Nano-BF3.SiO2 Catalyst-Promoted Michaelis-Arbuzov Reaction: Solvent-Free Synthesis and Antimicrobial Evaluation

Article abstract of DOI:10.1080/10426507.2014.996643

A simple, convenient synthetic route for the synthesis of novel dialkyl heteroaryl phosphonates by a reusable and green nano-BF₃.SiO₂ solid catalyst under solvent-free conditions through Michaelis-Arbuzov reaction with high yields is

Full text of DOI:10.1080/10426507.2014.996643

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Phosphorus, Sulfur, and Silicon and the
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Nano-BF3.SiO2 Catalyst-Promoted
Michaelis-Arbuzov Reaction: Solvent-
Free Synthesis and Antimicrobial
Evaluation
Mavallur Varalakshmia, Doddaga Srinivasulua & Venkata S. Kotakadib
a Department of Chemistry, Sri Venkateswara University, Tirupati,
517502, Andhra Pradesh, India
b DST-PURSE Centre, Sri Venkateswara University, Tirupati, 517502,
Andhra Pradesh, India
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Accepted author version posted online: 20 Jan 2015.
To cite this article: Mavallur Varalakshmi, Doddaga Srinivasulu & Venkata S. Kotakadi (2015) Nano-
BF3.SiO2 Catalyst-Promoted Michaelis-Arbuzov Reaction: Solvent-Free Synthesis and Antimicrobial
Evaluation, Phosphorus, Sulfur, and Silicon and the Related Elements, 190:9, 1518-1524, DOI:
10.1080/10426507.2014.996643
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Phosphorus, Sulfur, and Silicon, 190:1518–1524, 2015
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2014.996643
NANO-BF3.SiO2 CATALYST-PROMOTED
MICHAELIS-ARBUZOV REACTION: SOLVENT-FREE
SYNTHESIS AND ANTIMICROBIAL EVALUATION
Mavallur Varalakshmi,1 Doddaga Srinivasulu,1
and Venkata S. Kotakadi2
1Department of Chemistry, Sri Venkateswara University, Tirupati 517502, Andhra
Pradesh, India
2DST-PURSE Centre, Sri Venkateswara University, Tirupati 517502, Andhra
Pradesh, India
GRAPHICAL ABSTRACT
Abstract A simple, convenient synthetic route for the synthesis of novel dialkyl heteroaryl
phosphonates by a reusable and green nano-BF3.SiO2 solid catalyst under solvent-free con-
ditions through Michaelis–Arbuzov reaction with high yields is presented. All the newly syn-
thesized compounds were characterized by spectral data and screened for their antimicrobial
activity. Some of the compounds exhibited potent antibacterial activity against all the tested
pathogens, and warrant further investigation.
Keywords Michaelis–Arbuzov reaction; nano-BF3.SiO2; antimicrobial activity; multidrug
resistant activity (MDR); organophosphorus compounds; phosphonates
INTRODUCTION
Organophosphorus compounds bearing a phosphorus-carbon bond have played a key
role in numerous biologically active compounds, but the occurrences of these compounds
Received 14 September 2014; accepted 3 December 2014.
Address correspondence to Doddaga Srinivasulu, Department of Chemistry, Sri Venkateswara University,
Tirupati 517502, Andhra Pradesh, India. E-mail: doddaga s@yahoo.com
1518

NANO-BF3.SiO2 CATALYZED MICHAELIS–ARBUZOV REACTION
1519
are not widespread in nature. Phosphonates have a broad range of applications in various
fields such as medicinal and synthetic chemistry. Phosphonates represent a group of stable
organophosphorus compounds comprising a single P-C bond which makes their molecules
resistant to chemical and enzymatic hydrolysis, thermal decomposition,1 photolysis,2 and
inhibitors for certain biosynthetic pathways3 and they are widely used as flame retardants,
herbicides, detergents, pesticides, chelating agents for di- and trivalent metal ions, and
reagents for Wittig–Horner reactions.4-9 Arylmethyl and heteroarylmethyl phosphonate
compounds have been widely utilized as prominent precursors for the synthesis of stilbene
dendrimers10 and optical materials like alkyl(phenylvinylenes),11 trialkylamine-tethered
vinylenes,12 and thienylvinylenes.13
The Michaelis–Arbuzov reaction is considered as an efficient fundamental reaction
for the preparation of alkyl/aryl phosphonates from alkyl/aryl halides and trialkyl/aryl
phosphites.14 However, the reaction has some drawbacks such as the need for elevated
temperature, removal of the trialkyl phosphites used in excess, and weaker electrophiles
aryl/heteroaryl halides or vinyl halides that give lower yields. To avoid these problems,
researchers have been focusing considerable attention to enhance the efficiency of the
reaction by using new catalysts.
