Efficient synthesis of new 2,3-dihydrooxazole-spirooxindoles hybrids as antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2016.03.093

Two series of new 2,3-dihydrooxazole-spirooxindole derivatives were efficiently synthesized starting from N′-(2-oxoindolin-3-ylidene) benzohydrazide/N′-(2-oxoindolin-3-ylidene)-2-phenoxyacetohydrazide using designed synthetic route. Newly synthesized 2,3-

Full text of DOI:10.1016/j.bmcl.2016.03.093

Accepted Manuscript
Efficient synthesis of new 2,3-dihydrooxazole-spirooxindoles hybrids as anti-
microbial agents
Shailendra Tiwari, Poonam Pathak, Ram Sagar
PII:
S0960-894X(16)30325-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.093
BMCL 23736
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 January 2016
16 March 2016
25 March 2016
Please cite this article as: Tiwari, S., Pathak, P., Sagar, R., Efficient synthesis of new 2,3-dihydrooxazole-
spirooxindoles hybrids as antimicrobial agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.03.093
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Efficient synthesis of new 2,3-dihydrooxazole-spirooxindoles hybrids
as antimicrobial agents.
Shailendra Tiwari,a* Poonam Pathaka and Ram Sagarb
aDepartment of Chemistry, University of Allahabad, Allahabad (UP) 211002, India
bDepartment of Chemistry, Shiv Nadar University, NH-91 Dadri, GB Nagar (UP) 201314, India
Email: drshailendratiwariau@gmail.com
Abstract
Two series of new 2,3-dihydrooxazole-spirooxindole derivatives were efficiently synthesized
starting from N΄-(2-oxoindolin-3-ylidene) benzohydrazide/N΄-(2-oxoindolin-3-ylidene)-2-
phenoxyacetohydrazide using designed synthetic route. Newly synthesized 2,3-dihydrooxazole-
spirooxindole derivatives were screened for their antibacterial and antifungal activity against
different pathogenic strain of bacteria and fungi. The minimum inhibitory concentration (MIC),
minimum Bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were
determined for the test compounds as well as for reference standards. Compounds 4e, 4g, 7g
have shown good antibacterial activity whereas compounds 4f, 7b, 7d have displayed better
antifungal activity.
Keywords
2,3-dihydrooxazole-spirooxindole, Isatin, spirooxindole, hydrazide, schiff base, antimicrobial
antibacterial activity and antifungal activity.
Efficient synthesis of drug-like small molecules has been the focus of the research for medicinal
chemists and chemical biologists because, they play very important role in drug discovery
processes.1 These different drugs like bioactive compounds are broadly used to modulate enzyme
or receptor function and can serve as important leads for drug development.2 Isatin (1H-indole-
2,3-dione) and its derivatives have been proven as privileged core structures and are found in
many bioactive natural products and in various pharmaceutical agents.3 The synthetic versatility
of the isatin, due to its privileged scaffold, has led to the generation of a large number of
structurally diverse derivatives which include analogues derived from either C-3 carbonyl
modification with mono-, di-, and trisubstitution of the aryl ring and/or those obtained by
derivatisation of the nitrogen of isatin.4
The most fascinating derivatization of isatin analogues is undoubtly reaction on C-3 carbonyl
center instead of C-2 carbonyl. This selective and smooth derivatization is possible because C-3
prochiral carbonyl center is more reactive towards nucleophilic addition than C-2 carbonyl.5 The
reactions on the C-3 carbonyl group of isatins, mostly by nucleophilic additions or spiro
1

