Synthesis and Anti-Proliferative Activity of New α-Amino Phosphonate Derivatives Bearing Heterocyclic Moiety

Article abstract of DOI:10.1007/s11094-021-02404-1

Condensation of 4-chloroacetophenone 1 with phenyl hydrazine 2 afforded hydrazone 3. Further reaction of 3 with Vilsmeier reagent yielded the pyrazolaldehyde 4 in excellent yield. A one-pot, three-component reaction between aldehyde 4, triphenylphosphite 5, and appropriate amines in the presence of lithium perchlorate as Lewis acid catalyst gave the corresponding α-amino phosphonates 7-12(a-d) in good yields. The chemical structures of all new compounds were established by IR, ¹H NMR, and mass spectroscopy analysis. The anti-proliferative activity of the synthesized compounds against HCT-116, HepG2 and MCF-7 human cancer cells using the MTT assay was evaluated, and revealed higher anticancer activity when compared with reference drug doxorubicin. Among the tested compounds, pyrazole derivatives 4 and 9 exhibited the highest anticancer activity against breast (MCF-7), and colon (HCT-116) cancer cell lines with IC₅₀ = 2.7 and 3.3 μM, respectively.

Full text of DOI:10.1007/s11094-021-02404-1

DOI 10.1007/s11094-021-02404-1
Pharmaceutical Chemistry Journal, Vol. 55, No. 3, June, 2021 (Russian Original Vol. 55, No. 3, March, 2021)
SYNTHESIS AND ANTI-PROLIFERATIVE ACTIVITY OF NEW a-AMINO
PHOSPHONATE DERIVATIVES BEARING HETEROCYCLIC MOIETY
Ramzy E. Abdelwahed,1 Ahmed Habeeb Radhi,2 Hanem M. Awad,3
Ahmed A. El Gokha,1 Adel E-S. Goda,4 and Ibrahim El-Tantawy El Sayed1,*
Original article submitted October 2, 2019.
Condensation of 4-chloroacetophenone 1 with phenyl hydrazine 2 afforded hydrazone 3. Further reaction of 3
with Vilsmeier reagent yielded the pyrazolaldehyde 4 in excellent yield. A one-pot, three-component reaction
between aldehyde 4, triphenylphosphite 5, and appropriate amines in the presence of lithium perchlorate as
Lewis acid catalyst gave the corresponding -amino phosphonates 7-12(a-d) in good yields. The chemical
1
structures of all new compounds were established by IR, H NMR, and mass spectroscopy analysis. The
anti-proliferative activity of the synthesized compounds against HCT-116, HepG2 and MCF-7 human cancer
cells using the MTT assay was evaluated, and revealed higher anticancer activity when compared with refer-
ence drug doxorubicin. Among the tested compounds, pyrazole derivatives 4 and 9 exhibited the highest
anticancer activity against breast (MCF-7), and colon (HCT-116) cancer cell lines with IC50 = 2.7 and 3.3 M,
respectively.
Keywords: acetophenone; phenylhydrazine; Vilsmeier-Haack reaction; triphenylphosphite; anti-proliferative
activity.
1. INTRODUCTION
attention of many researchers to study its skeleton chemi-
cally and biologically [9–11].
Cancer constitutes a major public health problem world-
wide, since it is the second leading cause of death globally,
with 9.6 million deaths estimated in 2018. Due to the limita-
tions and side effects associated with available cancer treat-
ments now days, it is an urgent challenge for medicinal re-
searchers to develop more safe and selective anticancer
drugs [1]. Towards that end, pyrazole I and its derivatives II
are considered a pharmacologically important active scaffold
that possesses almost all types of pharmacological activities.
The presence of the pyrazole nucleus in different structures
leads to diversified applications in different areas such medi-
cine [2, 3]. In particular, they are described as inhibitors of
protein glycation, antibacterial [4], antifungal, anticancer [5],
antidepressant, anti-inflammatory [6], anti-tuberculosis, anti-
oxidant [7] as well as antiviral agents [8]. Owing to this di-
versity in the biological field, this nucleus has attracted the
Moreover, -amino phosphonates III are versatile class
of organophosphorus compounds because they are analogues
of the corresponding -amino acids IV and possess high
metabolic stability as well as insignificant toxicity [12-15].