Various reports were found in the literature for the formation of a C P bond with
different catalysts such as iodine,15 BF3.OEt2,16 NiCl2,17 Pd(OAc)2,18 CeCl3.7H2O,19 and
ionic liquids20 through Michaelis–Arbuzov reaction. There are some drawbacks using the
above mentioned catalysts. BF3.OEt2 requires longer reaction time, low yields, lack of
generality, and requires stoichiometric amount of the toxic and moisture sensitive catalyst.
NiCl2 and Pd(OAc)2 require vigorous conditions such as high temperature for completion
of the reaction. In order to avoid the above-mentioned problems and to enhance efficiency
22-24
21
of the reaction we tested BF3.SiO2
dialkyl heteroaryl phosphonates.
and nano BF3-SiO2 catalysts to synthesize the
Because of the broad range of applications of phosphonates in medicinal and synthetic
chemistry, we focused on designing new active phosphonate molecules and the development
of new methodologies for their synthesis in a simple and cost effective manner under
solvent-free conditions.
RESULTS AND DISCUSSION
Chemistry
The synthesis of dialkyl heteroaryl phosphonate 3(a–e) and 6(a–e) derivatives was
carried out effectively in the presence of nano-BF3.SiO2 under solvent-free conditions as
represented in Scheme 1.
3-Bromoquinoline (1) and triethylphosphite (2b) were selected as models for optimiz-
ing the reaction conditions. The model reaction was carried out in different solvents like
acetonitrile (ACN), dichloromethane (DCM), toluene, methanol (MeOH), 1,4-dioxane,
dimethyl formamide (DMF) and in solvent-free conditions in the presence of BF3.SiO2
and nano-BF3.SiO2 catalysts to synthesize compound (3b) and the yield is shown in
Table 1. The optimum yield of the product was observed when the reaction progressed
under solvent-free conditions. Also, the temperature effect, the amount of the catalyst
and the reusability of the catalyst up to five cycles were examined, revealing that the re-
◦
action was effectively progressed at 50 C in the presence of 20 mol% nano-BF3.SiO2
catalyst and no significant diminishing of the catalytic action on the reaction was ob-
served until the catalyst had been reused three times. After optimization of the reaction

1520
M. VARALAKSHMI ET AL.
Scheme 1 Synthesis of dialkyl heteroaryl phosphonates 3(a–e) and 6(a–e).
conditions, the generality of the reaction was checked by altering heteroaryl halides and tri
alkylphosphites.
Structures of the newly synthesized compounds 3(a–e) and 6(a–e) were confirmed
1
by IR, H, 13C, and 31P NMR, mass and CHN elemental analysis spectral data and are
described in the experimental section. In the IR spectra, the absorption bands in the regions
of 1155–1298 cm−1 correspond to P = O stretching frequency. In the 1H NMR spectra, the
chemical shift values in the region δ 1.56–1.58 ppm were assigned to –CH3, 3.52–4.01 ppm
was assigned to –OCH2 protons, 3.80–4.01 ppm was assigned to –CH protons of isopropyl
group (–CH–(CH3)2. Further, the structures of the titled compounds 3(a–e) and 6(a–e) were
confirmed by the corresponding 13C, 31P NMR chemical shift values, molecular ions, and
fragmentation ion peaks in mass spectra.
