Carbodiimides in the synthesis of enamino- and α-aminophosphonates as peptidomimetics of analgesic/antiinflammatory and anticancer agents

Article abstract of DOI:10.1002/ardp.201200142

Carbodiimide that was generated from the condensation reaction of iminophosphorane with phenylisocyanate was allowed to react with different phosphorus nucleophiles. Thus, the in situ resulted heterocumulene reacted with dialkylhydrogenphosphonates in tet

Full text of DOI:10.1002/ardp.201200142

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
1
Full Paper
Carbodiimides in the Synthesis of Enamino- and
a-Aminophosphonates as Peptidomimetics of
Analgesic/Antiinflammatory and Anticancer Agents
Wafaa M. Abdou, Reham F. Barghash, and Mohamed S. Bekheit
Chemical Industries Division, National Research Centre, Dokki, Cairo, Egypt
Carbodiimide that was generated from the condensation reaction of iminophosphorane with
phenylisocyanate was allowed to react with different phosphorus nucleophiles. Thus, the in situ
resulted heterocumulene reacted with dialkylhydrogenphosphonates in tetrahydrofuran (THF)/FeCl3/
H2O system to give fused pyrrole- (ꢀ14%) and pyrimidinephosphonates (ꢀ57%). On the other hand,
with tris-(dialkyl)aminophosphines, the reaction afforded the corresponding hexaalkylphosphinic
diamides as a water-sensitive fine powder, quite stable for a few days in a desiccator. When a
protonating agent was present in the reaction medium, the reaction was markedly accelerated
leading to the formation of the phosphamides. Next, some saturated and unsaturated Horner–
Emmons reagents were applied in situ to the same carbodiimide to obtain more phosphorylated
N-heterocycles. The analgesic and antiinflammatory activities of the newly synthesized compounds
were investigated and showed significant activities. Finally, we further estimated the antitumor
activity of five new phosphonates against four carcinoma cell lines.
Keywords: a-Aminophosphonates / Analgesic/antiinflammatory agents / Antitumor bioassay / Enaminophosphonates /
Horner–Emmons reagents / Pyrazoles
Received: April 5, 2012; Revised: June 29, 2012; Accepted: July 11, 2012
DOI 10.1002/ardp.201200142
Introduction
Kabachnik–Fields reaction, starting from substituted amines,
aldehydes, and tri-/dialkyl phosphites, in the presence of tetra-
hydrofuran (THF)–FeCl3 (10%) solution [12]. In this article, we
report a convenient approach to the synthesis of substituted
N-heterocylces enamino- and a-aminoposphonates from
the reaction of the in situ generated heterocumulene with
dialkylhydrogenphosphonates 5a–c, tris-(dialkyl)aminophos-
phines 10a,b, as well as some saturated 14a–d, and unsatu-
rated Horner–Emmons reagents 18, 20. The effects of selected
compounds on carrageenin-induced hind paw edema and
p-benzoquinone-induced writhing tests as well as their anti-
cancer activity against several different cancer cell lines were
investigated.
Due to the structural similarity of a-aminophosphonates
with a-amino acids the former have received an increasing
attention. The potential of a-aminophosphonates as enzyme
inhibitors and antibiotics as well as pharmacological agents
has been established [1–3]. Their inhibitory activity on
protein tyrosine phosphatases (PTPs) is proposed and a recent
review article in regard to heterocyclic glycopeptide-
mimetics as PTPs inhibitors has recently been reported [4].
Although a variety of synthetic methods of a-amino-
phosphonates are available, approaches to enaminophos-
phonates are somewhat even less known. In connection
with our interest in preparation of b-enamino- and a-amino-
phosphonates [5–11], we have recently reported a strategy Results and discussion
for the synthesis of a series of these compounds. The latter
arose from one-pot three-component synthesis, via
Synthesis of the required a-amino-/enaminophosphonates
is outlined in Schemes 1–6. First, the iminophosphorane 2
was obtained from the reaction of ethyl 4-amino-5-cyano-1-
phenyl-1H-pyrazole-3-carboxylate (1) [13] with in situ gener-
ated dichlorotriphenylphosphorane using a hexachloro-
ethane-triphenylphosphine-triethylamine reagent system.
Correspondence: Wafaa M. Abdou, Chemical Industries Division,
National Research Centre, Elbohouth St., D-12311 Dokki, Cairo, Egypt.
E-mail: wabdou@link.net
Fax: 20233370931
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Scheme 1. Preparation of the carbodiimide 4.
Subsequent treatment of 2 with phenylisocyanate (3) in THF
solution resulted in the formation of the carbodiimide 4 and
triphenylphosphine oxide (TPPO) via aza-Wittig reaction
(Scheme 1) [14].
The heterocumulene 4 is highly unstable, and should be
quickly freed from TPPO, followed by adding dialkylhydro-
genphosphonates 5a–c in THF–FeCl3 (aq.) solution. The reac-
tion mixture was refluxed for ꢀ6 h to give after the usual
workup the two phosphonates 8 and 9 (Scheme 2).
Scheme 2. Preparation of the phosphonates 8a–c and 9a–c.
The minor product 8a (14%) was analyzed correctly
for C16H19N4O5P: m/z (%): 378 (100) [Mþ], this is equivalent
to an additional product (4 þ 5a) minus 91 [MþꢁNPh]. The
structure of 8 was based on the spectroscopic data. The IR
spectra nmax, cmꢁ1 of 8a–c revealed, in each case, no bands at
ꢀ2220 cmꢁ1 due to the nitrile group (CN), and a showed
strong absorption band at ꢀ1710 assigned to the carboxylate
–
in tandem of the fission of the imino-double bond (C NPh)
–
[15, 16], giving rise to the intermediate 6, following intra-
molecular cyclization, and hydrolysis of the nitrile moiety
afforded 8 (due to the competitive interaction with the
carboxylate moiety [17, 18]). The presence of the FeCl3
solution may have provoked the reduction of the nitrile
group [14]. Furthermore, intramolecular cyclization of
the intermediate 6 through the interaction of CN in 4 has
previously been observed on similar occasions [19].
–
carbonyl (C O) stretching (these excluding any cycloaddition
–
reaction involving the carboxylate group). Moreover, the
1H NMR spectrum of 8a, for example, revealed two signals
at d ppm 1.31 (t) and 3.64 (q) due to the ethoxy (ester) protons.
The IR spectra of 9a–c showed strong absorption bands at
nmax, cmꢁ1: ꢀ2230, 1685, and 1590 assigned to nitrile (CN),
The phosphonate moiety [P(OCH3)2] was located as
a
doublet (3JP–H ¼ 11.6 Hz) at 4.38, whereas the amino group
exhibited two types of (H) protons, [(d (HA) ¼ 6.98 (br, 1H)
and d (HB) ¼ 9.01 (br, 1H)]. The different chemical shifts of
the NH2-protons are the spectroscopic evidence for the
presence of an intramolecular hydrogen bond between one
C O vibration of cyclic imide, and the imino (C N) stretch-
–
–
–
–
ing. The major product 9a (58%) gave microanalysis for the
formula C20H16N5O4P (m/z (%): 421(22), which is equivalent to
a 1:1 addition product minus 46 (C2H5OH). In the 1H NMR
spectrum (DMSO-d6) of 9a, the characteristic signal at dH
4.18 ppm was assigned to the methoxy-phosphorus ester
group while the aromatic protons were displayed in the
region 7.29–8.18 ppm. The 31P NMR spectra of 9 showed a
sharp singlet at dp (DMSO-d6) ꢀ27 ppm. The structure 9 was
assumed to be formed via the parallel intermediate 7, which
underwent intramolecular cyclization and subsequent aro-
matization accompanied with concomitant loss of an ethyl
alcohol molecule under the reaction conditions to afford the
–
of the NH -protons and the oxygen atom of the P O binding
2
–
in the phosphonate group. The 13C NMR spectra of 8
displayed, among others, three signals at ꢀ159, 128, 125
–
(d, JP–C ¼ ꢀ196.3 Hz) ppm assignable to C O (ester), C–NH ,
–
2
and C–P, respectively. Their 31P NMR spectra showed a
sharp singlet at dp (DMSO-d6) ꢀ24 ppm. The formation of
the products 8 is assumed to take place via the nucleophilic
addition of the phosphite-phosphorus to the carbodiimide 4
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Enamino- and a-Aminophosphonates versus Inflammatory and Cancer Diseases
3
final enaminone-phosphonate products 9a–c as depicted in
Scheme 2.
product. A similar treatment of 4 with triethyl methylphos-
phonate (14b), diethyl (2-amino-2-thioxoethyl)-phosphonate
(14c), or diethyl (methylthiomethyl)-phosphonate (14d),
the reaction yielded the respective analogs 16b–d in 66,
72, and 74% yield, respectively. The other possible structure
17 (based on a competition between the nitrile group and the
carboxylate moiety) was ruled out on the basis of the
spectroscopic data of the isolated products. For example,
the IR spectra nmax, cmꢁ1 of 16a–d showed the characteristic
bands due to the amino (ꢀ3480, 3370), the exocyclic imino
The carbodiimide 4 reacted also with tris-(dialkylamino)-
phosphines 10a,b in THF at room temperature (r.t.). The
reaction was rapid and exothermic at 5–108C. The resulting
crystalline 1:1 adduct (ꢀ84% yield) is formulated as tris-
(dialkylamino)methylenephosphoranes 11a,b, 11Aa,b, 11Ba,b
from the spectral data presented below. Evidently, the un-
–
–
–
saturated carbodiimide (–N C ) the carbon, but the result-
–
ing C-phosphonium-betaines 11 and/or 11B may rearrange
1
also to the cyclic structure 11A. In the H NMR spectrum of
–
(ꢀ1633), the two carbonyl esters (1708, 1694), and the P O
–
11a, the 18 1H of the six methyl groups attached to nitrogen
gave a doublet (JP–H ¼ 9.8 Hz) at 2.83 ppm; and the 31P NMR
shift of the amino-ylide 11a was displayed at dp 58.3 ppm,
which favor the open structure [20]. Compounds 11 are
water sensitive, quite stable for a few days in a desiccator
(Scheme 3). When a protonating agent (e.g., 1 mL H2O) was
present in the reaction medium, the reaction was markedly
accelerated leading to the formation of the phosphonic
diamides 13a,b (ꢀ80%). Furthermore, when compounds 11
were boiled in alcohol, 13a,b were again obtained. Obviously,
compounds 13a,b were formed through the initial formation
of 11, followed by quenching a molecule of H2O to give 13 via
the intermediate 12 with concomitant loss of a dimethyl
amine and an ethanol molecule (Scheme 3). The spectro-
scopic data of 13 confirmed the suggested structure, the
31P NMR signal was found at dp ꢀ 34 ppm [21].
