T.E.-S. Ali / European Journal of Medicinal Chemistry 44 (2009) 4539–4546
4545
and heated under reflux for 12 h. The reaction mixture was evap-
orated under reduced pressure, giving crude solids which were
crystallized from ethanol to give 16 and 21, respectively.
105 (100), 100 (56.98), 91 (45.87), 78 (22.65), 56 (31.56). Calcd for:
C5H9N6O2P (216.13): C, 27.78; H, 4.19; N, 38.88; Found: C, 27.53; H,
3.92; N, 38.63.
16: Pale brown crystals in 49% yield; mp 108–111 ꢁC. IR (KBr),
n
(cmꢀ1): 3212, 3146 (NH, NH), 2958 (C–Haliph), 1695 (C]Otria-
5.8. Synthesis of 4,8-dimethyl-3,3,3-triphenyl-2,3-dihydro-3-l5
-
[1,2,4]triazino[4,3-f][1,2,5,6,3]tetrazaphosphepin-7(1H)-one (23)
zinone), 1615, 1596 (C]N), 1287 (P]O), 1088 (P–O–C). 1H NMR
(DMSO),
d
: 1.12 (d, 3H, J ¼ 6.7 Hz, NCHCH3), 1.17 (t, 3H, J ¼ 7.5 Hz,
OCH2CH3), 2.13 (s, 3H, CH3), 3.77 (q, 2H, J ¼ 7.2 Hz, OCH2CH3),
A mixture of 17 (0.005 mol, 0.78 g) and acetyl triphenylphos-
phonium chloride (0.005 mol,1.70 g) in DMF (30 mL) containing few
drops of piperidine, was heated under reflux for 12 h, cooled and
then poured into ice. The obtained solid was filtered off and crys-
tallized from methanol. Brown crystals in 43% yield; mp 154–155 ꢁC.
5.38 (br, 1H, CH–P), 6.49 (br, 1H, NH), 8.24 (s, 1H, NH). 13C NMR
(DMSO),
d
: 16.4 (OCH2CH3, J ¼ 6.3 Hz), 16.7 (P–CH–CH3), 17.1
(CH3,triazine), 47.5 (CH–P, JC,P ¼ 147.2 Hz), 62.5 (OCH2CH3,
J ¼ 7.5 Hz), 150.2 (C]N), 151.9 (N]C–N), 172.3 (C]O). 31P NMR
(DMSO),
d
: þ21.5 ppm. MS (m/e, %): 261 (M þ 2, 0.35%), 259 (Mþ,
IR (KBr),
Haliph),1662 (C]Otriazinone),1601 (C]N).1H NMR (DMSO),
3H, CH3), 2.18 (s, 3H, CH3), 7.43–7.96 (m,15H, aromatic protons), 8.67
(s, 1H, NH), 10.0 (br s, 1H, NH). 13C NMR (DMSO),
: 16.0 (CH3,te-
n
(cmꢀ1): 3295, 3217 (NH, NH), 3096 (C–Harom), 2907 (C–
: 2.08 (s,
3.82), 214 (16.44), 213 (100), 176 (15.75), 103 (30.07), 77 (14.47),
64 (12.68), 51 (11.26). Calcd for: C8H14N5O3P (259.20): C, 37.07;
H, 5.44; N, 27.01; Found: C, 36.85; H, 5.23; N, 26.81.
d
d
21: Yellow crystals in 59% yield; mp 131–133 ꢁC. IR (KBr),
n
trazaphosphepine),17.0 (CH3,triazine),128.2–130.0 (phenylcarbons),145.9
(cmꢀ1): 3420 (br, NH2), 3250 (NH), 2950 (C–Haliph), 1665
(N]C–P, JC,P ¼ 62.5 Hz), 152.0 (C]N), 152.5 (N]C–N), 172.0 (C]O).
(C]Otriazinone), 1624 (C]N), 1210 (P]O), 1091 (P–O–C). 1H NMR
31P NMR (DMSO),
H, 5.23; N, 18.99; Found: C, 64.87; H, 5.05; N, 18.82.
d
: ꢀ22.3. Calcd for: C24H23N6OP (442.45): C, 65.15;
(DMSO),
d
: 1.12 (d, 3H, J ¼ 6.9 Hz, NCHCH3), 1.17 (t, 3H, J ¼ 7.5 Hz,
OCH2CH3), 2.13 (s, 3H, CH3), 3.72 (q, 2H, J ¼ 7 Hz, OCH2CH3), 5.38
(br, 1H, CH–P), 6.49 (s, 2H, NH2), 7.70 (s, 1H, NH). 13C NMR (DMSO),
5.9. Antibacterial activity
d
: 16.2 (OCH2CH3, J ¼ 6.3 Hz), 16.7 (P–C–CH3), 17.1 (CH3,triazine), 49.5
(CH–P, JC,P ¼ 145.2 Hz), 60.5 (OCH2CH3, J ¼ 6.9 Hz), 151.2 (C]N),
All the newly synthesized compounds were evaluated in vitro for
their antibacterial activity. The antibacterial activity is carried out
against four bacterial strains, S. aureus (MTCCB 737), S. epidermidis
(MTCCB 1824), E. coli (MTCCB 1652) and P. aeruginosa (ATCC 27853)
152.4 (N]C–N), 172.8 (C]O). 31P NMR (DMSO),
d: þ22.3 ppm. MS
(m/e, %): 275 (M þ 1, 1.34%), 274 (Mþ, 2.45), 257 (3.54), 228 (10.23),
211 (100), 110 (65.03), 102 (34.11), 84 (22.11), 74 (20.14), 56 (15.09).
