Phosphotungstic acid - Jeffamine? hybrid catalyst for one-pot Biginelli reaction starting from benzyl alcohol

Article abstract of DOI:10.1016/j.apcata.2020.117734

Herein, we report the catalytic action of phosphotungstic acid - Jeffamine? hybrid (PTA-Jeffamine?) for the synthesis of dihydropyrimidinones (DHPMs) starting from benzyl alcohol by Biginelli reaction in water in a one-pot system. Hydrogen peroxide was used as oxidizing agent instead of the conventional nitrate salts. A two-step and a one-step protocol was used to evaluate the catalytic activity. Both processes showed appreciable results with the one-pot one-step protocol demonstrating appreciable yield (87 percent) of DHPM within 5.5 h at pH 7.5. No acid was added and the pH was adjusted by controlling ratio of PTA/ Jeffamine?. Catalytic results were compared with literature reports for one-pot one-step Biginelli reactions starting from benzyl alcohol. Biginelli products were characterized by UV-visle–vis, FT-IR, NMR spectroscopy. DHPMs formed from the two-step process demonstrated appreciable anticancer activity. The present work may lead to the development of industry-friendly, non-toxic and scalable catalyst for one-pot Biginelli reactions.

Full text of DOI:10.1016/j.apcata.2020.117734

Journal Pre-proof
Phosphotungstic acid - Jeffamine® Hybrid Catalyst for One-Pot Biginelli
Reaction Starting from Benzyl Alcohol
Thangamani Suppan, Hema Priya Mahendran, Sankarganesh
Jeyaraj, Kallol Mohanta, Rama Ranjan Bhattacharje
PII:
S0926-860X(20)30327-6
DOI:
https://doi.org/10.1016/j.apcata.2020.117734
APCATA 117734
Reference:
To appear in:
Applied Catalysis A, General
Received Date:
Accepted Date:
14 April 2020
7 July 2020
Please cite this article as: Suppan T, Mahendran HP, Jeyaraj S, Mohanta K, Bhattacharje RR,
Phosphotungstic acid - Jeffamine® Hybrid Catalyst for One-Pot Biginelli Reaction Starting
from Benzyl Alcohol, Applied Catalysis A, General (2020),
doi: https://doi.org/10.1016/j.apcata.2020.117734
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®
Phosphotungstic acid - Jeffamine Hybrid Catalyst for One-Pot Biginelli Reaction Starting
from Benzyl Alcohol
Thangamani Suppan, Hema Priya Mahendran, Sankarganesh Jeyaraj, [b] Kallol Mohanta ,[a]
[
a]
[b]
and Rama Ranjan Bhattacharjee,[ a,c*]
[
[
a]
b]
PSG Institute of Advanced Studies, Coimbatore, Tamil Nadu 641 004, India
PSG Center for Molecular Medicine and Therapeutics, PSG Institute of Medical Sciences &
Research (Affiliated to the Tamil Nadu Dr MGR Medical University), Coimbatore, Tamil Nadu
India
[
*c]
Amity Institute of Nanotechnology (AINTK), Amity University Kolkata, West Bengal 700135,
India
Correspondence to: Rama Ranjan Bhattacharjee, E-mail: ramaranjanb89@gmail.com
*
Graphical Abstract:
Efficient conversion of Biginelli product in one-pot one-step process with Phosphotungstic acid
immobilized over Jeffamine as supported catalyst
1

Highlights

In the present manuscript we have for the first time successfully prepared Biginelli
®
compounds at almost neutral pH (7.5) with the PTA-Jeffamine catalyst in aqueous
medium in a one-pot one-step process.


The catalyst was effective both in the two-step as well as one-step one pot reaction
methodologies starting from BzOH.
Both processes showed appreciable results with the one-pot one-step protocol
demonstrated appreciable yield (87%) of DHPM within 5.5 h at pH 7.5.
No acid was added and the pH was adjusted by controlling ratio of PTA/ Jeffamine®.
The catalytic results were compared with literature reports for one-pot one-step Biginelli
reactions starting from benzyl alcohol.




The catalyst was easily separated from the products though a simple freezing process.
DHPM obtained from the one-pot two-step process demonstrated anti-cancer activities.
®
The PTA-Jeffamine catalyst showed better performance compared to other POMs having
Keggin structure.
1
13


The Biginelli products were well characterized with UV-Vis, FTIR and H and C NMR
spectroscopy.
The use of hydrogen peroxide as oxidant deserves a special mention in the present
manuscript. It can replace conventional oxidizing agents containing metal salts like
nitrates.
2


The work presented in this manuscript may pave ways to the development of industrial
catalysts and environmentally friendly reaction protocols.
Abstract
Herein, we report the catalytic action of phosphotungstic acid - Jeffamine® hybrid (PTA-
Jeffamine®) for the synthesis of dihydropyrimidinones (DHPMs) starting from benzyl alcohol by
Biginelli reaction in water in a one-pot system. Hydrogen peroxide was used as oxidizing agent
instead of the conventional nitrate salts. A two-step and a one-step protocol was used to evaluate
the catalytic activity. Both processes showed appreciable results with the one-pot one-step protocol
demonstrating appreciable yield (87%) of DHPM within 5.5 h at pH 7.5. No acid was added and
the pH was adjusted by controlling ratio of PTA/ Jeffamine®. Catalytic results were compared
with literature reports for one-pot one-step Biginelli reactions starting from benzyl alcohol.
Biginelli products were characterized by UV-visible, FT-IR, NMR spectroscopy. DHPMs formed
from the two-step process demonstrated appreciable anticancer activity. The present work may
lead to the development of industry-friendly, non-toxic and scalable catalyst for one-pot Biginelli
reactions.
Keywords: Phosphotungstic acid; Jeffamine; Catalyst; Biginelli;
Dihydropyrimidinones; One-pot reaction
INTRODUCTION
3

