Studies on the synthesis of anti-inflammatory drug...

Chapter I

HO

HO

O

NN

NH

O

HO

O

Studies on the synthesis of

anti-inflammatory drug,

Balsalazide

INTRODUCTION:

Ulcerative colitis is a common inflammatory disease of the colon and rectum which has an

estimated annual incidence of 0.002 to 0.006% (2 to 6 per 100 000 people) in the US.[1]

The

clinical symptoms of ulcerative colitis are bloody diarrhea, stool urgency, tenesmus and

abdominal discomfort; these events tend to wax and wane with time. The modern history for the

treatment of diarrhea, intestinal inflammation / infection begins with the development of several

oral rehydration agents, intestinal anti-infectives, adsorbents, antipropulsives (opioids),

intestinal anti-inflammatory agents, antidiarrheal micro-organisms and antidiarrheals which are

summarized in Table-1.1.

Ulcerative colitis is classified as mild, moderate or severe on the basis of clinical and

endoscopic findings. Salicylates, in either oral or topical formulation, are the drugs of first

choice for obtaining remission or maintenance of remission in patients with mild to moderate

MD. UMAR KHAN Thesis

Studies on the synthesis of anti-inflammatory drug, Balsalazide 2

Chapter - I

disease. Topical, oral or intravenous glucocorticoids, mercaptopurine, azathioprine and surgical

resection are reserved for patients affected with more severe forms of the disease.[1]

Table-1.1

Rehydration Oral rehydration therapy

Intestinal anti-

infectives

Antibiotics (Neomycin, Nystatin, Natamycin, Streptomycin, Polymyxin

B, Paromomycin, Amphotericin B, Kanamycin, Vancomycin, Colistin,

Rifaximin)

Sulfonamides (Phthalylsulfathiazole, Sulfaguanidine,

Succinylsulfathiazole)

Nitrofuran (Nifuroxazide, Nifurzide)

Imidazole (Miconazole)

Arsenical (Acetarsol)

Oxyquinoline (Broxyquinoline)

Intestinal

adsorbents

Charcoal, Bismuth, Pectin, Kaolin, Crospovidone, Attapulgite,

Diosmectite

Antipropulsives

(opioids)

Opium Tincture (Laudanum), Codeine, Morphine, Camphorated Opium

Tincture (Paregoric)

Crosses BBB (Diphenoxylate (Diphenoxylate/atropine), Difenoxin)

Does not cross BBB (Loperamide)

Intestinal anti-

inflammatory

agents

Corticosteroids acting locally (Prednisolone, Hydrocortisone,

Prednisone, Betamethasone, Tixocortol, Budesonide, Beclometasone)

Antiallergic agents, excluding corticosteroids (Cromoglicic acid)

Aminosalicylic acid and similar agents (Sulfasalazine, Mesalazine,

Olsalazine, Balsalazide, Ipsalazide)

Antidiarrheal

micro-organisms

Saccharomyces boulardii

Other

antidiarrheals

Albumin tannate, Ceratonia, Octreotide, Racecadotril

5-aminosalicylic acid (5-ASA), also known as mesalazine is an anti-inflammatory drug used to

treat inflammatory bowel disease, such as ulcerative colitis[2]

and mild-to-moderate Crohn's

disease.[3-5]

Mesalazine is a bowel-specific aminosalicylate drug that acts locally in the gut and

has its predominant actions there, thereby having few systemic side effects.[6]

As a derivative

of salicylic acid, mesalazine (2) is also thought to be an antioxidant that traps free radicals,

which are potentially damaging byproducts of metabolism.[7]

http://en.wikipedia.org/wiki/Anti-inflammatory

http://en.wikipedia.org/wiki/Medication

http://en.wikipedia.org/wiki/Inflammatory_bowel_disease

http://en.wikipedia.org/wiki/Ulcerative_colitis

http://en.wikipedia.org/wiki/Crohn%27s_disease

http://en.wikipedia.org/wiki/Crohn%27s_disease

http://en.wikipedia.org/wiki/Mesalazine#cite_note-1

http://en.wikipedia.org/wiki/Aminosalicylate

http://en.wikipedia.org/wiki/Mesalazine#cite_note-PharmGKB-2

http://en.wikipedia.org/wiki/Salicylic_acid

http://en.wikipedia.org/wiki/Antioxidant

http://en.wikipedia.org/wiki/Free_radicals

http://en.wikipedia.org/wiki/Mesalazine#cite_note-PharmGKB-2



Chapter - I

Table-1.2

Sr.

no.

Generic name

Systematic (IUPAC) name

Structure

1 Mesalazine (2)

5-amino-2-hydroxybenzoic acid

HO

O

HONH2

2 Sulfasalazine (3)

2-hydroxy-5-[(E)-2-{4-[(pyridin-2-

yl)sulfamoyl]phenyl}diazen-1-

yl]benzoic acid

HO

O

HON

N

SNH

O

O

N

3 Olsalazine (4)

5-[(2Z)-2-(3-carboxy-4-oxocyclo-

hexa-2,5-dien-1-ylidene)hydrazino]-

2-hydroxybenzoic acid

HO

O

HONH

N

O

OH

O

4 Ipsalazide (5)

(E)-5-((4-(((carboxymethyl)amino)-

carbonyl)phenyl)azo)-2-hydroxy

Benzoic acid

HO

O

HON

N

O

HN

O

OH

5 Balsalazide (1)

(E)-5-([4-(2-

carboxyethylcarbamoyl)phenyl]diaze

nyl)-2-hydroxybenzoic acid

HO

O

HON

N

O

HN

O

OH

Aminosalicylic acid and similar agents, which were approved by United States food and drug

administration (USFDA) and available in the market are given in Table-1.2.

Balsalazide is the generic name of 5-[(1E)-[4-[[(2-carboxyethyl)amino]carbonyl]phenyl]azo] 2-

hydroxybenzoic acid (1). It is an oral prodrug of 5-ASA (2), having an inert carrier molecule,

N-(4-aminobenzoyl)β-alanine (4-ABBA, 8).[8]

After oral administration, 1 is split into 2 and 8

via azo-reduction by the colonic micro flora.[9]

2 is then acts directly on the colon to reduce the

local inflammation of the colonic mucosa, responsible for the symptoms of ulcerative colitis.[10-

12] Balsalazide (1) appeared to be an effective treatment option when compared to Salazopyrin



Chapter - I

(3). The clinical use and dosage of 3 are limited by adverse reactions,[13-15]

several of which are

attributed to the absorption of the carrier metabolite sulphapyridine.[16]

Balsalazide (1) is an therapeutic agent for ulcerative colitis. It is used as disodium dehydrate

salt. It’s molecular formula is C17H13N3O6Na2.2H2O and molecular weight is 437.32amu. The

commonly used dose of balsalazide is 6.75g per day, which is equivalent to 2.3g of mesalamine

(2).[17]

REVIEW OF LITERATURE:

Balsalazide disodium and its complete synthesis was first disclosed by Chan[18]

in 1983,

assigned to Biorex Laboratories Limited, England, claiming product ‘Balsalazide’ and process

of its preparation. The synthesis involves converting 4-nitrobenzoyl chloride (6) to 4-

nitrobenzoyl-β-alanine (7), hydrogenating with Pd/C (5%) in ethanol and isolating by adding

diethyl ether to produce 4-aminobenzoyl-β-alanine (8). Thereafter, 4-aminobenzoyl-β-alanine

(8) was treated with hydrochloric acid and sodium nitrite to generate N-(4-diazoniumbenzoyl)-

β-alanine hydrochloride salt (9) which was reacted at low temperature with disodium salicylate

to furnish Balsalazide disodium insitu which was added to dilute hydrochloric acid at low

temperature to produce Balsalazide (1) (Scheme-1.1). Thus obtained Balsalazide was

recrystallized with hot ethanol and converted to pharmaceutically acceptable salt (disodium

salt).

H2N

O

NH

O

NH

NN

HO

O

HO

O

HO

HO

O

HO

HO

O

NN

NH

O

HO

O

O2N

Cl

O

O2N

O

NHHO

O

.Cl

Beta alanine

water / NaOH

methanol

Pd/C, H2

water, Con. HCl

Aq. NaNO2 solution

water

NaOH, Na2CO3

(6) (7) (8)

(9)(10)(1)

.....Scheme-1.1



Chapter - I

Optimization of this diazonium salt based process was performed by Huijun et al[19]

and

reported the preparation of the title compound in 64.6% overall yield. Zhenhau et al[20]

have

synthesized 1 from 4-nitrobenzoic acid (12) via chlorination, condensation, hydrogenation,

diazotization, coupling and salt formation with overall yield 73%. Li et al[21]

have given product

in 73.9% total yield starting from 4-nitrobenzoyl chloride (6), where as Yuzhu et al[22]

confirmed chemical structure of Balsalazide disodium by elemental analysis, UV, IR, 1H-NMR

and ESI-MS etc. Shaojie et al[23]

have also followed same process for its preparation. Yujie et

al[24]

synthesized 1 in this way; preparation of 4-nitrobenzoyl-β-alanine (7) under microwave

irradiation of 420 W at 52oC for 10sec., reduction in ethyl acetate in the presence of Pd/C

catalyst then diazotization, coupling and salt formation.

Eckardt et al[25]

have developed a process for the preparation of Balsalazide which comprises,

conversion of 4-aminobenzoyl-β-alanine (8) to 4-ammoniumbenzoyl-β-alanine sulfonate salt

using a sulfonic acid in water. This was treated with aq. sodium nitrite solution at low

temperature to generate 4-diazoniumbenzoyl-β-alanine sulfonate salt (11) which was quenched

with aq. disodium salicylate to furnish Balsalazide disodium solution. This was further acidified

to allow isolation of 1 and then conversion to disodium salt (Scheme-1.2) in 76% yield.

