancer cells. J. Med. Chem., 49,
3800–3808 (2006).
3)
Green DR, Evan GI. A matter of life and death. Cancer Cell, 1,
19–30 (2002).

Vol. 39, No. 4 (2016)
Biol. Pharm. Bull.
antimicrobial agents. Il Farmaco, 60, 727–730 (2005).
483
2
3) Liu ZC, Wang BD, Yang ZY, Li Y, Qin DD, Li TR. Synthesis,
crystal structure, DNA interaction and antioxidant activities of
35) Sperka T, Pitlik J, Bagossi P, Tözsér J. Beta-lactam compounds
as apparently uncompetitive inhibitors of HIV-1 protease. Bioorg.
Med. Chem. Lett., 15, 3086–3090 (2005).
2+
two novel water-soluble Cu complexes derivated from 2-oxo-
quinoline-3-carbaldehyde Schiff-bases. Eur. J. Med. Chem., 44,
4
477–4484 (2009).
36) Banik BK, Becker FF, Banik I. Synthesis of anticancer beta-
lactams: mechanism of action. Bioorg. Med. Chem., 12, 2523–2528
(2004).
2
4) Dimmock JR, Vashishtha SC, Stables JP. Anticonvulsant properties
of various acetylhydrazones, oxamoylhydrazones and semicarba-
zones derived from aromatic and unsaturated carbonyl compounds.
Eur. J. Med. Chem., 35, 241–248 (2000).
5) Verma A, Saraf SK. 4-Thiazolidinone—a biologically active scaf-
fold. Eur. J. Med. Chem., 43, 897–905 (2008).
6) Omar K, Geronikaki A, Zoumpoulakis P, Camoutsis C, Sokovic
M, Ciric A, Glamoclija J. Novel 4-thiazolidinone derivatives as po-
tential antifungal and antibacterial drugs. Bioorg. Med. Chem., 18,
37) Perez C, Pauli M, Bazerque P. An antibiotic assay by the agar-well
diffusion method. Acta Bio. Med. Exp., 15, 113–115 (1990).
38) Clinical and Laboratory Standards Institute. Performance standards
for antimicrobial susceptibility testing. 19th Info. Suppl., M100-S19,
Clinical and Laboratory Standards Institute, Wayne, PA (2009).
39) Clinical and Laboratory Standards Institute. Performance standards
for antimicrobial susceptibility testing. 20th Info. Suppl., M100-S20,
Clinical and Laboratory Standards Institute, Wayne, PA (2010).
40) Clinical and Laboratory Standards Institute. Performance standards
for antimicrobial susceptibility testing. 21st Info. Suppl., M100-S21,
Clinical and Laboratory Standards Institute, Wayne, PA (2011).
41) Ericsson HM, Sherris JC. Antibiotic sensitivity testing: report of an
international collaborative study. Acta Pathol. Microbiol. Scand. B:
Microbiol. Immunol., 217 (Suppl. 217), 1+ (1971).
2
2
426–432 (2010).
2
7) Babaoglu K, Page MA, Jones VC, McNeil MR, Dong C, Naismith
JH, Lee RE. Novel inhibitors of an emerging target in mycobacteri-
um tuberculosis; substituted thiazolidinones as inhibitors of dTDP–
rhamnose synthesis. Bioorg. Med. Chem. Lett., 13, 3227–3230
(2003).
2
2
3
8) Rawal RK, Tripathi R, Katti SB, Pannecouque C, De Clercq E. De-
sign synthesis and evaluation of 2-aryl-3-heteroaryl-1,3-thiazolidin-
42) National Committee for Clinical Laboratory Standards. Method
for dilution antimicrobial susceptibility test for bacteria that grow
aerobically. National Committee for Clinical Laboratory Standards,
Wayne, PA, 1997, Aprvd. Stand., M7-A4 (1997).
4
-ones as anti-HIV agents. Bioorg. Med. Chem., 15, 1725–1731
2007).
9) Kumar A, Rajput CS, Bhati SK. Synthesis of 3-[4′-(p-
chlorophenyl)-thiazol-2′-yl]-2-[(substituted
(
azetidinone/thiazolidi- 43) National Committee for Clinical Laboratory Standards. Reference
none)-aminomethyl]-6-bromoquinazolin-4-ones as anti-inflamma-
tory agent. Bioorg. Med. Chem., 15, 3089–3096 (2007).
0) Kaminskyy D, Zimenkovsky B, Lesyk R. Synthesis and in vitro
anticancer activity of 2,4-azolidinedione–acetic acids derivatives.
Eur. J. Med. Chem., 44, 3627–3636 (2009).
method for broth dilution antifungal susceptibility testing of yeast.
National Committee for Clinical Laboratory Standards, Wayne, PA,
1997, Aprvd. Stand., M27-A (1997).
44) Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D,
Warren JT, Bokesch H, Kenney S, Boyd MR. New colorimetric cy-
totoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst.,
82, 1107–1112 (1990).
45) Thompson WJ, Gaudino J. A general synthesis of 5-arylnicotinates.
J. Org. Chem., 49, 5237–5243 (1984).
3
1) Singh GS. β-Lactams in the new millennium. Part-I: monobactams
and carbapenems. Mini Rev. Med. Chem., 4, 69–92 (2004).
2) Singh GS. β-Lactams in the new millennium. Part-II: cephems, oxa-
cephems, penams and sulbactam. Mini Rev. Med. Chem., 4, 93–109
3
(
2004).
46) Kamal El-Dean AM, Geies AA, Mohamed TA. Synthesis of some
fused pyrazolo[3,4-b]pyridine heterocyclic compounds.. Indian J.
Chem. Sect. B: Organic Chemistry, Indian J. Chem. B, 30, 878–882
(1991).
47) Lacan M, Tabakovic K. Synthesis of new pyrazolo-, and tetrazolo-
pyridines. Croat. Chem. Acta, 47, 127–133 (1975).
3
3) Deshmukh AR, Bhawal BM, Krishnaswamy D, Govande VV,
Shinkre BA, Jayanthi A. Azetidin-2-ones, synthon for biologically
important compounds. Curr. Med. Chem., 11, 1889–1920 (2004).
4) Singh GS, Mmolotsi BJ. Synthesis of 2-azetidinones from 2-di-
azo-1,2-diarylethanones and N-(2-thienylidene)imines as possible
3