SYNTHESIS, SPECTRAL CHARACTERIZATION AND BIOACTIVITY OF NOVEL ORGANOPHOSPHORUS

COMPOUNDS

DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY

CANDIDATE: NEKHUMBE DIIMISENI EZEKIEL

SUPERVISOR: Prof. AR Opoku

Co-supervisor: Dr. C Sampath

INTRODUCTION TO ORGANOPHOSPHORUS COMPOUNDS

Organophosphorus compounds are chemical compounds containing carbon-phosphorus bonds.

Phosphorus-carbon bond formation has attracted much attention because of its application in organic synthesis and bioorganic chemistry.

Organophosphorus compounds are mostly esters, amides or thiol derivatives of phosphonic acid and constitute a large group of chemical compounds.

APPLICATIONS

VERTERINARY MEDICININE PHARMACEUTICALS

AGRICULTURE - INSECTICIDES

INTRODUCTION TO ALPHA-HYDROXYPHOSPHONATES

What are α-Hydroxyphosphonates?

α- Hydroxyphosphonates are class of Organophosphorus compounds of synthetic interest because of their biological activity.

R

HO

P

OR1

OR1

O

ALPHA CARBON

Their biological activities include: anti-cancer, anti-viral, anti-bacterial, anti-fungal, anti-leukemic and many other biological activities (Subba et al., 2012).

They are useful precursors of α- functionalized phosphonates such as:-

α-Aminophosphonates

α-Aminophosphonic acids Pudovik reaction(Pudovik, 1972)

Kabachnik-Field reaction(Fields, 1952)

The two main routes for synthesis of α-Hydroxyphosphonates Pudovik reaction Abramov reaction

In the Abramov reaction, an aldehyde or a ketone is heated with trialkyl or dialkylphosphite to obtain alpha-Hydroxyphosphonates (Subba et al., 2012).

AIM OF THE CURRENT RESEARCH PROJECT

To synthesize Novel α-Hydroxyphosphonates compounds and test their antibacterial and anticancer activities.

OBJECTIVES OF THE PROJECT

Design and synthesize eco-friendly novel α-Hydroxyphosphonates having biological importance.

Develop simple and effective method for synthesis of target molecules.

Characterize the structure of the newly synthesized α-Hydroxyphosphonates by elemental analysis, spectral techniques.

Assess the antibacterial and anticancer activity of the synthesized compounds.

MATERIALS AND METHODS

RCHO + P

O

OR1H

OR1P

O

OR1

OR1

OH

R

Dialkylphosphite Alpha Hydroxyphosphonates

Here R= Various aldehyedes R1= Phenyl, ethyl, methyl, iso propyl, butyl, etc

Reflux for 4-5 hours

1,4-Dimethyl piperizine

Synthesis of Alpha-Hydroxyphosphonates compounds by Abramov reaction.

RESULTS AND DISCUSSION

.COMPOUND 1

O2N CHO + PHO2N C P O

1,4-Dimethyl piperizine

THF (680C), Ref lux 4-5 hours

OH

H

O

4- Nitrobenzaldehyde Diphenyl phosphite

TLC system:- 7:3 Hexane and ethyl acetateYield:- 80%

O

O

Diphenyl (hydroxy(4-nitrocyclohexyl)methyl)phosphonate

O O

MP: 80-82oC

CHO

F

COMPOUND 2

+ PH

O

F

C

OH

H

P O

3-Flourobenzaldehyde Diphenyl phosphite

1,4- Dimethyl piperizine

THF (680C), Ref lux 4-5 hours

TLC system:- 7:3 Hexane and ethyl acetateYield:- 72%

O

OOO

diphenyl ((3-f luorocyclohexyl)(hydroxy)methyl)phosphonate

MP: 74-76oC

COMPOUND 3

NH

CHO

+ PH

O

NH

C HHO

P

O

1,4-Dimethly piperizine

THF (680C), Ref lux 4-5 hours

4-pyridinebenzaldehyde Diphenyl phosphite

TLC system:- 7:3 Ethly acetate and hexane (suitable system) 7:3 Hexane and ethyl acetate (not suitable system)Yield:- 78%

