Synthesis and in vitro antimicrobial, antioxidant, and antiproliferative activities of some new pyrano[2,3-c]pyrazoles containing 1,2-azaphospholes, 1,3,2-diazaphosphinines and phosphonate moieties

Article abstract of DOI:10.1080/00397911.2021.1939059

New 1,2-azaphosphole, 1,3,2-diazaphosphinine, and phosphonate derivatives containing pyrano[2,3-c]pyrazole moiety were achieved. The synthetic pathways depended on the reaction of 6-amino-3-methyl-1,4-diphenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile with some phosphorus reagents such as diethyl phosphite, phosphorus sulfides, and phenyl phosphorus halides under different conditions. Compounds 6 and 7 recorded potent antibacterial and antifungal activities with lower toxicity, while compounds 4 and 6 displayed promising antioxidative properties. Further, compounds 4 and 9 exhibited potent cytotoxic effects against MCF-7, HepG-2, and HCT-116 cancer cells. The early apoptotic cell death was observed by the compounds in all types of the treated cells. Compounds 3, 5, 7, 10, and 14 recorded low to moderate percentages of necrosis and late apoptosis toward all treated cells.

Full text of DOI:10.1080/00397911.2021.1939059

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Synthesis and in vitro antimicrobial, antioxidant,
and antiproliferative activities of some
new pyrano[2,3-c]pyrazoles containing 1,2-
azaphospholes, 1,3,2-diazaphosphinines and
phosphonate moieties
Tarik E. Ali, Mohammed A. Assiri, Hafez M. El-Shaaer, Somaia M. Abdel-
Kariem, Wafaa R. Abdel-Monem, Somaya M. El-Edfawy, Noha M. Hassanin,
Ali A. Shati, Mohammad Y. Alfaifi & Serag Eldin I. Elbehairi
To cite this article: Tarik E. Ali, Mohammed A. Assiri, Hafez M. El-Shaaer, Somaia M. Abdel-
Kariem, Wafaa R. Abdel-Monem, Somaya M. El-Edfawy, Noha M. Hassanin, Ali A. Shati,
Mohammad Y. Alfaifi & Serag Eldin I. Elbehairi (2021) Synthesis and inꢀvitro antimicrobial,
antioxidant, and antiproliferative activities of some new pyrano[2,3-c]pyrazoles containing 1,2-
azaphospholes, 1,3,2-diazaphosphinines and phosphonate moieties, Synthetic Communications,
51:16, 2478-2497, DOI: 10.1080/00397911.2021.1939059
To link to this article: https://doi.org/10.1080/00397911.2021.1939059
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2021, VOL. 51, NO. 16, 2478–2497
https://doi.org/10.1080/00397911.2021.1939059
Synthesis and in vitro antimicrobial, antioxidant, and
antiproliferative activities of some new pyrano[2,3-
c]pyrazoles containing 1,2-azaphospholes, 1,3,2-
diazaphosphinines and phosphonate moieties
Tarik E. Alia,b, Mohammed A. Assiria, Hafez M. El-Shaaerb, Somaia M. Abdel-
Kariemb, Wafaa R. Abdel-Monemb, Somaya M. El-Edfawyb, Noha M. Hassaninb
Ali A. Shatic, Mohammad Y. Alfaific, and Serag Eldin I. Elbehairic,d
,
b
aFaculty of Science, Department of Chemistry, King Khalid University, Abha, Saudi Arabia; Department
c
of Chemistry, Faculty of Education, Ain Shams University, Roxy, Cairo, Egypt; Faculty of Science,
d
Department of Biology, King Khalid University, Abha, Saudi Arabia; Cell Culture Lab, Egyptian
Organization for Biological Products and Vaccines (VACSERA Holding Company), Giza, Egypt
ABSTRACT
ARTICLE HISTORY
Received 6 April 2021
New 1,2-azaphosphole, 1,3,2-diazaphosphinine, and phosphonate
derivatives containing pyrano[2,3-c]pyrazole moiety were achieved.
The synthetic pathways depended on the reaction of 6-amino-3-
methyl-1,4-diphenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile
with some phosphorus reagents such as diethyl phosphite, phos-
phorus sulfides, and phenyl phosphorus halides under different con-
ditions. Compounds 6 and 7 recorded potent antibacterial and
antifungal activities with lower toxicity, while compounds 4 and 6
displayed promising antioxidative properties. Further, compounds 4
and 9 exhibited potent cytotoxic effects against MCF-7, HepG-2, and
HCT-116 cancer cells. The early apoptotic cell death was observed by
the compounds in all types of the treated cells. Compounds 3, 5, 7,
10, and 14 recorded low to moderate percentages of necrosis and
late apoptosis toward all treated cells.
KEYWORDS
Antimicrobial; antioxidant;
antiproliferative; 1,2-
azaphosphole; 1,3,2-
diazaphosphinines;
phosphonate;
pyrano[2,3]pyrazole
GRAPHICAL ABSTRACT
CONTACT Tarik E. Ali
tarik_elsayed1975@yahoo.com, tismail@kku.edu.sa
Department of Chemistry, Ain Shams
University, Cairo 11711, Egypt.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2021 Taylor & Francis Group, LLC

