chapter-3 synthesis, characterization and cytotoxicity...

Chapter-3

Synthesis, characterization and cytotoxicity

evaluation of nimesulide based

new glycolamide esters

1. Poster at International Conference on “Recent Advances in Drug

Discovery’’ organized by Kakatiya University, Warangal. Oct 22-24,

2008, Pg. No-69.

Chapter-3

106

3.1 INTRODUCTION

The glycolamide ester moiety has been found in many

pharmaceutically important prodrug molecules. Esters of

2-hydroxyacetamide (1) are known as glycolamide esters 2.

ArO

O

NR

R1

O

HO

O

NH2

Unsubstituted glycolamide = R, R1 = H

Monosubstituted glycolamide = R=H, R1=alkyl/aryl

Disubstituted glycolamide = R, R1 = alkyl/aryl

1 2

N,N-Disubstituted glycolamide esters have earlier been reported as

potentially useful biolabile carrier linked prodrug type for carboxylic

acids mainly for NSAIDs.1

Bundgaard and Nielsen have discovered that glycolamides are cleaved

with remarkable speed in human plasma as compared to methyl or ethyl

esters.2 The study was carried out on a series of benzoate esters of

various N-substituted glycolamides 3.

O

O

NR1

R2

O

3

A series of glycolamide ester prodrugs of 6-methoxy-2-napthylacetic

acid3 4 (metabolite of NSAID nabumetone) and 4-biphenyl acetic acid 5

(metabolite of NSAID fenbufen) were synthesized by masking the free

carboxyl group, mainly responsible for gastric damage.4

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107

OMe

O

O

N

O

R

R1

O

O

N

O

R

R1

Ph

4 5

Similarly glycolamide esters of aspirin5 6, ibuprofen6 7, niflumic

acid7 8, scutellarin8 9 as biolabile prodrugs of carboxylic acid agents were

disclosed in the literature.

OCOMe

O

O

NRR1

O

R1RN

O

O

O

Me

Me

Me

6 7

N

HN CF3

O

O

NRR1

O

OHOHO

OH

O

OO

O

OH

OOH

HO

R'RNO

8 9

Apart from the application of glycolamide esters as potent prodrugs,

they have multiple uses.

Glycolamide ester analogues of indomethacin were synthesized and

tested for their cyclooxygenase (COX-1 and COX-2) inhibition properties

in vitro by Smriti et al.9 Compound 10 displayed good anti-inflammatory

activity in vivo.

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108

NMe

Me

O

O

O

N

O

O

Cl

OMe

O

O

N

O

R

R1

Me

10 11

A series of glycolamide esters of naproxen 11 were synthesized by

Nalini et al.10 The prepared derivatives showed significant anti-

inflammatory, anticonvulsants and reduced ulcerogenic activity when

compared with naproxen.

N-benzhydryl glycolamide esters are used as carboxyl protecting

groups in peptide synthesis.11 Nipecotamide glycolamide esters are used

in the treatment of platelet mediated thrombosis disorder.12 Glycolamide

esters of ibuprofen were found to have comparable anti-inflammatory

and analgesic activity as parent ibuprofen.6 Colfenamate (12)

(carbamoylmethyl 2-(3-(trifluoromethyl)phenylamino)benzoate) having

analgesic, anti-inflammatory activity also belongs to the class of

glycolamide ester.13

F3CHN

O ONH2

O

12

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109

3.2 PRIOR ART ON SYNTHESIS OF GLYCOLAMIDE ESTERS Retrosynthesis of glycolamide ester (retrosynthetic route a & b)

indicate that the synthetic approach may consist of two parts: (i)

construction of amide bond and (ii) development of ester bond

irrespective of order (figure 3.1).

R O

O

NH

O

R'

ab

R O

O

O

C---N

-NH

R'

R'NH2R O

O

NH

O

R'

+RCOOCH2COCl

+

RCOOCH2COOHRCOOCH2CONH2ClCH2CONH2+RCOOH

(a)+

R O

O

NH

O

R'

C---O RCOOH

+

ClCH2CONHR' ClCH2COCl

(b)

+ R'NH2

Figure 3.1: Retrosynthetic pathway of glycolamide ester

Literature search revealed that reports are available for the synthesis

of glycolamide esters based on both strategies.

An overview on selected methods for the synthesis of glycolamide

esters.

A) Construction of ester followed by amide based on retrosynthetic route

a.2

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110

Gycolamide esters were synthesized by reaction of benzoyloxyacetyl

chloride (obtained by reaction between benzoyl glycolic acid and thionyl

chloride) with appropriate amines in benzene.

B) Construction of amide bond followed by ester linkage based on

retrosynthetic route b.2-10

Esterification of carboxylic acid which includes NSAIDs with

appropriate N-substituted 2-chloroacetamide in DMF at 25 °C 2,7 or 90 °C

3-8, 10 in presence of sodium iodide and TEA or in presence of catalytic

amount of DMAP9 using TEA as base.

3.3 OBJECTIVE OF THE PRESENT WORK

N,N-Disubstituted glycolamide esters of NSAIDs1 are extensively

used as carrier linked prodrug as they are hydrolysed extremely rapidly

in human plasma solutions, however, the rate of plasma catalysed

hydrolysis can be altered with the change of substituents on amide

nitrogen atom. Monosubstituted (-CO2CH2CONHR) or unsubstituted (-

CO2CH2CONH2) glycolamide esters were found to be more resistant than

N,N-disubstituted glycolamide esters (-CO2CH2CONRR').