Furthermore, incorporation of -amino phosphonate moiety
into different molecules have demonstrated to be a novel and
promising approach to design more potent and safer
anti-cancer drugs [16].
In continuation of our program on the utilization of nitro-
gen-containing heterocyclic scaffolds in multicomponent re-
actions for one-pot synthesis of new hybrid molecules incor-
porating -amino phosphonate scaffold, we would like to re-
port the synthesis of new -amino phosphonate derivatives
containing pyrazole heterocycle [17, 18]. This hybrid struc-
ture is important due to a range of biological activities and
1
Chemistry Department, Faculty of Science, Menoufia University, Shebin
El-Kom, Egypt.
2
Nursing Department, Al-Suwaria Technical Institute, Middle Technical
University, Baghdad Iraq.
3
Department of Tanning Materials and Leather Technology, National Re-
search Centre, Dokki, Giza, Egypt.
4
Tanta Higher Institute of Engineering and Technology, Tanta, Egypt.
*
e-mail: ibrahimtantawy@yahoo.co.uk
231
0091-150X/21/5503-0231 © 2021 Springer Science+Business Media, LLC

232
Ramzy E. Abdelwahed et al.
medicinal applications associated with them by combining
both moieties with the aim to have synergistc pharmacologi-
cal properties [19–21].
0.8 mmole), and appropriate amine 6a-6d and 11a-11d
(0.8 mmole) in acetonitrile (3 mL), lithium perchlorate
(10 mole %) was added. The reaction mixture was stirred at
room temperature until the starting materials were consumed
as monitored by TLC (3d). After the completion of the reac-
tion, the precipitated product was filtered off and crystallized
by using diethyl ether to give the desired product in good
yield.
Synthesis of diphenyl ((3-(4-chlorophenyl)-1-phenyl-
1H-pyrazol-4-yl) (pyridin-4-ylamino) methyl) phospho-
nate 7: Yield: (0.83 g., 70%), dark green solids, mp.
2. EXPERIMENTAL
198 – 200°C, IR (cm-1):
3395,
1670,
1227,
P=O
NH
C=N
2.1. Materials and Methods
1
671. HNMR ( , ppm):
1076, 752 and
POC
C-N
C-Cl
1HNMR spectra were recorded on Varian 400 MHz
5.45 – 5.53 (dd, 1H, CHP), 5.9 (s, 1H, NH), 7.22 – 9.31 (m,
24H, Ar.). MS: m/z = 593 (M+, C H ClN O P, 75%,),
spectrophotometer using DMSO-d solvent at the University
33 26
4
3
6
of Ulm (Germany) and the chemical shifts were expressed in
part per million ( , ppm) relative to the internal slandered
TMS (0PPM) for the center peak of residual DMSO
(2.49 ppm). The peak patterns are indicated as follows: s,
singlet; d, doublet; t, triplet. The coupling constants, J, are re-
ported in Hertz (Hz|). IR spectra were recorded on Shimadzu,
Japan spectrophotometer, Cairo University using anhydrous
KBr disc. Melting point (mp) was recorded on Stuart-scien-
tific melting point apparatus and was uncorrected. The bio-
logical activity was carried out at the division of Pharmaceu-
tical Industries, National Research Center, and Cairo, Egypt.
All reactions were followed by thin layer chromatography
(TLC) on kiesel gel F254 pre-coated plates (Merck). Starting
materials and solvents were purchased and used as received
without further purification.
m/z = 328
(M+-C H N O P,
100%),
m/z = 77
16 16
3
3
(M+-C H ClN O P, 34%), m/z = 187 (M+-C H NO P,
27 21
4
3
11 17
5
6%), m/z = 406 (M+-C H N O P, 63%).
21 19
4
3
Synthesis of diphenyl ((3-(4-chlorophenyl)-1-phenyl-
1H-pyrazol-4-yl)((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihyd-
ro-1H-pyrazol-4-yl)amino) methyl) phosphonate 8: Yield:
(0.96 g, 68%), orange solids, mp. 168 – 170°C; IR (cm-1):
3143,
2411,
1645,
1294,
1137,
POC C-N
NH
C=o
C=N
P=O
762 and
693. 1HNMR ( , ppm): 2.39 (s, 3H, CH ), 3.13
3
C-Cl
(s, 3H, N-CH ), 3.94 (s, 1H, NH), 5.88 (dd, 1H, CHP),
3
6.72 – 7.99 (m, 25H, Ar.). MS: m/z = 702 (M+
C H ClN O P, 33%), m/z = 388 (M+-C H N O P,
39 33
5
4
22 17
2
4
100%), m/z = 77 (M+-C H ClN O P, 55%), m/z = 314
33 27
5
4
(M+-C H ClN OP, 5%).