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Introduction
Organophosphorus compounds are important intermediates in organic synthesis and
have been widely used as a pharmaceutical,[1] agricultural,[2] and chemical agents.[3,4]
For example, phosphorus heterocycles[5] have received considerable interest because of
their unique biological activities as antimicrobial[6] and their anticancer effects.[7,8]
Ifosfamide and cyclophosphamide are two important examples of phosphorus hetero-
cycles that were launched on the market more than 30 years ago and are still used in
the treatment of cancer.[9,10] Also, phosphonate groups are chemically stable analogs of
phosphates and play numerous vital roles in the biochemistry of living organisms.
Phosphonates constitute an important class of bioisosteres for medicinal chemists and
chemical biologists.[11] The usefulness of this class of molecules has been highlighted by
the COVID-19 pandemic. Phosphonate nucleotide analogs, such as tenofovir and
cidofovir, can inhibit SARS-CoV-2 RNA polymerase[12] and may thus represent a
much-needed therapeutic weapon against this viral threat. On the other hand, pyr-
ano[2,3-c]pyrazole compounds are the major structural motif in several natural products
and synthetic compounds of widely recognized pharmacological properties.[13] In this
context, pyranopyrazole systems have well been recognized for their potent biological
activities including antimicrobial,[14] antioxidant,[15] antiviral[16], and antitumor.[17]
In view of the aforementioned details, and in extension to our efforts concerning
the design of new polyheterocycles containing phosphorus heterocycles or phospho-
nate groups with possible biological properties,[18–22] we herein display the synthesis
of some novel bioactive polycyclic compounds composed of pyrano[2,3-c]pyrazole
skeleton containing phosphorus heterocycles or phosphonate groups in one molecu-
lar frame which might show enhanced biological activity. The suggested synthetic
pathways depend on the reaction of the 6-amino-3-methyl-1,4-diphenyl-1,4-dihydro-
pyrano[2,3-c]pyrazole-5-carbonitrile with some phosphorus reagents such as diethyl
phosphite (in the presence or absence of electrophilic reagents), phosphorus sulfides,
and phosphorus halides under different reaction conditions. In addition, the isolated
products were screened for their antimicrobial, antioxidant, and antiprolifera-
tive activities.
Results and discussion
The known starting material, 6-amino-3-methyl-1,4-diphenyl-1,4-dihydropyrano[2,3-c] pyr-
azole-5-carbonitrile (3) was synthesized in good yield as reported in the literature[23]
through the reaction of 3-methyl-1-phenyl-5-pyrazolone (1) with 2-benzylidenemalononi-
trile (2) in distilled water containing a catalytic amount of sodium benzoate (Scheme 1).
Substrate 3 possesses one reactive electrophilic center (CꢀN) and another nucleophilic cen-
ter (NH2) which can be attacked by electrophilic and nucleophilic phosphorus reagents.
This provides a wide opportunity for the construction of phosphorus heterocycles fused
with pyranopyrazole system.
Phosphorus esters have gained significant interest as versatile reagents in the con-
struction of various novel phosphorus heterocycles.[19,22] Thus, diethyl H-phosphonate
reacted with substrate 3 at 80–90 ꢁC in the presence of BF3ꢂEt2O as a catalyst to yield
the nonisolable intermediate A via removal of the ethanol molecule. The latter

2480
T. E. ALI ET AL.
Scheme 1. The reaction of substrate 3 with diethyl H-phosphonate.
intermediate underwent an intramolecular heterocyclization through addition of
H-phosphorus atom on the nitrile group to give 6-ethoxy- 5-imino-3-methyl-6-oxido-
1,4-diphenyl-1,4,5,7-tetrahydropyrazolo[40,30:5,6]pyrano[3,2-d][1,2]azaphosphole
(4)
(Scheme 1). The Mþ peak for product 4 was recorded at m/z 420 in its mass spectrum,
1
while its 31P-NMR spectrum recorded a singlet at d 16.2 ppm. Moreover, the H-NMR
spectrum of compound 4 revealed specific one ethoxy group at d 1.03 (t, CH3) and
3.79–3.87 (m, OCH2) as well as the NH protons resonated as two singlets at d 9.94 and
11.52 ppm. Furthermore, its 13C-NMR spectrum exhibited some specific carbon atoms
at d 16.1 (CH3), 60.2 (OCH2), and a doublet signal at d 142.6 ppm (JPC ¼ 150.9 Hz)
for P–C¼NH.[24]
Dimethylformamide dimethylacetal (DMFDMA) is considered one of the very
important precursors in the construction of novel bioactive compounds.[25] Thus, heat-
ing of substrate 3 with DMFDMA in dry dioxane under reflux for 6 h, followed by add-
ition of diethyl phosphite and BF3ꢂEt2O then further heated under reflux for 10 h
afforded
diethylf[(5-cyano-3-methyl-1,4-diphenyl-1,4-dihydropyrano[2,3-c]pyrazol-6-
yl)imino]methylgphosphonate (5) in 30% yield (Scheme 2). When the previous reaction
was carried out using a two-fold excess of diethyl phosphite and a catalytic amount of
sodium metal, it furnished the interesting novel pyrrolo[30,20: 5,6]pyrano[2,3-c]pyrazolyl
bis(phosphonate) 6 as a sole product in 47% yield (Scheme 2).[26] The formation of
products 5 and 6 was explained through condensation of NH2 group of substrate 3
with the acetal carbon of the DMFDMA reagent giving the nonisolable N,N-dimethyl-
formimidamide intermediate B. The latter intermediate reacted with an equimolar
amount of diethyl phosphite in the presence of BF3ꢂEt2O or Na metal via a nucleophilic
addition of H-phosphorus atom at azomethine group to give the nonisolable