ANTIMICROBIAL ACTIVITY
Antibacterial Activity
The newly synthesized dialkyl heteroaryl phosphonates 3(a–e) and 6(a–e) were tested
for their antibacterial activity against two Gram +Ve bacterial strains, Staphylococcus au-
reus, Pseudomonas aeruginosa; two Gram –Ve bacterial strains, Klebsiella pneumoniae

NANO-BF3.SiO2 CATALYZED MICHAELIS–ARBUZOV REACTION
1521
Table 1 Solvent effect on the synthesis of dialkyl heteroaryl phosphonates 3(a–e) and 6(a–e) in the presence of
37% BF3.SiO2 and 37% nano-BF3.SiO2
Entry
Catalyst (g)
37% BF3.SiO2 (0.3)
37% BF3.SiO2 (0.3)
37% BF3.SiO2 (0.3)
37% BF3.SiO2 (0.3)
37% BF3.SiO2 (0.3)
37% BF3.SiO2 (0.3)
37% BF3.SiO2 (0.3)
37% Nano-BF3.SiO2 (0.3)
37% Nano-BF3.SiO2 (0.3)
37% Nano-BF3.SiO2 (0.3)
37% Nano-BF3.SiO2 (0.3)
37% Nano-BF3.SiO2 (0.3)
37% Nano-BF3.SiO2 (0.3)
37% Nano-BF3.SiO2 (0.3) (first run)
Solvent
Temperature (◦C) Time (h) Yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Methanol
DCM
Toluene
DMF
Reflux
45
8.0
6.0
7.5
6.5
7.0
6.0
5.0
6.0
5.0
6.5
6.0
6.0
5.5
3
30
68
62
50
68
45
80
78
74
70
68
64
68
88
87
85
78
65
Reflux
Reflux
Reflux
Reflux
50
ACN
1,4-dioxane
Solvent-free
Methanol
DCM
Toluene
DMF
ACN
1,4-Dioxane
Solvent-free
Reflux
45
Reflux
Reflux
Reflux
Reflux
50
37% Nano-BF3.SiO2 (0.3) (second run) Solvent-free
50
3
37% Nano-BF3.SiO2 (0.3) (third run)
37% Nano-BF3.SiO2 (0.3) (fourth run)
37% Nano-BF3.SiO2 (0.3) (fifth run)
Solvent-free
Solvent-free
Solvent-free
50
3
50
3
50
3
and Bacillus megaterium; two Gram –Ve antibiotic resistant bacterial strains, mutant Es-
cherichia coli (Streptomycin resistant) and donor E. coli (Rifampicin resistant) bacteria,
by the agar well diffusion method using Amoxiclav (SD063, Himedia) as a standard drug.
The diameter of inhibition zones in millimeters (DIZ) are represented in Table S1 (Sup-
plemental Materials) and the minimum inhibitory concentrations (MIC) of the synthesized
compounds are presented in Table S2 (Supplemental Materials).
Compounds 3a, 3b, 3c, and 3e have shown potent antibacterial activity against
all the tested pathogens whereas compound 3d has shown potent antibacterial activity
against Staphylococcus aureus and Pseudomonas aeruginosa. Compound 6a showed po-
tent antibacterial activity against Pseudomonas aeruginosa and Klebsiella pneumoniae.
Compound 6b has shown potent antibacterial activity against Staphylococcus aureus and
Klebsiella pneumoniae. Compound 6d showed potent antibacterial activity against Pseu-
domonas aeruginosa.
Antifungal Activity
The fungal strains Aspergillus niger and Penicillium spinulosum were used for testing
the antifungal activity of the newly synthesized compounds 3(a–e) by employing the poison
plate technique. Fluconazole (SD 114, Himedia) was used as a standard drug. The results
of the tested samples show that all the newly synthesized compounds exhibited potent to
good activity against the tested micro organisms as represented in Table S3.