Biginelli reaction is an industrially important process used for preparation of
dihydropyrimidinones (DHPMs), commonly known as Biginelli compounds. These are well
known for their pharmacological and biological applications [1, 2]. They are used in the field of
therapeutics [3], and bio-organic chemistry [4]. Most of the Biginelli derivatives are used for their
antifungal, antiviral, anticancer, antibacterial, anti-inflammatory, and antihypertensive properties
[
2]. In 1893, the Italian chemist Pietro Biginelli reported the acid-catalyzed cyclo condensation
reaction of benzaldehyde, ethyl acetoacetate and urea to form 3, 4-dihydropyrimidine-2 (1H)-one
DHPM) with 60-70% yield, in ethanol medium [5]. Concentrated HCl was used as catalyst. Since
(
then, Biginelli reactions have come up a long way with extensive literature reports in terms on the
effects of substituent, solvents, reactions conditions and catalysts. Notably like many other
reactions, the Biginelli reaction has its own drawbacks or limitations. The reaction suffers from
low yields, long reaction time and harsh reaction conditions [4]. Many research groups have tried
variety of acid catalysts like Lewis [6], Bronsted acids [7], ionic liquids [8,26], magnetic catalysts
[
9], magnetic ionic liquids [10] and also various natural catalysts like Pineapple Juice [11], apple,
pomegranate, grape juice [12], Garlic Glove [13], animal bone [14], heterogeneous catalysts
15,27,28,29,30] and zeolites [16]. Additionally, a large number of homogeneous catalysts have
been reported such as Mg (NO [17], Pb (NO [18], LaCl .7H O [19], P [20]. Biginelli
[
3
)
2
3
)
2
3
2
2
O
5
reactions in fruit juice medium was reportedly the most eco-friendly method for the synthesis of
DHPM [12,13].
With the advent of green chemistry, there is considerable emphasis on developing
environmental friendly protocols. Hence, an effective one pot protocol for Biginelli reaction can
be industrially feasible. Many one-pot reactions have been tested for Biginelli with various
catalysts [27, 31-35]. It has been observed in literature reports that for reactions starting from
4

benzyl alcohol (BzOH), the one-pot one-step process shows no yield at all [27, 31-35].
Alternatively, a one-pot two-step process has been shown to work well as it allows full conversion
of the BzOH into benzaldehyde (BzH) followed by the addition of urea and Ethyl acetoacetate
(EAA) [31-35]. Hence development of an efficient catalyst that favors one-pot one-step process
will be a great value addition to the well established Biginelli reactions.
Until now many metal based catalysts have been tested for one-pot one-step Biginelli
reaction. These catalysts often suffer from the leaching metals [36, 37]. It has been found that
presence ~ 5% metal leachates in the product can cause toxicity issues as Biginelli compounds are
more used in pharmaceutical studies. In this context, polyoxometalates (POMs) or inorganic
polyacids may be preferred over other metal based catalysts. These polyacids are interesting metal
based catalysts [21, 22]. The advantage of POMs over other inorganic/organic catalyst is the
stability of the metal center present in the cage. It does not show leaching of metal ions in the
reaction medium and hence can be superior compared to other metal-ligand complexes as catalysts
[
23]. Cost and scale of usage often limit their applications. In 2004 Tong-Shou Jin et al., have
reported phosphotungstic acid (PTA) catalyst in presence of ethanol solvent to produce the desired
Biginelli compound (Yield 94%) [24]. Mishra et al. have been reported in PTA used as a catalyst
for one-pot synthesis of Biginelli reaction under microwave irradiation (Yield 92%) [25]. Zhong
Zhang et al. highlighted [Cu12 (BTC)
0% yield in solvent free condition [38]. Recent report by Chavan et al. featured
40] as catalyst which on ultra sonication shows 93% yield where the reaction was
8
3
][H PW12O40] as catalyst for Biginelli reaction and showed
9
H
4
[
PMo11
1
V O
performed in ethanol solvent system [39]. Such homogeneous catalysis often suffers from
drawbacks related to separation and purification of products. Recently, various supported-POM
based catalysts were tested in Biginelli reaction. J. S. Ghomi et al. reported Biginelli reaction with
5

dendrimer-attached PTA nanoparticles immobilized on nano silica as catalyst under
ultrasonication [40]. Few of the listed POM based catalysts with their respective yield % are
following: H14NaP
5
W
29MoO110@SiO
2
(PASi) [82%], ZIF@9(NH
2
) [32%], PTA@ZIF 9(NH )
2
[
85%] [41] , PTA@MIL-101 [90%] [42] were reported. The advantage of supported POM catalyst
is the minimum usage of POM often dispersed all over the support. Hence a small amount of POM
is effective as supported catalyst and thus industrially affordable.
Though many reports have shown POMs working as efficient catalyst for Biginelli
reactions, there are very few reports where POM based catalyst have shown efficiency to catalyze
one-pot one-step process starting from BzOH. Hence in this manuscript, we focus our work on the
development of POM based catalyst that can show efficient catalytic effect in a one-pot one-step
process starting from BzOH. The efficiency of the catalyst was tested at various pH values in a
two-step and a one-step one-pot reaction methodology. The easy availability of the supporting
®
polymer (Jeffamine ) as industrial byproduct and the efficient utilization of POMs in the catalyst
formulation might help in the development of smarter industrial catalysts.
FIGURE 1. Structure of PTA and Jeffamine®.
RESULTS AND DISCUSSION
6