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Scheme 2. The reaction of substrate 3 with DMfDMA followed by diethyl phosphite.
a-aminophosphonate C (Scheme 2). In the case of pathway i, the intermediate C lost
one molecule of dimethylamine to yield the isolated product 5, while in case of pathway
ii, condensation of the second molecule of diethyl phosphite with the intermediate C
gave the bis(phosphonate) intermediate D with the elimination of dimethylamine mol-
ecule (Scheme 2). This intermediate D underwent ring closure via the addition of the
active CH–P group at the electrophilic CꢀN function to isolate the final product 6
(Scheme 2). The IR spectrum of product 5 displayed important absorption bands at
2195 (Cꢀ N) and 1200 (P¼O) cmꢃ1, while its mass spectrum exhibited the Mþ peak at
m/z 476 (27%). The 13C-NMR spectrum of product 5 exhibited a specific signal relevant
to CꢀN at d 111.8 ppm and a doublet signal at d 143.9 ppm (J¼140.5 Hz) attributable
1
to CH–P. Also, its H-NMR spectrum affirmed the proposed structure by recording two
ethoxy groups and the specific N¼CH–P proton as a doublet at d 8.58 with coupling

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T. E. ALI ET AL.
Scheme 3. The reaction of substrate 3 with triethyl orthoformate followed by diethyl phosphite.
1
constant (J¼22 Hz). On the other hand, the H-NMR spectrum of compound 6 exhib-
ited four ethoxy groups as two multiplets at d 0.86–1.31 (4 CH3) and 3.87–4.15 (4
OCH2) ppm, besides two broad signals at d 11.85 and 13.97 (2 NH) ppm. Also, its
mass spectrum displayed the expected Mþ peak at m/z 614 (24%). Additionally, its 13C-
NMR spectrum revealed the specific carbon atom of C–P at d 53.6 ppm as a doublet
signal (JPC¼142.6 Hz) and another signal relative to C¼NH at d 144.8 ppm.
Likewise, treatment of substrate 3 with triethyl orthoformate in dry dioxane followed
by addition of a solution of diethyl phosphite containing a catalytic amount of sodium
metal yielded diethyl f6-ethoxy-5-imino-3-methyl-1,4-diphenyl-4,5,6,7-tetrahydro-1H-
pyrrolo[30,20:5,6]pyrano[2,3-c]pyrazol-6-ylgphosphonate (7) and not the expected prod-
uct 5 (Scheme 3).[27] The formation of compound 7 is similar to the formation of com-
pound 6. The IR spectrum of product 7 recorded the presence of the important bands
of two NH and exocyclic C¼N functions at 3425, 3363, and 1624 cmꢃ1, respectively.

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Scheme 4. The reaction of substrate 3 with benzaldehyde followed by diethyl phosphite.
1
Moreover, its H-NMR spectrum recorded the chemical shifts of two NH protons as
two broad signals at d 11.82 and 13.98 ppm. In addition, its 13C-NMR spectrum exhib-
ited a doublet signal of C–P at d 67.0 ppm (JPC ¼ 144.6 Hz), while the carbon atom of
C ¼ NH was resonated at d 146.3 ppm.
In the same manner, treatment of substrate 3 with benzaldehyde and diethyl
phosphite in the presence of BF3ꢂEt2O as a catalyst in dry dioxane under the
Kabachnik-Field reaction[28] afforded the nonisolable a-amniophosphonate H. The latter
intermediate underwent an intramolecular cyclization via addition of the active hydro-
gen of CH–P group at the nitrile function followed by auto-hydrolysis to isolate diethyl
f3-methyl-5-oxo-1,4,6-triphenyl-4,5,6,7-tetrahydro-1H-pyrrolo[30,20:5,6]pyrano[2,3-c]pyr-
azol-6-ylgphosphonate (8) (Scheme 4). The IR spectrum of product 8 showed absorp-
tion bands for NH and C ¼ O functions at 3422 and 1691 cmꢃ1, respectively while its
31P-NMR spectrum recorded a singlet at d 22.0 ppm. Furthermore, its Mþ peak was