groups (1227, bonded) as well as the disappearance of the
band at ꢀ2233 cmꢁ1 corresponding to CN. The 1H NMR spec-
trum of 16a (dp ¼ 27.9 ppm) showed, as previously described,
two signals of NH2 protons [d (HA) ¼ 6.02 (br, 1H) and d
(HB) ¼ 9.67 ppm (br, 1H)]. The fused pyridines 16 were formed
via cyclization and transformations of the cyano group of the
initially 1:1 addition intermediate 15 (Scheme 4).
Conversely, the direct reaction of the in situ generated
carbodiimide
4 with the unsaturated Horner–Emmon
reagent, diethyl vinylphosphonoate 18 in dimethylform-
amaide (DMF) solution containing an excess of lithium
hydride (LiH) at r.t., produced the azaphosphone 19 (72%).
The formation of 19 can have resulted through Diels–Alder
addition accompanied with an extrusion of an ethanol mol-
ecule in a one-step reaction (Scheme 5) [16]. The azaphos-
phone 19 was proved by IR, NMR, and mass spectroscopic
analyses. Its IR spectrum exhibited absorption bands due to
The behavior of heterocumulene 4 toward some phos-
phonyl carbanions was next investigated, and the reactions
are displayed in Schemes 4–6. Thus, when the in situ gener-
ated carbodiimide 4 was treated with ꢀ1.3 equivalents of
diethylmethylphosphonoacetate (14a) in alcoholic sodium
ethoxide solution (or LiH/DMF) at r.t., the fused pyridine-
phosphonate derivative 16a (68%) was the only isolated
–
–
–
the stretching vibrations of CN, C O (ester), P O (free), and
–
P–O–C groups at nmax, cmꢁ1: 2222, 1712, 1260, and 1083,
respectively. Its 1H NMR spectrum (dp ¼ 15.3 ppm) showed
the H(3)- and H(4) of the azaphosphone ring as two doublets
at 7.55 (2JP–H ¼ 17.2 Hz) and 6.95 (3JP–H ¼ 6.7 Hz) ppm.
The latter’s were attested in its 13C NMR spectrum at 123.8
Scheme 3. Preparation of phosphonio-
methylamides, 11a,b, and phosphonic dia-
mides, 13a,b.
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In summary, we have developed a facile and efficient
regioselective method for the preparation of a new series
of a-amino-/enaminophosphonates in moderate to high
yields. Carbodiimide that was generated via the aza-Wittig
reaction of iminophosphorane with phenylisocyanate is
the key intermediate and could react with different types
of phosphorus reagents. This attractive method is effective
for the reaction of dialkylhydrogenphosphonates, trisamino-
phosphines as well as saturated and unsaturated phosphonyl
carbanions with carbodiimide, and provides excellent yields
of the products in a very short time, which makes it a novel
and economically viable process.
Pharmacological evaluation
Inflammation is a natural and beneficial reaction in response
to infections and trauma. Inflammation process begins when
an unknown antigen gains access to patient’s tissue and
combines with antibody in the joint. This activates an anti-
gen complement–antibody immune complex, which precipi-
tates in the synovial and joint fluid. In turn, it leads to
the release of chemical mediators that cause migration of
polymorphonuclear leukocytes phagocytizing the immune
complex, the lysosomal membrane discharges protease and
collagenate, causing continued inflammation, tissue destruc-
tion, and loss of physical properties of the connective tissue
and joints. Management of inflammatory disorder involves
the use of therapeutic agents for relieving pain and reducing
inflammation. Physical therapy, non-steroidal antiinflamma-
tory drugs (NSAIDs), disease modifying anti-rheumatic drugs
(DMARDs), and finally, immunosuppressive agents are the
generally accepted stepwise approach to achieve these goals.
Among different types of NSAIDs, pyrazoles and fused
pyrazole with six-membered rings [22, 23] occupy focus
position to be used as analgesic and antiinflammatory
agents. Furthermore, our previous findings with certain
pyrazole-based a-aminophosphonates resulted in derivatives
with potent in vivo antiinflammatory effects, with no toxic
symptoms [12]. Also, based on the previous findings that
indicated pyrazole-nucleus as suitable for anticancer activity
[24, 25], bioscreening of selected synthesized substituted
pyrazole phosphorus derivatives was determined. In sequel,
by keeping the pyrazole core structure intact, we studied
the effect of different phosphorus containing moieties side
chains: 9a,b, 13a,b, 16a–d, 19, and 21 on their analgesic,
antiinflammatory, and anticancer activities. Substrate 1
was also tested to reflect the effect of its transformations
to our products.
Scheme 4. Preparation of the phosphonates 16a–d.
Scheme 5. Preparation of the azaphosphone 19.
1
2
(d, JP–C ¼ 144 Hz, C(3)) and 138.63 (d, JP–C ¼ 8.5 Hz, C(4))
ppm, respectively.
Finally, coupling reaction of 4 with diethyl (2-methylallyl)-
phosphonate (20) in a mixture of LiOH/H2O/CHCl3 at
r.t. yielded ethyl 5-cyano-4-(3-(diethoxyphosphoryl)-4-methyl-
2-(phenylamino)-1H-pyrrol-1-yl)-1-phenyl-1H-pyrazole-3-carboxyl-
ate (21, 70%; Scheme 6). The 1H NMR spectrum of 21
displayed the methine proton of the pyrrole ring as a doublet
(4JP–H ¼ 4.2 Hz) at 6.24 ppm. Its 13C NMR spectrum
–
exhibited, among others, signals at 158.9 (C O, ester),
–
1
129.9 (d, JP–C ¼ 211.8 Hz, C–P), 116.2 [C(2)-pyrrole] ppm.
a-Aminophosphonate 21 may be regarded as a result of
(3 þ 2) cycloaddition as depicted in Scheme 6.
Analgesic evaluation
Evaluation of analgesic activity of the synthesized compounds
was assessed in vivo in mice using the p-benzoquinone-induced
writhing test [26]. Aspirin was used as the positive control in
Scheme 6. Preparation of the phosphonate 21.
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Enamino- and a-Aminophosphonates versus Inflammatory and Cancer Diseases
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Table 1. Analgesic and antiinflammatory effects of 9a,b, 13a,b, 16a–d, 19, and 21.
Test compds
Number of writhings ꢂ SEM
Swelling in thickness (ꢃ10ꢁ2 mm) ꢂ SEM (inhibtion of edema, (%))b)
(Analgesic activity, %)a)
90 min
180 min
270 min
360 min
Control
9aꢄ
28.2 ꢂ 2.8
38.0 ꢂ 2.0
50.0 ꢂ 3.0
74.0 ꢂ 3.0
77.0 ꢂ 3.0
11.5 ꢂ 3.6 (59.2%)ꢄ
12.2 ꢂ 4.2 (56.9%)ꢄ
24.0 ꢂ 3.4 (14.9%)ꢄꢄ
25.1 ꢂ 3.6 (10.9%)ꢄ
8.8 ꢂ 2.9 (68.9%)ꢄꢄꢄ
7.5 ꢂ 3.5 (73.4%)ꢄꢄꢄ
4.8 ꢂ 1.7 (83.2%)ꢄꢄꢄ
6.5 ꢂ 2.1 (77.0%)ꢄꢄꢄ
15.3 ꢂ 1.8 (45.7%)ꢄ
14.4 ꢂ 3.5 (48.9%)ꢄꢄ
26.4 ꢂ 3.6 (6.4%)
34.0 ꢂ 4.0 (10.5%)ꢄ
28.0 ꢂ 8.0 (44%)ꢄ
31.0 ꢂ 5.0 (58.1%)ꢄꢄꢄ 44.0 ꢂ 5.0 (42.8%)ꢄꢄꢄ
9bꢄ
33.5 ꢂ 5.8 (11.8%)ꢄꢄ 30.6 ꢂ 5.0 (38.8%)ꢄꢄ 58.0 ꢂ 4.0 (21.6%)ꢄꢄꢄ 45.0 ꢂ 4.0 (41.5%)ꢄꢄ
13aꢄ
37.2 ꢂ 5.0 (2.1%)ꢄ
36.0 ꢂ 6.1 (5.2%)ꢄꢄ
29.8 ꢂ 6.0 (21.5%)ꢄꢄ
46.0 ꢂ 4.0 (8%)ꢄꢄ
47.0 ꢂ 4.0 (6%)ꢄꢄꢄ
37.4 ꢂ 8.0 (25.2%)ꢄ
25.0 ꢂ 4.0 (50%)ꢄꢄ
27.0 ꢂ 5.0 (46%)ꢄꢄ
36.8 ꢂ 8.0 (26.4%)ꢄ
47.0 ꢂ 4.0 (6%)ꢄꢄ
35.0 ꢂ 4.0 (31.7%)ꢄ 41.0 ꢂ 3.0 (45.6%)ꢄꢄꢄ 42.0 ꢂ 4.0 (45.7%)ꢄꢄꢄ
69.6 ꢂ 5.0 (5.9%)ꢄꢄ
62.0 ꢂ 7.0 (19.4%)ꢄ
13bꢄ
16aꢄ
70.0 ꢂ 4.0 (5.4%)ꢄꢄꢄ
60.8 ꢂ 3.0 (21%)ꢄꢄꢄ
42.0 ꢂ7.0 (43.2%)ꢄꢄꢄ 38.0 ꢂ6.0 (50.6%)ꢄꢄꢄ
16bꢄ
16cꢄ
28.8 ꢂ 4.0 (24.2%)ꢄꢄꢄ 34.0 ꢂ ꢂ 6.0 (32%)ꢄꢄ 36.0 ꢂ 6.0 (51.3%)ꢄꢄꢄ 32.0 ꢂ 7.0 (58.4%)ꢄꢄꢄ
33.0 ꢂ 4.3 (13.1%)ꢄꢄꢄ
28.0 ꢂ 4.0 (26.3%)ꢄ
27.0 ꢂ 5.0 (28.9%)ꢄꢄ
34.0 ꢂ 3.0 (54%)ꢄꢄꢄ 28.0 ꢂ 4.0 (63.6%)ꢄꢄꢄ
16dꢄ
19
35.0 ꢂ 4.0 (52.7%)ꢄꢄꢄ 30.0 ꢂ 5.0 (61%)ꢄꢄꢄ
37.0 ꢂ 5.0 (50%)ꢄꢄꢄ
40.0 ꢂ 3.0 (48%)ꢄꢄꢄ
76.2 ꢂ 3.0 (1%)ꢄꢄꢄ
21
1
29.0 ꢂ 6.0 (23.6%)ꢄꢄ 35.8 ꢂ 8.0 (28.4%)ꢄꢄꢄ 42.0 ꢂ 7.0 (43.2%)ꢄꢄꢄ 44.0 ꢂ 6.0 (42.8%)ꢄꢄꢄ
40.0 ꢂ 6.1
68.0 ꢂ 6.0 (8.1%)ꢄꢄꢄ
Aspirin
Indomethacin
8.8 ꢂ 2.9 (68.9%)ꢄꢄꢄ
32.0 ꢂ 4.0 (15.5%)ꢄꢄ
Data obtained from animal experiments were expressed as means ꢂ SEM.
