Four-component, one-pot synthesis of spiropyrazolo pyrimidine derivatives by using recyclable nanocopper ferrite catalyst and antibacterial studies

Article abstract of DOI:10.1080/00397911.2016.1244271

A simple, efficient, ecofriendly, and cost-effective method has been developed for the synthesis of 16 spiropyrazolo pyrimidine derivatives (5a–5p) by a four-component, one-pot reaction of pyrimidine-2,4,6(1H,3H,5H)-trione, 3-oxo-3-phenylpropanenitrile, hydrazine, and isatins (4a–4f) by using nanocopper ferrite catalyst (20 mol%) in water with excellent yields (73–91%). The isatin with electron-withdrawing groups gave products in high yields (5h and 5p). The present methodology offers an environmentally benign, cost-effective, high-yielding method with recyclable catalyst. The drug likeness or “drugability” of all the synthesized compounds were tested through rule of five (RO5) parameters. Compounds 5f, 5h, 5n, and 5p have shown one RO5 violation each. The compounds were screened for their antibacterial activity against human pathogenic bacteria wherein two of the synthesized compounds were found to possess significant antibacterial activity. Compounds showing RO5 violations had negligible antibacterial activity.

Full text of DOI:10.1080/00397911.2016.1244271

Four Component One Pot Synthesis of Spiro Pyrazolo Pyrimidine Derivatives by
Using Recyclable Nano Copper Ferrite Catalyst and their Antibacterial Studies
Ravi kumar Ganta1, Ramgopal A1, Chatragadda Ramesh2, Raghu Babu K1, Murali
Krishna Kumar M3, Venkateswara Rao B1
1Departmaent of Engineering chemistry, A.U C E (A), Andhra University,
Visakhapatnam, India, 2Department of Ocean Studies and Marine Biology, Pondicherry
University, Andaman & Nicobar Islands, India, 3A U College of pharmaceutical sciences,
Andhra University, Visakhapatnam, India
Corresponding Author: Ravi kumar Ganta, E-mail: gantaravichem@gmail.com
Abstract
A simple, efficient, ecofriendly and cost effective method has been developed for the
synthesis of sixteen spiro pyrazolo pyrimidine derivatives (5a-5p) by four component one
pot reaction of pyrimidine-2, 4, 6(1H, 3H, 5H)-trione; 3-oxo-3-phenylpropanenitrile;
hydrazine and isatins(4a-4f) by using nano copper ferrite catalyst (20 mol%) in water
with excellent yields (73-91%). The Isatin with electron withdrawing groups gave
products in high yields (5h, 5p). The present methodology offers an environmentally
benign, cost effective, high yield w.r.t product formation and recyclable catalyst. The
drug likeness or drugability of all the synthesized compounds were tested through rule of
five (RO5) parameters. 5f, 5h, 5n and 5p compounds have shown one RO5 violation each.
The compounds were screened for their antibacterial activity against human pathogenic
bacteria where in two of the synthesized compounds were found to possess significant
antibacterial activity. Compounds showing RO5 violations were found to show negligible
antibacterial activity.
1

GRAPHICAL ABSTRACT:
INTRODUCTION
Hetero cyclic compounds are common structural units in marketed drugs and in
medicinal chemistry. Several heterocyclic compounds without bearing any substituent
possess excellent biological activity, which means that their heterocyclic core is
definitely part of the pharmacophore.[1] Even simple aliphatic heterocycles display
astonishing biological activities. Pyrazolopyramidine scaffolds are found to show
effective biological, pharmacological activities.[2] Multi component reactions (MCR) are
important synthetic tools for the synthesis of bio active hetero cyclic compounds.[3] They
are ecofriendly, have superior atom economy, less time consuming and involve easy
purification processes. MCRs have been broadly employed in the synthesis of
heterocyclic compounds due to the range of readily available starting materials, the
simplicity of one-pot procedures without the need for isolation of intermediates, and the
associated atom economy. The use of water as a green solvent for organic synthesis has
recently attracted considerable attention.[4]
Pyrimidines, pyrazoles, and spiro indoles are very important class of heterocyclic
compounds. Spiro indoles are key moieties in organic synthesis because of their evident
2