H2N

O

NH

O

NH

NN

HO

O

HO

O

HO

HO

O

HO

HO

O

NN

NH

O

HO

O

water, MSA

Aq. NaNO2 solution

water

NaOH, Na2CO3

.MSA

(8) (11)(10)

(1)

.....Scheme-1.2



Chapter - I

PRESENT WORK:

Present work uncovered a new synthetic process to prepare Balsalazide disodium consisting no

isolation of any intermediate which is cost effective, safe and industrially feasible. Hence, we

have developed and optimized new process, impurities formed in the process were identified,

prepared and characterized. A new process is developed for the preparation of Balsalazide (1)

and analogs starting from salicylic acid. Further, we have prepared different analogs, positional

isomers and metabolites.

RESULTS AND DISCUSSION:

Four synthetic approaches are described herein, among which approach A deals with 3-steps

preparation of Balsalazide in water. Process development and identification of impurities also

included in this approach. Approach B discloses almost twenty analogs of Balsalazide

preparation without isolation of any intermediate (in situ). Approach C depicted synthesis of

Balsalazide and analogs in four consecutive steps starting with salicylic acid and Approach D

uncovered reactions due to isomers of nitro benzoic acids and hydroxy benzoic acids.

APPROACH A:

The present investigation is directed towards an improved process to prepare Balsalazide

disodium (Scheme-1.3), by reacting 4-nitrobenzoic acid (12) with an halogenating agent[26-32]

selected from thionyl chloride, oxalyl chloride in an organic solvent such as toluene, diisopropyl

ether, cyclohexane or methylene chloride, to give 4-nitrobenzoyl chloride (6), which insitu was

reacted with sodium salt of β-alanine in water to give 4-nitrobenzoyl-β-alanine (7). The ESI

mass spectrum of 7 [(M-H)-] was observed at m/z 237 in negative ion mode, which suggested

the molecular weight of 7 was 238. IR stretching at 3379cm-1

indicates NH stretching of amide.

The 1HNMR spectrum of compound 7, in (DMSO-d6), β-alanine corresponding chemical shifts

at δ 2.54 ppm and δ 3.48 ppm were appeared. Similarly, shift at δ 8.89 ppm in 1HNMR

corresponds to amide of 7 observed. The assigned structure for compound 7 hence clearly

confirmed the above spectral data.



Chapter - I

4-nitrobenzoyl-β-alanine (7) was suspended in water in the presence of a base and hydrogenated

using palladium on carbon, and thereafter treated with an acid to give 4-aminobenzoyl-β-alanine

(8). The base was selected from sodium hydroxide, sodium carbonate, sodium bicarbonate,

potassium hydroxide or aqueous ammonia and acid was selected from hydrochloric acid,

sulfuric acid or acetic acid. The ESI mass spectrum of 8 [(M-H)-] was observed at m/z 207.3,

suggests the molecular weight of 8 was 208. IR stretching at 3418, 3334 and 3241cm-1

indicate

NH and NH2 stretching. In 1HNMR spectrum of compound 8, corresponding chemical shift of

NH2 at δ 5.60 ppm observed. Thus assigned structure for compound 8 was clearly confirmed by

the above spectral data.

4-aminobenzoyl-β-alanine (8) was treated with hydrochloric acid and sodium nitrite to generate

N-(4-diazoniumbenzoyl)-β-alanine hydrochloride salt (9) and treated with disodium salicylate to

furnish Balsalazide disodium. This was acidified with an acid to pH 4.2-5.0 and stirred at 50-

80oC, more preferably at 60-65

oC for four hours to give a mixture of Balsalazide (1) and its

monosodium salt [60:40 to 80:20]. The ESI mass spectrum of 1 as [(M-H)-]displayed peaks at

m/z 356.1, indicates the mass of the product was 357. IR stretching at 3371, 3039 indicates NH

and OH stretching. In 1HNMR spectrum of compound 1, chemical shifts corresponds to

salicylic acid at δ 6.95 ppm, δ 7.95 ppm and δ 8.34 ppm were observed. The assigned structure

for compound 1 was clearly confirmed by the above spectral data.

Thus obtained product was treated with sodium carbonate in water and disodium salt was

precipitated with alcohol, more preferably isopropyl alcohol at 5-35oC to obtain

pharmaceutically accepted salt (disodium salt).

The Balsalazide and its monosodium salt mixture obtained was purified using aqueous 1,4-

dioxane to yield highly pure Balsalazide acid and its monosodium salt mixture that was ideal for

the preparation of disodium salt. Purification occurs when 1,4-dioxane suspension was heated at

high temperature at 90-95oC and filtered at relatively high temperature 60-65

oC. Several

impurities go into solution by heating at 90-95oC. Structures of these impurities were given in

Figure-1.1.



Chapter - I

H2N

O

NH

O

NH

NN

HO

O

HO

O

HO

HO

O

HO

HO

O

NN

NH

O

HO

O

O2N

OH

O

O2N

O

NHHO

O

.Cl

Beta alanine

water / NaOH

water / NaOH

Pd/C, H2

water, Con. HCl

Aq. NaNO2 solution

water

NaOH, Na2CO3

SOCl2 / IPE

(12) (7) (8)

(9)(10)(1)

.....Scheme-1.3

PROCESS DEVELOPMENT AND IMPURITY PROFILE:

For the preparation of N-(4-nitrobenzoyl)-β-alanine (7), 4-nitrobenzoic acid (12) was selected as

a starting material because of its easy commercial availability and it was also well known in

chemical literature that acid group can be converted to amide conveniently via chloride

formation. Further, preparation of acid chloride has been described in literature, wherein acid

has been reacted with thionyl chloride[26]

/ oxalyl chloride[27]

/ phosphorous pentachloride[28]

/

phosphorous trichloride[29]

/ phosphorous oxychloride[30]

/ triphosgene[31]

etc[32]

to obtain acid

chloride, which on reaction with amines provides amides. We intended to prepare acid chloride

but owing to handling advantages decided to take it in situ for further conversion into amide.

Thus, 4-nitrobenzoic acid (12) was reacted with thionyl chloride in presence of DMF (catalyst)

in different solvents and after completion of reaction, isolated product, 4-nitrobenzoyl chloride

(6) was added to sodium salt of β-alanine. Results of the some batches are summarized in

Table-1.3.

It was clear from the above experiments that major impurity formed in above reaction was 4-

nitrobenzoic acid (12), which was forming due to decomposition of 4-nitrobenzoyl chloride (6)

with water. Subsequent to this observation, we tried to minimize the exposure of 6 to water.



Chapter - I

Figure-1.1

HO

HO

O

NN

OH

O

N

O

NHO

HN OH

O

N

HO

HO

O

HO

O

NN

NH

O

HO

O

OHN

N

NH

O

HO

O

HO

NN

NH

O

HO

O

HO

O

HO

NN

HNO

O

OH

NN

NH

O

HO

O

HO

O

HO

HNO

OH

O

NN

NH

O

HO

O

HO

N

N

HN

O

OH

O

NN

NH

O

HO

O

HO

N

N

HN OH

O

O

NH

OH

O O

(13) (14)

(15) (16)

(17) (18)

(19) (20)

Table-1.3

Solvent Thionyl

chloride

HPLC purity of

(6)

HPLC purity of

(7)

Impurity

formed (12)

Toluene 1.75 m. eq. 89.58% 84.87% 9.68%

n-Heptane 1.25 m. eq. 99.28% 95.89% 4.02%

Cyclohexane 1.25 m. eq. 99.86% 93.30% 6.62%

Cyclohexane 1.25 m. eq. 99.57% 93.47% 6.22%

Cyclohexane 1.25 m. eq. 99.46% 92.10% 7.69%



Chapter - I

Therefore, after formation of 4-nitrobenzoyl chloride (6), instead of isolation, it was reacted

with sodium salt of β-alanine. Results of some experiments are summarized in Table-1.4.

Table-1.4

Solvent Thionyl chloride HPLC purity of (7) Impurity formed

(12)

Methylene chloride 1.20 m. eq. 92.12% 7.59%

Toluene 1.20 m. eq. 95.87% 3.45%

Tetrahydrofuran 1.25 m. eq. 85.53% 13.40%

Tetrahydrofuran 1.25 m. eq. 88.07% 10.46%

1,4-Dioxane 1.25 m. eq. 85.69% 13.59%

Diisopropyl ether 1.25 m. eq. 99.48% 0.29%




However, 4-nitrobenzoic acid (12) mostly gets eliminated during filtration into mother liquor in

presence of diisopropyl ether. In one experiment, after reaction, diisopropyl ether layer was

separated first and then adjusted pH. The presence of 12 was observed more in 7.

It is known that 4-nitrobenzoic acid (12) would convert in to Des-β-alanine Balsalazide (13) and

may contaminate the final product. Therefore to fix a limit of 12 in 7 some experiments were

carried out to know its impact in product i.e., Balsalazide (1). Results of some experiments are

summarized in Table-1.5.

Table-1.5

(12) in (7) (27) in (8) (13) in (1)

1.38% 0.18% 0.07%

1.08% 0.07% Not detected





Chapter - I

Other impurity which forms in preparation of 7 at ~0.5% level was confirmed by LCMS as N-

(2-(3-hydroxypropylcarbamoyl)ethyl)-4-nitrobenzamide (21), does not have any adverse impact

on the final product quality. This impurity was eliminated during the isolation of 8 in the

reduction stage, which remains in the mother liquor owing to its higher solubility in water.

O2N

O

NHO

HN OH

O (21)

Formation of 7 was studied by varying the temperature and it was observed that slow addition

of 6 to sodium salt of β-alanine at < 10oC maintaining pH > 8 gave the best results. By these

conditions, we could obtain 7 close to 98% of chromatographic purity.

In the next step, which is well known in the chemical literature that nitro group can be

converted to amino by catalytic hydrogenation.[33]

Hydrogenation is a facile reaction, which is

carried by reacting 7 with hydrogen in presence of palladium on carbon. This reaction we tried

in different solvents at 25-35oC with varying quantities of palladium on carbon and at different

hydrogen pressure, which are summarized in Table-1.6.