Compounds 3a and 3e showed potent antifungal activity against both the tested
pathogens whereas compounds 3b and 3d showed potent antifungal activity against As-
pergillus niger. In addition, the newly synthesized compounds 3(a–e) and 6(a–e) also
showed potent activity against antibiotic resistant bacteria. We concluded that the newly
synthesized compounds 3(a–e) and 6(a–e) have multidrug resistant activity.

1522
M. VARALAKSHMI ET AL.
EXPERIMENTAL
All chemicals were purchased from Merck, Aldrich, and S. D. Fine-chem. (India) for
use without further purification. Solvents were distilled from the appropriate drying agents
and degassed before use. Melting points were determined in open capillaries on Guna
melting point apparatus and are uncorrected. IR spectra were recorded on JASCO FT-IR
5300 using KBr discs. 1H NMR, 31P NMR, and 13C NMR spectra were recorded on Bruker
AV-400 spectrometer. Mass spectra were recorded on an API 3000 mass spectrometer
(positive mode). The progress of the reactions was monitored by TLC on Merck silica
plates. Results are presented as chemical shift δ in ppm, multiplicity, J values in Hertz (Hz),
number of protons, and proton position. Multiplicities are shown as the abbreviations: s
(singlet), brs (broad singlet), d (doublet), t (triplet), and m (multiplet).
Synthesis of Dialkyl Heteroaryl Phosphonates 3(a–e) and 6(a–e)
To 3-bromoquinoline (1) (208.09 mg, 0.001 mol), triethylphosphite (2b) (166.16 mg.
◦
Nano-BF3.SiO2 (300 mg) was added and the reaction mixture was refluxed at 50 C for
3–5 h. After the completion of the reaction (confirmed by TLC), the reaction mixture was
dissolved in DCM (10 mL) and filtered to remove the catalyst. The filtrate was concen-
trated under vacuum and the resulting crude material was purified on short column of
silica gel using ethyl acetate:n-hexane (3:1) to obtain the final product diethyl quinolin-
3-ylphosphonate (3b). The same experimental procedure was utilized for synthesizing the
remaining title compounds. Complete characterization of the products and representative
1H, 13C, and 31P NMR spectra and mass spectra for 3a and 6a (Figures S1–S8) are presented
in the Supplemental Materials.
Diethyl Quinolin-3-ylphosphonate (3b)
Yield: 88%, mp: 201–203◦C. IR (KBr, cm−1): 1218 (P O, str), 1095 (P-C, str); 1H
NMR (DMSO-d6, 400 MHz): δ 1.59 (t, J = 6.8 Hz, 6H, H12, 14), 3.53–3.59 (q, 4H, H13,
14), 8.90 (s, 1H, H2), 8.66 (s, 1H, H4), 7.62–7.75 (q, 1H, H6), 7.76–780 (q, 1H, H7), 7.99
(d, J = 8.4 Hz, 1H, H5), 7.93 (d, J = 8.4 Hz, 1H, H8); 13C NMR (DMSO-d6, 100 MHz):
δ 17.1 (C12, 14), 79.0 (C11, 13), 151.4 (C10), 154.1 (C2), 137.8 (C4), 130.5 (C7), 129.2 (C8),
128.1 (C9), 127.8 (C5), 124.9 (C6), 122.5 (C3); 31P NMR (DMSO-d6, 161.9 MHz): δ 16.6.
M/s (positive mode) (m/z): 266 (M+H+). Anal. calcd. for C13H16NO3P: C 58.87, H 6.08,
N 5.28; found: C 58.85, H 6.12, N 5.26.
BIOLOGICAL ASSAY
Antibacterial Activity
Antibacterial activity of the newly synthesized dialkyl heteroaryl phosphonates 3(a–e)
and 6(a–e) was assayed against two Gram +Ve bacterial strains, S. aureus, P. aeruginosa;
two Gram –Ve bacterial strains, K. pneumoniae and B. megaterium; two Gram –Ve antibi-
otic resistant bacterial strains, mutant E. coli (Streptomycin resistant) and donor E. coli
(Rifampicin resistant) bacteria, by the agar well diffusion method25,26 using Amoxiclav
(SD063, Himedia) as a standard drug. The results are presented in Table S1.