O

O O

O

(R)-diphenyl (hydroxy(piperidin-4-yl)methyl)phosphonate

MP: 130-132oC

NH

+ HP

O

NH

C

OH

H

P

3-Pyridine aldehyde Diphenyl phosphie

COMPOUND 4

1,4-Dimethyl piperizine

THF (680C), Reflux 4-5 hours

TLC system:- 7:3 Hexane and ethyl acetate

OCHO

O O

O

O

Diphenyl (hydroxy(piperidin-3-yl)methyl)phosphonate

MP: 142-144oC

COMPOUND 5

Cl

CHO

+ PH

O

Cl

C HHO

P

O

1,4-Dimethyl piperizine

THF (680C), Reflux 4-5 hours

4-ChlorobenzaldehydeDiphenyl phosphite

TLC system:- 7:3 Hexane and ethyl acetateYield:- 71%

O O

OO

(R)-diphenyl ((4-chlorocyclohexyl)(hydroxy)methyl)phosphonate

MP: 60-69oC

COMPOUND 6

Cl

Cl CHO + HP

O

C P O

OH

H

Cl

Cl

1,4-Dimethyl piperizine

THF(680C), Reflux 4-5 hours

Diphenyl phosphite2,4-Dichlorobenzaldehyde

TLC sytem:- 7:3 Hexane and ethyl acetateYield:- 70%

O O

O

O

diphenyl ((2,4-dichlorocyclohexyl)(hydroxy)methyl)phosphonate

MP: 73-75oC

COMPOUND 7

O

O

CHO

+ PH

O O

O

C

H

OH

P O

1,4-Dimethyl piperizine

THF (680C), Ref lux 4-5 hours

Piperonaldehyde Diphenyl phosphite

TLC System:- 7:3 Hexane and ethly acetateYield:- 73%

O

O

OO

diphenyl ((hexahydrobenzo[d][1,3]dioxol-5-yl)(hydroxy)methyl)phosphonate

COMPOUND 8

NH

CHO

+ PH

O

NH

C

H

OH

P O

1,4-Dimethly piperizine

THF (680C), Reflux 4-5 hours

Diphenyl phosphite

Indole-3-aldehyde

TLC System:- 7:3 Hexane and ethyl acetateYield:- 76%

O

O

O

O

diphenyl ((3a,4,5,6,7,7a-hexahydro-1H-indol-3-yl)(hydroxy)methyl)phosphonate

MP: 125-127oC

THIN LAYER CHROMATOGRAPHY (TLC)

WASHING OF IMPURE SYNTHESIZED COMPOUNDS

SPECTROSCOPIC STUDIES

ANTIBACTERIAL ACTIVITY

TLC

Hexane

Ethyl acetate

ANTICANCER ACTIVITY

EIGHT SYNTHESIZED COMPOUNDS

IR SPECTRA RESULTS FOR:

COMPOUND 1

C P O

OH

H

Cl

Cl

O

O

COMPOUND 8

O

O

C

H

OH

P O

O

O

COMPOUND 9

ANTIBACTERIAL ACTIVITY

MICROBOTH DILUTION METHOD WAS USED TO TEST THE MINIMUM INHIBITORY CONCENTRATION AND MINIMUM BACTERICIDAL CONCENTRATION OF THE SYNTHESIZED COMPOUNDS

Staphylococcus aureasATCC-25925

Bacillus cereusATCC 10702

Vibrio fuvialisAL004

Escherichia coliATCC 10819

MINIMUM INHIBITORY CONCENTRATION (MIC)

Table 1: Antibacterial activities of 8 novel synthesized alpha-Hydroxyphosphonates.