Moreover, the glycolamide esters of aspirin failed to deliver aspirin in

human plasma when morpholine was the corresponding amine of amide

moiety.5

Attracted by these ideas, we became interested in the synthesis and

pharmacological evaluation of library of compounds. Our intention was

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111

to identify small molecule as cytotoxic agent having attractive chemical

scaffold for the development of new cytotoxic agent.

COX-2 inhibitor nimesulide shows anticancer effects in several

cancer cells and due to our interest in chemical modification of

nimesulide,14 we felt that nimesulide based substituted glycolamide ester

moiety might be worth exploring as it is capable of forming additional

hydrogen bonds.

We designed new glycolamide analogues 14 by using nimesulide (13)

as starting scaffold (figure 3.2).

Figure 3.2: Design of new nimesulide based glycolamide esters 14

The following few pages describe our efforts in this direction and

delineate successful synthesis of the nimesulide based glycolamide

esters.

3.4 RESULTS AND DISCUSSION

It was initially planned to synthesize target compounds 14 by a two

step process as shown in scheme 3.1.

N-(4-Amino-2-phenoxyphenyl)methane sulfonamide 15 was

prepared15 by reducing 13. Chloroacetylation of 15 in CHCl3 in presence

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112

of TEA gave the desired intermediate 2-chloro-N-(4-

methanesulfonylamino-3-phenoxy-phenyl)-acetamide (16) in good yields.

Scheme 3.1: Synthesis of nimesulide based glycolamide esters 14

The chloro compound 16 was well characterized by using mass, IR,

1H NMR, and 13C NMR spectroscopic techniques. Mass spectrum (figure

3.3) showed a protonated molecular ion peak at m/z 355. Appearance of

isotopic peak at m/z 357 confirmed the presence of one chlorine atom.

Figure 3.3: Mass (+Ve) spectrum of 16

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113

IR spectrum (figure 3.4) displayed a strong band at 1665 cm-1 due to

C=O stretching of amide group. The NH stretching frequency appeared at

ν 3325 and 3265 cm–1.

Figure 3.4: IR spectrum of 16

1H NMR spectrum in DMSO-d6 (figure 3.5) displayed two singlets at δ

4.20 and 2.96 ppm due to CH2 and CH3 groups respectively. When D2O

exchange experiment was performed two signals at δ 10.46 and 9.22

ppm disappeared which was in consistent with the presence of two labile

hydrogens (NH groups) in the structure.

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114

Figure 3.5: 1H NMR (DMSO-d6, 400 MHz) spectrum of 16

Figure 3.6: 13C NMR (CDCl3, 100 MHz) spectrum of 16

Appearance of total 13 singlets in 13C NMR spectrum (BB mode) of

compound 16 (figure 3.6) confirmed the presence of thirteen chemically

non equivalent carbon atoms. Peak at δ 164.7 ppm confirmed the amide

carbon. The signal for methyl group appeared at δ 39.4 ppm. The signal

at δ 52.7 ppm was due to CH2 group.

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115

Based on these spectral data and synthetic sequence the proposed

structure of 16 was confirmed. Detailed spectral data for 16 has been

given in the experimental section 3.7.1 of this chapter (page no. 126).

The second step, which is reaction of the resulting compound 16 with

commercially available acids (17a-l), was performed in the presence of

catalytic amount of DMAP9 using TEA as a base. To our surprise instead

of the desired glycolamide esters we isolated a white solid 18 with

melting point 264-265 °C irrespective of acid partner. All spectral data

which includes mass, IR, 1H NMR & 13C NMR were recorded and was

confirmed as 4-(dimethylamino)-1-(2-oxo-2-(4-methanesulfonylamino-3-

phenoxy phenylamino) ethyl) pyridinium salt (18) (scheme 3.2).

NHSO2Me

OPh

HN

O

Cl

NHSO2Me

OPh

HN

O

O R

O

NHSO2Me

OPh

HN

O

N

NMe2

+

RCOOH

DMAP, TEA rt

16 18

14

17

Scheme 3.2: Formation of pyridinium salt 18

Mass spectrum displayed molecular ion peak appeared at m/z 441

(figure 3.7).

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116

Figure 3.7: Mass spectrum of 18

Figure 3.8: IR spectrum of 18

IR spectrum of 18 (figure 3.8) displayed absorption at 1652 cm–1 due

to C=O stretching of amide group. In 1H NMR spectrum (figure 3.9),

appearance of singlet at δ 3.19 ppm, equivalent to 6H was indicative.

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117

Figure 3.9: 1H NMR (DMSO-d6, 400 MHz) spectrum of 18

Figure 3.10: 13C NMR (DMSO-d6, 100 MHz) spectrum of 18

In 13C NMR (figure 3.10) total seventeen signals appeared for

seventeen chemically non equivalent carbon atom including δ 164.7 for

amide carbon and δ 40.4 for geminal carbon atom of two methyl groups.

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118

Due to insolubility of the compound further nucleophilic attack by

carboxylate ion could not take place.