NANO-BF3.SiO2 CATALYZED MICHAELIS–ARBUZOV REACTION
1523
Determination of Minimum Inhibitory Concentration
The MIC represents the lowest concentration of the antimicrobial agent that prevents
the development of visible growth after overnight incubation.27 MIC measurements were
performed using the modified agar well diffusion method. MICs of the test samples are
represented in Table S2.
Antifungal Activity
Some of the compounds were tested for the antifungal activity against two fungal
strains A. niger, P. spinulosum by the poison plate method28 using Fluconazole (SD 114,
Himedia) as a standard drug. The results are shown in Table S3.
CONCLUSION
In this study, we reported the synthesis of novel dialkyl heteroaryl phosphonates
3(a–e) and 6(a–e), in an efficient method using nano-BF3.SiO2 catalyst, with shorter reac-
tion times, low temperatures and high yields under solvent-free conditions. Nano-BF3.SiO2
is an eco-friendly, easily available, less toxic, cost effective, reusable, and green solid cata-
lyst. The newly synthesized dialkyl heteroaryl phosphonates 3(a–e) and 6(a–e) have shown
potent to good activities against all the tested pathogens.
From this, we conclude that this method shows promise for the synthesis of biologi-
cally active phosphonate drugs or drug intermediates, and that compounds 3a, 3b, 3c, and
3e may prove useful as antimicrobial agents in future.
ACKNOWLEDGMENT
One of the authors, M. Varalakshmi, would like to thank Jeanne Gilbert, USA, for
providing technical support to the final manuscript.
FUNDING
One of the authors, M. Varalakshmi, expresses thanks to the University Grants Com-
mission, New Delhi, India, for providing financial support under the Rajiv Gandhi National
Fellowship Scheme [F. No: F1-17.1/2011-12/RGNF-SC-AND-1419 /(SA-III/Website)] for
carrying out this research work.
SUPPLEMENTAL MATERIAL
Supplemental data for this article can be accessed on the publisher’s website at
http://dx.doi.org/10.1080/10426507.2014.996643
REFERENCES
1. For a review, see: Freedman, L. D.; Doak, G. O. Chem. Rev. 1957, 57, 479-523.
2. Murai, T.; Tomizawa C. J. Environ. Sci. Health, Part B 1976, B11, 185-197.
3. Kononova, S. V.; Nesmeyanova, M. A. Biochemistry 2002, 67, 184-195.

1524
M. VARALAKSHMI ET AL.
4. Papazoglou, E. S.; Flame retardants for plastics. In: C. A. Harper (Ed), Handbook of Building
Materials for Fire Protection; McGraw-Hill: New York, 2004; pp. 4.1-4.88.
5. (a) Mori, I.; Fonne-Pfister, R.; Matsunaga, S.; Tada, S.; Kimura, Y.; Iwasaki, G.; Mano, J.;
Hatano, M.; Nakano, T.; Koizumi, S.; Scheidegger, A.; Hayakawa, K.; Ohta, D. Plant Physiol.,
1995, 107, 719-723 (b) Abeles, F. B.; Morgan, P. W.; Saltveit, M. E. in Ethylene in Plant Biology,
2nd ed., ch. 9, Academy Press, Inc: California, 1992, pp. 270.
6. Deluchat, V.; Lacour, S.; Serpaud, B.; Bollinger, J. C. Water Res. 2002, 36, 4301-4306.
7. Hasan, H. A. Microbiol. Res. 1999, 154, 95-102.
8. Acebal, S. G.; Grassi, R. L.; Vuano, B. M. An. Asoc. Quim. Argentina 1993, 81, 57-65.
9. Horner, L.; Hoffmann, H.; Klink, W.; Ertel, H.; Toscano, V. G. Chem. Ber. 1962, 95, 581-601.
10. Segura, J. L.; Gornez, R.; Martin, N.; Guldi, D. M. Org. Lett. 2001, 3, 2645-2648.
11. (a) Zhen, L.; Urian, R. C.; Liu, Y.; Jen, A. K. Y.; Pu, L. Chem. Mater. 2000, 12, 13-15.(b) Pu, Y.