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T. E. ALI ET AL.
Scheme 5. The reaction of substrate 3 with P4S10 in dioxane.
1
recorded at m/z 555 (12%) in its mass spectrum. The H-NMR spectrum of the cyclic
aminophosphonate 8 recorded additional multiplets for aromatic protons produced
from benzaldehyde reagent when compared with the starting material 3, besides a sing-
let relevant to NH proton at d 11.25 ppm. In its 13C-NMR spectrum, the carbon atom
of C–P moiety resonated as a doublet at d 68.4 ppm with a coupling constant 143.4 Hz
while the C ¼ O resonated as a singlet at d 165.4 ppm.
Lawesson’s reagent and phosphorus decasulfide are convenient thionating agents and
powerful platforms to construct novel compounds including bioactive phosphorus and
sulfur-based heterocycles. Thus, treatment of substrate 3 with phosphorus decasulfide in
dry dioxane afforded 2-sulfanyl-6-methyl-5,8-diphenyl-2-sulfido-4-thioxo-3,5,8-trihydro-
pyrazolo[40,30:5,6]pyrano[2,3-d][1,3,2]diazaphosphinine (9) (Scheme 5). The isolated
product 9 was formed through the addition of P4S10 reagent on the CꢀN group to give
the nonisolable thioamide intermediate J.[29] The latter intermediate underwent nucleo-
philic attack of the neighboring NH2 group at the electrophilic phosphorus atom of the
PS2 fragment, followed by an intramolecular cyclization (Scheme 5).[30] The IR spec-
trum of compound 9 showed the disappearance of CꢀN group and displayed broad
absorption bands at 3442, 3349 (two NH), 2664 (SH), and 1195 (C¼S) cmꢃ1. Moreover,
1
its H-NMR spectrum showed a singlet for SH proton at d 2.40 ppm and two broad sig-
nals at d 10.02 and 11.86 ppm for two NH protons. Further, the 13C-NMR spectrum of
product 9 displayed the carbon atom of C ¼ S at d 178.4 ppm. In addition, its mass

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Scheme 6. The reaction of substrate 3 with P4S10 in ethanol.
spectrum recorded the expected Mþ at m/z 456 which confirmed the sug-
gested structure.
In the same manner, when compound 3 was reacted with P4S10 in absolute ethanol,
it furnished the fused triheterocyclic system 10 in 55% yield (Scheme 6).[31] The mass
spectrum was great evidence for the formation of compound 10 which recorded its Mþ
1
at m/z 468. Its H-NMR spectrum revealed one ethoxy group at d1.01 (t, CH3) and 3.41
(q, OCH2) ppm. Furthermore, its 13C-NMR spectrum displayed the specific carbon
atoms of CH3, OCH2, and C¼S at d 17.2, 62.2, and 177.1 ppm, respectively. Besides, the
31P-NMR spectrum recorded a singlet at d 45.2 ppm. The proposed pathway suggested
that the chemically active O,O-diethyl dithiophosphoric acid intermediate (formed in
situ by refluxing P4S10 in absolute ethanol) underwent a nucleophilic attack of SH group
at the CꢀN group of substrate 3 forming the intermediate L. The latter intermediate
removed ethanol molecule through an intramolecular cyclization followed by Dimroth
rearrangement to produce the desired product 10 (Scheme 6).
On the other hand, treatment of compound 3 with Lawesson’s reagent (LR) in dry
dioxane gave the nonisolable intermediate N which underwent ring closure through a
nucleophilic attack of SH group at phosphorus atom of -SP(S)Ar fragment to form the
nonisolable intermediate O. Dimroth rearrangement of the latter intermediate led to the
formation of 2-(4-methoxyphenyl)-6-methyl-5,8-diphenyl-2-sulfido-4-thioxo-3,5,8-trihy-
dropyrazolo[40,30:5,6]pyrano[2,3-d][1,3,2]diazaphosphinine (11) (Scheme 7).[32,33] The
31P-NMR spectrum of product 11 displayed a signal at d 50.1 ppm. Also, its 13C-NMR
spectrum showed specific signals at d 54.4 and 175.9 ppm relative to MeO and C¼S
groups, respectively. In addition, the 1H-NMR spectrum recorded a singlet at d
3.86 ppm relevant to MeO protons and two broad signals at d 11.56 and 13.16 ppm for
two NH protons. The mass and IR spectra of product 11 were in accordance with the
suggested structure (see experimental section).

2486
T. E. ALI ET AL.
Scheme 7. The reaction of substrate 3 with LR in dioxane.
Furthermore, compound 3 reacted with P-(4-methoxyphenyl)-N,N0-bis(4-methyl-
phenyl) phosphonothioic diamide P (formed by the reaction of LR with p-toluidine in
molar ratio 1:4)[34] to produce the nonisolable intermediate Q with the removal of p-
toluidine molecule. Ring closure of the latter intermediate by attacking of NH group at
the CꢀN group led to the sole product 12 in 33% yield (Scheme 8). The IR spectrum
of product 12 showed characteristic absorption bands due to two NH and P ¼ S func-
tions at 3289, 3191, and 798 cmꢃ1, respectively. A strong confirmation of the structure
12 was its 1H-NMR spectrum which exhibited three singlets at d 2.02, 2.31, and
3.89 ppm relevant to two Me and one MeO groups. Also, other signals appeared at d
10.36 and 11.19 ppm for the two NH protons. In addition, its 13C-NMR spectrum dis-
played characteristic signals for carbon atoms of 2 Me, MeO, and C ¼ NH at d 12.1,
13.9, 55.0, and 154.9 ppm, respectively. The mass spectrum of product 12 recorded m/z
496 (Mþ–MeOC4H6, 12%).
Phenyl phosphorus halides play vital roles in the construction of bioactive organo-
phosphorus compounds.[35] This encouraged us to study the reactivity of substrate 3
toward some phenyl phosphorus halides. Thus, compound 3 condensed with P,P-
dichloro(phenyl)phosphine in dry pyridine to furnish 3-methyl-1,4-diphenyl-6-[(phenyl-
phosphanylidene)amino]-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (13) (Scheme
9). The IR spectrum of the latter product showed a characteristic band at 2223 cmꢃ1
relevant to the CꢀN function confirming the suggested structure. In addition, the car-
bon atom of CꢀN resonated at d 107.7 in its 13C-NMR spectrum. The mass spectrum
of compound 13 exhibited its Mþ peak at m/z 434 (6%).
Next, P-chlorodiphenylphosphine condensed with compound 3 in dry pyridine to
give the nonisolable intermediate S. The latter intermediate underwent ring closure
through the addition of the nucleophilic phosphorus atom at the electrophilic carbon
atom of CꢀN group giving the intermediate T which was rearranged into 5-imino-3-