ASA, aspirin, 100 mg/kg (body weight); INDO (A), indomethacin, 100 mg/kg (body weight); and all test drugs, 100 mg/kg (body weight)
were s.c. administered to mice (n ¼ 6–12) for a)pBQ induced writhing and b)CG-induced paw edema tests, respectively.
ꢄ
Statistical significance was evaluated from the control by one-way ANOVA post hoc Dunnett’s test (ꢄp < 0.05, ꢄꢄp < 0.01,
ꢄꢄꢄp < 0.001).
our experiments. The results shown in Table 1 indicate that
the substituted 4-aminopyridine phosphonate derivatives
16a–d are the compounds with the most potent analgesic
activity. Thus, 16c (83%) and 16d (77%) demonstrated higher
potency than aspirin at the same dose of 100 mg/kg. In
addition, compounds 16a and 16b resulted in comparable
activity at 68 and 73%, respectively, with respect to reference
aspirin with a small decrease in the inhibitory potential
of writhing movements. In the case of pyrimidinone-
phosphonates 9a and 9b there was a discrepancy in favor
of the substituted pyridine core that caused a decrease in
analgesic potency to 59 and 56%, respectively. However,
phosphonic diamide derivatives 13a and 13b failed to induce
analgesic activity and lacked the similar potency (ꢀ12%).
Nevertheless, azaphospholyl-2-oxide 19 and substituted
pyrrole phosphonate 21 restored the analgesic efficiency to
45 and 48%, respectively.
effects of compounds: The action on the first stage is attribu-
table to the inhibition of the release of chemical mediators
such as histamine, serotonin, and bradykinin [26]. However,
the second phase of edema might be related to the release
of arachidonic acid (AA) metabolites since it is potently
suppressed by aspirin, indomethacin, and other cyclooxy-
genases (COX-1 and COX-2) inhibitor drugs [28]. As shown
in Table 1, the most active derivatives showed a similar
pattern as indomethacin with an absolute gradual increase
in the second phase (after 270 min), therefore suggesting
that these compounds might exert their antiinflammatory
activities through the mechanisms involving the inhibition
of the formation of AA metabolites. Taken together, along
with our previous findings, we can conclude that a-amino-
phosphonate derivatives incorporating a pyrazole core can
be modified to potent analgesic/antiinflammatory drugs
for novel treatment of inflammation-related disorders.
%Inhibition of edema for the tested compounds at successive
intervals is displayed in Fig. 1.
Antiinflammatory bioassay
Antiinflammatory activities of the previous compounds 9a,b,
13a,b, 16a–d, 19, and 21 were also in good correlation with
the analgesic activity results, tested by using the carrageenin-
induced hind paw edema model [27] (Table 1). As shown, the
fused p-aminopyridine phosphonate derivatives (16a–d) as
well as fused pyrimdinone phosphonate derivatives (9a
and 9b) resulted in potent antiinflammatory activity, which
was comparable to indomethacin. It is claimed that edema
produced by carrageen is a biphasic event for the inhibitory
Acute toxicity
At the end of these experiments after 48 h of observation of
all animals, no morbidity or mortality was recorded.
Antitumor activity
Since the pro-inflammatory eicosanoids (i.e., leukotrienes
(LTs) and prostaglandins (PGs)), are evidenced to be visible
key regulators of cell proliferation and neo-angiogenesis, the
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W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Figure 1. %Inhibition of edema for the tested
compounds at successive intervals.
inhibitors of the pathways for their formation are
being investigated as potential anticancer drugs [29, 30].
Considering that our compounds displayed promising
in vivo antiinflammatory activities presumably by inhibiting
the formation of AA metabolites, we selected the above cell
lines which provide a suitable cellular model system to inves-
tigate the anticancer potential of these derivatives. The
results for each tested compound at 50 mM concentration
are reported.
DOX or CIS; the highest concentration of each compound
used was 50 mg/mL. Each concentration was evaluated
three times (each dose was incubated with the cells in
three different wells); thereby the data represent the
average of the total inhibition observed. The deviation in
the obtained data was ranged between: ꢄꢄꢄp < 0.001 and
ꢄp < 0.05 (Table 2).
The antitumor activity results displayed in Table 2 indicate
that the tested phosphonate derivatives 9a, 9b, 13a, 16a, and 16c
reflect good to moderate activity against the used human tumor
cells. The order of activity is 16c > 16a > 9a > 9b > 13a. It can
also be noticed that 16c exhibited remarkable antitumor
activity against the four tested carcinoma cell lines. Further
studies in experimental tumors in vivo for evaluating the
possible antineoplastic potential by these and the other
synthesized compounds are warranted.
Respectively, antitumor activity screening of 9a, 9b, 13a,
16a, and 16c in assays applying a human liver (HEPG2) and a
human colon (HCT116), a human breast carcinoma (MCF7)
and a human cervix (HeLa) cell line was investigated. The
evaluation was considered vc the known anticancer drugs:
doxorubicin (DOX) or cisplatin (CIS) by sulfo-rhodamine-B
stain (SRB) using the method of Skehan and Storeng [31].
The obtained results (Table 2) represent concentrations
(four different concs.: 5, 12.5, 25, and 50 mg/mL) of the used
investigated compounds resulting in growth inhibition
of 50% (IC50) for the tested human cell lines compared to
Finally, from the structure–activity relationship (SAR)
viewpoint, the data displayed in Tables 1 and 2 show that
the analgesic/antiinflammatory and antineoplastic activities
of p-aminopyridine-phosphorus derivatives were found to be
Table 2. Antitumor properties of the tested compounds DOX, CIS, 9a, 9b, 13a, 16a, and 16c.
Compd.
IC50, mg/mL (mM)
HEPG2
(liver cancer)
HCT116
(colon cancer)
MCF7
(breast cancer)
HeLa
(cervical cancer)
Doxorubicin (DOX)
Cisplatin (CIS)
9a
9b
13a
16a
16c
3.10 (5.7)ꢄ
—
6.40 (15.20)ꢄꢄꢄ
10.40 (23.16)ꢄ
20.20 (45.19)ꢄꢄ
5.62 (9.91)ꢄ
3.44 (6.19)ꢄꢄ
3.73 (6.86)ꢄꢄ
—
7.30 (17.33)ꢄ
11.60 (25.83)ꢄꢄꢄ
13.60 (30.42)ꢄ
5.23 (9.22)ꢄꢄꢄ
3.33 (6.00) ꢄꢄꢄ
4.4 (8.09)ꢄꢄꢄ
—
7.20 (17.10)ꢄꢄ
7.95 (17.70)ꢄ
15.90 (35.57)ꢄꢄ
3.88 (6.84)ꢄ
3.20 (5.76)ꢄꢄ
—
2.8 (9.30)ꢄꢄ
6.60 (15.67)ꢄ
7.80 (17.37)ꢄꢄꢄ
14.00 (31.31)ꢄ
4.16 (7.33)ꢄꢄ
2.66 (4.79)ꢄ
ꢄ
Deviation error: ꢄꢄꢄp < 0.001, ꢄꢄp < 0.01, p < 0.05; p is the percentage of inhibition.
(IC50, mM) ¼ IC50, mg/mL ꢃ 1000 M.Wt.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Enamino- and a-Aminophosphonates versus Inflammatory and Cancer Diseases
7
higher than those of the fused-pyrimidinone phosphonates-
based pyrazole as a core. In addition, we observed that there is
a close analogy among the potencies of the tested compounds
toward the analgesic/antiinflammatory or antineoplastic
effects. Thus, while 16a and 16c showed significant properties
toward the three activities, compounds 13a and 13b failed to
induce any effect. It can be deduced that there is a correlation
between the modes of action of this class of compounds
toward these types of diseases.