J.; Soma, M.; Kido, J.; Nishide, H. Chem. Mater. 2001, 13, 3817-3819. (c) Liao, L.; Pang, Y.;
Ding, L.; Karasz, F. E. Macromolecules 2008, 41, 5716-5722.
12. (a) Bordeau, G.; Larita, R.; Metge, G.; Fiorini-Debuisschert, C.; Charra, F.; Teulade-Fichou, M.
P. J. Am. Chem. Soc. 2008, 130, 16836-16837. (b) Shen, P.; Sang, G.; Lu, J.; Zhao, B.; Wan, M.;
Zou, Y.; Li, Y.; Tan, S. Macromolecules 2008, 41, 5716-5722.
13. (a) Elandaloussi, E. H.; Frere, P.; Richomme, P.; Orduna, J.; Grain, J.; Roncali, J. J. Am. Chem.
Soc. 1997, 119, 10774-10784. (b) Frere, P.; Rajmundo, J. M.; Blanchard, P.; Delaunay, J.;
Richomme, P.; Sauvajol, J. L.; Orduna, J.; Grain, J.; Roncali, J. J. Org. Chem. 2003, 68, 7254-
7265.
14. (a) Michaelis, A.; Kaehne, R. Chem. Ber. 1898, 31, 1048-1055. (b) Bhattacharya, A. K.; Thya-
garajan, G. Chem. Rev. 1981, 81, 415-430.
15. Yadav, V. K. Synth. Commun. 1990, 20, 239-246.
16. Renard, P. Y.; Varon, P.; Leclerc, E. Angew. Chem., Int. Ed. 2003, 42, 2389-2392.
17. Venkata Ramana, K.; Venkata Subbaiah, C.; Lokanatha, V.; Naga Raju, C. Der pharma Chemica
2011, 3(1), 181-188.
18. (a) Laven, G.; Stawinski, J. Synlett 2009, 2, 225-228. (b) Laven, G.; Kalek, M.; Jezowska, M.
New. J. Chem. 2010, 34, 967-975.
19. Subba Rao, D.; Thaslim Basha, S. K.; Naga Raju, C. Der pharmacia Lett. 2013, 5(3), 341-349.
20. Mataveeva, E. V.; Odinets, I. L.; Kozlov, V. A. Tetrahedron Lett. 2006, 47, 7645-7648.
21. Mirjalili, B. F.; Bamoniri, A.; Akbari, A. J. Iran. Chem. Soc. 2011, 8, 135-140.
22. Sadeghi, B.; Mirjalili, B. F.; Hashemi, M. M. Tetrahedron Lett. 2008, 49, 2575-2577.
23. Mirjalili, B. F.; Bamoniri, A.; Akbari, A. Tetrahedron Lett. 2008, 49, 6454-6456.
24. Sadeghi, B.; Mirjalili, B. F.; Hashemi, M. M. J. Iran. Chem. Soc. 2008, 5, 694-698.
25. Kazmi, S. U.; Ali, S. N.; Jamal, S. A.; Atta-ur-Rehman, J. Pharm. Sci. 1991, 4, 113-118.
26. Atta-ur-Rehman, Choudhary, M. I.; Thomson, W. J. Bioassay Techniques for Drug Development,
Harwood Academic Publishers: Amsterdam, 2001.
27. Greenwood, D.; Slack, R.; Peutherer, J. Medical microbiology, In: A Guide to Microbial Infec-
tions: Pathogenesis, Immunity, Laboratory Diagnosis and Control, 15th ed. ELST Publishers:
Edinburgh, 1997.
28. Rasheed, S.; Venkataramana, K.; Chandrasekhar, K.; Fareeda, G.; NagaRaju, C. Arch. Pharm.
Chem. Life Sci. 2012, 345, 294-301.