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Scheme 8. The reaction of substrate 3 with the intermediate P (formed in situ from LR and
p-toluidine).
Scheme 9. The reaction of substrate 3 with PhPCl2 in pyridine.

2488
T. E. ALI ET AL.
Scheme 10. The reaction of substrate 3 with (Ph)2PCl in pyridine.
methyl-1,4,6,6-tetraphenyl-1,4-dihydro-5H-6k5-pyrazolo[40,30:5,6]pyrano[3,2-d][1,2]aza-
phosphole (14) (Scheme 10). The IR spectrum of product 14 displayed absorption bands
1
for NH and exocyclic C¼N functions at 3354 and 1603 cmꢃ1, respectively. Its H-NMR
spectrum displayed a singlet relevant to NH proton at d 13.01 ppm, beside multiplet sig-
nals for twenty aromatic protons at d 6.98–7.62 ppm. In addition, the 13C-NMR spec-
trum revealed the existence of a doublet at d 150.5 ppm for C¼NH due to coupling
with phosphorus atom with JPC¼60 Hz.
Finally, addition of phenyl phosphonic dichloride to substrate 3 in dry pyridine fur-
nished 2,5,8-triphenyl-3,5,8-trihydropyrazolo[40,30:5,6]pyrano[2,3-d][1,3,2]diazaphosphi-
nine 15 in 72% yield (Scheme 11). The suggested pathway for this product based on
condensation between compound 3 and phosphorus reagent to give the nonisolable inter-
mediate U. The nucleophilic addition of chlorine atom at CꢀN group in the latter inter-
mediate afforded the 4-chloro-1,3,2-diazaphosphinine intermediate V. When the
intermediate V was treated with ice-water for 30 minutes, it gave the final product 15 in
the form of 15A and not the other tautomeric form 15B according to its spectroscopic data
(Scheme 11). The IR spectrum confirmed the suggested structure and showed characteris-
tic absorption bands at 3425, 3363, and 1679 cmꢃ1 for two NH and C¼O functions,
respectively. The expected Mþ peak of compound 15 was recorded at m/z 468 in its mass
spectrum while its 31P-NMR spectrum showed a singlet at d 34.31 ppm. The two NH pro-
tons resonated as two singlets at d 12.68 and 14.63 ppm in its 1H-NMR spectrum. Also, the
13C-NMR spectrum exhibited the important signal of C¼O at d 164.8 ppm.
Biological evaluations
Antimicrobial activity
The in vitro antimicrobial activities of the synthesized products were screened toward
three bacterial strains, namely Streptococcus pyogenes, Staphylococcus aureus, and

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Scheme 11. The reaction of substrate 3 with PhP(O)Cl2 in pyridine.
Escherichia coli, and three fungal strains, namely Aspergillus niger, Aspergillus clavatus,
and Candida albicans.[36,37] Minimum inhibitory concentration (MIC) of all synthesized
compounds was determined and given in Table 1. Ketoconazole and Ciprofloxacin as
standard drugs were used for the antifungal and antibacterial activities, respectively.
Variable antimicrobial activities toward the used microorganisms were recorded for the
synthesized compounds. Compounds 5, 9, and 13 did not record any acceptable inhibi-
tory effects toward all bacteria and fungi organisms in comparison with substrate 3 and
standard drugs. Compound 12 exhibited relatively moderate effects against all organ-
isms. On the other hand, products 10, 11, 14, and 15 showed good antibacterial and
antifungal effects except compound 11 that recorded excellent antifungal activities equal
to the standard drug. Furthermore, compounds 4, 6, 7, and 8 displayed excellent anti-
microbial activities toward most of the microorganisms except compound 8 that showed
moderate to good effects against the fungi organisms. Generally, the relationship
between the structure and activity indicated that the introduction of phosphorus hetero-
cycles and phosphonate groups to pyranopyrazole system can increase the antimicrobial
properties. Therefore, the presence of 1,2-azaphosphole ring fused with the pyranopyra-
zole system in compounds 4 and 14 exhibited extremely good to excellent antibacterial
and antifungal activities. In addition, the existence of 1,3,2-diazphosphinine rings