NOTE: ---- denotes the resistance towards the test antibacterial agent at a maximum test concentration of 5mg/ml.

 

Microorganisms Synthesized compounds

(alpha-Hydroxyphosphonates)

 

Controls

 

C1 C2 C3 C4 C5 C6 C7 C8 Ciprofloxacin (+)

water (-)

 

Staphylococcus

Aureus ATCC-25925

 

5mg/ml

 

----

 

----

 

5mg/ml

 

----

----

----

----

0.625µg/ml

 

na

 

Vibrio fluvialis AL004

 

5mg/ml

 

----

 

5mg/ml

 

5mg/ml

 

5mg/ml

 

----

 

5mg/ml

 

5mg/ml

 

2.5µg/ml

 

na

 

Bacillus cereus ATCC 10702

 

5mg/ml

 

----

----

 

----

 

----

 

----

 

5mg/ml

----

 

0.313µg/ml

 

na

 

Escherichia coli ATCC 10819

 

5mg/ml

 

----

 

----

 

----

 

----

 

----

 

5mg/ml

 

----

 

0.625µg/ml

 

na

MINIMUM BACTERICIDAL CONCENTRATION (MBC)

Table 2: MBC results for the novel alpha-Hydroxyphosphonates  

 

Microorganisms

Compounds  

Control(+) C1 C3 C4 C5 C7 C8 Ciprofloxacin

 

Staphylococcus

aureus ATCC-25925

> 5mg/ml

 

 

5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5µg/ml

 

Vibrio fluvialis AL004

 

> 5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5µg/ml

 

Bacillus cereus ATCC 10702

 

> 5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5µg/ml

 

Escherichia coli ATCC 10819

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

>5mg/ml

 

5µg/ml

ANTICANCER ACTIVITY

The discovery of lung cancer and other cancers can synthesize and secrete acetylcholine by these cancers (Song and Spindel, 2008).

The inhibition of the enzyme Acetylcholinesterase can decrease the risk of the development of cancer.

The anticancer activity was determined using the Acetylcholinesterase Assay Kit (MAK119).

AChE Activity (units/L) = (A412)final – (A412)initial x 200

(A412)calibrator – (A412)blank

ACETYLCHOLINESTERASE ACTIVITY

Table 1: The results showing the activity of Acetylcholinesterase at 1mg/ml of the compound (in 100 % DMSO).

Compound Acetylcholinesterase

activity

1 -33.63

2 -49.78

3 +14.80

4 +14.57

5 +9.64

6 -19.73

7 -81.84

8 -22.87

KEY:- No activity+ Activity

TABLE 2: The results showing the Acetylcholinesterase activity at 0.1mg/ml of the compound ( in 100% methanol).

Compound Acetylcholinesterase

activity

1 -27.62

2 -27.11

6 74.68

7 -23.02

8 -134.53

CONCLUSION

A convenient high-yielding one-pot, two-component reaction of various aldehydes with

dialkylphosphite was successfully accomplished via Abramov reaction without any by-

products.

1,4-Dimethylpiperazine was proved to be an efficient catalyst in all the reactions.

The highlighting advantages of Abramov reaction.

The results of the antibacterial and anticancer activities revealed that the 8 compounds

screened had the bioactivities that were performed.

Further work from this project

To perform some more bioactivities such as the anti-fungal and antiviral activities of the newly synthesized compounds.

Acknowledgements

I would like to thank the following people: Prof A.R Opoku (supervisor)

: Dr C Sampath (Co-Supervisor)

: Dr R.A Mosa and Dr D Penduka (f (for helping out with bioactivities)

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Reddy S. Subba, Ch. SyamaSundar, S. Siva Prasad, E. Dadapeer, C. Naga Raju and C. Suresh Reddy. (2012), Synthesis, spectral characterization and antimicrobial activity of α-hydroxyphosphonates, Der Pharma Chemica, 4(6):2208-2213.

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