17 16
3
Synthesis of diphenyl ((3-(4-chlorophenyl)-1-phe-
nyl-1H-pyrazol-4-yl)((4-oxo-2-thioxothiazoldin-3-yl)amino)
methyl)phosphonate 9: Yield: (1.1g, 85%), yellow solids,
2.2. Chemistry
mp.173 – 175°C; IR (cm-1):
3436,
1739,
C=O
Synthesis of 3-(4-chlorophenyl)-1-phenyl-1H pyrazo-
le-4-carbaldehyde (4). To a mixture of p-chloroacetophe-
none 1 (1.55 g, 0.01 mole), and phenyl hydrazine (1.08 g.,
0.01 mole) of 2 in 10 mL ethanol was refluxed in water bath
for 4 hrs. The reaction mixture was cooled, and the solid
formed was filtered and crystallized from diethyl ether to
form hydrazone 3. Further dropwise addition of a mixture of
NH
C=N
1601, 1288, 1117,
760 and
647 cm-1,
P=O
POC
C-N
C-Cl
1HNMR ( , ppm): 4.3(s, 2H, CH ), 5.88 (s, 1H, CHP),
7.4 – 9.27 (m, 21H, Ar.), MS: m/z = 647 (M+-C H N O ,
2
14 10
2
2
14%), m/z = 347 (M+-C H N O PS , 100%), m/e = 77
15 10
2
4
2
(M+-C H ClN O PS , 33%).
25 18
4
4
2
Synthesis of benzyl ((3-(4-chlorophenyl)-1-phenyl-1H-
pyrazol-4-yl)(diphenoxyphosphoryl)methyl)carbamate 10:
Yield: (1.13 g, 87%), orange solids, mp. 138 – 140°C; IR
DMF (0.73 g, 0.01 mole) and POCl (1.53 g, 0.01 mole) with
3
cooling under mechanical stirring for 5 h. The reaction mix-
ture was further refluxed for 6 h. at 70 – 80°C, then hydro-
lyzed on ice/water mixture , and neutralized by 5% NaOH
solution till pH= 4, then cooled. The solid formed was fil-
tered off, washed with water, dried and crystallization from
isopropanol to yield: (2.55 g, 90%), yellow solid, mp:
(cm-1):
3319,
1732,
1656,
1271,
P=O
NH
C=O
C=N
POC
1091,
756 and
685. 1HNMR ( , ppm): 4.57(s, 2H,
C-N
C-Cl
CH ), 4.71 – 4.78 (dd, 1H, CHP), 5.9 (s, 1H, -NH),
2
6.68 – 9.17 (m, 25H, Ar.), MS: m/z = 650 (M+C H N O ,
23 15
3
3
45%), m/z = 350 (M+-C H NO , 100%), m/z = 77
252 – 253°C. IR (cm-1):
and
731, 1HNMR ( , ppm): 10.13 (s, 1H, CHO), 9.31
(C-H Pyrazole), 6.54 – 7.95 (m, 8H, Ar-H), MS: m/z = 282
(M+, C H ClN O, 78%), m/z = 77(M+-C H N O, 100%),
m/z = 209 (M+-CHO, 15%), m/z = 237(M+-H, 74%),
m/z = 210(M+-CO, 20%).
1667,
1603,
1511
C=C
20 15
5
C=O
C=N
(M+-C H ClN O P, 42%), m/z = 226 (M+-C H ClN O P,
30 23
3
5
22 18
2
3
C-H
26%).
Synthesis 4-(((3-(4-chlorophenyl)-1-phenyl-1H- pyra-
zol-4yl)(diphenoxyphosphoryl)methyl) amino)benzoic
acid 12a: Yield: (0.76 g, 60%), white solids, mp.