annulations, transform it into various spiro-fused heterocyclic and carbocyclic frameworks
including important 2-oxindole derivatives. The 2-oxindoles, which are spiro-fused to other
cyclic frameworks have drawn remarkable interest of researchers around the globe working in
the area of medicinal chemistry, chemical biology and synthetic organic chemistry because they
are present in many bioactive natural products.6,7 Some of such 2-oxindole derivatives of isatin
are horsfiline, spirotryprostatins, gelseverine, gelsmine, rhynchophylline etc. These derivatives
have been known for various types of bioactivity such as progesterone receptors modulators,
anti- HIV, anticancer, antitubercular, antimalerial and MDM2 inhibitor.8,9
Indole nucleus itself is priviledged structure and has been reported for various biological
activities including fungicidal, bactericidal and herbicidal activities.10 The spirooxindoles is
among the most important class of naturally occurring substances, characterised by highly
pronounced biological properties, and is also the core structure of many synthetic
pharmaceuticals.11 The various biological activities of spirooxindoles derivatives have attracted
much attention from organic chemists and as a consequence a number of methods have been
reported for the preparation of spirooxindoles fused heterocycles. Isatin perhaps is the single
class of heterocyclic compound which has been most extensively employed as a starting material
for the synthesis of a wide number of spiro-cyclic oxindoles frameworks, both carbocyclic and
heterocyclic.12-14 Oxazole-benzamide derivatives also constitute an important class of
heterocyclic compounds and recently design, synthesis and structure-activity relationship of a
series of oxazole-benzamide derivatives reported alongwith their bacterial and pesticidal
activities.15
On the basis of the above literature abundance on bio-potentials of spiro-cyclic oxindoles and
oxazole-benzamide framework, we were confident that the hybrids spiro-cyclic oxindoles fused
with oxazole-benzamide framework would provide the important structural motifs for the
discovery of novel bioprobes or therapeutic agents. In continuation of our research on efficient
synthesis of drug like small molecules, we designed and synthesized spiro-cyclic oxindoles fused
with oxazole-benzamide as novel 2,3-dihydrooxazole-spirooxindoles hybrids as antimicrobial
agents.
Chemistry
For the development of efficient and facile methods for the synthesis of designed novel 2,3-
dihydrooxazole-spirooxindoles hybrids, we identified Isatin (indole-1H-2,3-dione) 1 as the key
starting material that can be transferred to our designed antimicrobial molecules in two step via
16-18
19-21
schiff base intermediate. The synthesis of intermediate schiff bases 3a-3g
and 6a-6g
was carried out by reacting Isatin 1 and corresponding commercially available analogues of
benzohydrazides 2a-2g and phenoxyacetohydrazides 5a-5g respectively. These schiff bases then
subsequently were coupled with benzoin at elevated temperature in presence of triethyl amine
which furnish desired 2,3-dihydrooxazole-spirooxindoles derivatives 4a-4g and 7a-7g
respectively in good yield (Scheme 1 and 2).
2

We initiated our synthetic protocol with commercially available Isatin 1 refluxing with diffrent
benzohydrazides 2a-2g in ethanol in the presence of catalytic amount of acetic acid which
furnished corresponding schiff bases 3a-3g as reported in literature.16-18 The schiff bases 3a-3g
were purified by crystallization and characterized using spectroscopic data. All the NMR and IR
data of these schiff bases 3a-3g were in consistant with the reported analytical data. After having
charatctrised and pure schiff base 3a in hand, it was refluxed with benzoin under various reaction
conditions using different solvents (MeOH, Toluene, THF etc) but ethanol under reflux in basic
condition for 6h was found better, which showed the formation of clean product (TLC). Reaction
mixture was cooled to room temperature and after usual workup (experimental section) and
purification it furnished 2,3-dihydrooxazole-spirooxindole derivative 4a in very good isolated
yiled (81%). Similarly other schiff bases 3b-3g were successfully transferred to their
corresponding 2,3-dihydrooxazole-spirooxindoles 4b-4g under reflux condition in ethanol using
triethyl amine in very good isolated yields (71-81%) (Scheme 1).
3
4
2
H
H
R
O
N
N
O
N
H2N
+
glacial AcOH/EtOH
90 oC, 3-5 h
O
O
2
O
N
R
N
H
3
H
4
1
3a-3g
2a-2g
3
R
a H
b 2-OH
c 4-OH
d 2-Cl
4
Ph
2
Ph
O
H
R
Benzoin/EtOH/Et3N
90 oC, 5-6 h, 70-81%
N
N
O
O
e 4-Cl
N
f 4-OMe
g 4-NO2
H
4a-4g
Scheme 1. Synthesis of 4a-4g.
In parallal synthesis approach using similar reaction protocols Isatin 1 refluxed with diffrent
phenoxyacetohydrazides 5a-5g in ethanol in the presence of catalytic amount of acetic acid
which furnished corresponding schiff bases 6a-6g as reported in literature.19-21 The schiff bases
6a-6g were purified by crystallization and characterized by comparing acquired analytical data
with data reported in literature. All the prepared schiff bases 3a-3g were refluxed with benzoin
under previous optimized condition in ethanol in presence of Et3N for 6 h which result in the
formation of nonpolar products on TLC. After usual workup and purification their corresponding
2,3-dihydrooxazole-spirooxindoles 7a-7g were isolated in 65-75% isolated yields (Scheme 2).
3

