1

Chapter 1

1.1 Introduction

Phosphorus, a non-metallic element, is a major essential element for living organisms. It

occupies a central position (VA group) in the periodic table. The electronic configuration of

phosphorus in the ground state is 01

z

1

y

1

x

2622 33p3p3p3s2p2s1s d . This distribution in its relevant

orbitals led to well-defined families of tri-, tetra-, penta- and hexa-co-ordinate derivatives of

phosphorus, in which ligands can be organic or inorganic. Phosphorus has empty‘d’ orbitals

which can readily accept electrons from good donors. By virtue of its orbital disposition,

phosphorus exhibits multi valency and facile convertibility from one valence state to the other,

in its reactions with both metals and non-metals (Dillon et al. 1998).

Phosphorus mainly exhibits trivalency and pentavalency. Trivalent phosphorus possesses

remarkable ability to form strong bonds with carbon, nitrogen, oxygen, sulfur and selenium,

leading to the formation of a variety of acyclic and cyclic organophosphorus compounds with

interesting structures and stereochemistry. There has been an impressive progress in the study

of organophosphorus chemistry during the last six decades. Phosphorus chemistry has attracted

much interest due to its extensive utilization in the fields of agrochemicals, pesticides,

insecticides and pharmacy.

Organophosphorus chemistry is one of the fastest growing fields in chemistry, since it has

profound impact on every sphere of the human endeavour. Organophosphorus heterocycles

have been attracting a great deal of interest because of the diversity in their structures and their

ubiquitous nature. Organophosphorus compounds are basically chemical compounds

containing carbon-phosphorus bonds. Due to these carbon-phosphorus bonds they have

attracted much attention in the novel applications in bioorganic chemistry and organic synthesis

(Reddy et al. 2012). Organophosphorus compounds have wide range of applications in the

areas of industrial, agricultural, and medicinal chemistry owing to their unique

2

physicochemical and biological properties. The chemistry of organophosphorus heterocycles

has attracted much attention in many biological activities such as anti-bacterial, anti-fungal,

anti-viral, anti-leukemic, systemic pesticides, anti-inflammatory, anti-parasitic, anti-cancer,

anti-oxidant, enzyme inhibitors, anti-hypertensive and many other biological activities. One of

the other useful properties of organophosphorus compounds is the relatively metabolic break

down and low stability in animal, plants organisms and in soil (Chaturvedi et al. 2014).

Some of the organophosphorus compounds such as sarin (A), tabun (B) and dimefore (C) are

deadly toxic and act as biological toxins and they were found to be useful as chemical warfare

agents (Clarke, 1969, Rose 1968, Robinson, 1967 and Meselson, 1970).

Infectious diseases caused by microorganisms are on more rampant world-wide than during

the last century. Scientist is working very hard to find new ways that will control this microbes

but trying to destroy this microbes is not an easy task (Patila et al, 2014). Currently, different

types of drugs for anti-bacterial activity such as the Ciprofloxacin and other such as the anti-

fungal, anti-parasitic and anti-viral drugs. The disease causing microbes are good in the

adaption in different environmental conditions. These microbes can develop new features that

make them resistant to the drugs that once kill them. Cancer is one of the other major problem,

many research fields are trying to find a new way of finding a way in destroying this cancerous

cells and many different approaches are been used to stop the tumor cells from growing.

Looking at these problems and the biological properties of organophosphorus compounds this

project was done to find new ways that can try to solve these problems. In this project a class

P

O

F(H3C)2HCO

H3CO

A

P

O

CNC2H5O

Me2N

B

P

O

FMe2N

Me2N

C

3

of organophosphorus compound which are α-hydroxyphosphonates compounds that have some

biological activities were the compounds of interest for performing some bioactivities. There

are different ways to synthesize this α-hydroxyphosphonates but in this project a reaction by

Abramov was selected to synthesize α-hydroxyphosphonates compounds.

4

Chapter 2

Literature Review

2.1 α -Hydroxyphosphonates

α-hydroxyphosphonates are a class of organophosphorus compounds of synthetic interest

because of their biological activity. Phosphonates are esters of phosphonic acid (H3PO3) and

phosphinic acid (H3PO2) respectively. α-hydroxyphosphonates are extremely important

because their wide range of applications in pharmaceutical chemistry (drug discovery) and are

also the multi-component, one pot reactions. Due to the carbon-phosphorus bond formation

this compounds have attracted much attention in novel applications in organic synthesis and

bioorganic chemistry. α-hydroxyphosphonates compounds may also be used as precursors for

the organophosphorus polymers processing corrosion-resistant, ion-exchange and also flame-

resistant properties (Reddy et al. 2012). Indeed, α-hydroxyphosphonates can also be used for

extracting or discovering of metal ions.