Table-1.6

Solvent Pd/C (w/w) Hydrogen pressure

Kg/cm2

Impurity A

(27)

Impurity B

(26)

Impurity C

(25)

Ethanol 4% 4 0.50% 0.31% 0.27%

Ethanol 5% 5 0.25% 0.58% 0.75%

Methanol 3% 5 0.05% 0.12% Not detected

Methanol 2% 10 0.08% 2.02% 0.21%

Based on the above data, we concluded that impurities were found in all experimental

conditions. We analyzed the sample, 8 by LCMS and found the related substances / side

reaction were formed exactly as reported in the literature (Figure-1.2).[34]

One experiment was



Chapter - I

carried out using ethanol as a solvent and added BHT (anti-oxidant) in reaction itself. The result

was similar as of the above.

Figure-1.2

O

HN OH

O

NN

O

NHHO

O

O

O

NHHO

O

O2N

O

NHHO

O

ON

O

NHHO

O

HOHN

O

NHHO

O

H2N

O

HN OH

O

NN

O

NHHO

O

O

HN OH

O

NH

HN

O

NHHO

O

(7)

(22)

(23)

(8)

(24)

(25)

(26)

Some experiments were carried out using methanol and modified the product isolation by using

anti solvents, which has no significant impact on product quality. The purification study was

also not encouraging to get the quality product.

We explored alternatives to hydrogenate 7 in less volume of solvent and conditions, where these

impurities may form in minimum amount. For that water was used as a solvent. As 7 was almost

insoluble in water, one mole equivalent of base was used to dissolve it as its sodium salt in

water and hydrogenated using Pd/C at 30-45oC. The reaction mass was filtered and after

removal of catalyst one mole equivalent of acid was added to precipitate the 8. Details of some

experiments are summarized in Table-1.7.



Chapter - I

Table-1.7

Solvent /

Volume

Pd/C

(w/w)

Hydrogen

Pressure

(Kg/cm2)

Time Impurity

A (27)

Impurity

B (26)

Impurity

C (25)

Water / 14 4% 15-20 30 min 0.06% ND ND

Water / 5 2% 18-22 1 hr 0.79% ND ND

Water / 5 2% 15-20 2 hr 2.75% 0.48% ND

Water / 5 2% 20-30 30 min ND 0.08% ND

Water / 5 2% 20-30 2 hr 30 min 0.05% ND ND

The advantages of hydrogenation reaction in water are:

(1) Higher purity and conversion of product and less formation of impurities,

(2) Less volume of reaction mass hence high throughput,

(3) Environment friendly,

(4) High productivity,

(5) Cost effective,

(6) Inherent safety where solvents are used and recovery are avoided.

The impurities except 27, formed in hydrogenation step were not capable of forming

hydrochloride salt, therefore these can be removed by filtration of 4-ABBA hydrochloride in

next step.

It is also well known that azo compounds are prepared by interaction of diazonium salt with a

phenol in the presence of sodium hydroxide or with an amine in the presence of sodium acetate.

The coupling is an electrophilic substitution reaction wherein diazonium ion reacts at the

position of greatest electron availability i.e., the position of ortho or para to the electron

releasing phenoxy or amino group (Figure-1.3).



Chapter - I

Figure-1.3

HO

N O2O

NO

NO

+-H2O

H2N

O

NHHO

O

slow

N

O

NHHO

O

NO H

H

-NO2

-H+

N

O

NHHO

O

NO

H

N

O

NHHO

O

HON

H+

tautomerization

N

O

NHHO

O

N

(8)

Keeping the like sequence we prepared the diazonium salt of 8 and added to disodium salt of

salicylic acid (10) using water as a solvent. 10 was used in 1.02 m. eq. with respect to 8 and

found conversion to product only 94% by HPLC. In another experiment with 1.0 m. eq. of

salicylic acid (10), product formation was reduced to only 86%. Upon increasing the mole ratio

to 1.2, product formation was ~97%, which was decreased again (93%) when 1.5 m. eq. of

salicylic acid (10) was used. Based on above observation optimization of coupling reaction has

been done with 1.2 m. eq. of 10.



Chapter - I

HO

HO

O

NN

OH

O

N

O

NHO

HN OH

O

N

HO

HO

O

HO

O

NN

NH

O

HO

O

OH

NN

NH

O

HO

O

HO

NN

NH

O

HO

O

HO

O

HO

NN

HNO

O

OH

NN

NH

O

HO

O

HO

O

HO

HNO

OH

O

NN

NH

O

HO

O

HO

N

N

HN

O

OH

O

NN

NH

O

HO

O

HO

N

N

HN OH

O

O

NH

OH

O O

O2N

Cl

O

O2N

OH

O

O2N

NH

O

HO

O

O2N

O

NHO

HN OH

O

H2N

OH

O

H2N

NH

O

HO

O

H2N

O

NHO

HN OH

O

NN

NH

O

HO

O

O

HN

O

OH

NN

NH

O

HO

O

O

HN

O

OH

NH

HN

NH

O

HO

O

O

HN

O

OH

O

N

OH

O

N

NH

O

HO

O

N

O

NHO

HN OH

O

N

NN

Cl

Cl

Cl

NN

NH

O

HO

O

HO

HO

O

H2N OH

O

PNB

COUPLING

HYDROGINATION

Pd-C / H2

Pd-C / H2Pd-C

/ H 2

DIA

ZO

CO

UP

LIN

G

NaNO2 / HCl NaNO2 / HCl NaNO2 / HCl

HO

HO

O

HO

HO

O

HO

HO

O

NaOH / Na2CO3

NaOH / Na2CO3

NaOH / Na2CO3

BALSALAZIDE PROCESS

HO

HO

O

NaOH / Na2CO3

DE

CA

RB

OX

YL

AT

ION

GOMBERG-BACHMANN

COUPLING

N

NH

O

HO

O

N

Cl

N

NH

O

HO

O

N

Cl

DECARBOXYLATION

GOMBERG-BACHMANN

COUPLING

N

NH

O

HO

O

N

Cl

.....Scheme-1.4

Analysis of Balsalazide (1) sample by LCMS found some unknown impurities. The presence of

impurities in an Active Pharmaceutical Ingredient (API) can have a significant impact on the

quality and safety of the drug products. Therefore, it was necessary to study the impurity profile



Chapter - I

of the API to be used in the manufacturing of a drug product. International Conference on

Harmonization (ICH) guidelines recommends identification and characterization of all

impurities which were present in API in a level of ≥0.05% (based on daily dose).[35]

In this

context, we have undertaken a comprehensive study to synthesize and characterize the

impurities in Balsalazide API (Scheme-1.4).

These impurities were: 5-[[4-[carboxy]phenyl]azo]-2-hydroxybenzoic acid (Des-β-alanine

Balsalazide, 13); 5-[4-[2-(2-carboxyethyl carbamoyl)ethyl carbamoyl]phenyl azo]-2-hydroxy-

benzoic acid (Balsalazide β-alanine, 14); 3-[[4-[[(2-carboxyethyl)amino]carbonyl]phenyl]-azo]-

2-hydroxybenzoic acid (Balsalazide 3-isomer, 15); 3-[4-(4-hydroxy phenylazo)benzoylamino]-

propionic acid (Decarboxy Balsalazide, 16); 2,4-bis-[[4-[(2-carboxyethyl)amino]carbonyl]-

phenyl]azo salicylic acid (Bis-azo salicylic acid, 17); 5-[2-[4’,5-bis[(2-

carboxyethyl)carbamoyl]-biphenyl-2-yl]diazenyl]-2-hydroxybenzoic acid (Biphenyl-azo

salicylic acid, 18); 3-[4-[5-[4-(2-carboxy ethyl carbamoyl)phenylazo]-2-hydroxy

phenylazo]benzoyl amino]propionic acid (Bis azo diacid, 19); 2,4-bis[[4[[(2-

carboxyethyl)amino]carbonyl]phenyl]azo]-3-[4-[[(2-carboxyethyl)-amino]-

carbonyl]phenyl]phenol (Bis-azo triacid, 20). In addition to these we have also synthesized and

characterized two metabolites[36]

namely 3-(4-acetylamino benzoylamino)-propionic acid (N-

acetyl-8) and 5-acetylamino 2-hydroxybenzoic acid (N-acetyl-2).

Des-β-alanine Balsalazide 13, a carryover impurity, originates due to the presence of 4-

nitrobenzoic acid 12 in intermediate 7 of the Balsalazide process. This impurity may also form

due to the amide hydrolysis of Balsalazide during the process. Des-β-alanine Balsalazide (13)

was prepared starting with 4-nitrobenzoic acid 12 according to the following scheme (Scheme-

1.5). Catalytic hydrogenation of 12 with Pd/C in methanol yielded 27, which was diazotized

with sodium nitrite in aqueous hydrochloride and coupled with sodium salicylate to give the

desired compound 13 in excellent yield. The ESI mass spectrum of 13 displayed peaks at m/z

285.2 [(M-H)+] in negative ion mode and as sodium ion adduct at m/z 307.0 [(M-H)+Na]. In

positive ion mode this compound appeared at m/z 287.2 [(M+H)+] in the mass spectrum. In



Chapter - I

1HNMR spectrum of compound 13, corresponding peaks of β-alanine at δ 2.61 ppm and δ 3.56

ppm were absent. Similarly in 13

CNMR spectrum peaks at δ 34.6 ppm and δ 36.6ppm due to β-

alanine moiety were also not observed. The assigned structure for compound 13 is clearly

confirmed by the above spectral data.

HO

HO

O

HO

HO

O

NN

OH

O

O2N

Oa b c

(10)

OH

H2N

O

OH

O

OH

NN

(12) (27) (28)

(13)

.Cl

Reagents and conditions: (a) Methanol, Pd/C, H2, RT, 2-3 h, 98%; (b) Water, conc. HCl , 0-5°C then aq. NaNO2

solution, 0-5°C, 1 h, ~100%;(c) Water , NaOH / Na2CO3, Salicylic acid, 0-5°C then addition of diazonium salt 28

solution, 1 h, ~100%.