198 – 200°C; IR (cm-1):
3291, 1794, 1674,
1254, 1092,
16 11
2
8
5
2
Synthesis of a-amino phosphonate derivatives
(7 – 12). General Procedure: To a mixture of aldehyde 4
NH
C=O
C=N
764 and
691, 1HNMR ( ,
P=O
POC
C-N
C-Cl
(0.23 g, 0.8 mmole), triphenylphosphite
5
(0.25 g.,
ppm): 5.49 (s, 1H, CHP), 6.73 – 7.74 (m, 15H, Ar.), 9.66 (S,

Synthesis and Anti-proliferative Activity
233
1H, -COOH). MS: m/z = 636 (M+-C H N O , 33%),
assay. After incubation, media were carefully removed,
40 L of MTT (2.5 mg/mL) were added to each well and
then incubated for an additional 4 h. The purple formazan
dye crystals were solubilized by the addition of 200 L of
DMSO. The absorbance was measured at 570 nm using a
Spectra Max Paradigm Multi-Mode micro plate reader. The
relative cell viability was expressed as the mean percentage
of viable cells compared to the untreated control cells.
34 26
4
3
m/z = 382
(M+-C H N P,
100%),
m/z = 77
15 10
2
(M+-C H ClN O P, 22%), m/z = 253 (M+-C H NO P,
29 22
3
3
20 17
5
6%).
Synthesis of diphenyl ((3-(4-chlorophenyl)-1-phenyl-
1H-pyrazol-4-yl) (p-tolylamino)methyl) phosphonate
12b: Yield: (0.92 g, 76%), black solids, mp. 103 – 105°C; IR
(cm-1):
3119,
1670,
1223,
1093,
752
C-N
NH
C=N
P=O
POC
and
686. 1HNMR ( , ppm): 3.34 (s, 3H, CH ),
C-Cl
3
2.4. Statistical Analysis
5.40 – 5.45 (dd, 1H, CHP), 5.72 (s, 1H, NH),6.73 – 9.16(m,
24H, Ar). MS: m/z = 606 (M+-C H ClN O P, 11%),
All experiments were conducted in triplicate and re-
peated on three different days. All values were represented as
35 29
3
3
m/z = 352
(M+-C H ClN ,
100%),
m/z = 77
15 10
2
(M+-C H ClN O P, 33%), m/z = 219 (M+-C H ClNO P,
29 24
3
3
20 18
3
mean SD. The values of IC were determined by probit
50
86%), m/z = 341 (M+-C H N O P, 87%).
18 20
3
2
analysis using SPSS Inc. probit analysis software (IBM
Corp., Armonk, NY, USA).
Synthesis of diphenyl ((3-(4-chlorophenyl)-1-phenyl-
1H-pyrazol-4-yl)(mesitylamino)methyl)phosphonate 12c:
Yield: (0.97 g, 76%), orange solids, mp. 183 – 185°C; IR
3. RESULTS AND DISCUSSION
(cm-1):
3329,
2862,
1652,
1269,
P=O
NH
C-C
C=N
POC
1088,
752 and
686.66, 1HNMR ( , ppm): 4.95 (s,
C-N
C-Cl
3.1. Chemistry
9H, CH ), 5.04 – 5.08 (dd, 1H, CHP), 6.57 (s, 1H, -NH),
3
6.93 – 8.35 (m, 22H, Ar.), MS: m/z = 634 (M+-C H N O ,
Aacetophenone hydrazone 3 was prepared in good yield
by heating p-chloroacetophenone 1 with phenylhydrazine 2
in methanol under reflux for 4 – 5 h. Then, Vilsmeier-Haack
23 17
3
4
33%), m/z = 382 (M+-C H N P, 100%), m/z = 77
15 10
2
(M+-C H ClN O P, 22%), m/z = 253 (M+-C H NO P,
29 22
3
3
20 17
5
(FH) reaction of hydrazone 3 using DMF and POCl af-
6%).
3
forded the corresponding pyrazole-4-carbaldehyde 4 in good
yield and high purity as depicted in Scheme 1.
Scheme 1
Synthesis of diphenyl (((4-aminophenyl)amino)(3-
(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)phos-
phonate 12d: Yield: (0.90 g, 73%), black solids, mp.