pharmacological and biological properties as well as synthetic intermediates for alkaloids
and drugs.[5-7] Pyrimidnes are a class of heterocyclic compounds of great importance in
biological activities owing to their anti fungal, [8] antimalarial, [9] antitumor, [10] and anti-
inflammatory [11] properties. Pyrazole derivatives have been subjected to medicinal
research because of their biological, pharmacological properties such as anticancer, [12]
anti microbial, [13, 14] antifungal, [15] anti-inflammatory, [16] and analgesic activity. [17]
Thus hybrid compounds of these three moieties could potentially lead to a series of
structurally and biologically interesting compounds. Spiro pyrazolo pyrimidine
derivatives are synthesized earlier [18] by using silica based tin complex. In the present
work, a novel green synthetic method is used to synthesize 16 spiro pyrazolo pyrimidine
derivatives by using recyclable nano copper ferrite catalyst.
In recent years, magnetic nano particles have emerged as a useful group of heterogeneous
catalysts. Separation of magnetic nano particles is simple and an attractive alternative to
filtration as it prevents loss of catalyst and enhances reusability without loss of catalytic
activity. The use of low-cost and readily available species as catalyst plays a significant
role for economical feasibility of the chemical processes. Copper ferrite nanoparticles
(CuFe2O4 NP) have the advantage of recyclability, easy work-up, and cleaner reaction
profiles apart from the lack of necessity for external ligands. This minimizes organic
waste generation when compared to the conventional catalytic systems. Moreover, they
also showed good air stability in various organic transformations. [19]
3

Drug development is expensive involving billions of dollars. A lead should a have
range of physico chemical properties that are consistent with the previous record of
discovery of orally active compounds. Literature survey reveals that the compounds with
poorer physico-chemical properties would fail in the pre-clinical trials. Hence a pass in
the physico-chemical properties would preserve both money and time. Before going for
antibacterial studies, we have explored drug likeness of the synthesized compounds by
following rule of five (RO5). Although the primary aim is to synthesize spiro pyrazolo
pyrimidine derivatives, we have focused on the physico-chemical properties to test the
drug likeness of these compounds so as to set the stage for next phase of development
from chemical lead to drug lead.
RO5 has four important parameters that include molecular weight (MW), partition
coefficient expressed as MlogP, number of H-bond donors (NH + OH) and number of H-
bond acceptors (N + O). [20, 21] Lipophilicity is expressed as log of the ratio of octanol
solubility to aqueous solubility as described by Moriguchi et al. [22 -25] An excessive
number of H-bond donors may impair permeability of the drug through membranes. [26,
27] An excessive number of H-bond acceptors may prevent permeability across a
membrane bi-layer. More than 90% of the oral drugs have a MW < 500, log P < 5, NH +
OH < 5 and N + O < 10. [20, 21] Two additional descriptors - total polar surface area
(TPSA) and total rotatable bonds are also evaluated. TPSA is a powerful descriptor in the
characterization of a drug regarding its absorption including gastro-intestinal tract and
bioavailability. [28] TRB is another descriptor that gives information about the
conformational flexibility of the molecule during the drug’s interaction with receptor
4

