R. Omrani, R.B. Ali, Y. Arfaoui et al.
Journal of Molecular Structure 1236 (2021) 130321
.85. IR (CHCl3, υ cm 1): (NH)= 3046.77, (C=C)= 1630.21, (PO)=
−
0
approved by the Ethics Committee of the Faculty of Medicine of
Tunis according to the standards of the International Council for
Laboratory Animal Science (ICLAS).
−
252.72. Anal. Calc. for C16 H N O P (318g.mol ): C, 60.37; H,
19 2 3
1
1
5
.97; N, 8.80; found C, 60.31; H, 5.96; N, 8.76%.
-
(4b/5b). (Z)-Dimethyl N’-benzyl-N-
cyclohexylcarbamimidoylphosphonate
6
.2.4. Animal experimentation design
1
The analgesic activity of newly synthesized phosphonate amide
Yellow solid; yield= 81%; mp 108.7°C; H NMR (CDCl , δ
3
H
derivatives was evaluated using the formalin test and the hot plate
test 15 minutes after an intra-peritoneal injection (IP) to rats di-
vided into 10 groups as follows:
ppm): 0.73-3.08 (m, 11H, H
), 3.45 (d, 10.8Hz, 6H, CH ), 4.30
3
hexyl
(
d, J=5.7Hz, 2H, CH ),7.02-7.73 (m, 5H, Harom), 6.8 (d, J=7.5Hz,
2
H, NH). 1 C NMR (CDCl , δ ppm): 172.73 (d, 148.5Hz, -C=N-
3
1
3
C
Group C: Control group treated with 0.9% saline solution (IP)
Group morph 5: treated with morphine at 5mg/kg dose (IP)
Group morph 7.5: treated with morphine at 7.5mg/kg dose (IP)
Group morph 10: treated with morphine at 10mg/kg dose (IP)
Group MAmP 5: treated by dimethylcyclohexylcarbamoylphos-
phonate at 5mg/kg dose (IP)
)
, 137.91 (s, N’-Cipso), 128.60 (d, J=1.3Hz, N’-C
), 127.78 (s, N’-
ortho
Cmeta), 127.38 (s, N’-Cpara), 52.72 (s, CH ), 50.08 (d, J= 6.4Hz, MeO),
2
4
31
2.63 (d, J=6.4Hz, N-C
), 31.04 (s, N-C
), 24.87 (s, N-Cpara).
ortho
ipso
P NMR (CDCl , δ ppm): (3c) = -1.28 (4c)= 0.91. IR (CHCl
3
P
3,
υ cm 1): (NH)= 2946.84, (C=C)=1630.83, (PO)= 1228.18. Anal.
−
−
H N O P (324g.mol ): C, 59.25; H, 7.71; N, 8.64;
25 2 3
1
Calc. for C16
Group MAmP 7.5: treated by dimethylcyclohexylcar-
bamoylphosphonate at 7.5mg/kg dose
found C, 5922; H, 7.73; N, 8.63%.
-
(4g/5g). (Z)-Dimethyl N’-benzyl-N-
Group MAmP 10: treated by dimethylcyclohexylcarbamoylphos-
phonate at 10mg/kg dose (IP)
(4-chloro)phenylcarbamimidoylphosphonate
Group EAmP 5: treated by diethylcyclohexylcarbamoylphospho-
nate at 5mg/kg dose (IP)
1
Orange oil; yield= 76%; H NMR (CDCl , δ ppm): 3.39 (d,
3
H
J=11.1Hz, 6H, CH ), 3.87 (s, 2H, CH ), 6.25-6.70 (m, 9H, Harom), 7.18
3
2
Group EAmP 7.5: treated by diethylcyclohexylcarbamoylphos-
phonate at 7.5mg/kg dose
13
(
d, J=7.6Hz, 1H, NH). C NMR (CDCl , δ ppm): 174.85 (d, J=148.2
3
C
Hz, C=N), 162.6 (d, J= 137.1Hz, N-C ), 141.6 (d, J=10 Hz, C
ipso
),
ipso
Group EAmP 10: treated by diethylcyclohexylcarbamoylphos-
phonate at 10mg/kg dose (IP)
1
32.71 (d, J=8Hz, N-Cmeta), 129.9 (s, C
), 127. 5 (s, Cmeta), 127.15
ortho
(
s, Cpara), 116.4 (d, J=3.9Hz, N-C
), 57.19 (d, J=6.4Hz, CH ), 53.79
ortho
2
31
(
d, J= Hz, MeO). P NMR (CDCl , δ ppm): (3g) = -2.09 (4g)= -
3
P
3
.16.