.....Scheme-1.5

Like Des-β-alanine Balsalazide 13, Balsalazide β-alanine 14 originates from N-(4-

nitrobenzoyl)di-β-alanine 21, which may present in intermediate 7 and carry through the

synthetic process to give this impurity in Balsalazide finish product.

Balsalazide β-alanine 14 was prepared by hydrogenating N-(4-nitrobenzoyl)di-β-alanine 21

with Pd/C in methanol to give the corresponding amine 29 ,which on diazotization followed by

coupling with salicylic acid in alkaline medium results the desired compound 14 (Scheme-1.6).

The ESI mass spectrum of 14 displayed peaks at m/z 427.2 [(M-H)-] and as sodium ion adduct at

m/z 449.0 [(M-H)-+Na] in -ve ion mode; in +ve ion mode this impurity appeared at m/z 429.2

[(M+H)+]. The

1H and

13C NMR spectra of compound 14 also support the assigned structure for

14 (Di-β-alanine analog of Balsalazide).

Balsalazide 3-isomer 15 is a positional isomer of Balsalazide 1 and can form due to the coupling

of diazonium salt 9 to 3-position of salicylic acid 10 instead of 5-position (Scheme-1.7).



Chapter - I

N-(4-Aminobenzoyl)-β-alanine 8 as hydrochloride salt was diazotized with aqueous sodium

nitrite solution at 0-5°C and further treated with sodium salicylate in water/ isopropyl alcohol

mixture at 0-5°C to yield compound 15 as a byproduct along with 1. The ESI mass spectrum of

compound 15 was showing equal mass value as that of Balsalazide. The 1H-NMR of 15 exhibits

three doublet of doublets at δ 6.61 ppm (dd, J = 7.7 Hz, 1H), 7.57 ppm (dd, J = 8.0 Hz and 1.7

Hz, 1H), and 7.87 ppm (dd, J = 8.0 Hz and 1.7 Hz, 1H) corresponding to three hydrogen at 4, 5

and 6 position of aromatic ring of salicylic acid. 13

C-NMR data also confirmed the assigned

structure of compound 15.

O2N

O

NHO

HN OH

O

H2N

O

NHO

HN OH

O

N

O

NHO

HN OH

O

N

N

O

NHO

HN OH

O

N

HO

HO

O

HO

HO

O

c

(10)

(21) (29) (30)

(14)

ba

Cl

Reagents and conditions: (a) Methanol, Pd/C, H2, RT, 2-3 h, 98%; (b) Water, Conc. HCl, 0-5°C aq. NaNO2

solution, 0-5°C, 1 h, 100%; (c) Water, NaOH /Na2CO3, Salicylic acid, 0-5°C then addition of diazonium salt 30

solution, 1 h, 82%.

.....Scheme-1.6

O

NH

NN

HO

O

(9)

(10)

HO

O

NN

NH

O

HO

O

(15)

OH

H2N

O

NHHO

O

(8)

a b

Cl

Reagents and condition: (a) Water, conc. HCl, 0-5°C aq. NaNO2 solution, 0-5°C, 1 h, ~100%; (b) Water, NaOH /

Na2CO3, Salicylic acid, 0-5°C then addition of diazonium salt 9 solution, 1 h, ~7%.

.....Scheme-1.7



Chapter - I

Decarboxy Balsalazide 16 was detected in 1 as a degradation product. Decarboxylation of

Balsalazide would result in this impurity. We have prepared this impurity from 4-

hydroxybenzoic acid 31, wherein N-(4-aminobenzoyl) β-alanine 8 was diazotized with aqueous

sodium nitrite solution / hydrochloric acid at 0-5°C and added to aqueous alkaline solution of 31

to obtain compound 16 (Scheme-1.8). The ESI mass spectrum of compound 16 displayed peak

at m/z 312.1 [(M-H)-] in -ve ion mode, which was 44 mass unit (amu) less than Balsalazide

(m/z356.2). In +ve ion mode its base peak appeared at m/z 314.0 [(MH) +

] and peak

corresponding to sodium ion adduct appeared at m/z 336.0 [(MH)+Na]. The 1H-NMR spectrum

of this compound showed signals at δ 6.97 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.88 (d,

J = 8.8 Hz, 2H), and 8.04 (d, J = 8.8 Hz, 2H), these values indicates that both aromatic rings are

para-substituted in addition 13

CNMR and DEPT spectra are also supporting the assigned

structure for compound in the structure, 116.1(CH), 121.9(CH), 125.2(CH), 128.4(CH).

O

NH

NN

HO

O

HO

(9) (31)

NN

NH

O

HO

O(16)

H2N

O

NHHO

O

(8)

a b

O

OH

HO

Cl

Reagents and conditions: (a) Water, conc. HCl, 0-5°C aq. NaNO2 solution, 0-5°C, 1 h, 100%; (b) Water, NaOH /

Na2CO3, salicylic acid, 0-5°c then addition of solution of diazonium salt 9, 1 h, 91%.

.....Scheme-1.8

Bis-azo salicylic acid 17 formed due to the coupling of Diazo intermediate 9 with Balsalazide 1

on ortho-position with respect to phenolic –OH group during its preparation, and carry through

the process to give this impurity in Balsalazide. The synthetic sequence for the preparation of

this impurity is depicted in Scheme-1.9. Using half mole of salicylic acid in diazo coupling

reaction yielded this impurity. But due to number of side reactions, formation of 17 was less.

The ESI mass spectrum of impurity 17 displayed peak at m/z 577.2 [(MH)+], 221 mass unit

(amu) more than Balsalazide (m/z 356.2) indicating attachment of Diazo intermediate 9 to

Balsalazide. The signals in 1H-NMR spectrum of this impurity at δ 7.90 (2d, J = 8.8 Hz, 4H)



Chapter - I

and 8.01 (2d, J = 8.8 Hz, 4H) represents two Diazo intermediate moieties, the remaining two

aromatic peaks at δ 8.15 (d, J = 2.5 Hz, 1H) and 8.51 (d, J = 2.5 Hz, 1H) due to benzene ring of

salicylic acid. The smaller coupling constant value (J = 2.5 Hz) indicates the meta-coupling in

the benzene ring, which authenticates the assigned structure for compound 17.

Base-promoted free radical coupling (Gomberg-Bachmann reaction)[37-39]

between the aryl

diazonium salt 9 and Balsalazide lead to the formation of Biphenyl-azo salicylic acid 18. The

synthetic sequence to prepare this impurity is illustrated in Scheme-1.9. Using half mole of

salicylic acid in reaction yielded this impurity. The formation of 18 was very less as there were

various side reactions. ESI mass spectrum of impurity 18 displayed peak at m/z 549.2 [(MH)+],

28 mass unit (amu) less than Bis-azo salicylic acid 17, indicating Diazo group detachment from

Bis-azo salicylic acid 17. 1HNMR data for compound 18 are 6.70 (d, J = 8.8 Hz, 1H), 7.58 (d, J

= 8.5 Hz, 2H), 7.60 (dd, J = 8.8 Hz and 2.5 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.93 (d, J = 8.5

Hz, 2H), 7.97 (dd, J = 8.8 Hz and 1.4 Hz, 1H), 8.05 (d, J = 1.4 Hz, 1H), 8.17 (d, J = 2.5 Hz,

1H).

O

NH

NN

HO

O

(9)

(10)

NN

NH

O

HO

O

H2N

O

NHHO

O

(8)

a b

HO

O

HO

NN

HNO

O

OH

NN

NH

O

HO

O

HO

O

HO

(17)

(18)

HNO

OH

O

Cl

Reagents and conditions: (a) Water, conc. HCl, 0-5°C aq. NaNO2 solution, 0-5°C, 1h, ~100%; (b) Water, NaOH /

Na2CO3, 0.5 mole eq. Salicylic acid, 0-5°C then addition of solution of 9, 1 h, 3-7%.

.....Scheme-1.9



Chapter - I

Bis-azo diacid 19 originates due to decarboxylation of the compound 17 and contaminate the

product. Bis-azo triacid 20 originates due to Gomberg-Bachmann type coupling of 9 and 19

(Scheme-1.10). The compound 19 was prepared by reacting diazonium salt 9 with alkaline

solution of para-hydroxybenzoic acid 31 (0.5 mole equivalent), whereas the compound 20 was

obtained by reacting 9 with alkaline solution of 0.25 mole equivalent of 31.

O

NH

NN

HO

O

(9)

(31)

NN

NH

O

HO

OH2N

O

NHHO

O

(8)

a b

HO

NN

NH

O

HO

O

HO

N

N

(19)

(20)

N

N

HN

O

OH

O

HN OH

O

O

NH

OH

O O

Cl

Reagents and conditions: (a) Water, conc. HCl, 0-5°C, aq. NaOH solution, 0-5°C, 1 h, ~100%; (b) Water, NaOH

/Na2CO3, 0.5 (for 19) / 0.25 (for 20) mole eq. 4-hydroxy benzoic acid 31, 0-5°C, solution of 9, 1 h, 19 (~32%) and

20 (~36%).

.....Scheme-1.10

Balsalazide disodium is freely soluble in water. After completion of the reaction, the mass was

acidified to different pH and filtered small sample at different temperature as Balsalazide (1).

Result of some experiments are summarized in Table-1.8.

Table-1.8

pH of

reaction

mass

Filtration

Temp

(oC)

Balsalazide-3-

isomer (15)

Bisazo

salicylic acid

(17)

Bis-azo

diacid

(19)

Bis-azo

triacid

(20)

HPLC

purity

0.5 2-5 0.43 0.21 0.16 0.16 96.53

0.5 2-5 0.39 0.19 0.17 0.21 97.26

2.0 2-5 0.44 0.33 0.17 0.26 96.18

2.0 25-30 0.42 0.09 0.11 0.10 98.40



Chapter - I

Isolation of Balsalazide by filtration was very slow and taken plenty of time. Due to poor

filterability, Balsalazide was extracted in organic solvents at lower pH and recrystallized from

the same solvent. Results of some experiments are summarized in Table-1.9.