sites. [29] RO5 parameters combined with TPSA and TRB collectively describe solubility
and permeability parameters of a drug.
RESULT AND DISCUSSIONS
Chemistry
In the present work, we began our investigations by screening the reaction of pyrimidine-
2, 4, 6(1H, 3H, 5H)-trione, 3-oxo-3-cphenylpropanenitrile, hydrazine and isatin` by using
CuFe2O4 NP catalyst in water. Initially, we have conducted a model reaction (Scheme 1)
without catalyst in different solvents at room temperature (30 oC), 60 oC, 80 oC and 100
oC. It is observed that no products were obtained even after 6 hr or 12 hr (Table 1 entry 1-
10). We have investigated the same reaction with 10 mol% of nano copper ferrite catalyst
at room temperature. Products in low yield (less than 10%, Table 1 entry 11-15) were
obtained in 12 hr. Increase of catalyst to 15 mol % resulted in slight increase of yield
(Table 1, entry 16-19). The yield was considerable at 20 mol% of catalyst. Further
increase of catalyst mol% did not result in any increase of yield. There was excellent
yield of products (table 1entry 20-23) at 20 mol% of catalyst, when the different solvents
are replaced with water and at 100 oC temperature (optimized). The model reaction may
be summarized as follows: Spiro pyrazolo pyrimidines are synthesized by reacting
pyrimidine-2, 4, 6(1H, 3H, 5H)-trione, 3-oxo-3-phenyl propane nitrile, hydrazine and
isatin using 20 mol% CuFe2O4 NP catalyst in water and obtained excellent yields at 100
oC. Continuing the success, different isatins were tested in our attempt to synthesize Spiro
pyrazolo pyramidine derivatives at the same reaction conditions (Scheme 2) and the
results are summarized in Table 2.
5

From the results as is evident from Table 2, we can conclude that the isatins with
electron withdrawing groups as substituent formed higher yields than isatins having
electron releasing substituent. The preferred positions of the substituent being 5- or 7- on
the isatin ring of the obtained condensation product. Maximum yields were obtained
with nitro group (89 & 91%) and fluorine (88 & 87%) as substituent on isatin (Table 2,
entry 5h, 5p, 5b, 5j). The yields (65 & 61%) are lower (Table 2, entry 5g, 5o) for isatins
with electron releasing group like methoxy group as substituent. These compounds were
confirmed by H1, C13 NMR and Mass spectra.
Over all, the developed method has several advantages like easy work up, cost
effectiveness, use of water as solvent, reusability of the catalyst and yields were better
than that of earlier reported method.[18]
The plausible mechanism for the formation of spiro pyrazolo pyramidine derivatives
from pyrimidine-2, 4, 6(1H, 3H, 5H)-trione, 3-oxo-3-cphenylpropanenitrile, hydrazine
and isatin` using CuFeNPS is shown in Figure 1. The reaction proceeds through the
formation of highly reactive intermediate6-((3-phenyl-1H-pyrazol-5-yl) amino)
pyrimidine-2, 4(1H, 3H)-dione. The above formed intermediate could not be isolated.
Reusability of the Catalyst
The reusability of CuFe2O4 is one of the most important advantages of this protocol that
makes it useful for practical commercial applications. We have examined the
6

recyclability of CuFe2O4 NP catalyst for the model reaction. Interestingly, the recovered
catalyst could be reused for up to six cycles (Table 3) under optimized reaction
conditions without leaching of the catalyst which is evident from the X-ray powder
diffraction (XRD) pattern as shown in Figure 2. The catalyst was separated by using an
external magnet after completion of the reaction and washed with water followed by
chloroform, dried in oven and reused for the next cycle.
Drug Likeness Through RO5
The drug likeness or drugability of all the synthesized compounds were tested through
RO5 parameters. 5f, 5h, 5n and 5p compounds have shown one RO5 violation each
(Table 4). The remaining compounds obeyed RO5. These values were correlated with
obtained antibacterial activities. It was found that compounds showing RO5 violations
have very poor or no antibacterial activity. The descriptors TPSA, TRB were included for
the sake of future interpretations and were not detailed here.
Biology (Antibacterial Activity)
Antibacterial activity assay was performed by Kirby-Bauer well diffusion method.
Antibacterial activity of synthesized compounds were screened against 10 human
pathogenic bacteria: Pseudomonas aeruginosa MTCC3216, Streptococcus pneumonia
MTCC655, Shigella sonnei NK4010, Shigella dysenteriae type 5NK2440, Shigella
flexneri type 2a503004, Salmonella typhi MTCC733, Vibrio parahaemolyticusserovar
O3:K6K5030, Vibrio cholera MTCC3905, Enterotoxigenic E. coli serotype O1151571,
Salmonella entericaserovar TyphimuriumB12101. Compounds 5l, 5m showed potential
7

