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.
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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
Chapter-3
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.
Chapter-3
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
Chapter-3
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
Chapter-3
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
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.
Chapter-3
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.
Chapter-3
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.
Chapter-3
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
Chapter-3
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
Chapter-3
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
Chapter-3
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.
Chapter-3
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