Table-1.9

Solvent Balsalazide-3-

isomer (15)

Bisazo

salicylic acid

(17)

Bis-azo

diacid (19)

Bis-azo

triacid (20)

HPLC

purity

Ethyl acetate 0.24 0.16 0.16 0.15 97.25

Ethyl acetate 0.33 0.05 0.13 0.14 98.07

Methyl ethyl

ketone

0.31 0.06 0.24 0.11 99.07

Methyl ethyl

ketone

0.22 0.07 0.19 0.18 98.68

It was observed from above experimental results that during extraction / recrystallization

Balsalazide-3-isomer (15) was decreasing in both ethyl acetate and methyl ethyl ketone,

whereas Bisazo salicylic acid (17) was decreasing only in ethyl acetate while remained almost

same in ethyl methyl ketone. Bis-azo diacid (19) and Bis-azo triacid (20) impurities were not

decreasing much in both the solvents. Requirement of high volume and higher temperature were

major drawbacks for use of both the solvents owing to lower solubility of Balsalazide in these

solvents. This process suffered from disadvantages such as use of large volume of solvents, it's

recovery and adverse cost impact and compromised safety. Moreover, the yield was also low.

Therefore to resolve this problem, the reaction mass was acidified to pH 4.2-5.0 and stirred at

50-80oC. Isolated a mixture of Balsalazide and its monosodium salt at 25-45

oC (sodium content

~3.54%, by ion chromatography).

This process improved not only filterability but also purity. Most of the impurities had also

reduced to reasonable levels. See Table-1.10.



Chapter - I

Table-1.10

pH of

the

reaction

mass

Filtration

temp.

(oC)

HPLC

Purity

Balsala

zide-3-

isomer

(15)

Bis-

azo

triacid

(20)

Bis-

azo

diacid

(19)

Des-β-

alanine

Balsalazi

de (13)

Bisazo

salicyl

ic acid

(17)

Yield

(w/w)

4.0 25-30 97.97 0.25 0.28 0.14 0.15 0.19 1.63

4.1 25-30 99.22 0.08 0.14 0.17 0.16 ND 1.56

4.2 25-30 99.58 ND 0.05 0.11 ND ND 1.45

4.2 40-45 99.34 0.05 0.10 0.13 0.05 0.05 1.27

4.5 40-45 99.46 0.06 0.06 0.15 0.07 0.07 1.46

4.5 40-45 99.58 0.04 0.03 0.12 ND 0.06 1.35

From the above table, we concluded that pH ~4.2 of the reaction mass and the filtration

temperature (25-35oC) was suitable for our process. Under these conditions most of the

impurities decreased and product quality as well as yield increased. In the case of pH (4.5) and

filtration temperature (40-50oC), results are same as that of above but decrease in yield was

noted. Yield was less due to pH as well as temperature owing to high solubility of Balsalazide at

particular pH (4.5) as it was in the form of a mixture of Balsalazide and its monosodium salt.

To know the behavior of these impurities in the final product, we converted Balsalazide (1)

having above impurities into Balsalazide disodium. 3-[4-[5-[4-(2-carboxy ethyl

carbamoyl)phenylazo]-2-hydroxy phenylazo]benzoyl amino]propionic acid (Bis azo diacid, 19);

2,4-bis[[4[[(2-carboxyethyl)amino]carbonyl]phenyl]azo]-3-[4-[[(2-carboxyethyl)-amino]-

carbonyl]phenyl]phenol (Bis-azo triacid, 20); 2,4-bis-[[4-[(2-carboxyethyl)amino]carbonyl]-

phenyl]azo salicylic acid (Bis-azo salicylic acid, 17) and 5-[[4-[carboxy]phenyl]azo]-2-

hydroxybenzoic acid (Des-β-alanine Balsalazide, 13) did not eliminate in mother liquor, hence

contaminating the product. So it was necessary to purify the product in this stage only. Different

solvents were tried to attain the good quality of Balsalazide. Results of some purification

attempts are summarized in Table-1.11. Values given in brackets are Input quantities.



Chapter - I

Table-1.11

Solvents Balsala

zide-3-

isomer

(15)

Bis-

azo

triacid

(20)

Bis-azo

diacid

(19)

Des-β-

alanine

Balsalazide

(13)

Bisazo

salicylic

acid

(17)

HPLC

purity

Aq. Ethanol (1:1) 0.03

(0.20)

0.13

(0.13)

0.21

(0.19)

ND

(ND)

ND

(0.06)

99.04

(99.36)

Aq. Methanol (20%

w/v)

0.08

(0.20)

0.13

(0.13)

0.20

(0.19)

0.12

(ND)

0.06

(0.06)

99.37

(99.36)

Aq. n-propanol (1:1) 0.06

(0.20)

0.12

(0.13)

0.22

(0.19)

ND

(ND)

0.05

(0.06)

99.47

(99.36)

Aq. THF (1:1) 0.03

(0.14)

0.07

(0.20)

0.22

(0.20)

ND

(ND)

0.03

(0.05)

99.65

(99.41)

Aq. Acetone (10%

w/w)

ND

-

0.32

(0.30)

0.16

(0.21)

1.01

(1.98)

ND

(ND)

98.32

(97.37)

Aq. acetic acid

(10%w/w)

0.05

(0.20)

0.16

(0.14)

0.16

(0.50)

ND

(ND)

0.22

(0.17)

99.09

(97.15)

Aq. acetic acid

(10%w/w)

0.12

(0.28)

0.11

(0.10)

0.17

(0.18)

ND

(ND)

0.08

(0.07)

99.42

(98.65)

Aq. DMF 0.43

(0.58)

0.72

(0.81)

0.14

(1.13)

0.74

(ND)

ND

(0.07)

97.50

(97.72)

Aq. ACN (10% w/v) 0.06

(0.58)

0.11

(0.81)

0.19

(0.13)

ND

(ND)

ND

(0.07)

99.55

(97.92)

Aq. 1,4-dioxane (10%

w/v)

0.21

(0.31)

0.14

(0.13)

0.19

(0.17)

0.05

(0.09)

0.05

(0.07)

98.97

(98.27)

Aq. 1,4-dioxane (5%

w/v)

0.16

(0.31)

0.07

(0.13)

0.11

(0.17)

ND

(0.09)

0.05

(0.07)

99.24

(98.27)

Aq. 1,4-dioxane (5%

w/v)

0.12

(0.31)

0.06

(0.13)

0.09

(0.17)

ND

(0.09)

0.05

(0.07)

99.24

(98.27)

DMF / Toluene 0.05

(0.05)

0.17

(0.17)

0.20

(0.16)

0.21

(0.27)

0.06

(0.22)

99.09

(98.78)

Acetonitrile, Water

(1:1)

0.17

(0.70)

0.05

(0.07)

0.16

(0.15)

ND

(ND)

ND

(ND)

99.25

(95.03)



Chapter - I

According to above study, 1,4-dioxane was slightly a better solvent to remove all impurities in

Balsalazide and its monosodium salt mixture. So based on above conclusion, we optimized the

purification in Aq. 1,4-dioxane (50%w/v) and the experimental results are summarized in

Table-1.12.

Table-1.12

Balsalazide-

3-isomer

(15)

Bis-azo

triacid

(20)

Bis-azo

diacid

(19)

Des-β-

alanine

Balsalazide

(13)

Bisazo

salicylic

acid

(17)

Highest

Unknown

HPLC

purity

Yield

(w/w)

ND

(0.06)

ND

(0.10)

0.01

(0.16)

0.06

(0.24)

0.04

(0.07)

ND

(0.29)

99.89

(98.91)

0.80

ND

(0.30)

ND

(0.05)

ND

(0.15)

ND

(0.05)

0.05

(0.06)

0.05

(0.10)

99.90

(99.30)

0.80

0.03

(0.48)

0.01

(0.12)

0.03

(0.19)

0.04

(0.15)

0.07

(0.11)

0.06

(0.14)

99.75

(98.53)

0.77

ND

(0.05)

ND

(0.04)

0.02

(0.23)

ND

(ND)

0.03

(0.07)

0.03

(0.08)

99.92

(99.39)

0.77

From the above tabulated results, we have concluded that Balsalazide purification was

necessary to remove all the impurities in this stage. The yield by the above purification method

is also higher compared to earlier purification method.

In next step, Balsalazide and its monosodium salt mixture was converted to disodium salt by

using sodium hydroxide, sodium carbonate or sodium bicarbonate etc.

Our aim was to achieve the maximum yield with good product quality using the mild condition

i.e., low temperature and mild base (sodium carbonate) for the preparation of Balsalazide

disodium. Therefore, Balsalazide and its monosodium salt mixture was taken in water pH was

adjusted to neutral (6.5) using aq. sodium carbonate to prepare a clear solution. The obtained

solution was filtered to remove any insoluble and isopropyl alcohol was added to the filtrate to

precipitate the product. The obtained yield was 1.15w/w. In this modified and optimized

process, extra impurity formation was not observed.



Chapter - I

APPROACH B:

Approach B discloses almost twenty analogs of Balsalazide preparation without isolation of any

intermediate (in situ). The synthetic process is same as of Approach A which directs towards a

green and improved process to prepare Balsalazide disodium and its analogs in situ three steps

in aqueous condition. It starts by reacting 4-nitrobenzoic acid (12) with an halogenating agent

such as thionyl chloride, in an organic solvent such as diisopropyl ether, to give 4-nitrobenzoyl

chloride (6), which in situ is reacted with sodium salts of various amino acids, selected from α,

β and γ amino acids (40[a-t], Figure-1.4) in water to give 4-nitrobenzoyl-amino acid sodium

salt (7[a-t]),. After completion of reaction, biphasic reaction mass was separated, and the lower

aq. layer, containing 4-nitrobenzoyl-amino acid sodium salt is hydrogenated using palladium on

carbon, to give 4-aminobenzoyl-amino acid sodium salt (8[a-t]), Scheme-1.11.