A large number of methods have appeared describing novel synthesis of organophosphorus

compounds. α-hydroxyphosphonates are usually synthesized with the reaction of aldehydes or

ketones and dialkyl or trialkyl phosphite in the presence of a catalyst. Several catalysts have

been reported for the synthesis of α-Hydroxyphosphonates such as MoO2Cl2, niobium

complexes, CaO, various metal complexes, titanium, aluminium, ytterbium and lanthanum

under thermal conditions (Kumari et al. 2013). Meanwhile, tris (trimethylsilyl) phosphite was

also used to synthesize α-Hydroxyphosphonates but it requires elevated temperature under

anhydrous reaction conditions. However, many of these methods are associated with various

drawbacks such as use of metal catalysts, tedious experimental procedures, unsatisfactory

yields, long reaction times and usage of expensive, moisture sensitive catalysts. Hence, there

is a need to develop a rapid and efficient protocol for the synthesis of α-Hydroxyphosphonates.

In addition, they are also useful intermediates in the synthesis of other phosphorous compounds

5

(Reddy et al. 2012). Because of their potential bioactivity against wide spectrum of disease

manifestations several methods are reported for their synthesis.

2.2. Several different strategies for the synthesis of α-Hydroxyphosphonates

There are two main routes that are most commonly used to synthesize α-hydroxyphosphonates

(1). One route is Pudovik reaction in which addition of dialkyl phosphites to carbonyl

compounds to generate α-hydroxyphosphonates. It is a powerful and direct method for the

construction of carbon-phosphorus bonds (Pudovik, 1979).

RCHO + H P

O OR1

OR1 R P

HOO

OR1

OR1

1

Another route is modified Abramov reaction. In the original Abramov reaction, (Abramov,

1954) an aldehyde or a ketone is heated with trialkyl phosphite under stringent conditions to

obtain α-hydroxyphosphonates. Under these stringent conditions dialkyl α-

alkoxyalkylphosphonates could also formed and hence Abramov reaction is modified to

overcome such difficulties. Silyl halide or oxalic acid can be used along with carbonyl

compounds and the phosphorus reagent. The Abramov reaction with such modifications is

called modified Abramov reaction.

Pokalwar et al. (2006) reported the synthesis of -hydroxyphosphonates (2) from 2-

chloroquinolin-3-carbaldehyde derivatives (3) through the modified Abramov reaction using

chloro (trimethyl) silane (TMSCl).

6

N

CHO

Cl

R1

R2

R3

3

P(OC2H5)3

TMSCl

Toluene,ref luxN

R1

R2

R3

OH

P

Cl

OOC2H5

OC2H5

2

Vahadat et al. (2008) reported the solvent-free synthesis of novel -hydroxyphosphonates

(4) via modified Abramov reaction in the presence of oxalic acid.

R1CHO + P

OCH3 Oxalic acid(10 mol %)

Solvent-free

800C, 3h

R1 CH

OH

P

OOCH3

OCH3

4

OCH3

H3CO

Suresh Kumar et al. (2012) reported the green synthesis of -hydroxyphosphonates (5) by the

addition of diethylphosphite to various aldehydes in the presence of piperazine as a catalyst

under neat condition by simple grinding process.

+ PHPiperazine

Grinding,rt, 3-10 min

R

OH

P

O OC2H5

OC2H5

5

RCHO

OC2H5

OC2H5

O

Enantio selective synthesis of -hydroxyphosphonates (6) using organo catalyst L-proline was

reported by Sampak Samanta et al. (2006).

7

R1 P

O

O OR2

OR2 +

ONH

COOH

(20 mol %)

P

R1 OHO

O OR2

OR2

6

2.3 Biological activities of α-hydroxyphosphonates

In recent years, the synthesis of -hydroxyphosphonates has attracted much attention due to

their important biological activities such enzyme inhibitors, anti-HIV, anti-oxidant, anti-

fungal, anti-bacterial and anti-viral properties (Reddy et al. 2012).

2.4. Aim

The aim of this project was to synthesize and characterize the novel -hydroxyphosphonates

and screen their anti-bacterial and anti-cancer activities.