®
FIGURE 2. Pictorial representation of the liquid-catalyst of PTA- Jeffamine mixture
For heterogeneous catalysis, often the disadvantage is the interface of the reaction phase
with that of catalyst [43]. We have recently reported a novel catalyst that works well in the interface
of aqueous and non-aqueous solvents. The catalyst is a hybrid of POM and a low molecular weight
co-polymer, Jeffamine [44]. The POM immobilized with Jeffamine as support demonstrated stable
structure-property relationship at a wide range of pH and temperature. Amphiphillic nature of the
polymer support allowed the POM to work as effective catalyst in both the aqueous and organic
phase. We showed that the Venturello ion formed due to a complex between the POM and peroxide
stabilized by Jeffamine participated in the reaction and performed well as a phase transfer catalyst
too [44]. The material was also shown to perform well as a multifunctional catalyst for various
types of reactions like selective oxidation of BzOH to BzH or the synthesis of porphyrin in water
[
44, 45]. In the current manuscript, we have tried to demonstrate the activity of the POM-polymer
catalyst and its effectiveness for Biginelli reactions.
Characterization of the catalyst
7

®
Jeffamine belongs to a class of green di-block copolymer with ethylene oxide (EO) and
propylene oxide (PO) moieties. The polymer is a class of low molecular weight polyetheramine
obtained as byproduct from industry. Jeffamine® consist of temperature dependant phase
separation properties, wide range of molecular weights, amine functionality, type of repetitive unit
distribution and good low temperature properties. The preparation process of the catalyst is easy
®
and scalable. Our group has reported that the PTA-Jeffamine can catalyze the selective oxidation
of BzOH to BzH in water. However, best conversion and selectivity of BzH was achieved at pH
®
7
.5 [44]. Strong electrostatic interactions between the ammonium cation of Jeffamine and anionic
oxygen of PTA may be responsible for the enhanced solubility and stability of PTA in water phase
®
[
14]. The final pH of the medium was dependent upon ratio of POM/Jeffamine . No additional
acid or base was added to the reaction medium.
The prepared catalytic mixtures of PTA-Jeffamine® were characterized by various
techniques. UV-Visible spectrophotometer was used for absorbance measurement in aqueous
medium in the range of 200-800nm. The structure of PTA remained almost unchanged during
catalyst preparation as evident in UV-Vis and FTIR data in Figure S5 (SI). Further structural
analysis was done with X-ray diffraction and Raman spectroscopy. The morphological studies by
FE-SEM (Figure S6) have already been reported in our recent work [44, 45]. Other POM-
®
Jeffamine catalyst were also prepared and characterized (Figure S7).
®
The XRD patterns of PTA and PTA-Jeffamine catalysts are shown in Figure 3. XRD
patterns of PTA represent the typical Keggin polyanion characteristics. The primary structure of
®
PTA and PTA-Jeffamine catalysts seemed identical when observed in their XRD patterns (Figure
®
3
). PTA-Jeffamine prepared at different pH may cause variations in the intensity of the peaks.
The XRD patterns for the catalyst prepared at pH 7.5 and 8.5 indicated presence of PTA crystallites
8

®
well dispersed in the Jeffamine . Sharp diffraction peaks at 2Ɵ values of 21°, 26°, 33° and 36°
were indexed with reported data (JCPDS 50-0304 and other reported data JCPDS 50-0657)
®
represents the cubic structure of Keggin PTA [46]. In Figure 3, the Jeffamine alone occurs that
an observable diffraction peak around 2휃 20° confirms the amorphous structure. A few not
noticeable peaks of PTA crystal and mixtures suggest that the attached PTA molecule has
®
monolayer dispersion on the surface of Jeffamine core [46].
®
FIGURE 3. XRD patterns of PTA and PTA-Jeffamine mixtures at different pH ratio.
®
PTA and PTA-Jeffamine catalysts were further characterized by Raman spectroscopy to
identify the structural changes of the prepared catalysts. The Raman spectrum of the Keggin
-1
heteropoly acid of pure PTA exhibits a strong band between 909 and 1005 cm (Figure 4). Similar
9

observation was reported by S .R. Matkovic et al. and co-workers [47]. The Raman band at 1005
-1
cm attributed to the symmetrical stretching of the terminal tungsten oxygen (W=O) within the
octahedral WO unit was observed in the spectrum. The presence of PW11 unit was indicated from
6
-1
the band at 909, 513 and 218 cm corresponding to Vs (W = Od), Vas (W-Od), Vas (W-Ob-W),
Vs (W-Oc-W) and V (W-Oa) respectively. Where Oa, Ob, Oc and Od are the oxygen atoms
®
attached to phosphorus and tungsten. In case of addition of Jeffamine to PTA, certain spectral
changes were observed (Figure 4). The red shifts of the Raman peaks may be caused by the surface
®
modification of PTA by Jeffamine . Peaks were observed in all mixtures in the spectral ranges
-1
between 113 and 191 cm . It shows the lattice vibration in crystals and strong Raman peaks.
1
0