Amino acids

(40[a-t])

O2N

O

AA

H2N

O

AA

N

O

AA

N

.Cl

HO

HO

O

NN

O

AAHO

HO

O

O2N

OH

O

water / NaOH

Pd/C, H2

water, Con. HCl

Aq. NaNO2 solution

water

NaOH, Na2CO3

SOCl2 / IPE

O2N

O

Cl

water / NaOH

(12) (6) (7[a-t])

(8[a-t]) (9[a-t])

(10)

(1[a-t])

.....Scheme-1.11

Thus obtained, 4-aminobenzoyl-amino acid was treated with hydrochloric acid and sodium

nitrite to generate N-(4-diazoniumbenzoyl)-amino acid hydrochloride salt (9[a-t]) and treated

with disodium salicylate to furnish Balsalazide disodium and its analogs (1[a-t]).



Chapter - I

This is acidified with an acid to pH 4.2-5.0 and stirred at 50-80oC, more preferably at 60-65

oC

for four hours to give a mixture of Balsalazide and its monosodium salt. Thus obtained product

is treated with sodium carbonate in water and disodium salt was precipitated with alcohol, more

preferably isopropyl alcohol at 5-35oC to obtain pharmaceutically accepted salt (disodium salt).

Figure-1.4 (40[a-t])

H2N

O OH

H2N

O OH

H2N

O OH

H2N

O OH

H2N

O OH

H2N

O OH

SH

H2N

O OHO

OH H2N

O OHO

NH2

H2N

O OH

OHH2N

O OH

Cl

H2N

O OH

NH O

OH

NH O

OH

HO

H2N

O OH

O

OH H2N OH

O

H2N

O

OHH2N

O

OH

H

H2N

O

OH

H

H2N

O

OH

H

H2N

O

OH

H

a b cd e

f g h i

j k l m

n o p q

r s t

The detailed experimental procedure with analytical data of all analogs are given in

experimental section.

APPROACH C:

Balsalazide and its analogs (1[a-t]) consisting four steps and starting from salicylic acid (10)

were prepared. The history starts with the conversion of salicylic acid (10) to its methyl ester

(32) using thionyl chloride with catalytic amount of DMF in methanol as a solvent. 4-

aminobenzoic acid (27) is treated with hydrochloric acid and sodium nitrite to generate an



Chapter - I

diazonium salt and treated with methyl salicylate (32) as obtained above to furnish an

intermediate (33, a key raw material in this approach) as shown in Scheme-1.12.

HO

HO

O

HO

O

O

Methanol

SOCl2

(10) (32)

H2N

O

OH

water, Con. HCl

Aq. NaNO2 solution

HO

O

O

NN

O

OH

(33)

(27)

.....Scheme-1.12

For the formation of peptide bond in the next stage, we treated 33 with methyl esters of different

amino acids (40[a-t]) using following strategies;

1) hydroxy group protection with acetic anhydride,

2) acid acivation with thionyl chloride,

3) acid activation with chloroformates,

4) acid activation with sulfonyl chlorides, and

5) using carbodiimides, as peptide coupling agent.

The results of these reactions are summarized schematically in Scheme-1.13.

It is clear from reaction Scheme-1.13 that, reaction is going to complete only by carbodiimide

reagents, without formation of any major impurity / by product.

Reaction of 33 with acetic anhydride under reflux gives impurity 35. Proton NMR of the

isolated product showing mixture of products 34 and 35 i.e., chemical shift observed at δ 2.35

ppm (s, 3H, COCH3), δ 3.88 ppm (s, 3H, OCH3) and δ 2.44 ppm (s, 1.6H, COCH3). This 1.6H

corresponds to impurity 35. Mass also conformed formation of di-acetylated impurity with mass

value is m/z 385, more 42 mass value than m/z 343 in positive ion mode [M+H]+. In -ve mode

it's showing m/z 341 as [M-H]- for 34.

Reaction of 33 with thionyl chloride in toluene under reflux condition with catalytic amount of

DMF confirms conversion to acid chloride 36 by TLC. This is reacted with methyl ester of



Chapter - I

amino acid salt (40o) with base to yield product 37 as a gummy mass with lots of impurities (as

monitored by TLC).

In another approach, 33 is activated with phenyl chloroformate and then reacted with methyl

ester of amino acid salts (40o). Analytical data supports the formation of product 37 with mass

value m/z 386 as [M+H]+, as well as impurity 38 with mass value was m/z 506. In

1H NMR

spectrum (DMSO-d6) the chemical shift observed are not clear due to mixture of products.

HO

O

O

NN

O

OH

O

O

O

NN

O

OH

O

O

O

O

NN

O

O

O

O

HO

O

O

NN

O

Cl

HO

O

O

NN

NH

O

O

O

O

O

O

NN

NH

O

O

O

OO

HO

O

O

NN

NH

O

O

O

O

O

O

NN

NH

O

O

O

S

O

O

HO

O

O

NN

NH

O

O

O

PRODUCT +

IMPURITIES (A

GUMMY MASS)

(34) (35)

(36)

(37)

(39)

(37)

(33)

(37)

(38)

+

+

+

ace

tic

an

hy

dri

de

thio

nyl chlo

ride

phenyl chloroformate

meth

an

e su

lfon

yl

chl o

rid

e

DCC/HOBt

.....Scheme-1.13



Chapter - I

In yet another approach, acid group of 33 is activated with methane sulfonyl chloride, in

methylene chloride using N,N-diisopropyl ethyl amine as base. After reaction with methyl ester

of amino acid salts (40o), we obtained gummy mass, which solidified on keeping. 1H-NMR and

mass disclosed formation of 37 with 39 as process impurity. This impurity has a mass value of

m/z 463.

Finally, we opted to use DCC, HOBt as peptide coupling reagent for coupling of 33 with methyl

ester of amino acid salts (40o), to got desired product 37 with good purity and yield. The 1H

NMR and mass spectral data confirms the structure of 37. In the mass spectrum of 37 the

[M+H]+ was observed at m/z 386, and [M-H]

- was observed at m/z 384.2, which suggested the

molecular weight of 37 was 385. In 1H NMR spectrum (DMSO-d6) the chemical shift observed

at δ 3.63 ppm and δ 3.94 ppm both singlets confirms two methyl groups as ester of 1.

Following this successful methodology, around 20 analogs (37[a-t]) were prepared as described

schematically in Scheme-1.14. Dimethyl ester 37 was converted to 1, by treating with aqueous

alkaline solution Scheme-1.15. The detailed experimental procedure with analytical data of all

analogs are given in experimental section.

HO

HO

O

HO

O

O

H2N

O

OH

Methanol

SOCl2

HO

O

O

NN

O

OH

Methyl ester

of

Amino acids

DCC / HOBt

HO

O

O

NN

O

AA Ester

NaOH

HO

HO

O

NN

O

AA

(1a-t)

(10) (32)

water, Con. HCl

Aq. NaNO2 solution

(27)

(33) (37 [a-t])

(40 [a-t])

.....Scheme-1.14



Chapter - I

APPROACH D:

Positional isomers of Balsalazide have been prepared by coupling o-, m-, and p-isomers of nitro

benzoic acid with 4-hydroxy benzoic acid and salicylic acid. This is summarized schematically

in Scheme-1.15.

H2N

O

OH

O2N

OH

OO2N

O2NO

OH HO

O

O2N

O

NHHO

O

water / NaOH

SOCl2 / IPE

water / NaOH

SOCl2 / IPE

water / NaOH

SOCl2 / IPE

O2N

O2N

NH

O

OHO

O

HN

O

OH

water / NaOH

Pd/C, H2

water / NaOH

Pd/C, H2

water / NaOH

Pd/C, H2

H2N

O

NHHO

O

H2NNH

O

OHO

H2N

O

HN

O

OH

HO

HO

O

HO

O

OH

water, Con. HClAq. NaNO2

solution

NaOH, Na2CO3


solutionNaOH, Na2CO3


solutionNaOH, Na2CO3HO

HO

O

HO

HO

O

HO

O

OH

HO

O

OH

OHOH

O

NN

NH

O

OH

O

OH

NN

NH

O

OH

O

N

NH

O

O

HO

N

OH

O

OH

N

NH

O

O

HO

N

OH

NN

N

O

OHO

(40o)

(12) (41) (42)

(7) (43) (44)

(8) (45)(46)

(10) (10)(10)(31) (31)

(31)

(1) (16) (47) (48)

(49)

.

....Scheme-1.15

Synthesis of 1 and Decarboxy Balsalazide, 16, were already discussed in approach A. 47 and 48

are the meta analogs of 1 and 16. Both of the isomers are prepared starting from 3-nitrobenzoic

acid, 41 and β-alanine 40o. After amide formation, catalytic hydrogenation of 43 with Pd/C in

water yielded 45, which was diazotized with sodium nitrite in aqueous hydrochloride and



Chapter - I

coupled with sodium salicylate to give the desired compound 47 in excellent yield. The ESI

mass spectrum of 47 displayed peaks at m/z 356 [(M-H)-] in negative ion mode, and in positive

ion mode this compound appeared at m/z 358 [(M+H)+], suggests the mass of the product is m/z

357. In 1HNMR spectrum of compound 47, corresponding peaks at 7.69 (dd, J = 8.8 Hz, 1H),

7.99 and 8.02 (2dd, J = 8.8 Hz and J = 2.5 Hz, 2H), and 8.33 (dd, J = 2.5 Hz, 1H), represents

meta coupling of protons as J value is 2.5 Hz. The assigned structure for compound 47 is clearly

confirmed the above spectral data.