activity with growth inhibition zones of 9mm, 8mm against Shigella sonnei; 12mm,
12mm against Shigella flexneri type 2a; 10 mm,12 mm against Enterotoxigenic E. coli
serotype O115 (Figure 3a, 3b, 3c) and the results were summarized in Table 5.
Structure Activity Relation (Sar)
An examination of antibacterial activity as shown in the Table 5 would reveal that
synthesized compounds (5l, 5m) with sulphur atom on pyramidine ring were more active
than compounds (5d, 5e) with oxygen atom. Among sulphur containing compounds; -
Cl, -Br substituted compounds on 5- position on the isatin were found to be more active.
The same was found to be true for oxygen containing compounds.
Simple and –F, -NO2 group substituted isatins showed no antibacterial activity
against the screened human pathogenic bacteria. Whereas –I, -OMe group substituted
isatins showed some antibacterial activity. The activity order of the groups was as
follows: -Cl ≥ -Br > -OMe > -I.
Over all the synthesized compounds were found to be active against three human
pathogenic bacterial cultures of the ten screened cultures.
CONCLUSION
In conclusion, a novel, efficient, four component one pot green synthetic method for the
preparation of Spiro pyrazolo pyrimidine derivatives in water by using nano copper
ferrite catalyst has been described. The utility of this method is demonstrated in the
8

synthesis. The advantages of this method include its simplicity of operation, cleaner
reaction, and good yields. The purification of the product is simple involving filtration.
The catalyst is easily separated by using external magnet and is reusable up to six cycles.
The drug likeness or drugability of all the synthesized compounds were tested through
RO5 parameters. 5f, 5h, 5n and 5p compounds have shown one RO5 violation each.
Furthermore, the compounds are screened for their antibacterial activity against human
pathogenic bacteria where in two of the synthesized compounds were found to possess
significant antibacterial activity. Interestingly, compounds showing RO5 violations were
found to show negligible antibacterial activity.
EXPERIMENTAL
Materials and Methods
All chemicals and reagents were obtained from Aldrich (Sigma-Aldrich), St. Louis, MO,
USA), Lancaster (Alfa Aeser, Johnson Matthey Company, Ward Hill, MA, USA), or
Spectrochem Pvt. Ltd. (Mumbai, India) and were used without further purification.
Progress of each reaction was monitored by TLC on silica gel glass plate containing 60
GF-254, and visualization was achieved by UV light or iodine indicator. 1H and 13C
NMR spectra were determined in DMSO-d6 by using Varian and Avance instruments.
Chemical shifts were expressed in parts per million ( in ppm) downfield from internal
TMS and coupling constants were expressed in Hz. 1H NMR spectroscopic data are
reported in the following order: multiplicity (s, singlet; brs, broad singlet; d, doublet; dd,
doublet of doublets; t, triplet; m, multiplet). ESI mass spectra were recorded on a Micro
mass Quattro LC using ESI+ software with capillary voltage 3.98 kV and an ESI mode
9

positive ion trap detector. Melting points were determined with an Electro thermal
melting point apparatus, and are uncorrected.
For calculating parameters relating to RO5 the following approaches were
adopted: m/z values from mass spectral data was used to arrive at MW; (NH + OH) and
(N+O) were arrived at by counting manually from the structures of the synthesized
compounds; MlogP values along with TPSA and TRB were exclusively taken from
molinspiration, an open access package, available on the web. The earlier three
parameters were also verified through molinspiration.
Typical Synthetic Procedure For Spiropyarazolo Pyramidine(5a-5p) Derivatives
Pyrimidine -2, 4, 6(1H, 3H, 5H)-trione (1mmol), 3-oxo-3-phenylpropanenitrile (1mmol),
hydrazine (1mmol), isatin(1mmol), 20 mol% of nano copper ferrite as catalyst and water
as solvent were taken in round bottomed flask. The reaction mixture was stirred at 100oc
for 6 hours and monitored by Thin Layer chromatography. After the completion of the
reaction, catalyst was separated by using external magnet and compounds purified by
column chromatography. Structures of compounds were confirmed by H1, C13 NMR and
Mass spectral analysis.
Spectral Data of 5a & 5m
3'-phenyl-8',9'-dihydrospiro[indoline-3,4'-pyrazolo[4',3':5,6]pyrido[2,3-d]pyrimidine]-
2,5',7'(1'H,6'H)-trione (5a)
10