®
FIGURE 4. Raman spectra of a) PTA b) Jeffamine c) pH 4.5, d) pH 6.5, e) pH 7.5 and f) pH 8.5
®
of PTA-Jeffamine mixtures.
®
Catalytic performance of PTA-Jeffamine in two step one-pot reaction system
In the two step reaction pathway, BzH was prepared in the first step by oxidation of BzOH
®
in water using PTA-Jeffamine catalyst and hydrogen peroxide as oxidant. After 1 h, to the same
1
1

mixture, proportionate amounts of urea and EAA were added. All the three components were
added in a one-pot condition as shown schematically in Figure 5. Subsequent addition of ethanol
to the reaction medium resulted in the formation of DHPM.
®
FIGURE 5. Schematic illustration of the catalytic behavior of PTA-Jeffamine mixtures in
dihydropyrimidinones synthesis via BzOH oxidation reaction process in two step one-pot
condition.
However the yield of the DHPM varied depending up on the pH of the medium, that is, the amount
®
of Jeffamine as shown in table 1. Effect of pH on the reaction was studied by keeping PTA
®
constant and varying the volume of Jeffamine . The reason behind the pH dependency may be
related to the structural changes of the catalyst as observed in our previous report at pH 7.5. The
phase transfer behavior of the catalyst at pH 7.5 may also contribute to the better yield.
®
Table 1. The yield calculations of 3, 4-dihydropyrimidinones at different PTA-Jeffamine pH ratio
1
2

S.No Different pH catalyst
DHPM
Yield %
M.P
Solutions
PTA-Jeffamine pH -3.5
PTA-Jeffamine pH -4.5
PTA-Jeffamine pH -5.5
PTA-Jeffamine pH -6.5
PTA-Jeffamine pH -7.5
PTA-Jeffamine pH -8.5
®
1
2
3
4
5
6
7
8
.
.
.
.
.
.
.
.
White crystal
White crystal
White crystal
White crystal
White crystal
White crystal
White crystal
White crystal
28%
41%
79%
74%
96%
76%
69%
32%
200˚C
196˚C
182˚C
202˚C
193˚C
190˚C
196˚C
165˚C
®
®
®
®
®
PTA
Jeffamine®
Characterization of DHPM
FT-IR spectra of DHPM obtained from Figure 6 shows two peaks for NH at first and third
-1
position in the range 3244 - 3115 cm . The ester carbonyl stretching frequency was observed in
-1
-1
the range of 1600- 1750 cm . FT-IR peak for C=O was observed in the range of 1450-1600 cm .
The C-N bond in dihydropyrimidinones ring corresponds to peaks in the range of 1150-1350 cm-
1
-1
and for C-O-C stretch at 1290.38 and1240 cm respectively. FT-IR spectroscopic data for DHPM
obtained at different pH is shown in Figure 6a. The experimental procedure of controlled reaction
is provided in SI and spectral data Figure S1. Table S1 shows the FT-IR spectral ranges for pure
DHPM and catalyzed reaction products of DHPMs at pH-7.5. The results from the reactions
®
separately done as controlled reactions with pristine PTA and Jeffamine are shown in table 1.
The products obtained from the controlled reactions were characterized by IR, UV and NMR
spectroscopic studies (Figure S3&S4).
Controlled reaction in absence of POM was also
performed (Table S2). In the UV spectrum, the λmax values of the DHPMs were observed at 284
and 245nm. The absorption maximum occurs at 284nm because the pair of electrons on nitrogen
atom is in conjugation with the pi bond system of the benzene ring. The peaks may originate from
n→π* and π→π* transitions from C=O group respectively. The pure DHPM after recrystallization
1
3

shows λmax at 284 nm. The absorption peak at 284 nm observed for pure DHPM and synthesized
DHPM products at different pH 3.5, 4.5, 5.5, 6.5, 7.5 and 8.5 are shown in Figure 6b.
NMR studies were performed to determine formation of DHPM and to determine if
impurities are present (Figure 6c). The quartet of two protons and the triplet of three protons were
seen around 4.08 ppm and 1.17 ppm, respectively. Both had a coupling constant (J) equal to 7.2
Hz, indicating presence of ethyl groups. The singlet of three hydrogen atoms around 2.34 ppm
indicated the presence of the isolated methyl group. At 8.17 ppm and 7.26 ppm, multiplets made
of two protons were indicative of an aromatic system, may be the pyrimidine aldehyde. A multiplet
of three protons was observed around 7 ppm, again suggesting aromaticity. The peak for 7 H
atoms at 7.26 ppm is due to peaks from the benzylic H and also from the two amide hydrogen. At
3
.7 ppm the observed singlet consisting of 6 protons was indicative of the two CH groups making
3
up the dimethylamino moiety, while the singlet around 2.3 ppm made up of 3 protons indicated
presence of an isolated methyl group. At 7 ppm aromatic protons around with each peak being
comprised of 2 H atoms. It should be noted that around 8 ppm and 7.32 ppm, where there should
be probable indication of singlet’s consisting of only one H-proton, there are small peaks; however,
their diminished sizes warranted no significant integration value, despite their apparent presence.
The products were analyzed with NMR spectroscopy for structural elucidation (Figure 6c). Table
1
3
S4 shows the Chemical shift values of DHPM pH 7.5. In the C NMR spectrum (Figure S5 ), the
chemical shift at δc 166.1 and 152.9 were due to carbonyl carbon, The chemical shift at δc 152.9,
1
48.6, 144.8, 128.9, 128.0 and 126.6, were on account of aromatic carbon. The chemical shift at
δc 100.1 indicated presence of C=C. The chemical shift at 54.3, due to C-4 carbon and δc 60.0 for
1
3
-
CH
2
carbon was observed. The chemical shifts at δc 18.0, and 14.3 due to methyl carbon. The C
NMR and GC-MS spectrum data were shown in Figure S2. Mass spectrum of the DHPM
1
4