2.5. Hypothesis

From the previous literature, there were many synthesized -hydroxyphosphonates and shown

varied biological activities such as the anti-bacterial, anti-fungal, anti-cancer, etc. But there is

still in need of expeditious and efficient protocol for the synthesis of novel α-

hydroxyphosphonates with anti-bacterial and anti-cancer activities.

2.6. Keeping in view of the above literature, the major objectives of this project were to:

Design and synthesize -hydroxyphosphonates having biological importance.

Develop simple and effective method for the synthesis of target molecules.

Characterize the structure of the newly synthesized -hydroxyphosphonates by spectral

techniques.

Assess the anti-bacterial and anti-cancer activity of the synthesized compounds.

8

Chapter 3

(See appendix A and B for more details)

Materials and methods

This chapter provides the description of the methods, equipment, reagents used to synthesize

-hydroxyphosphonates compounds, the anti-bacterial activity and the anti-cancer activity.

3.1. Equipment

Autoclave, 96-well microliter plate, pipette, micropipette, weighing machine, round-bottom

flask, laboratory condenser, laboratory spatula, incubator, petri-dish, schott bottle, test tube,

thin-layer chromatography plates, glass beaker, cotton wool, condenser, magnetic pellet,

magnetic stirrer with a hot plate, inoculating loop, Bunsen burner, vortex mixer, micro tube,

thermometer, water bath, Stuart® for melting point apparatus, Bruker-Tensor 27 for IR,

eppendorf tubes, microliter plate reader, rota-evaporator.

3.2. The bacteria strains

The selected bacteria were Staphylococcus aureus (ATCC 25925), Bacillus cereus (ATCC

10702), Vibrio Fluvialis (AL004), Escherichia coli (ATCC 10819) obtained from the

University of Zululand, Kwadlangezwa campus in the Department of Biochemistry and

Microbiology.

9

3.3. Chemicals and reagents

3.3.1. List of chemicals and reagents

Reagent/Chemical Name of supplier

4-Nitrobenzaldehyde Sigma-Aldrich

3-Fluoro aldehyde Sigma-Aldrich

4-Pyridine aldehyde Sigma-Aldrich

3-Pyridine aldehyde Sigma-Aldrich

4-Chloro aldehyde Sigma-Aldrich

2,4-Dichloro aldehyde Sigma-Aldrich

Dimethyl sulphoxide Sigma-Aldrich

Piperonaldehyde Sigma-Aldrich

Ethanol Merck

Indole-3-aldehyde Sigma-Aldrich

Ethyl acetate Sigma-Aldrich

Mueller-Hinton broth Merck

Mueller-Hinton agar Merck

Hexane Sigma-Aldrich

Acetone Sigma-Aldrich

Tetrahydrofuran Sigma-Aldrich

1,4-Dimethlypiperazine Sigma-Aldrich

Diphenylphosphite Sigma-Aldrich

Assay buffer Sigma-Aldrich

Methanol Merck

10

3.4. Methodology

3.4.1. General procedure for the synthesis of α-hydroxyphosphonates (3a-h)

A mixture of aldehyde (0.002 mol), diphenylphosphite (0.002 mol), and 1, 4-

dimethylpiperazine (0.002 mol) was refluxed for four hours in 20 mL of tetrahydrofuran (THF)

at 68 oC. The progress of the reaction was monitored by TLC analysis. After completion of the

reaction, as indicated by TLC (silica gel) using hexane and ethyl acetate (3:1) as a mobile phase,

the solvent was removed in a rota-evaporator and the crude product obtained was washed with

hexane and ethyl acetate repeatedly to afford the analytically pure 3a. Similarly, the compounds

3b to 3h were synthesized by adopting the above procedure.