For preparation of 48, taken 45, and was diazotized with sodium nitrite in aqueous

hydrochloride and coupled with sodium salt of 4-hydroxy benzoic acid, 31 to give the desired

compound 48 in excellent yield. The ESI mass spectrum of 48 displayed peak in positive ion

mode and compound appeared at m/z 314 [(M+H)+], suggests the mass of the product is m/z

313. In 1HNMR spectrum of compound 48, corresponding peaks at 6.96 (d, J = 8.8 Hz, 2H) and

7.82 (d, J = 8.8 Hz, 2H) represents decarboxylated compound, where as δ values at 7.64 (dd, J

= 8.8 Hz, 1H), 8.15 (dd, J = 8.8 Hz and J = 2.5 Hz, 1H), 8.18 (dd, J = 8.8 Hz and J = 2.5 Hz,

1H), 8.62 (dd, J = 2.5 Hz, 1H) represents meta coupling of protons as J value 2.5 Hz. The

assigned structure for compound 48 is clearly confirmed the above spectral data.

Interestingly, when we tried to prepare 50 and 51, ortho analogs of 1 and 16, starting with 2-

nitrobenzoic acid 42, and follow the sequence as described in Scheme-1.16, we got 49 from 46.

This is exactly what we got from Makino et al[40]

work.

H2N

O

HN

O

OH

NN

N

O

OHO

HO

HO

O

HO

O

OH





N

O

HN

O

OH

N

HO

O

HO

N

O

HN

O

OH

N

HO(46)

(10)

(31)

(49)

(50)

(51)

.....Scheme-1.16



Chapter - I

The ESI mass spectrum of 49 displayed peak in positive ion mode and appeared at m/z 220

[(M+H)+] and m/z 242.2 as [(M+Na)

+] suggests the mass of the product is m/z 219. Analytical

data are available in experimental section.

CONCLUSION:

We have developed an excellent process to prepare Balsalazide in aqueous medium without

isolating any reaction intermediate. To have a thorough understanding of impurity formation

pathways of the anti-inflammatory drug Balsalazide Disodium, it is essential to have detail

information about the various possible impurities, metabolites and their synthetic routes. In

view of regulatory importance of the impurities in active pharmaceutical ingredient, a detail

study on various impurities in Balsalazide API was conducted. Different process related

impurities, degradation product and metabolites in Balsalazide API were identified, synthesized

and characterized by using various spectroscopic techniques like LCMS, Mass, 1

HNMR,

13CNMR, Infrared. A new process was developed for the preparation of Balsalazide (1) and

analogs starting from salicylic acid. Further, we have prepared different analogs, positional

isomers and metabolites.

EXPERIMENTAL SECTION:

SYNTHESIS OF 7:

To a suspension of 4-nitrobenzoic acid, 12 (200 g, 1.20 mole) in diisopropyl ether (2000 mL) ,

added thionyl chloride (164 g, 1.38 mole) and DMF (4g) at 30-35oC. The contents were heated

slowly to reflux at 65oC and stirred at the same temperature to complete the reaction. After

completion of reaction, the reaction mass was cooled to 5-10oC , and added to β-alanine, 40o

(127.9 g, 1.44 mole) in DM water (6000 mL) containing sodium hydroxide (127.8 g) at 5-15oC

maintaining pH >8 and the reaction mass was stirred at this temperature to complete the

reaction. After completion of the reaction the pH of the reaction mass was adjusted to 1.8 using

aqueous hydrochloric acid at 8-12oC and thereafter, product was filtered and washed with

precooled water to obtain 4-nitrobenzoyl-β-alanine, 7. Yield 271 g (95% of theory).



Chapter - I

Chromatographic purity: 99.53 % (by HPLC, area normalization). IR (KBr, cm-1

): 3379 (N-H),

3072 (Ar-CH), 1725 (C=O), 1626 and 1595 (C=C ring), 1411 (aliphatic CH), 799 and 776 (Ar-

H, out of plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.54 (t, 2H), 3.48 (m, 2H), 8.06

(d, J = 8.5 Hz, 2H), 8.32 (d, J = 8.5 Hz, 2H), 8.89 (t, J = 5.5 Hz, 1H), 12.28 (brs, 1H). MS m/z

(ESI): 237 [(M-H)-], Calculated; m/z 238.

SYNTHESIS OF 8:

To 4-nitrobenzoyl-β-alanine, 7 (260 g, 1.09 mole) suspended in D M water (780ml), sodium

carbonate (58 g, 0.55 mole) was added at 20-25oC and stirred the contents at 20-30

oC to obtain

clear solution. The reaction mass was hydrogenated at 20-35oC using 10% w/w Pd/C (5.2 g,

wet) and at 15-20 Kg pressure. After completion of reaction the reaction mass was filtered

under nitrogen atmosphere. Thereafter, added concentrated sulfuric acid (53.53 g, 0.55 mole) to

the filtrate at 20-25oC and stirred the reaction mass further at 5-10

oC to complete the

precipitation. The solids were filtered, washed with water and dried to yield 4-aminobenzoyl-β-

alanine, 8. Yield 202 g (89% of theory). Chromatographic purity: 99.42 % (by HPLC, by area

normalization). IR (KBr, cm-1

): 3418, 3334, 3241 (N-H), 3061 (Ar-CH), 1716 (C=O), 1619 and

1595 (C=C ring), 1458 (aliphatic CH), 1212 (C-N), 1077 (C-O), 796 and 785 (Ar-H, out of

plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.48 (t, 2H), 3.40(m, 2H), 5.60(brs, 2H),

6.53 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H), 8.05 (t, J = 5.5 Hz, 1H), 12.18 (brs, 1H). MS

m/z (ESI): 207.3 [(M-H)-], Calculated; m/z 208.

SYNTHESIS AND PURIFICATION OF 1:

To a suspension of 4-aminobenzoyl-β-alanine, 8 (80 g, 0.385mole) in DM water (960 mL)

added concentrated hydrochloric acid (80 mL) at 20-35oC. Filtered the reaction mass and cooled

to 2 to -2oC. Added aqueous sodium nitrite solution (27.86g, 0.403mol dissolved in 120ml D M

water), prepared separately, to the filtrate maintaining temperature 2 to -2oC. The resulting

diazotized solution was added to a solution of salicylic acid, 10 (63.7 g, 0.462mole in 1280ml

D M water containing 30.77g sodium hydroxide and 81.54 g sodium carbonate) at 2 to -2 oC

maintaining pH > 8. After completion of reaction, reaction mass was heated to 60-70oC and pH



Chapter - I

adjusted to 4.5 using concentrated hydrochloric acid. After a further maintaining the reaction

mass at 60-70oC for 3 hours it was slowly cooled to 40

oC, filtered, washed with water and dried

to yield mixture of Balsalazide and its monosodium salt. Yield 121 g (88% of theory).

Chromatographic purity: 99.27 % (by HPLC, by area normalization).

Balsalazide and its monosodium salt mixture (120g) was added to 50%w/v aqueous 1,4-dioxane

(120 ml) and heated the contents to 90-95oC. After maintaining temperature 90-95

oC for 10

minutes, the suspension was slowly cooled to 60-65oC, filtered, washed with water and dried to

yield mixture of Balsalazide and its monosodium salt. Yield 100g (73% of theory).

Chromatographic purity: 99.78 % (by HPLC, by area normalization). IR (KBr, cm-1

): 3371 and

3039 (OH and NH), 1705 and 1699 (C=O), 1634 (C=O amide), 1590 and 1538 (C=C aromatic),

1464 and 1404 (aliphatic C-H), 1229 (C-N), 1073 (C-O), 773 and 738 (Ar-H out of plane bend).

1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.54 (t, 2H), 3.50 (m, 2H), 6.95 (d, J = 8.8 Hz, 1H),

7.87 (d, J = 8.5 Hz, 2H), 8.02 (d, J = 8.5 Hz, 2H), 7.95 (dd, J = 8.8 Hz and 2.5 Hz, 1H), 8.34 (d,

J = 2.5 Hz, 1H), 8.68 (t, J = 5.5 Hz, 1H), 12.12 (brs, 1H). MS m/z (ESI): 356 [(M-H)-],

Calculated; m/z 357.

SYNTHESIS OF 13:

To a suspension of 4-aminobenzoic acid, 27 (30 g, 0.22 moles) in DM water (360 mL),

concentrated hydrochloric acid (30 mL, ~36% w/w)) was added at 20-25°C and thereafter

resultant solution was cooled to 2 to -2°C. To this solution, aqueous solution of sodium nitrite

(15.86 g, 0.23 moles) in DM water (60 mL) was added at 2 to -2°C. Thus obtained diazotized

solution was added to pre-cooled alkaline solution of salicylic acid (36.26 g, 0.26 moles) in DM

water (630 mL) containing sodium hydroxide (17.52 g) and sodium carbonate (46.42 g) at 2 to -

2°C, maintaining pH ≥ 8. After completion of reaction, reaction mass was heated to 60-65°C,

acidified and stirred for 3 h maintaining same temperature. Thereafter, resulting slurry was

cooled to 40°C, filtered, washed with DM water and dried to give compound 2 as brown

crystalline solid. (62.6 g, ~100% yield). IR (KBr, cm-1

): 3425 (O-H), 3072 (Ar-CH), 1686

(C=O), 1602 and 1587 (C=C ring), 1457 (aliphatic CH), 1208 (C-N), 1077 (C-O), 799 and 776



Chapter - I

(Ar-H, out of plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 6.96 (d, J = 8.8 Hz, 1H),

7.90 (d, J = 8.5 Hz, 2H), 7.98 (dd, J = 8.8 Hz and 2.5 Hz, 1H), 8.11 (d, J = 8.5 Hz, 2H), 8.34 (d,

J = 2.5 Hz, 1H), 13.20 (brs, 1H). 13

C NMR and DEPT (DMSO-d6, 300 MHz, δ ppm): 116.6(C),

118.2(CH), 122.1 (CH), 126.5 (CH), 128.0 (CH), 130.6 (CH), 131.9 (C), 143.5 (C), 154.6 (C),

166.8 (C), 166.9 (C), 171.1(C). MS m/z (ESI): 285.2 [(M-H)-], 307.0 [(M-H)

-+Na].