Hence we decided to synthesize the glycolamide esters in a different

way.1 As shown in scheme 3.3 nimesulide based glycolamide esters 14

were synthesized by one pot sequential nucleophilic substitution of the

key intermediate 16 to 2-iodo-N-(4-methanesulfonylamino-3-phenoxy-

phenyl)-acetamide in the presence of potassium iodide and finally to the

glycolamide esters with appropriate and commercially available acids 17

in the presence of base TEA.

Scheme 3.3: Synthesis of 14

Acetic acid (17a) was chosen as acid partner to establish the

optimized condition. Then the acid (0.01 mol) was reacted with the key

intermediate 16 (0.01 mol) in presence of potassium iodide (0.001mol),

TEA (0.011 mol) and 10 mL DMF at 90 °C. The product (acetic acid (4-

methanesulfonylamino-3-phenoxy-phenyl carbamoyl)-methyl ester) (14a)

isolated was characterized by mass, IR and NMR spectroscopic analysis.

In mass spectrum (figure 3.11) protonated molecular ion peak

appeared at m/z 379 corresponding to the molecular formula

C17H18N2O6S.

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119

Figure 3.11: Mass (+Ve) spectrum of 14a

The presence of two carbonyl groups in the product was confirmed by

its IR spectrum which showed carbonyl stretching frequencies at ν 1742

cm–1 for ester and 1677 cm–1 for amide (figure 3.12).

Figure 3.12: IR spectrum of 14a

In 1H NMR spectrum (figure 3.13) the appearance of two singlets at δ

2.20 ppm for 3H & δ 4.64 for 2H confirmed the presence of –COMe group

and CH2 group respectively.

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120

Figure 3.13: 1H NMR (CDCl3, 400 MHz) spectrum of 14a

In 13C NMR (figure 3.14) appearance of 15 signals were highly

consistent with the 15 non equivalent carbon atoms of the product. Ester

and amide carbonyls appeared at δ 169.9 & 165.5 ppm respectively,

where as CH2 appeared at δ 62.4 ppm.

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121

Figure 3.14: 13C NMR (DMSO-d6, 100 MHz) spectrum of 14a

Having established the optimum reaction conditions, we then

decided to examine the reaction of 16 with other carboxylic acids 17b-l

including NSAIDs. All glycolamide esters 14a-l synthesized were isolated

in good to excellent yields and formations of no side products were

detected. The results are summarized in table 3.1.

Table 3.1 Comparison of time and yield of products 14a-l.

Entry Ar/R-COOH Ar/R= (17)

Products (14) Time (min)

Yield (%)

1

Me 17a

NHSO2Me

OPh

HN

O

O Me

O

14a

30 91

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122

2

Et 17b

NHSO2Me

OPh

HN

O

O Et

O

14b

30 95

3 Ph 17c

14c

20 80

4

17d

14d

30 90

5

17e

14e

30 70

6

17f

14f

15 75

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123

7

17g

14g

10

98

8

17h

14h

30

78

9

17i

14i

45 80

10

HN

Cl

Cl 17j

NHSO2Me

OPh

HN

O

O

O

HN

Cl

Cl 14j

15 92

11

17k

14k

30 90

12

17l

14l

15 90

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124

3.5 CYTOTOXIC ACTIVITY

The in vitro cytotoxic evaluation of the tested glycolamide ester

derivatives against human HCT-15 for colon cancer compared to that of

doxyrubicin as the reference drug is shown in table 3.2. It should be

noted that compound 14l exhibited the highest toxicity.

Table 3.2 Cytotoxic activity of synthesized compounds 14a-l

S.No Compounds % of cell death at various concentrations

1 µg

/mL

2 µg

/mL

5 µg

/mL

10 µg

/mL

25 µg

/mL

1 14a 0.94 6.89 8.15 8.77 20.30

2 14b 0.62 7.20 7.50 18.50 38.50

3 14c 0.31 12.5 2.50 7.52 31.00

4 14d 3.43 4.53 21.69 35.78 49.38

5 14e 1.88 6.58 13.16 17.55 28.80

6 14f 5.88 14.46 20.22 25.00 42.40

7 14g 6.00 7.96 14.46 18.62 41.91

8 14h 0.31 7.52 8.77 12.20 19.40

9 14i 7.35 12.37 14.95 16.29 48.89

10 14j 6.74 9.43 25.61 35.90 51.22

11 14k 0 5.96 12.20 30.70 55.79

12 14l 21.69 21.93 24.50 30.02 66.05

All the values are the average of the experiments done in triplicates. The cell line used was HCT-15 human colon cancer cell line. Doxorubicin [IC50 = 50µg /mL (0.09 µM)] was used as reference compound. IC50 value of 14k and l are 21.5 and 18.4 respectively (figure 3.15).

Chapter-3

125

14k 14l Figure 3.15: In vitro cytotoxic activity of few synthesized compounds

against HCT-15, human colon cancer cell line.

3.6 CONCLUSION

In conclusion, we have successfully accomplished the synthesis of

several new nimesulide based glycolamide esters in good yields.

Structures of the synthesized compounds were confirmed by

spectroscopic analysis. The new derivatives were examined in vitro for

their cytotoxic activities. Our results indicate that the compounds

possess low to moderate cytotoxic activity against HCT-15 human colon

cancer cell line.

3.7 EXPERIMENTAL SECTION

All the carboxylic acids used are commercially available.