H
H
O
N
O
O
N
N
O
O
H2N
+
2
3
glacial AcOH/EtOH
90 oC, 3-5 h
R
3
2
R
4
O
4
O
N
N
H
H
1
6a -6g
5a-5g
R
a H
Ph
H
Ph
b 2-CH3
c 3-CH3
d 4-CH3
e 2-Cl
f 4-Cl
g 2,4-Cl
Benzoin/EtOH/Et3N
90 oC, 5-6 h, 65-75%
N
N
O
3
2
O
R
4
O
O
N
H
7a -7g
Scheme 2. Synthesis of 7a-7g.
The mechanism of this transformation can be postulated as follows: hydroxy group of benzoin in
the presence of triethyl amine at elevated temperature attacking on imine carbon pusing imine
double bond towards nitrogen resulting an unstable intermediate I (Figure 1). Intramolecular
attack of hydrazine N-atom to carbonyl carbon of benzoin may resulting intermediate II which
under high temperature reaction condition looses water molecule and furnishing stable 2,3-
dihydrooxazole-spirooxindole derivative 7a (Figure 1). All the compounds and intermediates
were confirmed by spectral analysis.22
H
O
N
O
O
N
OH
Ph +
Ph
Ph
H
Ph
N
O
O
O
O
HN
N
O
H
O
Benzoin
6a
N
H
I
Ph
HO
Ph
H
Ph
H
Ph
H
N
N
N
O
N
O
O
O
- H2O
O
O
O
O
N
N
H
H
7a
II
Figure 1. Proposed Mechanism
Biology
The synthesized pure compounds were screened for antibacterial and antifungal activities
adopting standard protocols.23 The antibacterial activity, of prepared final pure compounds (4a-
4

4g and 7a-7g), was performed against Bacillus subtilis, Enterobacter and Klebsiella pneumoniae
using Ciprofloxacin as positive and DMSO as negative control. Minimum inhibitory
concentration (MIC) and minimum bateriacidal concentration (MBC) were determined and the
activity was reported in µg/ml. The nutrient broth, which contained logarithmic serially two fold
diluted amount of test compounds and controls were inoculated with approximately 5×105
c.f.u/ml of actively dividing bacteria cells. The cultures were incubated for 24 hour at 37°C and
the growth was monitored visually and spectrophotometrically. The antibacterial results are
summarized in table 1, only for those compounds which were found active against any strain of
bacteria. It is evident from table 1 that compounds 4b, 4e, 4f, 7a and 7g showed moderate
activity agaist gram-positive bacteria B. subtilis whereas, 4e, 4g, 7c, 7d and 7g were active agaist
gram-negative Enterobacter ranging from 30-50 µg/ml concentration and only one compound 7g
was found active against K. pneumoniae 25 µg/ml concentration (Table 1).
TABLE 1. Antibacterial activity of novel 2,3-dihydrooxazole-spirooxindole derivatives
Compounds
Gram-Positive bacteria
Gram-Negative bacteria
Enterobacter K. Pneumoniae
MIC MBC MIC MBC
B. subtilis
MIC
70
50
100
30
MBC
100
100
200
50
40
30
50
30
40
30
40
50
40
50
50
100
50
100
50
100
100
100
25
n.a.
50
n.a.
100
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
50
4b
4e
4f
4g
7a
7b
7c
7d
7g
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
25
50
100
n.a.
n.a.
n.a.
50
n.a.
n.a.
n.a.
25
Ciprofloxacin
DMSO
6.25
n.a.
12.50
n.a.
10.25
n.a.
6.25
n.a.
25
n.a.
n.a.
MIC (µg/ml), minimum inhibitory concentration i.e. the lowest concentration of the compound to inhibit the growth
of bacteria completely; MBC (µg/ml) minimum bacteriacidal concentration i.e. the lowest concentration of the
compound for killing the bacteria completely; n.a.-No activity detected.
The antifungal activity, of prepared final pure compounds, was performed against Pyricularia
oryzae, Pseudoperenospora cubensis, Sphaerotheca fuliginea and Phytophthora infestans using
Nystatin as positive and DMSO as negative control. Minimum inhibitory concentration (MIC)
was determined and reported in µg/ml. Antifungal activity was carried out through disk diffusion
method.24 All fungal cultures were routinely maintained on sabouraud dextrose agar (SDA) and
incubated at 28°C. The summarized data are presented in table 2. The antifungal activities are
summarized in table 2, only for those compounds which were found active against any of these
5