2490
T. E. ALI ET AL.
Table 1. The in vitro antimicrobial activities as minimum inhibitory concentration (MIC, mg/mL)a for
the synthesized compounds.
Bacterial strains
Fungal strains
Streptococcus
pyogenes
Staphylococcus
aureus
Aspergillus
clavatus
Compound
Escherichia coli Aspergillus niger Candida albicans
3
4
5
250
31.25
250
250
31.25
250
250
31.25
250
250
31.25
125
250
31.25
125
500
31.25
125
6
7
8
9
31.25
31.25
31.25
250
31.25
31.25
31.25
250
31.25
31.25
31.25
250
31.25
31.25
62.5
250
31.25
31.25
62.5
250
31.25
31.25
62.5
250
10
11
12
62.5
62.5
125
62.5
62.5
125
62.5
62.5
125
62.5
31.25
125
125
31.25
125
62.5
31.25
125
13
14
15
250
250
250
500
62.5
62.5
250
62.5
62.5
250
62.5
62.5
62.5
62.5
31.25
62.5
62.5
31.25
62.5
62.5
31.25
Ciprofloxacin
Ketoconazole
31.25
31.25
31.25
aAll results are expressed as mean SD from three experiments (n ¼ 3).
Table 2. The cytotoxcity of the products 4, 6, 7, and 8 using the
Brine Shrimp bioassay.
Compound
Lethal Concentration (LC50, mg/mL)a
4
6
7
8
1.65
7.32
5.67
2.36
4.51
Gallic acid
aAll results are expressed as mean SD from three experiments (n ¼ 3).
possess P¼S and P¼O groups as in products 10, 11, and 15 exhibited significant
improvements in the antimicrobial effects in comparison with the starting material 3.
Interestingly, the design of skeleton pyrrolo[30,20:5,6]pyrano[2,3-c]pyrazole bearing
diethyl phosphonate groups in compounds 6, 7, and 8 caused excellent inhibitory effects
nearly to the standard drugs toward all the microorganisms except compound 8 which
recorded good inhibitory effects against only fungal strains.
The cytotoxicity activity
The LC50 values of the most bioactive synthesized compounds 4, 6, 7, and 8 against
brine shrimp death were evaluated by the reported method using Gallic acid as a stand-
ard.[38,39] The results are summarized in Table 2. The lowest LC50 values were found in
the case of compounds 4 and 8 at 1.65 and 2.36 mg/mL, respectively, indicating their
higher cytotoxicity. On the other hand, compounds 6 and 7 displayed brine shrimp
death at 7.32 and 5.67 mg/mL, respectively. Therefore, both compounds 6 and 7 can be
active potent antibacterial and antifungal agents with lower cytotoxicity. So, we can con-
clude that skeleton pyrrolo[30,20:5,6]pyrano[2,3-c] pyrazole bearing diethyl phosphonate
groups is the most active potent antimicrobial agent with lower toxicity in host cells.

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Table 3. The in vitro antioxidant activities as inhibitory concentration
(IC50, mg/mL) for the synthesized compounds by using DPPH and
H2O2 methods.
Inhibitory concentration (IC50, mg/mL)
Compound
DPPH method
H2O2 method
3
4
5
6
7
8
9
10
11
12
13
14
16.21 0.29
11.86 0.31
35.12 0.29
10.12 0.52
13.25 0.23
14.67 0.66
25.87 0.00
18.38 0.33
15.63 0.23
24.36 0.39
19.35 0.21
12.43 0.21
36.42 0.76
10.23 0.23
22.53 0.51
17.05 0.51
40.23 0.54
16.69 0.31
19.36 0.62
21.49 0.34
30.53 0.00
26.13 0.26
24.65 0.29
28.52 0.27
29.14 0.50
20.84 0.33
50.46 0.25
18.62 0.52
15
Ascorbic acid
Antioxidant activity
The synthesized compounds were investigated for their in vitro antioxidant properties
[42]
by DPPH[40,41] and H2O2
methods. The ascorbic acid was used as standard con-
trol. The lower IC50 values indicated to a higher antioxidant activity. The measured
antioxidative properties of the synthesized compounds showed promising radical
scavenging abilities (Table 3). The results revealed that compounds 5, 9, 12, and 15
exhibited poor radical scavenging abilities in comparison with ascorbic acid.
However, remarkable activities were observed with products 3, 8, 10, 11, and 13.
Interestingly, compounds 4 and 14 exhibited significant antioxidant activities due to
the presence of 1,2-azaphosphole or 1,3,2-diazaphosphinine ring, two acidic NH
groups, and extended conjugation. Besides, the connection of the pyrrole ring with
NH and phosphonate group in compounds 6 and 7 caused promising antioxida-
tive properties.
Antiproliferative activity
The in vitro antiproliferative activities of the synthesized compounds were determined
by using the SRB assay[43] against three human cancer cell lines, mammary gland breast
cancer (MCF-7), liver cancer (HepG-2), and human colon cancer (HCT-116) in com-
parison with doxorubicin as reference drug. The IC50 values of cytotoxic activities of
the synthesized compounds 3-15 are presented in Table 4. It is obvious that the thirteen
tested compounds displayed variable degrees of inhibitory effects on the tested human
tumor cell lines. Compounds 3, 5, 12, 13, and 14 revealed no acceptable cytotoxic activ-
ities on all tumor cell lines. For action on MCF-7, the highest cytotoxic effects were
demonstrated by compounds 4, 6, 8, 9, 10, and 15 which showed the IC50 values in
ranges from 3.7 to 8.4 lg/mL in comparison with doxorubicin (IC50 ¼ 1.4 lg/mL).
Moderate cytotoxic effects were also recorded by compounds 7 and 11 with values of
IC50 ¼ 16.9 and 14.5 lg/mL, respectively. The HepG-2 cell line was demonstrated to be