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126

3.7.1 Synthesis of 2-chloro-N-(4-methanesulfonylamino-3-phenoxy-

phenyl)-acetamide (16)

NHSO2Me

OPh

HN

O

Cl

1 g (3.6 mmol) of compound 15 was taken into a round bottom flask.

To this, 20 mL of CHCl3 and 0.6 mL (4.3 mmol) of TEA were added. This

mixture was stirred and then cooled to 0 oC. To this mixture 0.3 mL (3.6

mmol) of X-chloroacetyl chloride was added drop wise. The mixture was

then allowed to come to room temperature and stirring was continued for

an additional 30 minutes. After completion of the reaction as monitored

by TLC, the reaction mixture was quenched with 10-15 mL of cold water,

which was then extracted with CHCl3. The organic layers were collected,

combined, washed with water, dried over anhydrous Na2SO4 and

concentrated under reduced pressure. The crude was recrystallised with

aqueous EtOH.

DESCRIPTION: Off white solid.

M.P: 135-136 °C.

RF: 0.45 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3325, 3265, 1665, 1611, 1589.

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127

1H NMR (400 MHz, DMSO-d6): δ 10.30 (s, 1H, NH, D2O exchangeable),

9.25 (s, 1H, NH, D2O exchangeable), 7.50-7.40 (m, 2H), 7.38-7.26 (m,

2H), 7.21-7.14 (m, 2H), 7.10 (d, J ═ 8.8 Hz, 2H), 4.20 (s, 2H), 3.00 (s,

3H).

13C NMR (100 MHz, CDCl3): δ 164.7, 155.4, 148.2, 134.8, 130.2, 124.7,

124.4, 123.1, 118.8, 115.6, 110.1, 52.7, 39.4.

MASS (m/z): 355 [M+H]+, (47.5 %).

ELEMENTAL ANALYSIS found C, 50.84; H, 4.02; N, 7.73.

C15H15ClN2O4S requires C, 50.78; H, 4.26; N, 7.90 %.

3.7.2 General procedure for synthesis of glycolamide esters, 14a-l

A mixture of N-chloroacetamide (16) (3.54 g, 10 mmol), appropriate

acid (17) (10 mmol), KI (0.166 g, 1 mmol) and TEA (1.53 mL, 11 mmol) in

10 mL DMF was stirred at 90 °C. The progress of the reaction was

monitored by TLC. After completion, the reaction mixture was poured

into water (50 mL) and extracted with ethyl acetate (3×50 mL). The

combined organic extracts were washed with aqueous sodium

bicarbonate (2 %, 50 mL) and water (3×50 mL). The organic layer was

dried over anhydrous sodium sulphate and evaporated under reduced

pressure to get solid residue which was purified by column

chromatography to get the corresponding glycolamide esters 14a-l.

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128

3.7.2.1 Acetic acid (4-methanesulfonylamino-3-phenoxy-phenyl

carbamoyl)-methyl ester (14a)

NHSO2Me

OPh

HN

O

O Me

O

DESCRIPTION: Light orange solid.

M.P: 116-120 °C.

RF: 0.38 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3277, 1742, 1677, 1616, 1591.

1H NMR (400 MHz, CDCl3): δ 7.78 (bs, 1H, NH, D2O exchangeable), 7.58

(d, J ═ 8.8 Hz, 1H), 7.41-7.37 (m, 2H), 7.32 (d, J ═ 2.1 Hz, 1H), 7.21-7.13

(m, 2H), 7.01 (d, J ═ 7.9 Hz, 2H), 6.76 (s, 1H), 4.62 (s, 2H), 2.98 (s, 3H),

2.20 (s, 3H).

13C NMR (100 MHz, DMSO-d6): δ 169.9, 165.5, 155.8, 151.1, 137.8,

130.0, 127.8, 123.9, 122.9, 119.3, 114.0, 108.8, 62.4, 40.3, 20.4.

MASS (m/z): 379 [M+H]+, (100 %).

ELEMENTAL ANALYSIS found C, 53.84; H, 4.82; N, 7.33. C17H18N2O6S

requires C, 53.96; H, 4.79; N, 7.40 %.

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129

3.7.2.2 Propionic acid (4-methanesulfonylamino-3-phenoxy-phenyl

carbamoyl)-methyl ester (14 b)

NHSO2Me

OPh

HN

O

O Et

O

DESCRIPTION: Light orange solid.

M.P: 102-104 °C.

RF: 0.52 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3278, 2928, 1747, 1674, 1612.

1H NMR (400 MHz, CDCl3): δ 7.72 (bs, 1H, NH, D2O exchangeable), 7.59

(d, J ═ 8.5 Hz, 1H), 7.41-7.37 (m, 2H), 7.34 (s, 1H), 7.19-7.17 (m, 1H),

7.13 (dd, J ═ 8.5, 1.8 Hz, 1H), 7.01 (d, J ═ 8.3 Hz, 2H), 6.78 (bs, 1H, NH,

D2O exchangeable), 4.63 (s, 2H), 2.98 (s, 3H), 2.48 (q, J ═ 7.6 Hz, 2H),

1.20 (t, J ═ 7.6 Hz, 3H).

13C NMR (100 MHz, DMSO-d6): δ 173.2, 165.5, 155.9, 151.0, 137.4,

130.0, 127.8, 123.9, 122.9, 119.2, 114.0, 108.8, 62.3, 40.3, 26.4, 8.8.