Preparation of carbodiimide 4
To a solution of 1.2 g iminophosphorane 2 (2.3 mmol) in 15 mL
dry THF was added 0.28 g phenylisocyanate (3, 2.3 mmol) under
nitrogen at r.t. After the reaction mixture was stirred for 12 h at
0–58C, the solvent was removed under reduced pressure and 20 mL
ether/petroleum ether (1:2) was added to precipitate TPPO.
After filtration the solvent was removed to give carbodiimide 4,
which was generally used directly without further purification.
Compound 4 was also isolated, for analytical purposes, from the
reaction mixture by column chromatography on silica gel.
Ethyl 5-cyano-1-phenyl-4-{[(phenylimino)methylene]-
amino}-1,4-dihydropyrazorol-3-carboxylate (4)
Experimental
White material, mp: 110–1118C (ligroin), yield: (0.65 g, 77ꢁ%1).
IR: nmax, cmꢁ1: 2233 (CN), 1709 (C O), 1665, 1595, 1490 cm
.
General
–
–
1H NMR (CDCl3):
d
1.36 (t, JH–H ¼ 7.2 Hz, 3H, H3CC), 4.33
Melting points were determined with open capillary tubes on an
electrothermal (variable heater) melting point apparatus and
were uncorrected. IR spectra were recorded on a PerkinElmer
297 grating IR spectrophotometer using KBr pellets. NMR spectra
were measured with a JEOL E.C.A-500 MHz (13C: ꢀ125 MHz,
1H: ꢀ500 MHz, and 31P: ꢀ200 MHz) spectrometer. 31P NMR
spectra were recorded with H3PO4 (85%) as external reference.
1H and 13C NMR spectra were measured using SiMe4 as an
internal reference in CDCl3 or DMSO-d6. Chemical shifts (d)
are given in ppm. Mass spectrometry was performed on a
JEOL JMS-AX 500 spectrometer. The appropriate precautions in
handling moisture-sensitive compounds were considered.
Solvents were dried by standard techniques. Thin-layer chroma-
tography (TLC) used Merck 0.2 mm silica gel 60 F254 analytic
aluminum plates. Elemental analyses were carried out at the
Micro Analytical Centre, Cairo University, Cairo, Egypt, using
Elemental C, H, N Analyzer Vario EL III Germany. All inter-
national principles and local regulations concerning the care
and use of laboratory animals were considered during the
pharmacological screening.
(q, JH–H ¼ 7.2 Hz, 2H, H2CO), 7.23–7.37 (m, 10H, H–Ph), 13C NMR
(CDCl3): d 164.2, 161.6, 147.6, 144.7, 136.0, 135.4, 132.4, 129.8, 129.4,
126.1, 124.8, 120.4, 61.2, 60.2, 14.5, 14.3. EI–MS m/z (%): 357 (100)
(Mþ], 328 (84), 302 (60), 77 (37). Anal. calcd. for C20H15N5O2 (357.1):
C, 67.22; H, 4.23; N, 19.60. Found: C, 67.27; H, 4.18, N, 19.67.
Reaction of carbodiimide 4 with dialkylhydro-
genphosphonates 5a–c
Preparation of the phosphonate derivatives 8a–c and 9a–c
General Method: To the solution of 0.8 g 4 (2.2 mmol) prepared
above, a solution of 2.7 mmol dimethyl- (5a), diethyl- (5b), or
diisopropylhydrogenphosphite (5c) in 15 mL THF containing
10% FeCl3, and 1 mL H2O was added. The reaction mixture
was refluxed for 3–6 h. After completion of the reaction (TLC),
10 mL AcOEt was added to the mixture. The organic phase was
separated, washed with 20 mL distilled water, and dried over
anhydrous sodium sulfate. Solvents were evaporated under
vacuum, and the residue was crystallized from the proper sol-
vent to give the corresponding phosphonates 8a–c, 9a–c, which
had been separated by fractional crystallization.
Synthesis
Preparation of the iminophosphorane 2
Ethyl 6-amino-5-(dimethoxyphosphoryl)-1-phenyl-1,4-
dihydropyrrolo[3,2-c]pyrazole-3-carboxylate (8a)
The procedure reported by Liu et al. [13] for the preparation of 4
was modified as follows: To a mixture of 2.04 g ethyl 4-amino-5-
cyano-1-phenyl-1H-pyrazole-3-carboxylate (1, 8 mmol), 3.14 g
PPh3 (12 mmol), and 2.84 g C2Cl6 (12 mmol) in 40 mL dry
THF, was added dropwise 2.42 g NEt3 (24 mmol) at r.t. The color
of the reaction mixture quickly turned yellow. After stirring for
4–6 h, solvents were removed under reduced pressure to give the
iminophosphorane 2.
Colorless crystals, mp 240–2428C (EtOH), yield: (120 mg, 14%).
IR: nmax, cmꢁ1: 3415–3323 (NH & NH ), 1717 (C O, ester), 1233
–
–
2
(P O, bonded), 1090 (P–O–C). 1H NMR [DMSO-d6]:
d 1.31
–
–
(t, JH–H ¼ 7.2 Hz, 3H, H3CC), 3.64 (q, JH–H ¼ 7.2 Hz, 2H, H2CO),
3
4.38 (d, JP–H ¼ 11.6 Hz, 6H, (H3CO)2P), 6.98 (br, 1H, HAN), 7.29–
7.79 (m, 5H, H–Ph), 9.01 (br, 1H, HBN), 9.88 (br, 1H, HN, pyrrole)
13
ppm. C NMR [DMSO-d ]: d 159.6 (C O), 136.5 (C N), 143.1,
6
–
–
–
128.7, 121.2, 120.3, 119.9, 111.8 (C–Ph, C-pyrazole), 128.5
–
Ethyl 5-cyano-4-[(triphenylphosphoranylidene)amino]-1,4-
dihydropyrazol-3-carboxylate (2)
2
1
(d, Jp–c ¼ 17.6 Hz, C–NH2), 125.4 (d, Jp–c ¼ 196.3 Hz, C–P),
2
60.9 (OCH2), 52.3 (d, Jp–c ¼ 14.8 Hz, CH3OP), 14.1 (CH3, ester)
Colorless crystals, mp: 126–1288C (cyclohexane), yield: (3.6 g,
ppm. 31P NMR [DMSO-d6]: dp 23.4ppm. EI–MS: m/z (%): 378 (100)
[Mþ], 360 (68) [Mþꢁ18, H2O], 349 (11) [Mþꢁ29, C2H5], 222 (24)
[Mþꢁ156, [P(O)(OMe)2)]þH2OþC2H5], 109 (18) (P(O)(OMe)2), 77
(66). Anal. calcd. for C16H19N4O5P (378.1): C, 50.80; H, 5.06; N,
14.81; P, 8.19. Found: C, 50.76; H, 5.01; N, 14.78; P, 8.25.
87%). IR: nmax, cmꢁ1: 2232 (CN), 1709 (C O). 1H NMR [DMSO-
–
–
d6]: d 1.36 (t, JH–H ¼ 7.2 Hz, 3H, H3CC), 3.69 (q, JH–H ¼ 7.2 Hz,
2H, H2CO), 7.43–8.01 (m, 20H, H–Ph) ppm. 13C NMR [DMSO-d6]:
–
–
–
d 160.6 (C O), 136.5 (C N), 143.6, 137.1, 133.4, 132.3, 131.7, 129.8,
–
129.6, 129.4, 128.8, 124.4 (C–Ph), 121.0 (CN), 107.4 (C–CN), 60.9
(CH2O), 14.1 (CH3CO) ppm. 31P NMR [DMSO-d6]: dp 21.4 ppm.
EI–MS: m/z (%): 516 (100) [Mþ], 487 (88), 499 (82), 262 (66), 254
(48), 237 (65), 77 (44). Anal. calcd. for C31H25N4O2P (516.2):
C, 72.08; H, 4.88; N, 10.85; P, 6.00. Found: C, 72.06; H, 4.85;
N, 10.83; P, 6.03.
Ethyl 6-amino-5-(diethoxyphosphoryl)-1-phenyl-1,4-
dihydropyrrolo[3,2-c]pyrazole-3-carboxylate (8b)
Colorless crystals, mp 222–2248C (EtOH), yield: (110 mg, 12%). IR:
nmax, cmꢁ1: 3374–3327 (NH & NH ), 1710 (C O, ester), 1233 (P O,
–
–
–
–
2
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
3
bonded), 1090 (P–O–C). 1H NMR [DMSO-d6]: d 1.36–1.45 (m(2t), 9H,
H3C(ester) & 2H3C-phosphonate), 3.59 (q, JH–H ¼ 7.2 Hz, 2H,
6.94 (br, 1H, HAN), 7.27–7.87 (m, 5H, H–Ph), 9.11 (br, 1H, HBN),
16.5 (d, Jp–c ¼ 7.6 Hz, (MeCO)2P) ppm. 31P NMR [DMSO-d6]:
dp 26.9 ppm. EIþ–MS: m/z (%): 449 (13) [Mþ], 423 (100) [Mþꢁ26,
3
þ
–
–
H2C, ester), 4.37 (dq, JH–H ¼ 7.4, JP–H ¼ 4.8 Hz, 4H, (CH2O)2P),
CN], 395 (9) [M ꢁ(26þ28), (CNþC O)], 286 (22) [M ꢁ(26þ137)
(CNþP(O)(OEt)2)], 137 (30) (P(O)(OEt)2), 77 (72). Anal. calcd.
for C22H20N5O4P (449.1): C, 58.80; H, 4.49; N, 15.58; P, 6.89.