3.4.2. Biological assays

3.4.2.1. Anti-bacterial activity

3.4.2.1.1. Determination of minimum inhibitory concentration (MIC)

The MIC’s of the α-Hydroxyphosphonates were determined using the micro broth dilution

method as described by Penduka et al. (2011), in 96-well microtiter plates. The starting

concentration of each α-Hydroxyphosphonates was 5 mg/mL and it was dissolved in 5%

DMSO with sterile distilled water. A 100 µL volume of double strength Mueller-Hinton broth

was pipetted to all 96 wells. The synthesized compound was then serial diluted in a double

strength Mueller-Hinton broth to make different test concentrations of the compound in the

wells. A volume of 20 µL of the test bacteria was introduced in column 1 to column 9. A

standard antibiotic ciprofloxacin was used as a positive control in column 10 (it was serial

diluted to different concentration) and also 5% DMSO was also introduced in column 11 for

any bacterial activity and lastly in column 12 sterility wells were included. The plate was

incubated over night at 37 oC for 24 h. After incubation the results were read visually with a

naked eye by pipetting 40µL volume of iodonitrotetrazolium violet (INT) in all wells. A colour

11

change form colourless to a purple colour indicated that there was bacterial growth based on

the oxidation-reduction reaction in which electrons are transferred from NADH to INT which

is purple. The MIC’s were then recorded in lowest concentration of α-hydroxyphosphonates

and also the antibiotic ciprofloxacin after 24 h.

3.4.2.1.2. Determination of the minimum bactericidal concentration (MBC)

The MBC was determined from the MIC micro broth dilution technique by sub culturing 15µL

of volume from the wells that did not show any growth after 24 h of incubation. They were

inoculated into fresh Mueller-Hinton agar plates, there after they were streaked and incubated

overnight for 24 h at 37 oC. The MBC results were recorded to the lowest concentration at

which the α-hydroxyphosphonate and ciprofloxacin kills the bacteria.

3.4.3. Anti-cancer activity

3.4.3.1 Acetylcholinesterase Activity Assay

1 mg/mL of samples of all α-Hydroxyphosphonates was prepared and it was dissolved with

100 % DMSO. 80 mg of the reagent was prepared together with the 4 mL of assay buffer and

they were mixed with a vortex. The first column (in duplicates) in the 96-well plate was

pipetted with 200 µL distilled water (assay blank) and 200 µL of the Calibrator (positive

control) in the second column. Therefore, the third well was pipetted with 10 % DMSO. The

samples (10 µL) were added into separate wells of the 96 well plates. 190 µL of the freshly

prepared working reagent was transferred to all sample wells and the plate was tapped briefly

to mix. The samples were incubated at room temperature for 2 min and there after the initial

absorbance measured at 412 nm was recorded. The plate was further incubated for 10 min to

take the final measurement (A412) final.

12

Chapter 4

Results

RCHO +PH

O

O O

1a-h 2

THF, 4 h

1, 4-Dimethylpiperazine

68 oC

R P

HOO

O O

3a-h

N

CHO

N

CHO

F

CHO

CHO

CHO

Cl

Cl

Cl

NO2OHC

O

OOHC

NH

CHO

RCompound

1a

1b

1c

1d

1e

1f

1g

1h

Scheme1 Synthesis of novel α-Hydroxyphosphonates (3a-h)

13

Table 4.1. Physical, analytical and spectral data for the synthesized α-Hydroxyphosphonates

(3a-h)

Compound

Melting

point

(oC)

Yield

(%)

IR

(cm-1)

LC-MS

Diphenyl (hydroxyl (4-nitrocyclohexyl)

methyl) phosphonate (3a)

80-82

80

3159 (OH),

1206 (P=O),

1024 (C-O-P)

391.35

Diphenyl ((3-fluorocyclohexyl)

(hydroxy) methyl) phosphonate) (3b)

74-76

72

3250 (OH),

1220 (P=O),

1020 (C-O-P)

364.38

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

methyl) phosphonate (3c)

130-132

78

3410 (OH),

1217 (P=O),

1024 (C-O-P)

347.35

Diphenyl (hydroxy (piperidin-3-yl)

methyl) phosphonate (3d)

142-144 76 3374 (OH),

1235 (P=O),

1038 (C-O-P)

347.35

(R)-diphenyl ((4-chlorocyclohexyl)

(hydroxy) methyl) phosphonate (3e)

67-69

71

3305 (OH),

1242 (P=O),

1042 (C-O-P)

380.09

Diphenyl ((2, 4-dichlorocyclohexyl)

(hydroxy) methyl) phosphonate (3f)

73-75

70

3312 (OH),

1217 (P=O),

1042 (C-O-P)

415.25

Diphenyl ((hexahydrobenzo[d] [1, 3]

dioxol-5-yl) (hydroxy) methyl)

phosphonate (3g)

Liquid

73

3341 (OH),

1235 (P=O),

1046 (C-O-P)

390.37

Diphenyl ((hexahydrobenzo[d] [1, 3]

dioxol-5-yl) (hydroxy) methyl)

phosphonate (3h)

125-127

76

3174 (OH),

1242 (P=O),

1049 (C-O-P)

385.39

14

4.2. The anti-bacterial activity

4.2.1 MIC determination

The MIC results all the synthesized α-Hydroxyphosphonates and the antibiotic are shown in

Table 4.2.1.1. The maximum MIC concentration was 10 mg/mL for all the α-

Hydroxyphosphonates and 10 µg/mL for the standard antibiotic ciprofloxacin. The 5 % DMSO

didn’t show any activity against the test bacteria.