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T. E. ALI ET AL.
Table 4. The IC50 (mg/mL) of the synthesized compounds against different tumor
cell lines.
IC50 (mg/mL)
Compound
MCF-7
HepG-2
HCT-116
3
4
5
6
7
8
9
10
11
12
13
14
23.6 0.5
8.3 0.4
73.5 1.6
8.4 0.3
16.9 0.5
5.04 0.3
3.7 0.7
5.6 1.03
14.5 0.4
85.9 3.1
46.3 5.2
388.4 9.9
8.1 0.9
120.8 6.6
10.5 0.5
102.5 4.1
18.8 0.8
20.3 0.3
14.5 2.2
6.4 0.2
34.4 2.5
47.5 2.1
45 3.2
1732.3 6.6
97.3 5.2
87.1 3.8
1.6 0.04
46.8 2.4
3.7 0.7
27.9 0.9
7.7 0.6
10.7 1.3
22.1 1.5
10.6 0.7
30.7 1.5
7.6 0.5
52.8 1.5
361.1 5.9
536.1 4.2
38.2 3.6
2.0 0.03
15
Doxorubicin
1.4 0.07
the least sensitive among the tested carcinoma cells. However, the highest sensitivity to
compound 9 as its growth inhibitory effect was the displayed value of IC50 at 6.4 lg/
mL. The moderate activity was shown by compounds 4, 6, 7, and 8 which have IC50
values at 10.5, 18.8, 20.3, and 14.5 lg/mL, respectively. Compounds 10 and 11 revealed
weak cytotoxicity properties. In addition, the HCT-116 cancer cell line was weakly
affected by compounds 8, 10, and 15. However, a significant growth inhibitory effect
was noticed by compounds 4, 6, and 11 with IC50 values 3.7, 7.7, and 7.6 lg/mL,
respectively, compared with doxorubicin (IC50¼2.0 lg/mL). The residual compounds 7
and 9 showed moderate antiproliferative activities on HCT-116 cell line with IC50 values
10.7 and 10.6 lg/mL, respectively. These results indicated a specific therapeutic index
for those phosphorus compounds that have an auspicious perspective to be substances
for the candidate for anticancer.
In this research, the following relationships of structure with activity were assumed
by comparing the structures of the synthesized phosphorus compounds to their experi-
mental cytotoxicity. In general, the study revealed that the new molecular frames exhib-
ited cytotoxicity properties more slightly than the starting substrate 3. The presence of
phosphorus heterocycles or phosphonate groups combined with the pyranopyrazole
moiety may be causing a broad spectrum of antiproliferative effects on various carcin-
oma cells (MCF-7, HepG-2, and HCT-116). The presence of phosphoryl moiety such as
P(O)(OEt)2 and N ¼ P-Ph as directly attached on the pyranopyrazole system in com-
pounds 5 and 13 reduced the cytotoxic effects in comparison with the substrate 3. The
construction of 1,3,2-diazaphosphinine ring fused with pyranopyrazole moiety in prod-
ucts 9, 10, 11, 12, and 15 exhibited acceptable antiproliferative properties. Remarkably,
the existence of C¼S and P¼S group in the 1,3,2-diazaphosphinine unit as compounds
9, 10 and 11, enhanced the cytotoxic activity more than C¼NH and C¼O groups of
1,3,2-diazaphosphinine in compounds 12 and 15. Interestingly, the design of 1,2-aza-
phosphole ring fused with pyranopyrazole core as in compounds 4 and 14 led to vari-
able toxic activities. This indicated that the presence of cyclic NH-P(O)OEt system
increased the cytotoxic activity more than N¼P(Ph)2 system. On the other hand, the
introduction of pyrrole ring bearing diethyl phosphonate groups to pyranopyrazole core