Cream white solid; mp: >300 ˚C: 1H NMR (300 MHz, DMSO-d6): δ 6.51 (d, J = 7.55 Hz,
1H), 6.61 (d, J = 6.98, 2H) 6.84(t, J = 8.12 & 14.91 Hz, 1H), 7.00 (d, J = 6.98 Hz, 1H),
7.058-7.17(m, 3H), 7.29 (t, J = 7.55 & 14.91 Hz, 1H), 8.32(s, 1H), 9.10 (s, 1H), 9.99(s,
1H), 11.70 (s, 1H). 13C NMR (75 MHz, DMSO-d6): δ 47.99, 79.63, 100.65, 109.39,
121.81, 123.83, 126.10, 128.10, 128.23, 128.79, 129.03, 129.21, 137.54, 139.95, 142.80,
145.30, 147.12, 159.58, 173.89, 178.85. HRMS (ESI): m/z calcd. for C21H14N6O3:
399.1249 (M+H)+. Found: 399.1243
6-bromo-3'-phenyl-7'-thioxo-6',7',8',9'-tetrahydrospiro[indoline-3,4'-pyrazolo [4', 3' :5,
6] pyrido[2,3-d] pyrimidine]-2,5'(1'H)-dione (5m)
White solid; mp: >300 ˚C;1H NMR (300 MHz, DMSO-d6): δ 6.33(d, J = 8.12Hz, 1H),
6.66(d, J = 6.98 Hz, 2H), 7.18(t, J = 7.36 & 15.10 Hz, 2H), 7.31(m, 2H), 7.39 (dd J =
1.88 & 6.23 Hz, 1H), 9.11(s, 1H), 10.12(s, 1H), 11.69(s, 1H),12.04(s, 1H), 12.62(s, 1H).
13C NMR (75 MHz, DMSO-d6): δ 47.43, 83.76, 89.09, 99.68, 111.30, 126.13, 127.79,
128.27, 128.57, 128.66, 130.21, 131.55, 136.08, 139.11, 139.45, 141.52, 142.01, 146.78,
159.15, 173.41, 177.82. HRMS (ESI): m/z calcd. for M.F: C21H13 BrN6O2S: 494.8699
(M+H)+. Found: 494.8694
ACKNOWLEDGEMENT
The Corresponding author is grateful to CSIR, New Delhi for supporting through
fellowship (JRF&SRF), and Department of Engineering chemistry, AUCE (A), Andhra
11

University, and Visakhapatnam for providing general lab facilities. The author is also
thankful to Prof. B. Venkateswara Rao for his valuable and constant support.
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Table 1. Optimized Reaction conditions
Entry
Solvent
Temperature
Catalyst (mol %)
Time (hr)
Yield %
in 0c
RT
1
–
–
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
6
–
2
CH3CN
Methanol
Ethanol
Water
–
RT
RT
RT
RT
100
80
–
–
3
–
–
4
–
-
5
–
–
6
–
–
7
CH3CN
Methanol
Ethanol
Water
–
–
–
8
80
–
–
9
80
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
100
RT
RT
RT
RT
RT
100
80
–
–
10
10
10
10
10
15
15
15
15
10
15
20
–
CH3CN
Methanol
Ethanol
Water
–
<10
<10
<10
<10
32
42
60
62
75
83
85
CH3CN
Methanol
Ethanol
Water
Water
Water
80
6
80
6
100
100
100
4
4
6
15