Table 4.2.1.1. MIC determination of α-Hydroxyphosphonates against some test bacteria.

NOTE: R- denotes the resistance towards the test antibacterial agent at a maximum test

concentration of 5 mg/mL and NA- also represents no activity against the test bacteria.

Test bacteria

Test anti-bacterial agent (mg/mL)

(α-Hydroxyphosphonates)

Controls

3a

3b

3c

3d

3e

3f

3g

3h

Ciprofloxacin

(µg/mL )

5%

DMSO

Staphylococcus

Aureus (ATCC

25925)

5

R

R

5

R

R

R

R

0.625

NA

Vibrio fluvialis

(AL004)

5

R

5

5

5

R

5

5

2.5

NA

Bacillus cereus

(ATCC 10702)

5

R

R

R

R

R

5

R

0.313

NA

Escherichia coli

(ATCC 25925)

5

R

R

R

R

R

5

R

0.625

NA

15

4.2.2. MBC determination

The results for the MBC determination are shown in Table 4.2.2.1, in which the compounds

that showed activity in the MIC concentration were tested for their bactericidal activity at

5mg/mL and the standard antibiotic ciprofloxacin was also tested for its bactericidal activity at

different obtained MIC concentrations (in µg/mL).

Table 4.2.2.1. MBC determination of the α-Hydroxyphosphonates and ciprofloxacin against

some test bacteria.

Note: > -denotes that the MBC concentration is greater than 5mg/mL of the test anti-bacterial

agent and 5µg/mL of antibiotic, ND- represent that the MBC was not determined.

Test bacteria

Test anti-bacterial agent (mg/mL)

Control(+)

3a 3c 3d 3e 3g 3h Ciprofloxaci

n (µg/mL)

Staphylococcus

Aureus (ATCC 25925)

> 5

5

>5

ND

ND

ND

> 5

Vibrio fluvialis

(AL004)

> 5

>5

>5

> 5

> 5

> 5

> 5

Bacillus cereus

(ATCC 10702)

> 5

ND

ND

ND

> 5

ND

> 5

Escherichia coli

(ATCC 10819)

> 5

ND

ND

ND

> 5

ND

5

16

4.3 Anti-cancer activity

4.3.1. Acetylcholinesterase Activity

(See Appendix D for more details)

The results Acetylcholinesterase activity inhibition are show in the Table 4.3.1.1 and Table

4.3.1.2 , by which all the synthesized compounds were screened for its inhibition. 1 mg/mL of

the compound was used and it was dissolved in 100 % DMSO (Table 4.3.1.1) and 0.1 mg/mL

of the compound with different solvent (100 % methanol) was also used to the compounds

which had a negative number. A negative number (-) in this case shows that the compound was

effective to the inhibition of the activity of Acetylcholinesterase and a positive number shows

that the reaction is not inhibited.

Table 4.3.1.1 Acetylcholinesterase activity at 1 mg/mL of the compound

Compound Acetylcholinesterase activity

3a -33.63

3b -49.78

3c 14.80

3d 14.57

3e 9.64

3f -19.73

3g -81.84

3h -22.87

Table 4.3.1.2 Acetylcholinesterase activity at 0.1 mg/mL of the compound

Compound Acetylcholinesterase activity

3a -27.62

3b -27.11

3f 74.68

3g -23.02

3h -134.53

17

Chapter 5

5.1 Discussion

The α-hydroxyphosphonates (3a-h) were synthesized by the reaction of aldehyde with various

diphenylphosphite was refluxed for 4h at 60-68oC in dry tetrahydrofuran (THF) in the presence

of 1, 4-dimethylpiperazine as a strong base. The progress of the reaction was monitored by

TLC analysis. After completion of the reaction, as indicated by TLC (silica gel) using hexane

and ethyl acetate (3:1) as a mobile phase, the solvent was removed in a rota-evaporator and the

crude product obtained was purified by using hexane and ethyl acetate with repeated washing

to afford the analytically pure 3a. Similarly, the compounds 3b to 3h were prepared by adopting

the above procedure.