MASS (m/z): 393 [M+H]+, (100 %).

ELEMENTAL ANALYSIS: found C, 55.39; H, 5.02; N, 7.01. C18H20N2O6S

requires C, 55.09; H, 5.14; N, 7.14 %.

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130

3.7.2.3 Benzoic acid (4-methanesulfonylamino-3-phenoxy-phenyl

carbamoyl)-methyl ester (14c)

NHSO2Me

OPh

HN

O

O Ph

O

DESCRIPTION: White solid.

M.P: 127-128 °C.

RF: 0.72 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3266, 2933, 1736, 1672, 1611, 1548.

1H NMR (400 MHz, CDCl3): δ 8.07 (d, J ═ 7.4 Hz, 2H), 7.77 (s, 1H), 7.65-

7.61 (m, 2H), 7.59-7.50 (m, 2H), 7.48-7.36 (m, 3H), 7.20-7.16 (m, 1H),

7.10 (dd, J ═ 8.7, 2.2 Hz, 1H), 7.01 (d, J ═ 8.0 Hz, 2H), 6.73 (s, 1H), 4.88

(s, 2H), 2.97 (s, 3H).

13C NMR (100 MHz, DMSO-d6): δ 165.4, 155.8, 151.1, 137.4, 133.6,

130.0, 129.3, 129.1, 128.8, 127.9, 123.9, 122.9, 119.3, 114.0, 108.7,

63.0, 40.3.

MASS (m/z): 441 [M+H]+, (100 %).


requires C, 59.99; H, 4.58; N, 6.36 %.

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131

3.7.2.4 2-Methyl-benzoic acid (4-methanesulfonylamino-3-phenoxy

phenylcarbamoyl)-methyl ester (14d)

NHSO2Me

OPh

HN

O

O

O Me


M.P: 139-140 °C.

RF: 0.51 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3274, 3236, 1727, 1677, 1613, 1224, 1153.

1H NMR (400 MHz, CDCl3): δ 7.95 (dd, J ═ 8.7, 1.5 Hz, 1H), 7.78 (s, 1H),

7.60 (d, J ═ 8.2 Hz, 1H), 7.48 (dd, J ═ 7.6, 1.5 Hz, 1H), 7.41 -7.37 (m,

3H), 7.35-7.29 (m, 2H), 7.17 (t, J ═ 8.1 Hz, 1H), 7.10 (dd, J ═ 8.7, 2.0 Hz,

1H), 7.02 (d, J ═ 7.7 Hz, 2H), 6.72 (s, 1H), 4.86 (s, 2H), 2.97 (s, 3H), 2.68

(s, 3H).

13C NMR (100 MHz, CDCl3): δ 180.0, 165.7, 165.3, 155.5, 148.1, 140.9,

134.7, 133.0, 132.1, 131.5, 130.2, 127.9, 126.0, 124.6, 123.0, 118.6,

115.8, 110.6, 63.3, 39.4, 21.8.

MASS (m/z): 455 [M+H]+, (100 %).


requires C, 60.78; H, 4.88; N, 6.16 %.

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132

3.7.2.5 4-Methoxy-benzoic acid (4-methanesulfonylamino-3-

phenoxy phenylcarbamoyl)-methyl ester (14e)

NHSO2Me

OPh

HN

O

O

O

OMe


M.P: 198-200 °C.

RF: 0.64 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3340, 3307, 2843, 1710, 1604.

1H NMR (400 MHz, CDCl3): δ 8.03 (d, J ═ 8.8 Hz, 2H), 7.76 (bs, 1H, NH,

D2O exchangeable), 7.60 (d, J ═ 8.4 Hz, 1H), 7.39 (t, J ═ 8.5 Hz, 3H),

7.20-7.17 (m, 1H) 7.09 (dd, J ═ 8.8, 2.2 Hz, 1H), 7.01 (d, J ═ 8.6 Hz, 2H),

6.97 (d, J ═ 8.8 Hz, 2H), 6.71 (bs, 1H, NH, D2O exchangeable), 4.86 (s,

2H), 3.89 (s, 3H), 2.97 (s, 3H).

13C NMR (100 MHz, DMSO-d6): δ 165.6, 165.0, 163.3, 155.8, 151.1,

137.4, 131.5, 130.0, 127.9, 123.9, 122.9, 121.3, 119.3, 114.0, 108.8,

62.7, 55.5, 40.3.

MASS (m/z): 471 [M+H]+, (100 %).


requires C, 58.71; H, 4.71; N, 5.95 %.

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133

3.7.2.6 4-Nitro-benzoic acid (4-methanesulfonylamino-3-phenoxy-

phenylcarbamoyl)-methyl ester (14f)

NHSO2Me

OPh

HN

O

O

O

NO2


M.P: 206-208 °C.

RF: 0.49 (CHCl3 : EtOAc = 7 : 3).

IR (KBr) νmax/cm–1: 3273, 3096, 2949, 1728, 1682, 1663.


9.26 (s, 1H, NH, D2O exchangeable), 8.37 (d, J ═ 8.8 Hz, 2H), 8.22 (d, J ═

8.7 Hz, 2H), 7.43 (t, J ═ 7.8 Hz, 2H), 7.36-7.27 (m, 2H), 7.22 (d, J ═ 3.0

Hz, 1H), 7.18 (t, J ═ 7.3 Hz, 1H), 7.07 (d, J ═ 7.3 Hz, 2H), 4.92 (s, 2H),

3.32 (s, 3H).