Table 2. Synthesis of spiro pyrazolo pyramidine derivatives in optimized conditions
Entry
5a
5b
5c
5d
5e
5f
R
X
O
O
O
O
O
O
O
O
S
Time
6
Yield (%)
85
H
5-F
7-F
Cl
6
88
6
84
6
86
Br
6
79
I
6
78
5g
5h
5i
OMe
NO2
H
6
65
6
89
6
76
5j
5-F
7-F
Cl
S
6
87
5k
5l
S
6
81
S
6
86
5m
5n
5o
5p
Br
S
6
76
I
S
6
73
OMe
NO2
S
6
61
S
6
91
16

Table 3. Recyclability of catalyst
Reaction cycle
Yield (%)
1st
91
2nd
89
3rd
86
4th
84
5th
83
6th
81
17

Table 4. Solubility and permeability parameters
Compound Different no. of Rule of five parameters
TPSA TRB
Code
substituents at atoms
M
log
P
MW
N
+
NH No of
5/6/7'
+
OH
5
violations
positions
7'-O; 5-H
7'-O; 5-F
7'-O; 7-F
7'-O; 5-Cl
7'-O; 5-Br
7'-O; 5-I
O
5a
5b
5c
5d
5e
5f
30
31
31
31
31
31
32
33
30
31
31
31
31
31
32
33
2.04 398.38 9
2.19 416.37 9
2.04 416.37 9
2.58 432.83 9
2.71 477.28 9
2.99 524.28 9
1.96 428.41 10
1.86 443.38 12
2.27 414.45 8
2.41 432.44 8
2.39 432.44 8
2.93 448.89 8
3.06 493.35 8
3.33 540.35 8
2.01 446.49 9
2.21 459.45 11
0
135.53 1
135.53 1
135.53 1
135.53 1
135.53 1
135.33 1
144.77 2
181.36 2
118.46 1
118.46 1
118.46 1
118.46 1
118.46 1
118.46 1
127.69 2
164.28 2
5
0
5
0
5
0
5
0
5
1*
0
5g
5h
5i
7'-O;5-OMe
7'-O; 5-NO2
7'-S; 5-H
7'-S; 5-F
5
5
1*
0
5
5j
5
0
5k
5l
7'-S; 7-F
5
0
7'-S; 5-Cl
7'-S; 5-Br
7'-S; 5-I
5
0
5m
5n
5o
5p
5
0
5
1*
0
7'-S; 5-OMe
7'-S; 5-NO2
5
5
1*
* Nearly 10% of orally active drugs have 1 or more RO5 violations [20]
18

Table 5. Antibacterial studies of synthesized compounds
S.
Solubi- Volume of Growth inhibition zones (in mm) against human pathogenic bacteria
a
b
c
d
e
f
g
h
i
j
No
sample
lity
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
Ethanol 100 µl
DMSO 100 µl
Ethanol 100 µl
Ethanol 100 µl
Ethanol 100 µl
Ethanol 100 µl
Ethanol 100 µl
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9
7
-
-
-
-
-
-
9
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11
9
-
-
-
-
-
-
12
12
-
10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9
11
-
-
-
-
-
-
10
12
2
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Ethyl
100 µl
Ethanol 100 µl
DMSO 100 µl
Ethanol 100 µl
Ethanol 100 µl
Ethanol 100 µl
Ethanol 100 µl
Ethanol 100 µl
Ethyl
100 µl
a).Pseudomonas aeruginosa, b). Streptococcus pneumonia, c).Shigella sonnei,
d).Shigelladysenteriae type5, e) Shigella flexneri type 2a, f) Salmonella typhi , g) Vibrio
parahaemolyticusserovar O3: K6, h). Vibrio cholera, i) Enterotoxigenic E.coli serotype
O115, j) Salmonella entericaserovar Typhimurium
19

Scheme 1. General Reaction
20

Scheme 2. Model Reaction
21

Figure 1. Reaction pathway for the formation of spiro pyrazolo pyramidine derivatives
22

Figure 2. a) XRD of Native form of CuFe2O4 b) XRD pattern after 6th cycle
23

Figure 3. antibacterial activity
24