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S. M. Sondhi et al.
CHCl3 gave 4,4,6-trimethyl-1,4-dihydropyrimidine-2(3H)thione (6).
Further elution with CHCl3/ethyl acetate (1:1) gave 3,3,4a-
trimethyl-3,4,4a,5-tetrahydropyrimido[6,1-b]pyrido[2,3-d]imidazole
(7) (Found: C, 58.0; H, 6.7; N, 22.6; C12H16N4S requires C, 58.1; H, 6.
5; N, 22.6%). The filtrate from the above reaction mixture was
extracted with ethyl acetate (50×5 mL). The combined ethyl-acetate
extracts were washed with water and dried over sodium sulfate. Ethyl
acetate was distilled off and the solid product left behind was washed
with chilled methanol to give 2,2′-thiodipyrido[2,3-d]imidazole (8)
(Found: C, 53.9; H, 2.9; N, 31.2; C12H8N6S requires C, 53.7; H, 3.0; N,
31.3%).
prior to the phenylquinone challenge. Writhing was again recorded for
each mouse in a group and the percentage protection was calculated
using the following formula, where NTM refers to the number of writhes
in the treated mouse, and NUM refers to the number of writhes in the
untreated mouse:
% protection = 100–[(NTM)/(NUM)×100]
This was taken as percentage of analgesic response and was averaged in
each group of mice.
The percentage of animals exhibiting analgesia was determined
with each dose. Compounds (2a–f), (7) and (8) were screened for
analgesic activity. Compounds (2a), (2d) and (7) showed good analgesic
activity (80, 90 and 80%, repectively, at 100 mg/kg p.o.; 60, 50 and
60%, respectively, at 50 mg/kg p.o.; and 20, 20 and 30%, respectively,
at 25 mg/k.g p.o.). Compounds (2b), (2c), (2e), (2f) and (8) showed
repectively 60, 60, 20, 20 and 40% analgesic activity at 100 mg/kg p.o.;
25, 40, 20, 10 and 20% at 50 mg/kg p.o.; and 25, 20, 0, 0 and 0% at 25
mg/k.g. p.o.
Condensation of 2,3-Diaminopyridine with
4-Isothiocyanato-4-methylpentan-2-one
2,3-Diaminopyridine (218 mg, 2 mmol) was dissolved in DMF
(0.5 mL), and 4-isothiocyanato-4-methylpentan-2-one (0.33 mL, 2
mmol) was added. The reaction mixture was heated in a water bath for
8 h and then the solvent was removed under vaccum. Chilled methanol
(5 mL) was added to the semi-solid residue left behind, and the solid
product that separated out was filtered off and washed with chilled
methanol (1 mL). The solid product so obtained was air-dried to give
compound (7). The filtrate was concentrated and subjected to column
chromatography over silica gel. Elution with CHCl3/ethyl acetate (1:4)
gave 2-amino-3-(4,4,6-trimethyl-2-thioxo-1,2,3,4-tetrahydropyrimid-
in-1-yl)pyridine (9).
Anti-Amoebic Activity Screening
In vitro testing against Entamoeba histolytica.[27–29]
Activity
against Entamoeba histolytica (strain HM-1: IMSS) in vitro was
assessed using the microplate method. Dimethyl sulfoxide (DMSO) (40
µl) was added to the sample (1 mg) followed by enough culture medium
to obtain a concentration of 1 mg/mL. Samples were dissolved or
suspended by mild sonication in a sonicleaner bath (Julabo, USRI, West
Germany) for a few minutes and then further diluted with medium to a
concentration of 0.1 mg/mL. Twofold serial dilutions were made in the
wells of a 96-well microtitre plate (Nunc) in 170 µL of medium. Each
test included metronidazole as a standard amoebicidal drug. Control
wells (culture medium plus amoebae) were prepared from a confluent
culture by pouring off the medium, adding 2 mL of medium and chilling
the culture on ice to detach the organisms from the side of the flask. The
number of amoeba per mL was estimated with a haemocytometer, and
Trypan Blue exclusion was used to confirm viability. Fresh culture
medium was added to dilute the suspension to 105 organisms, and
170 µL of this suspension was added to the test and control wells in the
plate so that the wells were completely filled (total volume, 340 µL). An
inoculum of 1.7×104 organisms/well was chosen so that confluent, but
not excessive, growth took place in the control wells. Each plate was
sealed with expanded polystyrene (0.5-mm thick), secured with tape,
placed in a modular incubating chamber (Flow Laboratories, High
Wycombe, U.K.) and gassed for 10 min with nitrogen before incubation
at 37oC for 72 h.