13C NMR (100 MHz, CDCl3 + DMSO-d6): δ 163.9, 163.0, 155.3, 149.5,

148.9, 136.0, 133.8, 130.0, 128.9, 125.3, 122.8, 122.7, 122.5, 117.8,

114.0, 109.1, 62.6, 39.1.

MASS (m/z): 486 [M+H]+, (100 %).


requires C, 54.43; H, 3.94; N, 8.66 %.

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134

3.7.2.7 3-Phenyl-acrylic acid (4-methanesulfonylamino-3-phenoxy-

phenylcarbamoyl)-methyl ester (14g)

NHSO2Me

OPh

HN

O

O

O

Ph

DESCRIPTION: Buff solid.

M.P: 198-200 °C

RF: 0.62 (CHCl3 : EtOAc = 8 : 2).

IR (KBr) νmax/cm–1: 3251, 3092, 1719, 1682, 1509.


9.25 (s, 1H, NH, D2O exchangeable), 7.75-7.68 (m, 3H), 7.45-7.40 (m,

4H), 7.35-7.28 (m, 3H), 7.23 (d, J ═ 2.4 Hz, 1H), 7.18 (t, J ═ 7.3 Hz, 1H),

7.07 (d, J ═ 7.4 Hz, 2H), 6.72 (d, J ═ 16.1 Hz, 1H), 4.72 (s, 2H), 2.96 (s,

3H).

13C NMR (100 MHz, DMSO-d6 + CDCl3): δ 174.3, 165.0, 164.7, 155.3,

148.8, 144.8, 136.1, 133.1, 129.6, 129.0, 128.0, 127.12 125.2, 122.9,

122.7, 117.9, 116.2, 114.2, 109.3, 61.8, 39.7.

MASS (m/z): 467 [M+H]+, (100 %).


requires C, 61.79; H, 4.75; N, 6.00 %.

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135

3.7.2.8 2-(6-Methoxy-naphthalen-2-yl)-propionic acid (4-methane

sulfonylamino-3-phenoxy-phenylcarbamoyl)-methyl ester

(14h)

NHSO2Me

OPh

HN

O

O

O

Me

OMe


M.P: 196-198 °C

RF: 0.84 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3464, 3386, 3060, 2936, 1742, 1692, 1607.

1H NMR (400 MHz, CDCl3): δ 7.70 (d, J ═ 9.1 Hz, 2H), 7.67 (bs, 1H, NH,

D2O exchangeable), 7.41-7.33 (m, 4H), 7.21-7.12 (m, 4H), 6.91 (d, J ═

8.0 Hz, 2H), 6.82 (s, 1H), 6.58 (s, 1H), 5.81 (dd, J ═ 8.8, 2.2 Hz, 1H),

4.87 (d, J ═ 15.8 Hz, 1H), 4.38 (d, J ═ 15.7 Hz, 1H), 3.98 (q, J ═ 7.0 Hz,

1H), 3.96 (s, 3H), 2.89 (s, 3H), 1.65 (d, J ═ 7.3 Hz, 3H).

13C NMR (50 MHz, DMSO-d6): δ 184.6, 173.5, 164.2, 162.5, 158.3,

157.1, 146.4, 135.4, 133.2, 129.2, 128.3, 126.8, 126.3, 125.7, 121.2,

118.6, 116.6, 115.8, 112.4, 105.7, 102.9, 62.7, 55.1, 52.2, 44.1, 18.5.

MASS (m/z): 549 [M+H]+, (100 %).


requires C, 63.49; H, 5.14; N, 5.11 %.

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136

3.7.2.9 2-(4-Isobutyl-phenyl)-propionic acid (4-methanesulfonyl

amino-3-phenoxy-phenylcarbamoyl)-methyl ester (14i)

NHSO2Me

OPh

HN

O

O

O

Me

Me

Me


M.P: 48-50 °C.

RF: 0.50 (CHCl3).

IR (KBr) νmax/cm–1: 3336, 3284, 2954, 2931, 2868, 1746, 1693, 1612,

1590, 1537.

1H NMR (400 MHz, CDCl3): δ 7.50 (d, J ═ 8.7 Hz, 1H), 7.38 (t, J ═ 7.7

Hz, 2H), 7.26-7.07 (m, 7H), 6.97 (d, J ═ 7.6 Hz, 2H), 6.66 (s, 1H), 6.60

(d, J ═ 7.7 Hz, 1H), 4.82 (d, J ═ 15.7 Hz, 1H), 4.41 (d, J ═ 15.7 Hz, 1H),

3.81 (q, J ═ 7.0 Hz, 1H), 2.93 (s, 3H), 2.42 (d, J ═ 6.9 Hz, 2H), 1.85-1.79

(m, 1H),1.54 (d, J ═ 7.3 Hz, 3H), 0.89 (d, J ═ 6.2 Hz, 6H).

13C NMR (50 MHz, CDCl3): δ 172.5, 165.2, 155.6, 147.6, 141.5, 137.1,

134.6, 130.2, 129.9, 127.1, 124.5, 122.9, 118.2, 115.6, 110.7, 62.4,

44.9, 44.8, 39.3, 30.2, 22.3, 17.7.