Calcd for: C8H15N6O3P (274.21): C, 35.04; H, 5.51; N, 30.64; Found:
C, 34.87; H, 5.32; N, 30.52.
employing the nutrient agardisc diffusion method[25] at 100 mg/mL
concentration. DMSO was used as blank exhibited no activityagainst
anyof the used organisms. Theantibacterial activity was determined
by measuring the inhibition zone (Table 1), after 16–20 h of incu-
bation at 37 ꢁC for bacterial strains. Tetracycline was used as refer-
5.6. Synthesis of 3-[ethylideneamino]-6-methyl-2-oxido-2-phenyl-
2,3-dihydro[1,2,4,3] triazaphospholo[5,1-c][1,2,4]triazin-7-one (18)
ence drug against bacterial strains at 30 mg/mL concentration.
A
mixture of 17 (0.005 mol, 0.78 g) and acetaldehyde
(0.005 mol, 0.22 g) in THF (30 mL) containing triethylamine
(0.01 mol, 1.01 g) was warmed for 10 min, then added to a solution
of phenylphosphonic dichloride (0.005 mol, 0.97 g) in THF (3 mL)
and heated under reflux for 4 h. The reaction mixture was evapo-
rated under reduced pressure, giving crude solid which was crys-
tallized from ethanol. Yellow crystals in 77% yield; mp 196–198 ꢁC.
5.10. The minimum inhibitory concentration (MIC)
A current definition of the minimum inhibitory concentration
MIC is ‘‘the lowest concentration which resulted in maintenance or
reduction of inoculum viability’’. The determination of the MIC
involves
a semi-quantitative test procedure which gives an
IR (KBr),
(C]Otriazinone), 1644, 1609 (C]N), 1284 (P]O). 1H NMR (DMSO),
2.13 (s, 3H, CH3), 2.50 (d, 3H, CH3CHN), 5.77 (br, 1H, N]CH), 7.44–
7.99 (m, 5H, aromatic protons), 8.29 (s,1H, NH). 13C NMR (DMSO),
16.8 (CH3–C–N), 17.0 (CH3,triazine), 128.5–130.1 (phenyl carbons),
142.3 (N]CH–CH3), 151.1 (C]N), 152.2 (N]C–N), 172.2 (C]O). 31
NMR (DMSO),
n
(cmꢀ1): 3180 (NH), 3077 (C–Harom), 2961 (C–Haliph), 1694
approximation to the least concentration of antimicrobial agent
needed to prevent microbial growth. The method displays tubes of
growth broth containing a test level of preservatives, into which an
inoculum of microbes was added. The end result of the test was the
minimum concentration of antimicrobial agents. The tube dilution
technique [26] was applied for the determination of MIC of the
tested compounds against. Dilution series were set up with 3.12,
d:
d:
P
d
: þ26.4 ppm. Calcd for: C12H13N6O2P (304.24): C,
47.37; H, 4.30; N, 27.62; Found: C, 47.12; H, 4.17; N, 27.45.
6.25, 12.5, 25 and 50 mg/mL of nutrient broth medium to each tube,
100 m
L of standardized suspension of the test microbes (107 cell/
5.7. Synthesis of 2,7-dimethyl-2-oxido-1,2,3,4-tetrahydro-8H-
[1,2,4]triazino[4,3-e][1,2,4,5,3] tetrazaphosphinin-8-one (22)
mL) were added and incubated at 37 ꢁC for 24 h (Table 2).
5.11. Cytotoxicity bioassay
A mixture of 17 (0.005 mol, 0.78 g) and bis(dimethylamino)-
methylphosphonate (0.005 mol, 1.03 g) in THF (30 mL) containing
few drops of concentrated hydrochloric acid, was heated under
reflux for 5 h. The reaction mixture was evaporated under reduced
pressure, giving crude solid which was crystallized from ethanol.
Brine shrimp lethality bioassay [33,34] is a recent development
in the assay which indicates cytotoxicity as well as a wide range of
pharmacological activities (e.g. antimicrobial, anticancer, antiviral,
insecticidal, pesticidal, AIDS, etc.). In this method, the eggs of the
brine shrimp, Artemia salina leach, were hatched for 48 h to mature
shrimp. 38 g of sea salt was weighed, dissolved in 1 L of distilled
water, filtered off and was kept in a small tank. The eggs were then
added to the divided tank. Constant oxygen supply was provided
and temperature 37 ꢁC was maintained for 48 h to hatch and
mature the shrimp called as nauplii (Larvae). Samples of the tested
compounds were prepared by dissolving 10 mg of each compound
in 2 mL of DMSO. From this stock, a series of solutions 80, 40, 20, 10
Pale brown crystals in 81% yield; mp 175–177 ꢁC. IR (KBr),
3533 (br, NHNH), 3146 (NH), 2957, 2925 (C–Haliph), 1690 (C]Otria-
zinone), 1618 (C]N), 1269 (P]O). 1H NMR (DMSO),
: 2.13 (s, 3H,
CH3), 2.50 (s, 3H, CH3–P), 5.34 (br, 1H, NH), 6.48 (br, 1H, NH), 7.68 (s,
1H, NH). 13C NMR (DMSO),
: 16.6 (CH3,tetrazaphosphinine), 17.0
n
(cmꢀ1):
d
d
(CH3,triazine), 149.9 (C]N), 150.4 (N]C–N), 173.2 (C]O). 31P NMR
(DMSO),
d
: þ27.1 ppm. MS (m/e, %): 219 (M þ 3, 0.06%), 216 (Mþ,
1.09), 208 (13.45), 176 (23.09), 165 (34.76), 162 (23.90), 149 (45.98),