compound catalyzed by pH 7.5 recorded and its molecular ion at m/z 256, 213, 185, 171, 129, 73,
0, 43, 41.
6
FIGURE 6. a) IR spectra of compound DHPM and different pH catalyzed reaction products of
DHPMs b) UV-Visible spectra of compound DHPM and different pH catalyzed reaction products
of DHPMs c) NMR spectra of compound DHPM and different pH catalyzed reaction products of
DHPMs.
1
5

The above mentioned FT-IR and NMR analysis of the products obtained from the two step
®
one pot reaction suggest that PTA-Jeffamine derivative was more effective than only PTA as
catalyst. Because in only presence of PTA in oxidation reaction there was no formation of
benzaldehyde and the detailed section was reported in our previous work [44]. It was assumed that
may be PTA was working efficiently towards the first step of the reaction (i.e., oxidation) in
®
presence of Jeffamine , it was also capable to formulate the second step (Multicomponent) more
efficient. The controlled reaction also carried out without any catalyst for 5.5h, monitored and
found that there is no product formation. The reaction mixture was characterized by UV-visible,
1
13
FT-IR, H and C NMR spectroscopy mentioned in SI (Figure S1).
Anti-cancer analysis
Non-small cell lung carcinoma (NSCLC) is a devastating cancer arising in the lung
epithelial cells. NSCLC accounts for about 85% of lung cancer [48]. This cancer remains
aggressive with increasing incidence and poor survival rate despite of current multimodal
treatment. It usually grows and spreads slowly compared to small cell lung cancer and remains as
a common lung carcinoma [49]. DHPM compound and its derivatives were mostly tested on
various cancer cell lines including breast, liver, ovarian, gastric, kidney, skin, colorectal, prostate,
central nervous system, cervical, endothelial, pancreas, blood, lymphoma, myeloma,
Pheochromocytoma and lung [50]. The U251 and OVCAR-03 were found to be more sensitive to
DHPM derivatives which affected the cell growth at IC50 <10µg/mL [51]. In this manuscript, the
®
DHPM synthesized by the PTA-Jeffamine catalyst (pH 7.5) was tested against A549 cell lines as
a model for NSCLC. This was also done to test the efficiency of the synthesized DHPM. Results
of the DHPM test compound produced a dose and time dependent cell cytotoxicity against A549
1
6

cell line. After 24 h and 48 h of treatment, the compound induced up to 50% cell death at different
concentrations in A549 cell line. A significant decrease in cell viability at higher concentrations
(i.e., above 200µM) was observed in A549 cell line in Figure 7. To establish the optimal treatment
concentration for the drug, its IC50 values were calculated and compared. The half maximal
inhibitory concentration of DHPM compound against A549 cells was shown at 202.6µM, with an
2
R value of 0.944. Analyzing the percentage of viability and percentage of growth inhibition of
DHPM compounds shows that the test compounds are dose dependent and decreases the cell
viability as the dose is increased. PBMCs viability was found to remain more than 70% even after
7
2 h of drug treatment. The percentage of cell viability at different concentrations of DHPM at
PBMCs was calculated to identify the specificity of DHPM compound with cancer cells (Figure
). A549 non-small cell lung cancer cell line untreated control cells and cells treated with 100µM
7
DHPM were shown in Figure 7a & b. There was no significant higher cytotoxic effect observed in
PBMCs at IC50 concentration against A549 cells. Rather, the percentage of viability remained
higher in PBMCs at all concentrations compared to untreated A549 cells. Though there were few
variations in the pattern of cell growth inhibition at different concentrations, the percentage ranged
between 20% - 35% (rarely >35%). The time dependent cytotoxicity of DHPM compound was
evaluated by treating the cells with drugs for 24 and 48 h. Cell deaths in 48 hrs treatment were
higher when compared to 24 h treatment as shown in Figure 7d.
1
7