Found: C, 58.75; H, 4.47; N, 15.53; P, 6.82.
6
9.83 (br, 1H, HN, pyrrole) ppm. 13C NMR [DMSO-d ]: d 159.8 (C O),
–
–
136.4 (C N), 143.3, 129.2, 122.1, 120.3, 119.1, 110.9 (C–Ph,
–
–
2
C-pyrazole), 128.2 (d, Jp–c ¼ 17.8 Hz, C–NH2), 126.7 (d,
1Jp–c ¼ 198.6 Hz, C–P), 63.2 (d, Jp–c ¼ 8.7 Hz, (CH2O)2P), 60.8
2
Diisopropyl 3-cyano-7-oxo-2,6-diphenyl-6,7-dihydro-2H-
pyrazolo[4,3-d]pyrimidin-5-ylphosphonate (9c)
3
(OCH2, ester), 15.9 (d, Jp–c ¼ 7.6 Hz, CH3COP), 14.3 (CH3, ester)
ppm. 31P NMR [DMSO-d6]: dp 23.8 ppm. EI–MS: m/z (%): 406 (88)
[Mþ], 388 (100) [Mþꢁ18, H2O], 377 (16) [Mþꢁ29, C2H5], 222 (33)
[Mþꢁ184, [P(O)(OEt)2)]þH2OþC2H5], 137 (27) (P(O)(OEt)2), 77 (60).
Anal. calcd. for C18H23N4O5P (406.1): C, 53.20; H, 5.70; N, 13.79;
P, 7.62. Found: C, 53.17; H, 5.64; N, 13.71; P, 7.69.
Colorless crystals, mp 250–2528C (MeCN), yield: (0.6 g, 57%).
IR: nmax, cmꢁ1: 2233 (CN), 1685 (C O), 1560 (C N), 1258 (P O),
–
–
–
–
–
–
1077 (P–O–C). 1H NMR [DMSO-d6]: d 1.56 (dd, JH–H ¼ 6.6 Hz,
4JP–H ¼ 5.8 Hz, 12H, (Me2CHO)2P), 4.77 (dsept, JP–H ¼ 12.4 Hz,
3
2H, (HCO)2P), 7.43–7.98 (m, 10H, H–Ph) ppm. 13C NMR
[DMSO-d6]: d 176.9 (d, Jp–c ¼ 165.1 Hz, C–P), 153.9 (C O), 151.5,
1
–
–
141.7, 138.8, 132.5, 129.8, 129.9, 128.1, 127.4, 123.3 (C-pyrazole,
Ethyl 6-amino-5-(diisopropoxyphosphoryl)-1-phenyl-1,4-
dihydropyrrolo[3,2-c]pyrazole-3-carboxylate (8c)
2
C–Ph), 121.8 (CN), 108.3 (C–CN), 72.4 (d, Jp–c ¼ 9.5 Hz, (CHO)2P),
3
24.6 (d, Jp–c ¼ 7.9 Hz, (Me2CO)2P) ppm. 31P NMR [DMSO-d6]:
Colorless crystals, mp 278–2808C (EtOH), yield: (124 mg, 13%).
IR: nmax, cmꢁ1: 3388–3330 (NH & NH ), 1723 (C O, ester), 1223
–
dp 27.2 ppm. EI–MS: m/z (%): 477 (18) [Mþ], 451 (100) [Mþꢁ26,
–
2
þ
þ
(P O, bonded), 1110 (P–O–C). 1H NMR [DMSO-d6]:
d
1.14
–
–
–
CN], 423 (11) [M ꢁ(26þ28), (CNþC O)], 286 (66) [M ꢁ(26þ165)
–
(CNþP(O)(OC3H7)2)], 166 (17) [P(O)(OCHMe2)2], 77 (65). Anal. calcd.
for C24H24N5O4P (477.2): C, 60.37; H, 5.07; N, 14.67; P, 6.49. Found:
C, 60.33; H, 5.02; N, 14.59; P, 6.45.
(t, JH–H ¼ 7.2 Hz, 3H, H3C, ester), 1.31 (dd, JH–H ¼ 6.6 Hz,
4JP–H ¼ 5.8 Hz, 2 ꢃ 6H, (Me2CHO)2P), 3.66 (q, JH–H ¼ 7.2 Hz, 2H,
3
H2CO, ester), 4.77 (dsept, JP–H ¼ 12.4 Hz, 2H, (HCO)2P), 6.64
(br, 1H, HAN), 7.54–7.81 (m, 5H, H–Ph), 9.06 (br, 1H, HBN),
10.07 (br, 1H, HN, pyrrole) ppm. 13C NMR [DMSO-d6]: d 159.2
Reaction of carbodiimide 4 with tris-(dialkylamino)-
phosphines 10a,b
–
–
(C O), 136.7 (C N), 143.0, 127.6, 122.8, 121.5, 119.2, 111.6
2
–
–
(C–Ph, C-pyrazole), 128.2 (d, Jp–c ¼ 17.8 Hz, C–NH2), 126.7
1
2
Preparation of 11a,b and 13a,b
(d, Jp–c ¼ 201.5 Hz, C–P), 75.0 (d, Jp–c ¼ 9.5 Hz, (CHO)2P), 60.9
General Method: The aminophosphine 10a (R2 ¼ Me) or 10b
(R2 ¼ Et; 2.3 mmol) in 4 mL dry THF was added dropwise to
0.8 g 4 (2.2 mmol) in 15 mL of the same solvent, and the reaction
mixture was stirred at r.t., for ꢀ10 h (TLC). The solvent was
evaporated under vacuum; the residue was collected, washed
with light petroleum, and crystallized from the proper solvent to
give compounds 11a,b.
3
(CH2O, ester), 23.7 (d, Jp–c ¼ 7.9 Hz, (Me2CO), 14.3 (CH3, ester)
ppm. 31P NMR [DMSO-d6]: dp 25.2 ppm. EI–MS: m/z (%): 434 (72)
[Mþ], 416 (100) [Mþꢁ18, H2O], 405 (18) [Mþꢁ29, C2H5], 375 (45)
[Mþꢁ59, Etþ2Me], 222 (33), 165 (27) [P(O)(OCHMe2)2], 77 (60).
Anal. calcd. for C20H27N4O5P (434.2): C, 55.29; H, 6.26;
N, 12.90; P, 7.13. Found: C, 55.23; H, 6.30; N, 12.82; P, 7.19.
Dimethyl 3-cyano-7-oxo-2,6-diphenyl-6,7-dihydro-2H-
pyrazolo[4,3-d]pyrimidin-5-ylphosphonate (9a)
Colorless crystals, mp 226–2288C (acetone), yield: (540 mg, 58%).
5-Cyano-3-(ethoxycarbonyl)-1-phenyl-1-H-pyrazol-4-yl)-
(phenylimino)(tris-(dimethylamino)-phosphonio)-
methylamide (11a)
IR: nmax, cmꢁ1: 2234 (CN), 1690 (C O), 1599 (C N), 1262 (P O,
–
–
–
–
–
–
free), 1054 (P–O–C). 1H NMR [DMSO-d6]: d 4.18 (d, 3JP–H ¼ 11.4 Hz,
Straw yellow, mp 173–1758C (MeCN), yield: (0.98 g, 86%).
IR: nmax, cmꢁ1: 2221 (CN), 1715 (C O), 1320, 838 (P[N(Me )] ).
6H, (H3CO)2P), 7.29–8.18 (m, 10H, H–Ph) ppm. 13C NMR [DMSO-d6]:
–
–
2
3
1H NMR [DMSO-d6]: d 1.34 (t, JH–H ¼ 7.2 Hz, 3H, H3CC, ester),
d 174.2 (d, 1Jp–c ¼ 178.6 Hz, C–P), 150.8 (C O), 150.5, 141.8, 138.8,
–
–
137.3, 132.5, 129.9, 129.8, 128.1, 127.5, 123.3 (C-pyrazole, C–Ph),
3
2.83 (d, JP–H ¼ 9.8 Hz, 18H, (Me2N)3–P), 3.64 (q, JH–H ¼ 7.2 Hz,
2H, H2CO, ester), 7.28–8.15 (m, 10H, H–Ph) ppm. 13C NMR [DMSO-
2
122.2 (CN), 103.2 (C–CN), 54.5 (d, Jp–c ¼ 7.6 Hz, (CH3O)2P) ppm.
6
d ]: d 159.7 (C O), 143.2, 137.6, 136.2, 129.0, 127.4, 127.1, 124.4,
31P NMR [DMSO-d6]: dp 28.3 ppm. EI–MS: m/z (%): 421 (22) [Mþ], 395
–
–
122.7, 118.8 (C-pyrazole, C–Ph), 123.9 (CN), 98.5 (C–CN), 60.9
þ
þ
–
–
(100) [M ꢁ26, CN], 367 (14) [M ꢁ(26 þ 28), (CNþC O)], 286 (56)
1
2
[Mþꢁ(26 þ 109), (CNþP(O)(OMe)2)], 109 (28) (P(O)(OMe)2), 77 (65).
Anal. calcd. for C20H16N5O4P (421.1): C, 57.01; H, 3.83; N, 16.62;
P, 7.35. Found: C, 56.9; H, 3.92; N, 16.53; P, 7.29.
(CH2O),), 57.6 (d, Jp–c ¼ 166.3 Hz, C–P), 34.2 (d, Jp–c ¼ 25.2 Hz,
(Me2N–P), 14.0 (CH3CO) ppm. 31P NMR [DMSO-d6]: dp 58.3 ppm.
EI–MS: m/z (%): 520 (21) [Mþ]. Anal. calcd. for C26H33N8O2P (520.3):
C, 59.99; H, 6.39; N, 21.53; P, 5.95. Found: C, 59.95; H, 6.38;
N, 21.50; P, 5.92.