MASS (m/z): 525 [M+H]+, (100 %).


requires C, 64.10; H, 6.15; N, 5.34 %.

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137

3.7.2.10 [2-(2, 6-Dichloro-phenylamino)-phenyl]-acetic acid (4-

methanesulfonylamino-3-phenoxy-phenylcarbamoyl)-

methyl ester (14j)

NHSO2Me

OPh

HN

O

O

O

HN

Cl

Cl


M.P: 122-123 °C.

RF: 0.57 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3475, 3382, 3246, 1718, 1666, 1228, 1160.

1H NMR (400 MHz, CDCl3): δ 7.51 (d, J ═ 8.7 Hz, 1H), 7.38-7.33

(m, 4H), 7.28-7.14 (m, 4H), 7.06-6.87 (m, 6H), 6.69 (s, 1H, NH, D2O

exchangeable), 6.42 (d, J ═ 8.2 Hz, 1H), 6.28 (s, 1H, NH, D2O

exchangeable), 4.69 (s, 2H), 3.92 (s, 2H), 2.95 (s, 3H).

13C NMR (100 MHz, CDCl3): δ 180.1, 169.7, 164.7, 155.7, 147.7, 142.4,

137.0, 134.5, 130.8, 130.2, 129.9, 128.9, 128.7, 124.9, 124.5, 122.9,

122.7, 122.2, 118.5, 117.9, 115.7, 110.3, 62.9, 39.4, 38.2.

MASS (m/z): 614 [M+H]+, (100 %).

ELEMENTAL ANALYSIS: found C, 56.34; H, 4.01; N, 6.52.

C29H25Cl2N3O6S requires C, 56.68; H, 4.10; N, 6.84 %.

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138

3.7.2.11 2-(2, 3-Dimethyl-phenylamino)-benzoic acid (4-

methanesulfonyl amino-3-phenoxy-phenylcarbamoyl)-

methyl ester (14k)

NHSO2Me

OPh

HN

O

O

O HN

Me

Me

DESCRIPTION: Pale yellow solid.

M.P: 140-141 °C.

RF: 0.50 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3338, 2925, 1687, 1608, 1508, 1327, 1218, 1156,

1098.

1H NMR (400 MHz, CDCl3): δ 9.08 (1H, NH), 7.96 (dd, J ═ 8.0, 1.1 Hz,

1H), 7.80 (bs, 1H, NH, D2O exchangeable), 7.61 (d, J ═ 8.4 Hz, 1H), 7.41-

7.05 (m, 9H), 7.01 (d, J ═ 7.7 Hz, 2H), 6.75-6.68 (m, 3H), 4.88 (s, 2H),

2.97 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H).

13C NMR (50 MHz, DMSO-d6): δ 167.2, 165.5, 155.7, 151.0, 148.6,

138.0, 137.9, 137.3, 134.7, 131.6, 131.5, 129.9, 127.8, 126.7, 126.0,

123.9, 122.9, 122.7, 119.2, 116.3, 113.9, 113.3, 109.9, 108.8, 62.7,

40.7, 20.2, 13.5.

MASS (m/z): 560 [M+H]+, (100 %).


requires C, 64.39; H, 5.22; N, 7.51 %.

Chapter-3

139

3.7.2.12 2-Acetoxy-benzoic acid (4-methanesulfonylamino-3-

phenoxy-phenylcarbamoyl)-methyl ester (14l)

NHSO2Me

OPh

HN

O

O

O O Me

O


M.P: 147-148 °C.

RF: 0.39 (CHCl3 : EtOAc = 9 : 1).

IR (KBr) νmax/cm–1: 3249, 3154, 3099, 2944, 1758, 1730, 1674, 1610,

1555, 1507.


9.26 (s, 1H, NH, D2O exchangeable), 8.00 (d, J ═ 6.5 Hz, 1H), 7.71 (t, J ═

6.5 Hz, 1H), 7.45-7.16 (m, 8H), 7.07 (d, J ═ 8.0 Hz, 2H), 4.81 (s, 2H),

2.96 (s, 3H), 2.24 (s, 3H).

13C NMR (100 MHz, DMSO-d6): δ 169.1, 165.1, 163.5, 155.8, 152.9,

151.1, 150.2, 137.3, 134.4, 130.1, 127.9, 126.3, 124.2, 124.0, 122.9,

122.4, 119.3, 113.9, 108.7, 63.1, 40.3, 20.7.

MASS (m/z): 499 [M+H]+, (100 %).


requires C, 57.82; H, 4.45; N, 5.62 %.

Chapter-3

140

3.7.2.13 4-(Dimethylamino)-1-(2-oxo-2-(3-phenoxy phenylamino)

ethyl) pyridinium (18) (scheme 3.2)

NHSO2Me

OPh

HN

O

N

NMe

Me

+


M.P: 264-265 °C.

IR (KBr) νmax/cm-1: 3408, 3175, 3045, 1652, 1607, 1332, 1221.


9.30 (bs, 1H, NH, D2O exchangeable), 8.20 (d, J ═ 7.6 Hz, 2H), 7.32-7.29

(m, 3H), 7.17-6.98 (m, 5H), 6.85 (d, J ═ 7.9 Hz, 2H), 5.13 (s, 2H), 3.19 (s,

6H ), 2.96 (s, 3H).