The structures of the title compounds (3a-h) were established by their spectroscopic data. The

characteristic IR stretching absorptions were observed in the regions 3281-3245 cm-1 (OH),

1240-1205 cm-1 (P=O), and 1061-1009 cm-1 (P-O-Caliphatic) (Veera et al. 2009). In the 1H NMR

spectra, the P-CH group appeared as a doublet at δ 4.52-5.23 (J=9.56-11.56 Hz) due to its

coupling with phosphorus (Veera et al. 2009). The 13C NMR spectral data of the title

compounds (3a-h) showed characteristic chemical shifts for aromatic carbons. The P-C carbon

signals were observed at δ 72.4-76.8 and the data of other carbon signals were observed in the

expected region. 31P NMR chemical shifts of the title compounds (3a-h) appeared in the

expected region at δ 20.24-22.90. Liquid chromatography/mass spectrometry (LC-MS) spectra

gave molecular ions and diagnostic daughter ion peaks at their expected m/z values.

18

Biological activity

Anti-bacterial activity

Preliminary anti-bacterial activity of the title compounds (3a-h) were evaluated (Table 4.2.1.1

and 4.2.2.1). All the title compounds were screened against Gram positive bacteria

(Staphylococcus aureus (ATCC 25925), Bacillus cereus (ATCC 10702)) and Gram negative

(Vibrio Fluvialis (AL004), Escherichia coli (ATCC 10819)) bacteria by the microbroth

dilution method for their MIC and MBC and the results were compared with the standard drug

ciprofloxacin. The results revealed that majority of the synthesized compounds showed varying

degrees of inhibition against the tested microorganisms. In general, the compound 3a showed

higher activity against the selected Gram positive bacteria and Gram negative bacteria at a

maximum concentration of 5mg/mL the activity of this compound can be due to the presence

of the electron-withdrawing (deactivating) group such as the Nitro atom attached to its benzene

ring, whereas compounds 3c, 3d, 3e, 3g and 3h showed moderate activity. Compounds 3b and

3f were not effective at 5 mg/mL to all the selected bacteria. Only compound 3c showed a

bactericidal activity (able to kill the bacteria). The lowest concentration (highest dilution)

required to arrest the growth of bacteria was known as minimum inhibitory concentrations

(MIC). The standard antibiotic ciprofloxacin (positive control) was effective against all the

selected bacteria at MIC concentration at a range of 0.039 to 5 mg/mL. The 5 % DMSO

(negative control) did not show any activity against the bacteria.

19

Anti-cancer activity

The discovery that lung cancer and other cancers can synthesize and secrete Acetylcholine

which acts as an autocrine growth factor. Acetylcholine secreted by these cancers stimulate

growth of the cancerous cells (tumor growth) (Song and Spindel, 2008). All the title

compounds (3a-h) were tested for Acetylcholinesterase activity. Among them, the compound

3g at a concentration of 1 mg/mL (100% DMSO) was the most effective (stop the reaction

completely) due to the heteroatoms attached to the benzene ring, even compounds 3a, 3b, 3f

and 3h did show some activity whereas the rest of the compounds did not show any activity.

In the concentration of 0.1 mg/mL (100% Methanol) compound 3h emerged as the most

effective, this is due to its Indole moiety attached to the benzene ring. The effectiveness of this

compounds was showed by their negative values (Table 4.3.1.1 and 4.3.1.2), whereas the

positive values of the compounds showed no activity (they weren’t able to inhibit the reaction).

The results were also compared visually (with naked eye) with the positive control (Calibrator),

the clear wells represented the inhibition of the reaction and the wells that showed a light yellow

colour, meaning that there was an activity of the enzyme Acetylcholinesterase. The inhibition

of the activity of Acetylcholinesterase by this compounds can be due to the presence of

electron-withdrawing deactivating groups such as the nitro, fluoro and chloro atoms present on

the benzene ring. The inhibition of the enzyme Acetylcholinesterase can result in the decrease

of the growth of tumor cells in the body, since this enzyme activates this growth of tumor.