Diethyl 3-cyano-7-oxo-2,6-diphenyl-6,7-dihydro-2H-
pyrazolo[4,3-d]pyrimidin-5-ylphosphonate (9b)
Colorless crystals, mp 214–2168C (EtOH), yield: (583 mg, 59%).
5-Cyano-3-(ethoxycarbonyl)-1-phenyl-1H-pyrazol-4-yl)-
((phenylimino)(tris-(diethylamino)phosphonio)-
methylamide (11b)
IR: nmax, cmꢁ1: 2225 (CN), 1687 (C O), 1586 (C N), 1256 (P O,
–
–
–
–
–
–
free), 1080 (P–O–C). 1H NMR [DMSO-d6]: d 1.31 (dt, JH–H ¼ 6.6 Hz,
4JP–H ¼ 5.3 Hz, 2 ꢃ 3H, (H3CCO)2P), 4.09 (dq, 3JP–H ¼ 10.7 Hz, 4H,
(CH2O)2P), 7.43–7.98 (m, 10H, H–Ph) ppm. 13C NMR [DMSO-d6]:
Straw yellow, mp 150–1528C (CH2Cl2), yield: (1.12 g, 84%).
IR: nmax, cmꢁ1: 2227 (CN), 1709 (C O), 1317, 863 (P[N(Et )] ).
–
–
2
3
–
1H NMR [DMSO-d6]: d 1.34 (t, JH–H ¼ 7.2 Hz, 3H, H3CC, ester),
d 175.2 (d, 1Jp–c ¼ 179.4 Hz, C–P), 154.8 (C O), 151.0, 141.6, 139.5,
–
138.8, 132.5, 129.8, 129.7, 128.1, 127.0, 123.3 (C-pyrazole, C–Ph),
4
1.28 (dt, JP–H ¼ 5.8 Hz, 18H, [MeC]2N)3–P), 3.45–3.61 (m, 14H,
2
[H2C]2N)3, H2CO, ester), 7.34–8.15 (m, 10H, H–Ph) ppm.
122.6 (CN), 105.2 (C–CN), 60.2 (d, Jp–c ¼ 8.7 Hz, (CH2O)2P),
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Enamino- and a-Aminophosphonates versus Inflammatory and Cancer Diseases
9
13
6
C NMR [DMSO-d ]: d 160.5 (C O), 143.1, 137.4, 136.7, 129.6,
–
described in the general procedure to yield the phosphonate
derivatives 16a–c.
–
128.1, 127.4, 124.1, 123.9, 122.6 (C-pyrazole, C–Ph), 117.8 (CN),
1
102.5 (C–CN), 59.7 (OCH2),), 56.5 (d, Jp–c ¼ 169.6 Hz, C–P), 37.8
2
3
(d, Jp–c ¼ 25.7 Hz, CH2–N–P), 17.0 (d, Jp–c ¼ 8.8 Hz, MeCN–P),
14.0 (CH3CO) ppm. 31P NMR [DMSO-d6]: dp 58.5 ppm. EI–MS: m/z
(%): 604 (17) [Mþ]. Anal. calcd. for C32H45N8O2P (604.3): C, 63.56; H,
7.50; N, 18.53; P, 5.12. Found: C, 63.53; H, 7.45; N, 18.51; P, 5.08.
When the above reaction (4 þ 10a,b) was carried out in
ethanol (or THF þ catalytic amount of water), phosphamides
13a and 13b were obtained after the usual working up.
3-Ethyl-6-methyl-7-amino-6-(diethoxyphosphoryl)-
1-phenyl-5-(phenylimino)-5,6-dihydro-1H-pyrazolo[4,3-b]-
pyridine-3,6-dicarboxylate (16a)
Yellow substance, mp 180–1828C (MeCN), yield: (0.85 g, 68%). IR:
nmax, cmꢁ1: 3482, 3379 (NH ), 1708, 1694 (2C O), 1633 (C NPh,
–
–
–
exocyclic), 1585 (C N), 1227 (P O, bonded), 1084 (P–O–C).
–
2
–
–
–
–
1H NMR [DMSO-d6]: d 1.24–1.58 (m(2t), 9H, (H3CC, ester &
(H3CCO)2P), 3.48 (s, 3H, H3CO, ester), 3.78 (q, JH–H ¼ 6.5 Hz,
[3-Cyano-7-oxo-2,6-diphenyl-6,7-dihydro-2H-
pyrazolo[4,3-d]pyrimidin-5-yl]-N,N,N,N-tetramethyl-
3
2H, H2CO, ester), 4.32 (dq, JP–H ¼ 11.3 Hz, 4H, (H2CO)2P), 6.02
(br, 1H, HAN), 7.32–7.89 (m, 10H, H–Ph), 9.67 (br, 1H, HBN) ppm.
2
13C NMR [DMSO-d6]: d 177.3 (d, JP–C ¼ 11.1 Hz, C NPh), 162.3,
–
phosphonic diamide (13a)
Colorless crystals, mp 150–1528C (CH2Cl2), yield: (0.8 g, 82%).
–
159.5 (2C O), 157.8, 154.4, 147.9, 143.7, 129.2, 128.5, 123.3,
–
–
IR: nmax, cmꢁ1: 2221 (CN), 1685 (C O), 1533 (C N), 1256 (P O),
111.4 (C–Ph, C-pyrazole), 119.5 (d, JP–C ¼ 22 Hz, C–NH2), 63.1
2
–
–
–
–
–
–
1314, 859 (P[N(Me2)]2). 1H NMR [DMSO-d6]:
d
2.83 (d,
(d, JP–C ¼ 8.7 Hz, (CH2O)2P), 61.8 (CH2O), 52.1 (CH3O), 47.8
2
3JP–H ¼ 10.4 Hz, 12H, (Me2N)2–P), 7.34–8.14 (m, 10H, H–Ph)
ppm. 31P NMR [DMSO-d6]: dp 33.6 ppm. EI–MS: m/z (%): 448 (62)
[Mþþ1], 421 (100) [Mþꢁ26, CN], 286 (87) [Mþꢁ(26þ135),
(CNþOP[NMe2]2)], 270 (15), 135 (48) OP[N–(Me2)2], 77 (74). Anal.
calcd. for C22H22N7O2P (447.2): C, 59.06; H, 4.96; N, 21.91; P, 6.92.
Found: C, 58.98; H, 4.9; N, 21.85; P, 6.88.
(d, JP–C ¼ 148.4 Hz, C–P), 16.3 (d, JP–C ¼ 7.6 Hz, (CH3CO)2P), 14.2
(CH3CO) ppm. 31P NMR [DMSO-d6]: dP 27.9 ppm. EI–MS: m/z (%) 568
(16) [Mþþ1], 567 (16) [Mþ], 523 (28) [Mþꢁ44, (MeþEt)], 432 [Mþꢁ135,
(MeþEtþNPh)], 416 (55) [Mþꢁ151, (MeþEtþNPhþNH2)], 279 (100)
[Mþꢁ288, (MeþEtþNPhþNH2þP(O)(OEt)2], 212 (50), 137 (53)
(P(O)(OEt)2), 119 (17), 77 (66). Anal. calcd. for C27H30N5O7P (567.2):
C, 57.14; H, 5.33; N, 12.34; P, 5.46. Found: C, 57.12; H, 5.31; N, 12.32;
P, 5.43.
1
3
[3-Cyano-7-oxo-2,6-diphenyl-6,7-dihydro-2H-
pyrazolo[4,3-d]pyrimidin-5-yl]-N,N,N,N-tetraethyl-
phosphonic diamide (13b)
Diethyl 7-amino-6-(diethoxyphosphoryl)-1-phenyl-5-
(phenylimino)-5,6-dihydro-1H-pyrazolo[4,3-b]pyridine-3,6-
dicarboxylate (16b)
Colorless crystals, mp 118–1208C (cyclohexane), yield: (0.86 g,
78%). IR: nmax, cmꢁ1: 2221 (CN), 1682 (C O), 1604 (C N), 1254
–
–
–
–
(P O), 1317, 863 (P[N(Et2)]2). 1H NMR [DMSO-d6]:
d
1.05
Yellow substance, mp 152–1548C (CH2Cl2), yield: (0.84 g, 66%). IR:
–
–
4
nmax, cmꢁ1: 3477, 3350 (NH ), 1715, 1700 (2C O), 1633 (C NPh,
–
–
2
–
(dt, JH–H ¼ 6.4 Hz, JP–H ¼ 4.2 Hz, 12H, (Me2CN)2–P), 3.21
–
3
–
exocyclic), 1590 (C N), 1225 (P O, bonded), 1050 (P–O–C).
–
–
(q, JP–H ¼ 10.8 Hz, 8H, CH2–N–P), 7.43–7.98 (m, 10H, H–Ph)
–
ppm. 31P NMR [DMSO-d6]: dp 34.2 ppm; EI–MS: m/z (%): 504 (55)
[Mþþ1], 503 (50) [Mþ], 477 (100) [Mþꢁ26, CN], 286 (88)
[Mþꢁ(26þ191), (CNþOP[NEt2]2)], 270 (15), 191 (48) OP[N(Et)2)2],
77 (70). Anal. calcd. for C26H30N7O2P (503.2): C, 62.02; H, 6.01;
N, 19.47; P, 6.15. Found: C, 61.96; H, 5.94; N, 19.42; P, 6.12.