FIGURE 7. A549 non-small cell lung cancer cell line a) Untreated control cells b) Cells treated
with 100µM DHPM. c) Dose dependent cytotoxicity of DHPM against A549 and PBMC cell lines
d) Time dependent cytotoxicity of DHPM against A549 cell lines. [(***- extremely statistically
significant, **- statistically very significant)]
An in vitro examination of cytotoxicity induced by DHPM compound was performed.
Results indicate that the drugs induce a dose dependent cell death. The cell viability decreased
gradually as the concentration has been increased. The percentage of cell viability and half
maximal inhibitory concentration significantly reduced in 48 h treatment compared to 24 h
treatments. We used healthy PBMCs to evaluate any cytotoxicity of DHPM against healthy host
cells. DHPM analogue was non-toxic to PBMCs on comparing the activity of DHPM to A549. In
1
8

the published work of Amany S. Mostafa, Khalid B. Selim, 2018, the DHPM derivatives exhibited
lower toxicity against MRC-5 cells at IC50 44.16 and 32.04µM [52]. But the toxicity of our novel
synthesized DHPM remained low even at the half maximal inhibitory concentration (<20%) and
more than 80% of cells remained viable after the treatment of 72 h. In the research work published
by Venugopala et al., the DHPM derivatives exhibited up to 20% growth inhibition at 50µg/mL
[
53].
Comparing the DHPM derivatives treated against breast cancer cell line MCF7 and MDA-
MB-231, the novel synthesized DHPM molecule has proven to show significant decrease in cell
viability gradually in all concentrations tested [54]. The half maximal inhibitory concentrations
have also fallen between 200µM - 250µM, whereas in the research work published by Guido et
al., the concentrations used were up to 1Mm [54]. The DHPM derivatives exhibited over 80% of
cell growth inhibition with IC50 around 6 to 35µM. The monastrol mimic Biginelli DHPM
derivatives exhibited cytotoxicity against HepG2 with half maximal inhibitory concentration of
1
20.62µg/mL and it exhibited weak toxicity towards HeLa cell lines with IC50 200µg/mL [55].
®
Though the DHPM compound obtained by the PTA-Jeffamine catalyst was found to have
moderate activity against cancer cell lines at lower concentrations, it is more specific only to cancer
cells. At the same concentrations, the compound exhibited very low toxic effect on normal cells
(PBMCs). In addition to these, further studies can be done in order to elucidate the molecular
pathways in NSCLC cancer cell line death induced by DHPM compound.
1
9

Catalytic reaction with other POMs
®
In order to compare the catalytic activity of PTA based Jeffamine catalyst with other
Keggin structure POMs, the following reactions were performed as shown in table 2. The two step
one-pot Biginelli reaction starts with 1.9M of BzOH, 30% H
2
2
O (3.9 M) in presence of POM-
®
0
Jeffamine catalyst with refluxing temperature of 90 C and time 1.5 h. In that same pot condition,
addition of 2.1M of ethyl acetoacetate and 1.4M of urea was done. After 2h of the reaction, 20ml
of ethanol was added to the above reaction mixture and again reflux at 90˚C for 2 h. The yellow
color of the homogeneous reaction mixture was kept for crystallization. The PMA-Jeffamine®
catalyst produces the 37% yield and the other two POM catalysts showed no product formation.
®
The results indicated that the Keggin structure of PTA supported on Jeffamine is the better
catalyst for DHPM production in water.
®
Table 2. The yield calculations of 3, 4-dihyropyrimidinones at different POM-Jeffamine pH ratio
S.No Different pH; catalyst
DHPM
Yield %
M.P
Solutions
PMA-Jeffamine pH-5.7
Phosphomolybdic acid
®
1
2
3
.
.
.
White crystal 37%
No product formation
No product formation
196˚C
®
STA-Jeffamine pH-7.8
Silicotungstic acid
H
3
PMO12.SiO
2
-Jeffamine®
pH-6.7
®
Catalytic performance of PTA-Jeffamine in one-step one-pot reaction system
®
After successfully testing two step one-pot process, the PTA-Jeffamine catalyst was tested
for the one-pot one-step process starting from BzOH. The reaction was initiated with the addition
of 1.9M of BzOH, 30% H
2
2
O (3.9 M), 2.1M of ethyl acetoacetate, 1.4M of urea and catalyst in a
2
0

0
single reaction vessel followed by refluxing at 90 C for 5.5 h (Figure 8). Three different ratio of
®
PTA-Jeffamine was tested as catalyst separately. PTA was kept constant and the amount of
®
Jeffamine was varied resulting in three different pH (6.5, 7.5 & 8.5). No acid was added and the
®
pH was adjusted by controlling ratio of PTA/ Jeffamine . The clear yellow colored homogeneous
reaction mixture was kept overnight for crystallization. The white crystal mixture obtained was
washed thoroughly with ethanol and water mixture (2:1) until the product turned pure white and
was further recrystallized from ethanol.
®
FIGURE 8. Schematic illustration of the catalytic behavior of PTA-Jeffamine mixtures in DHPM
synthesis via BzOH oxidation reaction process in one-pot one-step protocol.
Recrystallized compound was characterized by IR, NMR and mass spectroscopy (Figure S9a &
b). The yield % calculated for the three reactions indicate product formation only at pH
7
.5.Repeated reactions reproduced the same results. Such selective formation of DHPM can be
2
1