20

Chapter 6

6.1. Conclusion

A convenient high-yielding one-pot, two-component reaction of aldehyde (1a-h), and

diphenylphosphite (2) by using 1, 4-dimethylpiperazine as a catalyst for the synthesis of novel

α-hydroxyphosphonates (3a-h) was accomplished. 1, 4-dimethylpiperazine was proved to be

an efficient catalyst in this reaction. The notable advantages of the present synthetic protocol

are shorter reaction times, higher yields of final products without any by-products and easy

reaction work-up procedure. It is expected that the present methodology will find application

in organic synthesis. The results of the anti-bacterial and anti-cancer activities reveal that

among the 8 compounds screened, 3a compound showed anti-bacterial activity and 3a, 3b, 3g

and 3h compounds exhibited to display highest anti-cancer activity.

6.2 Further work from this project

A future research work from this project can be performing some bioactivities like anti-fungal

and anti-viral activities of the synthesized α-hydroxyphosphonates.

21

References

Abramov V. S. (1954), Synthesis, Dokl Aqlkd Nauk SSSR, (95): 191.

Chaturvedi Kalpana, Ashish Kumar and Abhinav Mishra. (2014), Synthesis, antibacterial and

antifungal properties of novel organophosphorus compounds, Der Pharma Chemica, 6(3):27-

32.

Clarke R. (1969), We all fall down, The Prospects of Biological and Chemical Warfare Pelican,

London.

Dillon K.B, Mathey, F and Nixon, J.F. (1998), Phosphorus. The Carbon Copy, John Wiley. &

Sons, New York.

Kumari Shweta, Amiya Shekhar and Devendra D. Pathak. (2014), A New Catalyst and Solvent-

free Green Synthesis of α-Hydroxy Phosphonates and α-Aminophosphonates 3(1): 45-54.

Meselson M.S., (1970) Chemical and Biological Weapons, Sci. Am., 222 (5) 15.

Pudovik A.N, (1979), Synthesis, 81.

Patila S. Nilesh, Ganesh B. Deshmukha, Sambhaji V. Patila, Avinash D. Bholayb, Nitin D.

Gaikwada.(2014), Synthesis and biological evaluation of novel N-aryl maleimide derivatives

clubbed with α-hydroxyphosphonates, Journal of medicinal chemistry. 83: 490–497.

Penduka Dambudzo, Omobola O. Okoh and Anthony I. Okoh. (2011), In-Vitro Antagonistic

Characteristics of Crude Aqueous and Methanolic Extracts of Garcinia kola (Heckel) Seeds

against Some Vibrio Bacteria, (16): 2754-2765.

Pokalwar U Rajkumar, Rajkumar V.Hangarge, Prakash V. Maske and Muralidhar S. Shingare.

(2006) Synthesis and antibacterial activities of α-hydroxyphosphonates and α-

acetyloxyphosphonates derived from 2-chloroquinoline-3-carbaldehyde, Arkivoc, (xi), 196.

22

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.

Rose S. (1968) Chemical and Biological Warfare, Harrap, London.

Sampak Samanta and Cong-Gui Zhao. (2006), J Am Chem Soc., 23, 128, 7442.

Song Pingfang and Eliot R. Spindel (2008), Basic and clinical aspects of non-neuronal

Acetylcholine: expression of non-neuronal acetylcholine lung cancer for therapy, Journal of

pharmalogical sciences, (106): 180-185.

Suresh Kumar, C. Bhupendra Reddy, M. Veera Narayana Reddy, C. Radha Rani and C. Suresh

Reddy. (2012), Organic Commun. 50

Vahdat S.M. R. Baharfar, Md. Tajbakhsh, A. Heyadri, S. Meysam Baghbanian and S. Khaksar.

(2008), Organocatalytic synthesis of α-hydroxy and α-aminophosphonates, Tetrahedron Lett.

(49): 6501- 6504.

Veera Narayana Reddy M, Balakrishna A, Anil Kumar M, Chandra Sekhar Reddy G. Uma

Ravi Sankar A, Suresh Reddy C, Murali Krishna T, (2009). One-Step Synthesis and Bioassay

of N-Phosphoramidophosphonates, Chem. Pharm. Bull., (57): 1391-1395.

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Appendix A

A.1 Preparation of media

A1.1 Mueller-Hinton broth

4.2 g of Mueller-Hinton broth was weighed and it was also dissolved in 200 mL Schott bottle

with distilled water. I was dispensed into test tubes and then the tubes with the Mueller-Hinton

broth were autoclaved (15 min at 121 oC). This preparation was done for the cultivation of the

bacteria.