1H NMR [DMSO-d6]: d 1.22–1.56 (m (3t), 12H, 4CH3CO), 3.90–
3
4.00 (m(2q), 4H, 2H2CCO, 2ester), 4.27 (dq, JP–H ¼ 10.4 Hz, 4H,
(H2CO)2P), 6.02 (br, 1H, HAN), 7.38–7.75 (m, 10H, H–Ph), 9.67 (br,
1H, HBN) ppm. 13C NMR [DMSO-d6]: d 177.1 (d, 2JP–C ¼ Hz, C NPh),
–
–
–
162.3, 159.8 (2C O), 157.8, 154.5, 147.9, 143.7, 129.2, 128.5,
–
2
123.3, 111.4 (C–Ph, C-pyrazole), 119.3 (d, JP–C ¼ 37 Hz, C–NH2),
2
63.1 (d, JP–C ¼ 8.7 Hz, (CH2O)2P), 61.8, 59.9 (2CH2O), 47.4
Reactions of carbodiimide 4 with phosphonates
14a–d, 18, and 20
1
3
(d, JP–C ¼ 211 Hz, C–P), 16.3 (d, JP–C ¼ 7.6 Hz, (CH3CO)2P), 14.2,
14.0 (2CH3CO) ppm. 31P NMR [DMSO-d6]: dP 26.9 ppm. EI–MS: m/z
(%): 582 (14) [Mþþ1], 581 (11) [Mþ], 523 (23) [Mþꢁ58, 2C2H5], 432
[Mþꢁ149, (2EtþNPh)], 416 (58) [Mþꢁ165, (2EtþNPhþNH2)], 279
(100) [Mþꢁ302, (2EtþNPhþNH2þP(O)(OEt)2], 212 (48), 137 (53)
(P(O)(OEt)2), 119 (26), 77 (60). Anal. calcd. for C28H32N5O7P (581.2):
C, 57.83; H, 5.55; N, 12.04; P, 5.33. Found: C, 57.81; H, 5.53; N, 12.02;
P, 5.31.
Preparation of phosphonate derivatives 16a–d, 19, and 21
General Procedure: To the in situ 0.8 g carbodiimide 4 (2.2 mmol) a
solution of the appropriate phosphonate 14a–d, 18, or 20
(2.5 mmol) containing 4.0 mmol of the assigned base was added,
as indicated below. The reaction mixture was stirred at r.t. for
ꢀ6–10 h (TLC). After the completion of the reaction, the product
mixture was cooled, poured onto iced-water, and acidified
with conc. HCl to pH ꢀ 6, followed by extraction with AcOEt
(3 ꢃ 50 mL), and the combined organic phase was dried over
anh Na2SO4. After removal of the solvent under vacuum, the
resulting residue was washed several times with light petroleum
(40–608C), and crystallized from the proper solvent to give the
corresponding product 16a–d, 19, or 21, respectively.
Ethyl 7-amino-6-carbamothioyl-6-(diethoxyphosphoryl)-
1-phenyl-5-(phenylimino)-5,6-dihydro-1H-pyrazolo[4,3-b]-
pyridine-3-carboxylate (16c)
Colorless needles, mp 186–1888C (MeCN), yield: (0.89 g, 72%).
IR: nmax, cmꢁ1 3461, 3349 (2NH ), 1710 (C O), 1628 (C NPh,
–
–
–
–
2
ꢁ1
– –
exocyclic), 1605 (C N), 1232 (P O, bonded), 1074 (P–O–C) cm .
– –
Preparation of 16a–c
Reagents: Operation as above, 0.8 g compound 4 (2.2 mmol),
diethyl methylphosphonoacetate (14a) (0.52 g, 2.5 mmol),
triethyl phosphonoacetate (14b) (0.56 g, 2.5 mmol), or diethyl
(2-amino-2-thioxoethyl)-phosphonate (14c) (0.53 g, 2.5 mmol)
in 20 mL of absolute ethanol containing 103 mg sodium
(4.5 mmol), at r.t. The product mixture was worked up as
1H NMR [DMSO-d6]: d 1.13 (t, JH–H ¼ 6.5 Hz, 3H, CH3CO), 1.28 (dt,
JH–H ¼ 6.5, 4JP–H ¼ 3.8 Hz, 6H, (H3CCO)2P), 3.88 (q, JH–H ¼ 6.5 Hz,
3
2H, H2CO, ester), 4.31 (dq, JP–H ¼ 9.5 Hz, 4H, (H2CO)2P), 6.22
(br, 1H, HAN), 7.14–7.76 (m, 10H, H–Ph), 8.00 [br, 2H,
H2N–C(S)], 9.67 (br, 1H, HBN) ppm. 13C NMR [DMSO-d6]: d 194.3
2
–
–
–
–
–
(C S), 180.3 (d, JP–C ¼ Hz, C NPh), 162.3 (C O), 158.0, 154.7,
–
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

10
W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
3
147.9, 144.4, 129.2, 129.1, 128.5, 123.9, 123.3, 111.4 (C–Ph,
2
7.66 (m, 10H, H–Ph) ppm. 13C NMR: d 164.3 (d, JP–C ¼ 6.8 Hz,
2
–
C NPh), 157.9 (C O), 153.2, 142.5, 135.0, 128.2, 127.7, 127.4,
–
–
–
C-pyrazole), 127.5 (d, JP–C ¼ 37 Hz, C–NH2), 62.2 (d, JP–C ¼ 8.7 Hz,
1
(CH2O)2P), 61.8 (CH2O), 54.8 (d, JP–C ¼ 211 Hz, C–P), 16.3
121.8, 120.9, 119.9 (C-pyrazole, C–Ph), 120.2 (CN), 138.63
2
3
1
(d, JP–C ¼ 7.6 Hz, (CH3CO)2P), 14.2 (CH3CO, ester) ppm.
[d, JP–C ¼ 8.5 Hz, C(4)], 123.8 (d, JP–C ¼ 144 Hz, C–P),
31P NMR [DMSO-d6]: dP 27.5 ppm. EI–MS: m/z (%): 569 (13)
[Mþþ1], 568 (15) [Mþ], 550 (13) [Mþꢁ18, H2O], 521 [Mþꢁ47,
(EtþH2O)], 461 (76) [Mþꢁ107, (H2OþEtþC(S)NH2)], 370 (68)
[Mþꢁ198, (H2OþEtþC(S)NH2þNPh)], 233 (100) [Mþꢁ335,
(H2OþEtþNPhþC(S)NH2þP(O)(OEt)2], 137 (48) (P(O)(OEt)2), 119
(24), 212 (38), 77 (65). Anal. calcd. for C26H29N6O5PS (568.16):
C, 54.92; H, 5.14; N, 14.78; P, 5.45; S, 5.64. Found: C, 54.90;
H, 5.12; N, 14.76; P, 5.43; S, 5.62.
96.1 (C–CN), 60.9 (CH2O), 60.6 (d, JP–C ¼ 8.7 Hz, CH2OP), 16.8
2
(d, JP–C ¼ 7.6 Hz, CH3COP), 14.05 (CH3CO) ppm. 31P NMR
3
[DMSO-d6]: dP 15.3 ppm. EI–MS: m/z (%) 475 (18) [Mþ], 449 (26)
[Mþꢁ26, CN], 404 (28) [[Mþꢁ71, (CNþOEt)], 331 (100) [Mþꢁ144,
(CNþOEtþCO2Et)], 240 (91), 212 (23), 119 (16), 77 (44). Anal. calcd.
for C24H22N5O4P (475.1): C, 60.63; H, 4.66; N, 14.73; P, 6.51. Found:
C, 60.61; H, 4.63; N, 14.71; P, 6.53.
Preparation of 21
Preparation of 16d
Reagents: Compound 4 (0.8 g, 2.2 mmol) and a solution of
diethyl (2-methylallyl)phosphonate (20) (0.48 g, 2.5 mmol) in
30 mL CH3Cl were treated with 5 mL of an aqueous LiOH
solution (0.5 N), followed by heating under reflux for 6 h.
The product mixture was then extracted with 2 ꢃ 20 mL of
CH3Cl. The combined extracts were washed with 40 mL of
distilled H2O, dried, and the solvent was removed under reduced
pressure to give phosphonate 21.
Reagents: Compound 4 (0.8 g, 2.2 mmol), diethyl (methylthio-
methyl)-phosphonate (14d) (0.49 g, 2.5 mmol), LiH (44 mg,
5.5 mmol), and DMF (20 mL) were stirred at r.t, for 6 h. The
product mixture was worked up as described in the general
procedure to afford the phosphonate derivative 16d.
Ethyl 7-amino-6-(diethoxyphosphoryl)-6-(methylthio)-
1-phenyl-5-(phenylimino)-5,6-dihydro-1H-pyrazolo[4,3-b]-
pyridine-3-carboxylate (16d)
Ethyl 5-cyano-4-(3-(diethoxyphosphoryl)-4-methyl-2-
(phenylamino)-1H-pyrrol-1-yl)-1-phenyl-1H-pyrazole-3-
carboxylate (21)
Yellow substance, mp 143–1458C (cyclohexane), yield: (0.9 g,
74%). IR: nmax, cmꢁ1 3497, 3381 (NH ), 1715 (C O), 1618
–
–
(C NPh, exocyclic), 1598 (C N), 1230 (P O, bonded), 1097
2
–
–
–
White needles, mp 154–1568C (CH2Cl2), yield: (0.84 g, 70%).
–
–
–
IR: nmax, cmꢁ1 ¼ 3378 (NH), 2234 (CN), 1708 (C O), 1227 (P O),
(P–O–C). 1H NMR [DMSO-d6]:
d
1.23 (t, JH–H ¼ 6.5 Hz, 3H,
–
–
–
–
1084 (P–O–C). 1H NMR [DMSO-d6]: d 1.14 (t, JH–H ¼ 6.5 Hz, 3H,
4
H3CCO, ester), 1.31 (dt, JH–H ¼ 6.5, JP–H ¼ 3.8 Hz, 6H,
4
CH3CO), 1.34 (dt, JH–H ¼ 6.5 Hz, JP–H ¼ 4.3 Hz, 6H, (H3CO)2P),
(H3CCO)2P), 2.33 (s, 3H, H3CS), 3.68 (q, JH–H ¼ 6.5 Hz, 2H,
4
3
2.10 (d, JP–H ¼ 4.5 Hz, 3H, CH3), 3.77 (q, JH–H ¼ 6.5 Hz,
H2CCO), 4.28 (dq, JP–H ¼ 9.8 Hz, 4H, (H2CO)2P), 6.32 (br, 1H,
3
HAN), 7.14–7.65 (m, 10H, H–Ph), 9.67 (br, 1H, HBN) ppm.