®
related to the activity of PTA-Jeffamine catalyst prepared at pH 7.5. To explain the observation
we consider a few factors that may have influenced the reaction. We may assume that the
Venturello ion has a considerable effect on stabilizing the product as has been shown in our
previous report on the selective conversion of BzOH to BzH. Secondly we understand that the
very fast production of BzH from BzOH in the reaction medium due to the catalyst can contribute
substantially to the yield [44]. Following Le Chatelier's principle, if the concentration of substrate
at any point of time in the reaction medium is high, the forward reaction also proceeds fast to
completion. As the rate determination step for Biginelli reaction is either the formation of the
iminium ion or the carbenium ion intermediate, the fast build up of BzH in the reaction medium
can influence the equilibrium leading up to the intermediate formation [56,57]. High
concentrations of the intermediates may subsequently control the overall kinetics of the Biginelli
process. The stabilizing effect of the Venturello ion against over oxidation of BzH to BzAc
provides effectively high concentrations of BzH in the reaction medium that favors the may favor
the above equilibrium [44]. Further we may also predict that the worm-like micelles of the catalyst
formed at pH 7.5 (as shown in our previous reports 44, 45) may be the reason for the catalyst to
be more active for the one step one-pot reaction. Table 3 shows a comparative study with literature
reports on one-pot one-step processes starting from benzyl alcohol. Though the yield of our
reaction is high compared to the other reports and our reactions conditions are more
environmentally friendly (pH 7.5) compared to other reaction conditions as shown in table 3.
Table 3. Reaction starting from benzyl alcohol studies on two-step and one-step one pot system.
2
2

S.No
Catalyst
pH
Time/temp Oxidizing Yield % Ref
agent
°
C
Two-step one pot system
1
2
3
4
5
6
[MIMPS]
3
PW12
O
40 acidic 10-20 min, NaNO
3
90
81
94
82
93
96
[31]
[32]
[33]
[34]
[35]
7
00W
CuSO
4
x H
2
O in
5.2
24 h
60
-
PBS
[Hmim]HSO
4
acidic 2-4 h
NaNO
3
8
0
Bi(NO
3
)3.5H
2
O
-
3h
Bi(NO
3 3
)
9
0
Al(NO
3
)
3
· 9 H
2
O
-
15 min,
-
8
0
PTA-Jeffamine®
pH -7.5
7.5
5.5 h
90
H
O
O
Present
2
2
manuscript
One-step one pot system
7
PTA-Jeffamine®
pH -7.5
7.5
5.5h
90
H
2
2
87
Present
manuscript
2
3

Characterization of DHPM from one-pot one-step process
FTIR, NMR and ESI-Mass spectra were carried out to characterize the Biginelli product obtained
from one-pot one-step process performed at pH 7.5 FTIR and NMR spectral characterization data
were provided in Figure S9. The ESI-Mass spectra of pH 7.5 catalyzed reaction product of DHPM
were shown the molecular ion at m/z 102, 165, 187, 188, 203, 266, 351, 360 and their mechanistic
representations of the mass spectra were shown in Figure 9.
FIGURE 9. ESI Mass spectra of one-pot DHPM
®
Characterization of the PTA- Jeffamine catalyst after reaction
After the Biginelli reaction was over, the phase separated aqueous part containing the PTA-
®
Jeffamine catalyst was kept under observation in normal laboratory condition. The aqueous part
separated after first reaction cycle dried and used for characterization (Figure 10a). Due to strong
®
electrostatic interactions with the PTA molecules, Jeffamine can remove most of the PTA
molecules from organic part and bring them into the aqueous part. FTIR spectroscopy was used to
2
4

®
detect the presence of PTA along with Jeffamine in the phase separated aqueous part (Figure
-1
-1
1
0b). FTIR data shows peaks at 1247 cm (ether) and 3435 cm (amine) that indicate presence of
®
-1
Jeffamine along with four characteristic bands of PTA at 1087,948,883,842 cm . The four FTIR
peaks of PTA were observed to be slightly blue shifted as compared to that of pure PTA-
®
Jeffamine catalyst. The shift in FTIR peak position indicates changes in characteristics of the
bridge-oxygen present in PTA. The characteristic changes observed in the FTIR data of PTA can
be attributed to the presence of trace amounts of BzA in the aqueous part. BzA can be detected in
-1
the phase separated aqueous part from FTIR data. Sharp FTIR peak at 1714 cm corresponding to
–
CO of COOH. Presence of BzA resulted in lower pH values of the extracted aqueous part from
the first reaction cycle (pH 7.5). The same may also interfere in the catalytic process resulting in
lower conversion of BzOH and lower selectivity towards BzH. After recover of catalysts from
separation, the Tollen’s tests performed with the used catalyst present in the phase separated
organic part in Figure S8.
2
5

FIGURE 10. a) Catalyst separation process b) IR spectra of the aqueous and organic part of the
catalyst recovery process.
CONCLUSIONS
Literature reports have suggested that Biginelli reactions are mostly acid catalyzed. Such
dependency on acidic medium may affect the performances of the Biginelli products in anticancer
and other biological applications. In the present manuscript we have for the first time successfully
®
prepared Biginelli compounds at almost neutral pH (7.5) with the PTA-Jeffamine catalyst in
aqueous medium in a one-pot one-step process. The catalyst was effective both in the two-step as
well as one-step one pot reaction methodologies starting from BzOH. Both processes showed
appreciable results with the one-pot one-step protocol demonstrated appreciable yield (87%) of
DHPM within 5.5 h at pH 7.5. No acid was added and the pH was adjusted by controlling ratio of
®
PTA/ Jeffamine . The catalytic results were compared with literature reports for one-pot one-step
Biginelli reactions starting from benzyl alcohol. The catalyst was easily separated from the
products though a simple freezing process. DHPM obtained from the one-pot two-step process
®
demonstrated anti-cancer activities. The PTA-Jeffamine catalyst showed better performance
compared to other POMs having Keggin structure. The Biginelli products were well characterized
1
13
with UV-Vis, FTIR and H and C NMR spectroscopy. The use of hydrogen peroxide as oxidant
deserves a special mention in the present manuscript. It can replace conventional oxidizing agents
containing metal salts like nitrates. It also helps in forming the active Venturello ion in association
with the catalyst that stabilizes BzH. The work presented in this manuscript may pave ways to the
development of industrial catalysts and environmentally friendly reaction protocols.
EXPERIMENTAL
2
6