Assessment of anti-amoebic activity. After incubation, the growth
of amoeba in the plate was checked with a low-power microscope. The
culture medium was removed by inverting the plate and shaking it
gently. The plate was then immediately washed once in sodium chloride
solution (0.9%) at 37°C. This procedure was completed quickly, and the
plate was not allowed to cool in order to prevent the detachment of
amoebae. The plate was allowed to dry at room temperature, the
amoeba were fixed with methanol and, when dry, stained with (0.5%)
aqueous eosine for 15 min The stained plate was washed once with tap
water and then twice with distilled water and allowed to dry. A 200-µL
portion of 0.1 M sodium hydroxide solution was added to each well to
dissolve the protein and release the dye. The optical density of the
resulting solution in each well was determined at 490 nm with a
microplate reader (Labsystem Multiskane Bichromatic, U.K.). The
percentage inhibition of amoeba growth was calculated from the optical
densities of the control and test wells, and was plotted against the
logarithm of the dose of the drug tested. Linear regression analysis was
used to determine the best-fitting straight line from which the IC50 value
was found.
Acid-Catalysed Condensation of
4-Isothiocyanato-4-methylpentan-2-one
4-Isothiocyanato-4-methylpentan-2-one (1 mL, 6 mmol) and acetic
acid (10 mL) were heated under reflux for 11 h, the solvent was
removed under reduced pressure and the solid residue left behind was
washed with aqueous sodium carbonate (10%, 10 mL) and then with
water. The crude product was dissolved in methanol and treated with
charcoal to remove coloured impurities. Removal of the solvent gave a
white solid, which was recrystallized from methanol to give
4,4,6-trimethylpyrimidine-1,4-dihydro-2(3H)thione (6) (350 mg,
40%). The spectroscopic data and m.p. for this compound were
identical to those reported in Table 1.
Anti-Inflammatory Activity Screening[25]
Anti-inflammatory activity testing was carried out using
carrageenin-induced paw oedema in albino rats. Oedema in one of the
hind paws was induced by injection of 0.1 mL of a 1% carrageenin
solution into planter aponeurosis. The volume of the paw was measured
plethysmographically both immediately after and 3 h after injection of
the irritant. The difference in volume gave the amount of oedema that
had developed. Percentage inhibition of the oedema between the control
group and the group treated with test compound was calculated and
compared with the group receiving the standard drug. At 100 mg/kg
p.o., compounds (2b) and (7) possessed good (43 and 34%,
respectively) anti-inflammatory activity, whereas compounds (2a),
(2c), (2d) and (8) inhibited the carrageenin-induced hind paw oedema
by 6, 24, 12 and 16%, respectively, as compared with the standard drug
ibuprofen (which showed 51% activity at 50 mg/kg p.o.).
[26]
Analgesic Activity Screening
Analgesic activity was evaluated in albino mice using the
phenylquinone writhing assay. Female swiss mice (15–20 g), bred in the
Animal House of Central Drug Research Institute, Lucknow, and
maintained under standard laboratory conditions, were used in the
study. Mice were injected with 0.2 mL of 0.02% aqueous
phenylquinone (2-phenyl-1,4-benzoquinone) and writhing was
observed for 20 min. The number of writhes produced by each mouse
was counted during this period. A minimum of 10 writhes produced by
a mouse was considered positive and these mice were used in the
analgesic testing on the following day.
Compounds (2a–f), (7) and (8) were screened for anti-amoebic
activity against Entamoeba histolytic (strain HM-1: IMSS) and their
IC50 (50% inhibition concentration) values were determined. The most
promising IC50 value was shown by compound (2b) [0.32µg/mL, as
compared with the standard drug metronidazole (IC50 0.21µg/mL)].
These mice, five per group and showing significant writhing, were
given orally 50 and 100 mg/kg p.o. doses of test compounds 15 min
Sondhi, Sham M.