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as in compounds 6, 7, and 8, increased the antiproliferative activities. Further, the pres-
ence of two diethyl phosphonate groups improved the antiproliferative potency of more
than one diethyl phosphonate group.
Apoptotic effect
Apoptosis is a form of programmed cell death that occurs in multicellular organisms.
Biochemical events lead to characteristic cell changes (morphology) and death. These
changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensa-
tion, chromosomal DNA fragmentation, and global mRNA decay.
The treated tumor cell lines MCF-7, HepG-2, and HCT-116 were stained with acrid-
ine orange and ethidium bromide for 48 h. After, their examination under a fluorescent
microscope, the cells appeared in the form of four colors as follows: living cells (normal
green nucleus with normal, round, intact nuclei, and cytoplasm that indicates the viabil-
ity of the cell control), early apoptotic (early-stage apoptosis is represented by changes
and ultimate loss of the mitochondrial membrane potential, they appeared as a bright
green nucleus with fragmented chromatin), necrotic cells (necrosis is caused by factors
external to the cell or tissue, such as infection, or trauma which result in the unregu-
lated digestion of cell components, they appeared as uniformly bright red-stained cell
nuclei) and late apoptotic (late-stage apoptosis that can be identified by looking at the
defragmentation of DNA, they appeared as red-stained nuclei with chromatin fragmen-
tation) (Supplementary Figures 1–3).[44,45] The highly early apoptotic cell death was
observed in all types of treated tumor cells in all compounds. The considerable necrotic
activity compared with other compounds was displayed only in compound 10, while
the other compounds did not cause the death of the necrosis pathway in all cancer cells.
The late apoptosis was obviously observed with compounds 3, 5, and 7 especially
against MCF-7 and HepG-2 cell lines, while compounds 3, 7, and 14 were observed in
the case of HCT-116 cell line (Supplementary Figures 4–6). The obtained results were
referenced to the control that absence of any cell death manifestations.
Experimental
The melting points were determined in an open capillary tube on a digital Stuart SMP-
3 apparatus. Infrared spectra were measured on FT-IR (Nicolet IS10) spectrophotometer
1
using KBr disks and Perkin-Elmer 293 spectrophotometer using KBr disks. H- and
13C-NMR spectra were measured on Gemini-300BB spectrometer (400 and 100 MHz),
using DMSO-d6 as a solvent and TMS (d) as an internal standard. 31P-NMR spectra
were measured on a Bruker (162 MHz) spectrophotometer using DMSO-d6 as a solvent,
TMS as an internal standard, and 85% H3PO4 as an external reference. Mass spectra
were recorded on direct probe controller inlet part to single quadrupole mass analyzer
in (Thermo Scientific GCMS). Elemental microanalysis was performed on Perkin-Elmer
2400II at the Chemical War department, Ministry of Defense. The purity of the synthe-
sized compounds was checked by thin-layer chromatography (TLC) and elemental
microanalysis. The biological evaluations were measured in the King Khalid University,
Abha, KSA.

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T. E. ALI ET AL.
Synthesis of 6-ethoxy-5-imino-3-methyl-6-oxido-1,4-diphenyl-1,4,5,7-
tetrahydropyrazolo [40,30:5,6]pyrano[3,2-d][1,2]azaphosphole (4)
A mixture of diethyl phosphite (1.5 mL, 10 mmol) and compound 3 (1.64 g, 5 mmol) in
the presence of trifluoroboron etherate (0.1 mL) was fused on water bath at 80–90 ꢁC
for 6 h. The excess of reagent was evaporated under vacuum. The formed semi-solid
was treated with n-hexane. The isolated solid was filtered off and crystallized from
EtOH to give yellow solid in 42% yield (0.88 g); mp 166–168 ꢁC. IR (KBr), (v max,
cmꢃ1): 3267 (br, 2NH), 3068 (C–Harom), 2915, 2852 (C–Haliph), 1658 (C ¼ NH), 1604,
1
1581 (C ¼ C), 1213 (P ¼ O), 1081 (P–O–C). H-NMR (400 MHz, DMSO-d6): 1.03 (t,
3H, J ¼ 6.8 Hz, CH3), 1.97 (s, 3H, CH3), 3.79–3.87 (m, 2H, OCH2), 4.95 (s, 1H, H-4),
7.11–7.86 (m, 10H, Ph–H), 9.94 (s, 1H, NH), 11.52 (s, 1H, NH). 13C-NMR (100 MHz,
DMSO-d6): 12.0 (CH3), 16.1 (CH3), 34.2 (C-4), 60.2 (OCH2), 81.1 (C-4a), 117.7 (C-3a),
120.4 (C-20,60phenyl), 124.5 (C-400phenyl), 126.0 (C-40phenyl), 127.5 (C-300,500phenyl), 128.3 (C-
200,600phenyl), 129.3 (C-30,50phenyl), 135.9 (C-100phenyl), 140.0 (C-10phenyl), 142.6 (d,
J ¼ 150.9 Hz, C-5), 144.9 (C-3), 151.2 (C-8a), 159.5 (C-7a). 31P-NMR (162 MHz, DMSO-
d6): 16.2 ppm. MS (m/z, I %): 420 (Mþ, 16%). Anal. Calcd. for C22H21N4O3P (420.21):
C, 62.85%; H, 5.03%; N, 13.33%. Found: C, 62.59%; H, 4.88%; N, 13.01%.
Conclusion
We suggested a simple, efficient, one-pot, three-component method for the construction
of new functionalized pyrano[2,3-c]pyrazolyl and pyrrolo[30,20:5,6]pyrano[2,3-c]pyrazolyl
phosphonates, 2-sulfido(oxido)-pyrazolo[40,30:5,6]pyrano[2,3-d][1,3,2]diazaphosphinines
and pyrazolo[40,30:5,6]pyrano[3,2-d][1,2]azaphospholes. The suggested pathway
depended on reaction substrate 3 with some phosphorus reagents under different condi-
tions. The synthesized compounds were screened in vitro for their antimicrobial, anti-
oxidant, and anticancer activities. Both compounds 6 and 7 recorded potent
antibacterial and antifungal activities, while compounds 4 and 6 showed promising anti-
oxidative properties. For antiproliferative activity, compounds 4 and 9 showed the most
potent cytotoxic effects.
Full experimental details and spectral data of the synthesized compounds can be
accessed on the publisher’s website.
Funding
The authors extend their appreciation to the deanship of scientific research at King Khalid
University for funding this work through research groups program under grant number (RGP.2/
2/42).
ORCID
Noha M. Hassanin
http://orcid.org/0000-0001-8380-8432

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