13C NMR (100 MHz, DMSO-d6): δ 164.7, 155.9, 155.8, 151.1, 143.3,

137.4, 130.0, 127.8, 124.0, 123.1, 119.2, 114.0, 108.9, 107.1, 58.3,

40.4, 38.9.

MASS (m/z): 441 (M+, 100 %).

3.8 REFERENCES

1. Bundgaard, H.; Nielsen, N. M. J. Med. Chem., 30, 1987, 451–454.

2. Nielsen, N. M.; Bundgaard, H. J. Pharm. Sci., 77, 1988, 285–298.

3. Wadhwa, L. K.; Sharma, P. D. Ind. J. Chem., 34B, 1995, 408–415.

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141

4. Sharma, P. D.; Singh, K. J.; Gupta, S.; Chandiran, S. Indian. J.

Chem., 43B, 2004, 636–642.

5. Nielsen, N. M.; Bundgaard, H. J. Med. Chem., 32, 1989, 727–734.

6. Bansal, A. K.; Khar, R. K.; Dubey, R.; Sharma, A. K. Drug Dev. Ind.

Pharm., 27, 2001, 63–70.

7. Gadad, A. K.; Bhat, S.; Tegli, V. S.; Redasani, V. V. Arzneimittel-

Forschung, 52, 2002, 817–821.

8. Cao, F.; Gueo, J. X.; Ping, Q. N.; Liao, Z. G. Eur. J. Pharm., 29,

2006, 385–393.

9. Smriti, K.; Manjula, M.; Akhila, V.; Rahul, B.; Ram, T.; Basha, S.

K. J. S.; Ramesh, M.; Rao, C. S.; Pal, M. Bioorg. Med. Chem., 14,

2006, 4820–4833.

10. Nalini, C. N.; Ramachandran, S.; Kavitha, K.; Saraswathi, V. S. Int.

J. Res. Pharm. Biomed. Sci., 2, 2011, 1112–1117.

11. Amblard, M.; Rodriguez, M.; Martinez, J. Tetrahedron, 44, 1988,

5101–5108.

12. Hoekstra, W. J. US 6066651.

13. Boltze, K. H.; Kreisfeld, H. Arzneimittel-Forschung, 27, 1977, 1300–

1312.

14. (a) Kavitha, K.; Reddy, V. R.; Mukkanti, K.; Pal, S. J. Braz. Chem.

Soc., 21, 2010, 1060–1064. (b) Abir, B.; Ghosh, S.; Kavitha, K.;

Reddy, V. R.; Mukkanti, K.; Pal, S.; Mukherjee, A. K. Chem. Phy.

Lett., 493, 2010, 151–157. (c) Kavitha, K.; Varsha, P.; Mukkanti,

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142

K.; Reddy, V. R.; Pal, S. Molbank, 2011, M740; doi:

10.3390/M740.

15. Pericherla, S.; Mareddy, J.; Rani, D. P. G.; Gollapudi, P. V.; Pal, S.

J. Braz. Chem. Soc., 18, 2007, 384–390.

3.9 SOME IMPORTANT SPECTRA OF THE COMPOUNDS

Figure 3.16: Mass (+Ve) spectrum of 14b

Figure 3.17: IR spectrum of 14b

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143

Figure 3.18: 1H NMR (CDCl3, 400 MHz) spectrum of 14b

Figure 3.19: 13C NMR (DMSO-d6, 100 MHz) spectrum of 14b

Chapter-3

144

Figure 3.20: 1H NMR (CDCl3, 400 MHz) spectrum of 14c

Figure 3.21: 13C NMR (DMSO-d6, 100 MHz) spectrum of 14c

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145

Figure 3.22: 1H NMR (CDCl3, 400 MHz) spectrum of 14d

Figure 3.23: 13C NMR (CDCl3, 100 MHz) spectrum of 14d

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146

Figure 3.24: IR spectrum of 14e

Figure 3.25: 1H NMR (CDCl3, 400 MHz) spectrum of 14e

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147

Figure 3.26: 1H NMR (DMSO-d6, 400 MHz) spectrum of 14f

Figure 3.27: 13C NMR (CDCl3 + DMSO-d6, 100 MHz) spectrum of 14f

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148

Figure 3.28: 1H NMR (DMSO-d6, 400 MHz) spectrum of 14g

Figure 3.29: 13C NMR (CDCl3 + DMSO-d6, 100 MHz) spectrum of 14g

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149

Figure 3.30: 1H NMR (CDCl3, 400 MHz) spectrum of 14h

Figure 3.31: 13C NMR (DMSO-d6, 50 MHz) spectrum of 14h

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150

Figure 3.32: 1H NMR (CDCl3, 400 MHz) spectrum of 14i

Figure 3.33: 13C NMR (CDCl3, 50 MHz) spectrum of 14i

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151

Figure 3.34: 1H NMR (CDCl3, 400 MHz) spectrum of 14j

Figure 3.35: 13C NMR (CDCl3, 100 MHz) spectrum of 14j

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152

Figure 3.36: 1H NMR (CDCl3, 400 MHz) spectrum of 14k

Figure 3.37: 13C NMR (DMSO-d6, 50 MHz) spectrum of 14k

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153

Figure 3.38: 1H NMR (DMSO-d6, 400 MHz) spectrum of 14l

Figure 3.39: 13C NMR (DMSO-d6, 100 MHz) spectrum of 14l

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