For the double strength Mueller-Hinton broth, 8.4 g was weighed and it was dissolved in

the Schott bottle (200 mL) with distilled water. It was then autoclaved (15 min. at 121 oC).

A1.2 Mueller-Hinton agar

9.5 g of Mueller-Hinton Agar was weighed and dissolved in 250 mL Schott bottle with distilled

water. It was autoclaved (15 min at 121oC). After autoclaving it was let to cool down (avoiding

it to solidify) and then it was prepared in sterile petri-dishes.

A2. Preparation of iodonitrotetrazolium (INT)

0.04 g of INT was weighed and it was dissolved in 200 mL of sterile distilled water for the

visualisation of the bacterial growth

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Appendix B

Details of the methods

B1. Preparation of the compounds for anti-bacterial activity.

0.01 g of the each compound was weighed and it was dissolved in 50 µL of DMSO together

with 950 µL distilled water and they were all mixed together in the eppendorf tube using a

vortex mixer.

B2. Preparation of the MIC

Firstly, 1 mL of the bacteria was diluted into the Mueller-Hinton broth.100 µL of double

strength Mueller-Hinton broth was pipetted into all 96-wells of the microtiter plate. 100µL of

the compound (in triplicates) was pipetted also pipetted into the wells excluding column 10, 11

and 12. 20 µL of the microbe was pipetted from column 1 to column 9. The antibiotic

ciprofloxacin was used as a positive control and the volume of 10 mg/mL was added in column

10. 50 µL of DMSO was added in column 11 and lastly distilled water was also used in column

12 for their activities against the bacteria.

B3Acetylcholinesterase Activity Assay Kit

A3.1 Preparation of the working reagent

0.08 g of the reagent was weighed and then it was mixed together with 4000 µL of Assay Buffer

into a centrifuge tube and they were dissolved using a vortex.

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Appendix C

Spectral studies

Infrared (IR) results for the synthesized α-Hydroxyphosphonates (1a-h)

C1. Diphenyl (hydroxyl (4-nitrocyclohexyl) methyl) phosphonate

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C2. Diphenyl ((3-fluorocyclohexyl) (hydroxy) methyl) phosphonate

C3. (R)-diphenyl (hydroxy (piperidin-4-yl) methyl) phosphonate

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C4. Diphenyl (hydroxy (piperidin-3-yl) methyl) phosphonate

C5. (R)-diphenyl ((4-chlorocyclohexyl) (hydroxy) methyl) phosphonate

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C6. Diphenyl ((2, 4-dichlorocyclohexyl) (hydroxy) methyl) phosphonate

C7. Diphenyl ((hexahydrobenzo[d] [1, 3] dioxol-5-yl) (hydroxy) methyl) phosphonate

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C8. Diphenyl ((3a, 4, 5, 6, 7, 7a-hexahydro-1H-indol-3-yl)(hydroxy)methyl) phosphonate

30

Appendix D

Calculation of Acetylcholinesterase Activity

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

(A412)calibrator – (A412)blank

200 = equivalent activity (units/L) of the Calibrator when Assayed is read at 2 minutes and 10

minutes

(A412) calibrator = Absorbance of the calibrator at 10 minutes. (A412) blank = Absorbance of the

blank at 10 minutes.

Table D1: The microtiter plate reader results after incubation in 2 and 10 minutes in two

concentrations (1 mg/ml and 0.1 mg/mL).

1 mg/mL in 100% DMSO

Incubation time

0.1 mg/mL in 100 % Methanol

Incubation time

2 minutes 10 minutes 2 minutes 10 minutes

Blank: 0.04 Blank: 0.089 Blank:0.051 Blank:

Calibrator: 0.2195 Calibrator: 0.981 Calibrator:0.457 Calibrator:

DMSO: 0.877 DMSO: 0.491 Methanol:0.604 Methanol:

3a: 1.622 3a: 1.472 3a: 0.685 3a: 0.631

3b: 2.232 3b: 2.01 3b: 0.899 3b: 0.846

3c: 0.796 3c: 0.862 3f: 0.887 3f: 1.033

3d: 0.135 3d: 0.2 3g: 0.301 3g: 0.256

3e: 0.255 3e: 0.298 3h: 1.456 3h: 1.193

3f: 2.504 3f: 2.416

3g: 2.295 3g: 1.93

3h: 1.836 3h: 1.734

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