218 – 220°C; IR (cm-1):
3422,
3321,
1625,
C=N
NH2
NH
1269, 1092,
756 and
687. 1HNMR ( ,
P=O
POC
C-N
C-Cl
ppm): 5.38 (dd, 1H, CHP), 6.06 (s, 1H, -NH),), 6.8 (s, 2H,
-NH ), 7.04 – 8.34 (m, 24H, Ar.). MS: m/z = 607
2
(M+C H N O , 52%), m/z = 293 (M+-C H ClN P,
24 19
3
4
17 16
2
100%), m/z = 78 (M+-C H ClN O P, 63%), m/z = 254
28 22
4
3
(M+-C H N O P, 13%).
19 17
2
3
2.3. Antiproliferative Activity
In-vitro cytotoxic activity. Cell culture of HCT-116 (hu-
man colorectal carcinoma), HepG2 (human liver carcinoma)
and MCF-7 (human breast adenocarcinoma) cell lines were
purchased from the American Type Culture Collection
(Rockville, MD) and maintained in DMEM medium which
was supplemented with 10% heat-inactivated FBS (fetal bo-
vine serum), 100U/ml penicillin and 100U/ml streptomycin.
The cells were grown at 37°C in a humidified atmosphere of
The structure of compound 4 was elucidated on the basis
1
of IR, HNMR and mass spectral analysis. In the structure
prove of the aldehyde 4 a characteristic signal appeared in
HNMR at delta 10.13 ppm which corresponds to the aldehy-
dic proton (CHO), furthermore; a broad peak appeared in IR
spectrum at 1667 cm-1 corresponds to carbonyl absorption of
the aldehyde group. This in addition to the mass spectrum
analysis showed the molecular ion peak of 4 at m/z 282. A
plausible mechanism for cyclization along with formylation
of pyrazole is outlined in (scheme 2). The mechanism in-
volves an initial electrophilic attack of VH reagent A on
hydrazone 1 yielded the intermediate B which subsequently
losses amolecule of HCl to provide intermediate C, the
nucleophilic attack by N-H group initiates the cyclisation
5% CO .
2
MTT cytotoxicity assay. The cytotoxicity activity
against HCT-116, HepG2 and MCF-7 human cancer cell
lines was estimated using the 3-[4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, which is
based on the reduction of the tetrazolium salt by mitochon-
drial dehydrogenases in viable cells [22 – 24]. Cells were
dispensed in a 96 well sterile microplate (5 104 cells/well),
and incubated at 37°C with series of different concentrations,
in DMSO, of each tested compound or Doxorubicin (positive
control) for 48 h. In a serum free medium prior to the MTT
and the resulting pyrazole intermediate losses Me NH to
2
give the more stable pyrazole derivative D. The pyrazole D
react with another molecule of VH reagent A in an electro-

234
Ramzy E. Abdelwahed et al.
Scheme 2
philic substitution process giving an iminium salt E, which is
hydrolysed to corresponding 4-formyl pyrazole 2 as depicted
in Scheme 2. In summary the electrophilic attack of first VH
complex at the probable attacking site of hydrazones results
into cyclisation. While electrophilic attack of second com-
plex forms formyl product after hydrolysis. Finally, intra-
molecular (1, 5) hydrogen shift, cyclisation and elimination
ture of 7, 8, 9 and 10 was elucidated on the basis of IR,
1HNMR and mass spectral analysis. In the structure prove of
-amino phosphonates 7, 8, 9 and 10 exhibited characteristic
IR stretching frequencies in the region 1288 – 1291,
1117 – 1137 and 3436 – 3143 cm-1 for P=O, POC and N-H
respectively. The P-C-H proton signal appeared as multiplets
at 5.45 – 5.53 and the N-H proton signal appeared at 5.9
for N-protected -amino phosphonates 7. While the P-C-H
proton signal appeared at 5.88 and the N-H proton signal
of NHMe to give the corresponding aldehyde 4.
2
Furthermore, the three one-pot reaction of aldehyde 4,
triphenylphosphite 5, appropriate amine such as 4-amino-
pyridine 6a, 4-amino-antipyrine 6b, 3-amino-Rhodanine 6c
and benzyl carbamate 6d, in acetonitrile (3 mL), in the pres-
ence of lithium perchlorate (10 mole%) as a Lewis acid cata-
lyst. The reaction mixture was stirred at room temperature
until the starting materials were consumed as monitored by
TLC (3 days). After the completion of the reaction, the pre-
cipitated product was filtered off and crystallized by using
diethyl ether, to product the compound 7, 8, 9and 10 in good
yield and high purity as depicted in (Scheme 3). The struc-
appeared at 3.94, -CH proton signal appeared at 4.3 for
2
the free -amino phosphonates 8, 9. The P-C-H proton signal
appeared as multiplets at 4.71 – 4.78 and the N-H proton
signal appeared at 5.9, -CH proton signal appeared at
2
5.57 for the free -amino phosphonates 10. This, in addition
to the mass spectrum analysis, showed the molecular ion
peaks of 7, 8, 9 and 10 at m/z 593,702,647 and 650.