strain of fungi. It is evident from table 2 that compounds 4b, 4e-4g, 7c-7d and 7g showed
antifungal activity agaist used strains of fungi ranging from 8-18 µg/ml in concentration and
compound 7c was the only active compound against P. oryzae and P. infestans in 20 and 12
µg/ml resepectively. Compound 7g was found active against S. fuliginea 14 µg/ml concentration
(Table 2).
TABLE 2. Antifungal activity of novel 2,3-dihydrooxazole-spirooxindole derivatives
Compounds
Fungal Species and MIC (µg/ml)
P. oryzae
16
P. cubensis
S. fuliginea
P. infestans
12
12
14
18
12
8
n.a.
12
n.a.
18
n.a.
12
12
8
14
14
14
12
14
18
12
14
n.a.
18
n.a.
4b
4e
4f
4g
7a
7b
7c
7d
7g
14
12
16
15
17
20
8
n.a.
20
12
16
12
n.a.
12
14
18
n.a.
Nystatin
DMSO
n.a.
MIC (µg/ml), minimum inhibitory concentration i.e. the lowest concentration of the compound to inhibit the growth
of fungi. n.a.-No activity detected.
The structure-activity relationship (SAR) of the tested compounds for antibacterial activity can
be summarized as follows: i) in the series of the 2,3-dihydrooxazole-spirooxindoles, the
substitution at phenyl ring is important for better antibacterial activity; ii) 4-Cl and 4-NO2
substitution in this series shown better activity than other groups against B. subtilis and K.
neumoniae; iii) Phenoxyacetohydrazides condensed 2,3-dihydrooxazole-spirooxindoles do not
show any better antibacterial activity. Antifungal activity of these compounds was very much
comparable to Nystatin a known antifungal agent. The mechanism responsible for the
antibacterial activity of examined compounds is not known at the moment; work is in progress to
to investigate the mechanism of antibacterial action, and synthesis of more effective compounds.
In summary, an efficient, clean and convenient synthesis of novel 2,3-dihydrooxazole-
spirooxindole derivatives has been developed. The procedure offers several advantages including
simple reaction conditions as well as simple experimental and product isolation procedures, thus,
making the synthesis of series of novel 2,3-dihydrooxazole-spirooxindoles in good to very good
yields. These 2,3-dihydrooxazole-spirooxindoles were designed through the recombination of,
cheap and readily available starting materials, benzoin and schiff bases of isatin, with
corresponding analogues of benzohydrazides and phenoxyacetohydrazides. Subsequent
preliminary anti-bacterial and anti-fungal screening done on these compounds which revealed
6

that newly generated compounds possess antimicrobial activity and these can be elaborated as
potential antimicrobial agents. The complete library realization of these novel 2,3-
dihydrooxazole-spirooxindole derivatives and associated detailed biological evaluations will be
reported in due course of time.
Acknowledgements
We are thankful to the Head, Department of Chemistry, University of Allahabad, Allahabad for
necessary laboratory facilities. The authors would also like to thank SAIF, CDRI Lucknow and
SAIF, Punjab University, Chandigarh for spectral and analytical data and Centre of
Biotechnology, University of Allahabad, Allahabad for antimicrobial activity data.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://.
References and notes
1. Schreiber, S. L. Science 2000, 287, 1964.
2. Hoon, S.; Smith, A. M.; Wallace, I. M.; Suresh, S.; Miranda, M.; Fung, E.; Proctor, M.;
Shokat, K. M.; Zhang, C.; Davis, R. W.; Giaever, G.; Stonge, R. P.; Nislow, C. Nat. Chem.
Biol. 2008, 4, 498.
3. Wang, Y.; Chan, F. Y.; Sun, N.; Lui, H. K.; Sp, P. K.; Yan, S. C.; Chan, K. F.; Chiou, J.;
Chen, S.; Abagyan, R.; Leung. Y. C.; Wong, K. Y. Chem. Biol. Drug Des. 2014, 84, 685.
4. Singh,G.S.; Desta, Z. Y. Chem. Rev. 2012, 112, 6104 and references cited therein.
5. Dou, X.; Yao, W.; Wen, S.; Lu, Y. Chem. Commun. 2014, 50, 9469.
6. Santos, Maria M.M. Tetrahedron 2014, 70, 9735.
7. Cheng, D.; Ishihara, Y.; Tan, B.; Barbas, Carlos F. ACS Catal. 2014, 4, 743.
8. Badillo, J. J.; Hanhan, N. V.; Franz, A. K. Curr.Opin. Drug Discovery Dev. 2010, 13, 758. b)
Pandeya, S. N.; Smitha, S.; Jyoti, M. Sridhar, S. K. Acta Pharm. 2005, 55, 27.
9. Tiwari, S.; Tiwari, N.; Agrawal, T.; Khan, M.; Hussain, N. Indian J. Chem., Sect B, 1995 34B,
1010.
10. Yu, B.; Yu, D.-Q.; Liu, H.-M. Eur. J. Med. Chem. 2015, 97, 673 and references cited therein.
11. Tiwari, S.; Ahamad, A.; Alauddin, Indian J. Chem., Sect B, 2014 53B, 352.
12. S. Sun, Y.; Sun, J.; Yan, C. G. Beilstein J. Org. Chem. 2013, 9, 8.
13. Mohammadizadeh, M. R.; Firoozi, N. Tetrahedron Lett. 2010, 51, 5467.
14. Stokes, N. R.; Baker, N.; Bennett , J. M.; Chauhan, P. K.; Collins, I.; Davies, D. T.;
Gavade, M.; Kumar, D.; Lancett, P.; Macdonald, R.; MacLeod, L.; Mahajan, A.; Mitchell, J.
7