Preparation of PTA-Jeffamine®
®
0
.1 M of Jeffamine was taken and titrates against the 0.01M of PTA, the pH of the solution was
adjusted for values of 3.5, 4.5, 6.5, 7.5 & 8.5 and then the mixture was stirred overnight.
®
Figure 1 shows the structures of PTA and Jeffamine . In the present work, the catalytic effect of
®
PTA on synthesis of dihydropyrimidinones was studied in presence of Jeffamine in water.
Procedure for the two-step one-pot synthesis of dihydropyrimidinones
®
An aqueous solution of PTA (0.01 M) was drop wisely added to 0.1M of Jeffamine under
®
continuous stirring. pH of the medium depended on the volume of added Jeffamine as the amount
of PTA was kept constant. 1.9M of BzOH, 30% H
2
2
O (3.9 M) was added to the catalyst solution
0
and refluxed at 90 C for 1.5 h. In that same reaction vessel (one-pot), 2.1M of ethyl acetoacetate
and 1.4M of urea were added. After 2h, 20ml of ethanol was added to the above reaction mixture
and again refluxed at 90˚C for 2 h. The yellow color of the homogeneous reaction mixture slowly
turns to yellowish white solid. The reaction was monitored by TLC (ethyl acetate/ n-hexane). The
solid product was kept in the refrigerator overnight for crystallization. Sometimes the reaction
mixture was kept at room temperature overnight. The white crystal mixture was washed
thoroughly with ethanol and water mixture (2:1) until the product turned pure white and
recrystallized from ethanol. The solid product was characterized by FT-IR, UV-Visible
1
13
spectroscopy, and H, C NMR spectroscopy.
Material and methods
2
7

Benzyl alcohol, hydrogen peroxide, ethyl acetoacetate, urea was purchased from LOBA chemie
®
and were used without purification. Jeffamine was obtained as gift sample from Huntsman India
Ltd. Phosphotungstic acid (PTA), Phosphomolybdic acid (PMA), Silicotungstic acid (STA) was
purchased from Sigma and Fluka used as received without further purification. Ethanol was
purchased from China chemicals. Milli-Q water was used in all preparation. The standards and
the reaction mixture were spotted on TLC Silica gel 60 F254 purchased from Merck chemicals.
The cytotoxicity analysis materials and methodology mentioned in SI.
Characterization methods
The reaction mixture was spotted on TLC aluminium oxide 150 F254 neutral (Solvent:
Hexane/chloroform, 9:1) purchased from Merck. The solvent system is 9:1 Hexane/chloroform.
UV-Visible spectrum was recorded with Shimadzu UV-1800 spectrophotometer. The samples
were placed on sample holder with quartz window of 1cm path-length. The scanning range is 200-
8
6
00 nm. FT-IR spectra were recorded using Shimadzu IR affinity series 1S in the region of 4500-
-1
00cm . Samples were analyzed with KBr and ATR accessory. The products were analyzed with
1
13
NMR spectroscopy for structural elucidation. H and C NMR spectra were analyzed using Bruker
BioSpin GmbH. DMSO and CDCl was used as a solvent for analysis. X ray-Diffraction analysis
3
was performed using CuKα radiation, (k = 1.5406 A° at 40 kV and 30 mA, Empyrean, Malvern
Panalytical) for the 2θ measurement and scanned from 10-80° for patterning of the samples.
Raman spectroscopy was used for the structural elucidation of molecular structure of the samples.
-1
The spectral range was scanned from 200-3500 cm (WiTec alpha 300, Germany).
Declaration of interests
2
8

The authors declare that they have no known competing financialinterestsor personal relationships that
could have appeared to influence the work reported in this paper.
Acknowledgements
RRB thanks DST-WTI [DST/TM/WTI/2K15/60(G) – A] for providing financial support. RRB
®
thanks HUNTSMAN INDIA Ltd. for gifting the Jeffamine sample. We also thank PSG Institute
of Advanced Studies for providing basic research facilities.
References
[
[
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[
[
[
3] C. D. Graaff, E. Ruijter and Romano V. A. Orru.,Chem. Soc. Rev., 2012, 41, 3969–4009.
4] H. G. O. Alvim, E. N. D. Silva Juniorand B. A. D. Neto., J. Name., 2013, 00, 1-3.
5] G. C Tron, A. Minassi, and G. Appendino, European Journal of Organic Chemistry,
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011, (28), 5541–5550.
[
6] C. O. kappe, Acc. Chem. Res., 2000, 33, 879-888.
2
9