6
.2.5. Hot Plate Test
The Hot plate test is a behavioral model of nociception which
6
6
.2. Biological activities assays
is commonly employed to screen analgesic drug effects. During a
hot plate test, rats display several noxious-evoked patterns as well
as exploratory and self-care responses.
.2.1. Evaluation of antibacterial and antifungal activities
The synthesized compounds have been screened for following
The Hot plate test uses the reflexes of the thermal pain due
to the contact of the paws with a heated surface. The rats were
placed on a heated plate maintained at a constant temperature of
activity by the paper-disc method with a diameter of 6.0 mm.
The antibacterial activity was determined by disc diffusion method
on Mueller-Hinton agar [34]. These were evaluated by measur-
ing the inhibition zone in the presence of increasing concentra-
tions of phosphonoamidates and phosphonoamidines from 10 to
4
8°C. During the hot plate test period we noted the latency time
before the rat elevated and licked its paws and jumped along with
the time when the rat fled out of the heated plate [17,24].
4
0mg/mL. The antibacterial study was focused on standard bac-
terial strains of Staphylococcus aureus (ATCC29213), Pseudomonas
aerogunosa (ATCC27859) and Escherichia coli (ATCC 25922). The an-
tifungal study was focused on standard strains of Candida parap-
silosis (CECT13009)
6.2.6. Formalin Test
The formalin test is used to evaluate nociception, applied
mainly to rats and mice, and involves moderate continuous pain,
generated by the inflamed tissues. In this test, a solution of 5% for-
malin was injected subcutaneously in a posterior paw of the rat to
produce a biphasic pain. During the 60 min of the test, the initial
response to the pain (the early phase) was evaluated from 1 to 5
min after the injection of formalin. The response to pain (the late
phase) was evaluated 50 min after the pain induction during 10
min [15,16].
6
.2.2. Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay
The DPPH assay was based on the method reported by Blois
[
23]. The phosphonoamidate and phosphonoamidine compounds
were added at increasing concentrations (from 1 to 40 mg/mL) to 1
mL of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) dissolved in methanol
to 100 μM. The mixture was incubated at room temperature for
3
0 min and then the absorbance of stable DPPH was determined
To evaluate the rat pain degree we attributed a score as follows:
Score 0 - The injected paw is pressed firmly on the floor and
obviously bears the animal’s weight. Score 1 - The injected paw
rests lightly on the floor or wall and is definitely in contact with
it, but little if any weight is placed on the paw; during locomotion
there is a definite limp. Score 2 -The injected paw is elevated and
is not in contact with any surface. Score 3 - The animal licks, bites,
or shakes the affected paw.
at 517 nm using a UV– visible spectrophotometer type UNIVER-
SAL 320UV/VIS. The concentration of DPPH was calculated using a
standard curve. The free radical scavenging activity was expressed
∗
as follows: % Inhibition = ((C0–Csample)/C0) 100
(
C0
=
Concentration of DPPH in the control and Csam-
ple = Concentration of DPPH in the sample
6
.2.3. Animals
Male Wistar rats were housed in pairs in cages (25 × 50 cm)
and maintained at 23°C, 12/12h light/dark cycle under specific
pathogen-free conditions. The rats were allowed to acclimatize in
the experimental medicine unit for a period of one week before
the beginning of the study. During the experiment period, they re-
ceived a commercial pellet diet and water. Rats weighing 200 g
were used for the experiments. All experimental procedures were
6.2.7. Statistical analyses
The results are presented as averages ± standard deviation. All
analyses were carried out with Biostat software for Windows. Sig-
nificant differences between treatment effects were determined by
the U Man Whitney test for multiple comparisons with statistical
significance when p < 0.05.
9