P.; Nayal, N.; Nayal, Y. N.; Pitt, G. R. W.; Singh, M.; Yadav, A.; Srivastava, A.;
Czaplewski, L. G.; Haydon, D. J. Bioorg. Med. Chem. Lett. 2014, 24, 353 and references
cited therein.
15. Liang, Z.; Zhang, D.; Ai, J.; Chen, L.; Wanga, H.; Kong, X.; Zheng, M.; Liu, H.; Luo, C.;
Geng, M.; Jiang, H.; Chen, K. Bioorg. Med. Chem. Lett. 2011, 21, 3749.
16. Gürsoy A.; Karali, N.; Büyüktimkin, S.; Demirayak, S.; Ekinci, A. C.; Ozer, H.
Farmaco. 1996 51, 437.
17. Ali, R.; Mishra, B.; Nizamuddin Indian J. Chem., Sect B, 1989, 28B, 526.
18. Khattab, M. A.; Abdel-Megeed, M. F.; El-Baradie, H. Y. E.; Issa, R. M. Proceedings of the
Pakistan Academy of Sciences 1981, 18, 1.
19. Pandey, V. K.; Dwivedi, A.; Pandey, O. P.; Sengupta, S. K. J. Agric. Food
Chem. 2008, 56, 10779.
20. Karah, N.; Terzioĝlu, N.; Gürsoy, A. Arzneimittel-Forschung. 1998, 48,75
21. Singh, K.; Tiwari, N.; Nizamuddin Indian J. Chem., Sect B, 1993, 32B, 1086.
22. Compound 4a. Amorphous solid (1.85 g, 81%). IR (KBr, cm-1): 1450, 1500, 1580 (C=C str)
1
1665, 1690 (C=O str), 2950 (=C-H str), 3200 (N-H str); H NMR (400 MHz, DMSO-d6) δ:
5.99-6.06 (m, 2H, ArH), 7.19-7.27 (m, 3H, ArH), 7.31-7.45 (m, 6H, ArH), 7.52-7.63 (m, 3H,
ArH), 7.78 (t, 1H, J = 7.5 Hz, ArH), 7.91 (d, 2H, J = 7.5 Hz, ArH), 7.92 (d, 2H, J = 7.5 Hz,
13
ArH); C NMR (100 MHz, DMSO-d6) δ: 118.2, 127.7, 128.1, 128.9, 129.0, 129.3, 129.9,
130.0, 131.9, 132.7, 133.7, 135.1, 136.0, 140.2 195.3 (C=O), 195.6 (C=O); FAB HRMS m/z:
calcd for C29H21N3O3 [M + H]+, 460.1661; found, 460.1660.
23.Khan, S. A.; Kumar, P.; Joshi, R. Iqbal, P. F.; Saleem, K. Eur. J. Med. Chem. 2008, 43, 2029.
24. National Committee for Clinical Laboratory Standard, 2008, 28, 1-25. Reference method for
broth dilution antifungal susceptibility testing of yeast, approved standard. Document M27-
A3. National Committee for Clinical Laboratory Standards, Wayne, PA, USA.
8

Graphical Abstract
3
H
Ph
O
4
R
Ph
2
Ph
H
N
O
O
5a-5g
Ph
H2N
H
O
R
N
O
R
HN
N
N
3 R
4
2
O
N
i)
i)
O
H2N
2a-2g
O
O
O
O
O
N
ii) Benzoin
65-75%
ii) Benzoin
70-81%
N
H
N
H
H
7a -7g
4a-4g
1
MIC = 30-100 g/mL against B. subtitles, Enterobacter and K. Pneumoniae,
= 8-20 g/mL against P. oryzae, cubensis, infestans and S. fuliginea.
9