Moreover, aldehyde 4 and triphenylphosphite 5 were
converted to compounds 12a-d when reacted with 4-amino-

Synthesis and Anti-proliferative Activity
235
Scheme 3
1
benzoic acid 11a, p-toluidine 11b, 2,4,6-trimethylaniline 11c
and benzene-1,4-diamine11d, Dissolve all the mixture in
acetonitrile (3 mL), in the presence of lithium perchlorate
(10 mole%) as a Lewis acid catalyst. The reaction mixture
was stirred at room temperature until the starting materials
were consumed as monitored by TLC (3 days). After the
completion of the reaction the precipitated product was fil-
tered off and crystallized by using diethyl ether, The struc-
were elucidated on the basis of IR, HNMR and mass spec-
tral analysis. In the structure prove of -amino phosphonates
12a, 12b, 12c and 12d exhibited characteristic IR stretching
frequencies in the region 1254 – 1269, 1088 – 1093 and
3422 – 3291 cm-1 for P=O, POC and N-H respectively. The
P-C-H proton signal appeared at 5.49 and the -COOH pro-
ton signal appeared at 9.66 for -amino phosphonates 12a.
While the P-C-H proton signal appeared as multiplet at
5.40 – 5.45, 5.04 – 5.08 and the N-H proton signal appeared
1
tures were confirmed on the basis of IR, H NMR and M.S
spectral data according to Scheme 4. The structures of 12a-d
Scheme 4

236
Ramzy E. Abdelwahed et al.
Scheme 5
manner (Figs. 1 – 3). In case of HCT-116 human colorectal
carcinoma cells, both (Fig. 1 and Table 1) show that, six
compounds (7, 12b, 9, 12a, 8, and 12c) were more potent
cytotoxic compounds; three compounds (10, 12d, and 4) had
significant comparable cytotoxic activities to that of
doxorubicin. In case of MCF-7 human breast cancer cells,
four compounds (4, 9, 12a and 12c, respectively) were more
potent cytotoxic compounds; two compounds (12d and 7)
had significant comparable cytotoxic activity to that of
doxorubicin (Fig. 2 and Table 1). The three other compounds
were slightly less active against MCF-7 cancer cells. In case
of HepG2 human liver cancer cells, seven compounds (9,
12b, 8, 12a, 10, 12c and 7) were more potent cytotoxic com-
pounds; the two other compounds (12d and 4) had signifi-
cant comparable cytotoxic activity to that of doxorubicin
(Fig. 3 and Table 1).
at 5.72, 6.57 for the free -amino phosphonates 12b, 12c.
The P-C-H proton signal appeared at 5.38 and the N-H pro-
ton signal appeared at 6.06 for the free -amino phospho-
nates 12d. This in addition to the mass spectrum analysis
showed the molecular ion peak of 12a, 12b, 12c and 12d at
m/z 636, 606, 634 and 607.
A plausible mechanism for the formation of -amino
phosphonates compounds is outlined in (Scheme 5). The
mechanism of the reaction is believed to be following the
path of activation of aldehyde 4 as well as the in situ gener-
ated imine (Schiff base) F by Lewis acid in the first step.
This facilitates nucleophilic addition of the amino group of
the carbamate component to adehyde 4 as well as the
nucleophilic addition of trimethyl- or triphenyl phosphite to
the polar C=N bond of imine F to give the phosphonium salt
G as an intermediate. In final step, this phosphonium salt get
decomposed by water liberating product K through the
hydroxyl phosphite intermediate H as shown in Scheme 5.