2H, H2CO), 4.23 (dq, JP–H ¼ 9.7 Hz, 4H, (H2CO)2P), 6.24
4
13C NMR [DMSO-d6]: d 173.9 (d, JP–C ¼ 14.2 Hz, C NPh), 162.3
2
–
(d, JP–H ¼ 4.2 Hz, 1H, HC–pyrrole), 7.22–8.11 (m, 10H, H–Ph),
–
(C O, ester), 155.5, 143.7, 129.2, 128.5, 128.2, 123.3, 123.2, 122.9
13
6
9.68 (br, 1H, HN) ppm. C NMR [DMSO-d ]: d 158.9 (C O), 143.2,
–
–
–
–
2
141.8, 129.4, 128.3, 127.4, 122.5, 120.0, 119.3 (C–Ph, C-pyrazole),
135.7 (d, 2JP–C ¼ 14.0 Hz, C–NHPh), 129.9 (d, 1JP–C ¼ 211.8 Hz, C–P),
120.3 (CN), 118.2 (d, 2JP–C ¼ 14.8 Hz, C–Me), 116.2 [d, 3JP–C ¼ 8.8 Hz,
(C-pyrazole, C–Ph), 119.5 (d, JP–C ¼ 17.4 Hz, C–NH2), 63.3 (d,
2JP–C ¼ 8.7 Hz, (CH2O)2P), 61.8 (CH2O), 51.5 (d, JP–C ¼ 144.6 Hz,
1
3
C–P), 16.2 (d, JP–C ¼ 8.6 Hz, (CH3CO)2P), 16.0 (CH3–S), 14.29
2
(CH3C–O) ppm. 31P NMR [DMSO-d6]: dP 26.9 ppm. EI–MS: m/z
(%): 556 (17) [Mþþ1], 555 (12) [Mþ], 526 (18) [Mþꢁ29, Et], 508
(11) [Mþꢁ47, SMe], 479 (70) [Mþꢁ76, (EtþSMe)], 388 [Mþꢁ167,
(EtþSMeþNPh)], 372 [Mþꢁ183, (EtþSMeþNPhþNH2)], 235 (100)
[Mþꢁ320, (EtþSMeþNPhþNH2þP(O)(OEt)2], 212 (38), 137 (52)
(P(O)(OEt)2), 119 (30), 77 (68). Anal. calcd. for C26H30N5O5PS (555.2):
C, 56.21; H, 5.44; N, 12.61; P, 5.57; S, 5.77. Found: C, 56.20; H, 5.42;
N, 12.63; P, 5.55; S, 5.75.
C(2)-pyrrole], 98.5 (C–CN), 62.7 (d, JP–C ¼ 8.7 Hz, (CH2O)2P), 60.9
(CH2O), 15.9 (d, 3JP–C ¼ 7.6 Hz, (CH3CO)2P), 14.0 (CH3CO), 13.0 (CH3)
ppm. 31P NMR [DMSO-d6]: dP 29.4 ppm. EI–MS: m/z (%) 547 (21) [Mþ],
532 (31) [Mþꢁ15, Me], 521 (18) [Mþꢁ26, CN], 506 (58) [Mþꢁ41,
(MeþCN)], 414 (44) [Mþꢁ133, (CNþMeþNHPh)], 277 (100)
[Mþꢁ270, (CNþMeþNHPhþP(O)(OEt)2)], 212 (33), 137 (42)
(P(O)(OEt)2), 119 (18), 77 (39). Anal. calcd. for C28H30N5O5P (547.2):
C, 61.42; H, 5.52; N, 12.79; P, 5.66. Found: C, 61.40; H, 5.50;
N, 12.77; P, 5.63.
Preparation of 19
Reagents: A mixture of 0.8 g of compound 4 (2.2 mmol) in
10 mL DMF, and 0.41 g diethyl vinylphosphonate (18) (2.5 mmol)
was treated, at r.t., with 44 mg LiH (5.5 mmol) in 20 mL DMF. The
product mixture was worked up as described in the general
procedure to afford the phosphonate derivative 19.
Pharmacological screening
Animals
Male Swiss albino mice (25–30 g) were housed in a room with
controlled temperature (22 ꢂ 18C), humidity (55 ꢂ 10%) and
photoperiod (12:12 h) light–dark cycle for at least a week before
being used. They were maintained on standard pellet diet and
water ad libitum throughout the experiment. A minimum of six
animals were used in each group. Throughout the experiments,
the animals were processed according to the suggested inter-
national ethical guidelines for the care of laboratory animals of
animal ethics.
Ethyl-5-cyano-4-(2-ethoxy-5-(phenylimino)-2,5-dihydro-
1H-1,2-azaphospholyl-2-oxide)-1-phenyl-1H-pyrazole-3-
carboxylate (19)
Colorless needles, mp 103–1108C (pentane), yield: (0.75 g, 72%.
IR: nmax, cmꢁ1 ¼ 2222 (CN), 1712 (C O, ester), 1632 (C NPh),
–
–
–
–
1608 (C N), 1260 (P O, free), 1083 (P–O–C). 1H NMR [DMSO-d6]:
–
–
–
–
d 1.24 (t, JH–H ¼ 6.7 Hz, 3H, CH3CO), 1.29 (dt, JH–H ¼ 6.5 Hz,
4JP–H ¼ 4.3 Hz, 3H, H3COP), 4.22 (q, JH–H ¼ 6.7 Hz, 2H, H2CO),
Analgesic activity
Analgesic activity was evaluated using the p-benzoquinone
(p-BQ)-induced writhing test in mice [26]. Thirty minutes after
3
4.07 (dq, JH–H ¼ 6.5 Hz, JP–H ¼ 5.0 Hz, 2H, H2COP), 6.95
3
2
(d, JP–H ¼ 6.7, 1H, H(4)), 7.55 (d, JP–H ¼ 17.2, 1H, H(3)), 7.27–
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–12
Enamino- and a-Aminophosphonates versus Inflammatory and Cancer Diseases
11
the subcutaneous administration of a test sample in dimethyl
sulfoxide (DMSO; 100 mg/kg body weight), the mice were intra-
peritoneally injected with 0.1 mL/10 g body weight of 2.5% w/v
(p-BQ) solution in distilled water. Control animals received an
appropriate volume of dosing vehicle DMSO. The mice were then
kept individually for the observation and the total number of the
abdominal contractions (writhing movements) was counted for
the following 15 min, starting 5 min after the pBQ injection.
The data represent the average of the total number of writhes
observed. Analgesic activity was then expressed as the percentage
change from writhing controls.
of cells to the wall of the plate; (ii) different concentrations of
each compound under test (5, 12.5, 25, and 50 mg/mL) were
added to the cell monolayer triplicate wells that prepared for
each individual dose. Each concentration is evaluated three
times (each dose is incubated with the cells in three different
wells) and the monolayer cells were incubated with the com-
pounds for 48 h at 378C and in atmosphere of 5% CO2; (iii) after
48 h, cells were fixed, washed and stained with SRB stain;
(iv) excess stain was washed away with acetic acid and the
attached stain was recovered with Tris-EDTA buffer (pH 7.4);
(v) color intensity was measured in an ELISA plate reader;
(vi) the relation between surviving fraction and drug concen-
tration is displayed in Table 2.
ðNc ꢁ NtÞ
% Potency ¼
ꢃ 100
Nc
IC50 mg=mL
M:Wt:
Calcualtions : ðIC50 mMÞ ¼
ꢃ 1000
where Nc and Nt are the total number of writhing movements
at time 0 and after 15 min, respectively.
Antiinflammatory activity
Authors like to thank the Central Lab, School of Pharmacy, Alexandria
University, Egypt, and NY Cancer Institute, NY, USA for carrying out the
pharmacological screening.
Carrageenin-induced hind paw edema model was utilized for
evaluation of antiinflammatory activity [27]. Each experimental
group contained seven animals minimally. Thirty minutes after
the subcutaneous administration of a test sample in DMSO
(100 mg/kg body weight) or dosing vehicle, each mouse was
injected with 0.1 mL/10 g body weight of freshly prepared sus-
pension of carrageen (0.5 mg/25 mL) in physiological saline into
sub-plantar tissue of the right hind paw. As a control group,
25 mL saline was injected into the left hind paw. Paw edema was
measured in 90, 180, 270, and 360 min after carrageenin injec-
tions. Measurement of paw volume: The volume of the hind paw
of the mouse up to the ankle joint was measured plathysmo-
graphically by the mercury displacement method. The ankle
joint of the mouse was marked with a skin marking pencil
and the paw was dipped into the mercury, so that the mark
on the paw coincides with a prefixed line kept constant on the
syringe. The level of the mercury was every time brought to
the level of this line by adjusting the height of the displaced
mercury. Mean values of each treated group were compared with
the control group and analyzed by using statistical methods.
The antiinflammatory activity was expressed as percentage inhi-
bition of edema volume in the treated animals in comparison
with the control group.
The authors have declared no conflict of interest.
References
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ðVc ꢁ VtÞ
% Inhibition of edema ¼
ꢃ 100
[8] W. M. Abdou, A. A. Sediek, M. D. Khidre, Monatsh. Chem. 2008,
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Vc
where Vc and Vt are the volumes of edema for the control and
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