3.3. Structure-Activity Relationships (SARs)
Results of the anti-proliferative activity assay of -ami-
no phosphonate derivatives are summarized in Table 1, along
with the data of the anticancer drug doxorubicin. The
cytotoxicity test of the -amino phosphonate derivatives and
their chemically modified analogues indicated a synergistic
effect of the substituents at the pyrazole core structure. In
case of human colon cancer and human liver cancer, one can
conclude that, the anti-proliferative activity increased by the
introducing the following groups: amino-pyridine, amino-an-
3.2. In Vitro Anti-Proliferative Activity
Nine compounds were examined in vitro for their activ-
ity against HCT-116, HepG2 and MCF-7 human cancer cells
using the MTT assay. The percentages of viable cells were
calculated and compared to those of the control. Activities of
these compounds against the three carcinoma cell lines were
compared to the activity of doxorubicin as well. All com-
pounds suppressed the three cancer cells in a dose-dependent

Synthesis and Anti-proliferative Activity
237
Fig. 1. Dose dependent cytotoxic activities of nine compounds against HCT-116 cancer cells according to the MTT assay.
Fig. 2. Dose dependent cytotoxic activities of nine compounds against MCF-7 cancer cells according to the MTT assay.
Fig. 3. Dose dependent cytotoxic activities of nine compounds against HepG2 cancer cells according to the MTT assay.
tipyrine, amino-Rhodanine, benzyl carbamate, aminobenzoic
acid, p-toluidine and 2,4,6-trimethylaniline, respectively.
However, introducing benzene-1,4-diamine moiety did not
cause any change in the activity compared to compound 4.
Surprisingly, in case of human lung cancer, introducing the
same groups caused decrease in the anti-proliferative activity
compared to compound 4. On the other hand, compound 9
showed a strong anticancer activity against all three cancer
in addition, compounds 9, 12b, 8, 12a, 10, 12c, 7 and 12d
also exhibited a strong potency with IC = 7.3, 8.6, 8.7, 9,
50
9.4, 9.6, 9.8 and 11.3 M against (HePG-2) cell lines. From
the above mentioned results, it is obtained that all the nine
tested compounds are strong cytotoxic compounds on both
human colon cancer and human liver cancer. Further varia-
tions in substituents and substitution pattern may be neces-
sary to obtain more potent and selective anticancer lead com-
pounds.
cell lines tested in this study with IC = 5.6, 3.3 and 7.3 M
50
In conclusion, the present study fosters a simple and eas-
ily scalable approach for the preparation of a new series of
-amino phosphonate derivatives containing pyrazole
heterocycle. All the newly synthesized compounds were
for HCT-116, MCF-7 and HePG-2 cancer cell lines respec-
tively. Moreover, products 7, 12b, 9, 12a, 8, 12c, 10, and 12d
showed a higher activity with IC = 4.4, 4.7, 5.6, 5.9, 6.4,
50
7.5, 7.9 and 8.4 M against colorectal carcinoma (HCT-116)

238
Ramzy E. Abdelwahed et al.
TABLE 1. IC50 of Nine Compounds against Three Cancer Cell Lines According to MTT assay
IC50 ( M) SD
Compound
R
HCT-116
4.4 2.5
MCF-7
HepG2
N
7
7.0 2.5
9.8 4.1
CH3
8
6.4 3.1
9.2 4.1
8.7 4.5
N
O
N
CH3
9
S
5.6 2.4
3.3 1.1
7.3 2.9
N
S
O
10
7.9 3.5
9.0 3.5
9.4 3.1
O
C
O
HOOC
12a
12b
5.9 2.4
4.7 2.6
3.4 2.1
7.2 3.8
9.0 3.5
8.6 3.2
CH3
12c
12d
CH3
7.5 3.1
4.8 2.3
9.6 3.5
H3C
CH3
8.4 4.1
6.6 3.0
11.0 5.1
H2N
Aldehyde 4
9.2 3.9
9.4 3.9
2.7 1.1
6.7 2.1
11.3 6.5
10.4 3.1
Doxorubicin
screened for their in vitro antitumor activity against three hu-
man carcinoma cell lines, namely colorectal carcinoma
(HCT-116), breast carcinoma (MCF-7) and liver carcinoma
(HepG-2) using MTT cytotoxicity assay at different concen-
trations. The results showed that all compounds possessed
good antitumor activity against all cell lines. Accordingly,
this class of compounds could be considered as excellent
templates for future optimization or modification to obtain
potent antitumor agents.
ACKNOWLEDGMENTS
We would like to acknowledge financial support of the
Menoufia University throughout project No Ib-2013.
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