SYNTHESIS OF 14:

To a suspension of N-(4-aminobenzoyl) di-β-alanine (6 g, 0.02 mole) in DM water (72 mL),

concentrated hydrochloric acid (6 mL, ~36% w/w)) was added at 20-25°c and resultant solution

was cooled to 2 to -2°C. To this solution, aqueous solution of sodium nitrite (1.4 g, 0.02 mole)

in DM water (6 mL) was added at 2 to -2°C. Thereafter, resulting diazotized solution was added

to pre-cooled solution of salicylic acid (3.2 g, 0.02 mole) in (120 mL) DM water containing

sodium hydroxide (1.55 g) and sodium carbonate (4.15 g) at 2 to -2°C, maintaining pH ≥ 8.

After completion of reaction, resultant mass was heated to 60-65°c and acidified. Resulting

suspension was cooled to 30-35°C, filtered, washed with DM water and dried to obtain desired

product 3. (7.7 g, ~82% yield). IR (KBr, cm-1

): 3375 (O-H), 3019 (Ar-CH), 1675 (C=O), 1600

and 1577 (C=C ring), 1455 (aliphatic CH), 1212(C-N), 1067 (C-O), 795 and 770 (Ar-H, out of

plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.40 (m, 4H, 2CH2), 3.26 (m, 2H, CH2),

3.47 (m, 2H, CH2), 6.77 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.85 (d, J = 8.5 Hz, 2H),

7.97 (d, J = 8.5 Hz, 2H), 8.02 (t, J = 5.5 Hz, 1H), 8.28 (d, J = 2.5 Hz, 1H), 8.62 (t, J = 5.5 Hz,

1H), 12.21(brs, 1H). 13

C NMR and DEPT (DMSO-d6, 300 MHz, δ ppm): 34.8 (CH2), 35.6

(CH2), 36.2 (CH2), 37.1 (CH2), 118.9 (CH), 119.8 (C), 122.5 (CH), 123.4 (C), 127.2 (CH),

127.8 (CH), 129.2 (CH), 135.9 (C), 134.1 (C), 154.7 (C), 166.5 (C), 170.8 (C), 171.3 (C), 173.7

(C). MS m/z (ESI): 427.2 [(M-H)-], 449.0 [(M-H)

-+Na].

SYNTHESIS OF 15:

Concentrated hydrochloric acid (10 mL, ~36% w/w)) was added to a suspension of N-(4-

aminobenzoyl) β-alanine (10 g, 0.05 mole) in DM water (145 mL) at 20-25°C. Obtained clear

solution was cooled to 0 to -2°C and a solution of sodium nitrite (3.5 g, 0.05 mole) in DM water



Chapter - I

(20 mL) was added to it. Thus obtained, diazotized solution was added to a solution of salicylic

acid (8 g, 0.06 mole) in water / isopropyl alcohol mixture (1:1, 150 mL) containing sodium

carbonate (18 g) at 2 to -2°C, maintaining pH ≥ 8. After completion of reaction, reaction mass

acidified, filtered, washed and dried. Thereafter, desired impurity Balsalazide 3-isomer 4 was

purified by flash chromatography (ethyl acetate / methanol as eluant) to get brown solid. (1.2 g,

7% yield). IR (KBr, cm-1

): 3426 and 3185 (OH and NH), 1717 and 1711 (C=O), 1631(C=O

amide), 1572 and 1545 (C=C aromatic), 1444 and 1399 (aliphatic C-H), 1229 (C-N), 1071 (C-

O), 771 and 736 (Ar-H out of plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.52 (m,

2H, CH2), 3.47 (m, 2H, CH2), 6.61 (dd, J = 7.7 Hz, 1H), 7.57 (dd, J = 8.0 Hz and 1.7 Hz, 1H),

7.86 (d, J = 8.2 Hz, 2H), 7.87 (dd, J = 8.0 Hz and 1.7 Hz, 1H), 8.00 (d, J = 8.5 Hz, 2H), 8.70 (t,

J = 5.5 Hz, 1H), 18.43 (brs, 1H). 13

C NMR and DEPT (DMSO-d6, 300 MHz, δ ppm): 35.0

(CH2), 36.7 (CH2), 115.1 (CH), 119.3 (CH), 122.8 (CH), 123.5 (C), 129.1 (CH), 135.0 (CH),

136.1 (C), 142.1 (C), 155.0 (C), 165.5 (C), 166.4 (C), 171.4 (C), 174.1 (C). MS m/z (ESI): 356.2

[(M-H)-], 378.0 [(M-H)

-+Na].

SYNTHESIS OF 16:

To a suspension of N-(4-aminobenzoyl)-β-alanine (20 g, 0.1 mole) in DM water (300 mL)

concentrated hydrochloric acid (20 mL, ~36% w/w) was added at 20-25°c, and resulting clear

solution was cooled to 2 to -2°C. To the obtained solution, aqueous solution of sodium nitrite

(6.88 g, 0.1 mole) in DM water (20 mL) was added at 2 to -2°c. Thus obtained, diazotized

solution was added slowly to pre-cooled solution of 4-hydroxybenzoic acid (13.53 g, 0.1 mole)

in DM water (200 mL), containing (8.15 g) of sodium hydroxide and (15.9 g) of sodium

carbonate at 2 to -2°C, maintaining pH ≥ 8. Thereafter, after completion of reaction, reaction

mass was acidified, filtered, washed and dried to obtain the desired Impurity 5. (27.38 g, 91%

yield). IR (KBr, cm-1

): 3336 (OH and NH); 3048 (Ar-H); 2969 (aliphatic C-H); 1707 and 1606

(C=O); 1589 and 1543 (C=C aromatic); 1464 (aliphatic C-H); 1228 (C-N) 1087 (C-O), 774 and

724 (Ar-H out of plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.54 (m, 2H, CH2), 3.50

(m, 2H, CH2), 6.97 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H), 8.04



Chapter - I

(d, J = 8.8 Hz, 2H), 8.72 (t, J = 5.5 Hz, 1H), 10.47 (brs, 1H), 12.30 (brs, 1H). 13

C NMR and

DEPT (DMSO-d6, 300 MHz, δ ppm): 33.8 (CH2), 35.7 (CH2), 116.1 (CH), 121.9 (CH), 125.2

(CH), 128.4 (CH), 135.6 (C), 145.3 (C), 153.6 (C), 161.5 (C) , 165.6 (C), 172.9 (C). MS m/z

(ESI): 314.0 [(MH)+], 336.0 [M

++Na].

SYNTHESIS OF 17:

To a suspension of N-(4-aminobenzoyl)-β-alanine (20 g, 0.1 mole) in DM water (250 mL)

concentrated hydrochloric acid (20 mL, ~36 % w/w) was added at 20-25°C. Resulting clear

solution was cooled to 2 to --2°C and (6.7 g, 0.1 mole) of sodium nitrite in DM water (30 mL)

was added to it .Thus obtained, diazotized solution was added to a pre-cooled solution of

salicylic acid (6.6 g, 0.05mole) in DM water (300 mL) containing sodium hydroxide (7.68 g)

and sodium carbonate (15.26 g) at 2 to -2°C and maintaining pH above 8. After addition,

reaction mass was acidified, filtered, washed with DM water and dried. Thereafter, desired

impurity was purified by flash chromatography. IR (KBr, cm-1

): 3440 and 3178 (OH and NH),

2968 and 2975 (aliphatic C-H), 1713 and 1631 (C=O), 1548 and 1493 (C=C aromatic), 1451

(aliphatic C-H), 1242 (C-N), 1074 (C-O), 772 and 739 (Ar-H out of plane bend). 1H NMR

(DMSO-d6, 300 MHz, δ ppm): 2.47 (m, 4H, 2CH2), 3.48 (m, 4H, 2CH2), 7.90 (2d, J = 8.8 Hz,

4H), 8.01 (2d, J = 8.8 Hz, 4H), 8.15 (d, J = 2.5 Hz, 1H), 8.51 (d, J = 2.5 Hz, 1H), 8.70 and 8.74

(2t, J = 5.2 Hz, 2H), 18.89 (brs, 1H). 13


(CH2), 35.9 (CH2), 113.7 (CH), 121.2 (C), 121.7 (CH), 122.2 (CH), 128.3 (CH), 128.8 (CH),

135.2 (C), 135.6 (C), 139.5 (C), 142.4 (C), 153.7 (C), 154.0 (C), 165.5 (C), 169.5 (C), 171.2

(C), 173.2 (C). MS m/z (ESI): 577.2 [(MH)+], 599.0 [M

++Na].

SYNTHESIS OF 18:

This related substance was prepared in laboratory as described in previous example. IR (KBr,

cm-1

): 3446 and 3180 (OH and NH), 2960 and 2971 (aliphatic C-H), 1720 and 1635 (C=O),

1552 and 1490 (C=C aromatic), 1443 (aliphatic C-H), 1248 (C-N), 1076 (C-O), 775 and 731

(Ar-H out of plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.54 (m, 4H, 2CH2), 3.49 (m,

4H, 2CH2), 6.70 (d, J = 8.8 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.60 (dd, J = 8.8 Hz and 2.5 Hz,



Chapter - I

1H), 7.70 (d, J = 8.8 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.97 (dd, J = 8.8 Hz and 1.4 Hz, 1H),

8.05 (d, J = 1.4 Hz, 1H), 8.17 (d, J = 2.5 Hz, 1H), 8.64 (t, J = 5.2 Hz, 1H), 8.77 (t, J = 5.2 Hz,

1H), 18.34 (brs, 1H). 13


(CH2), 116.9 (CH), 119.2 (CH), 119.9 (C), 126.2 (CH), 127.5 (CH), 128.6 (CH), 129.4 (CH),

130.4 (CH), 131.3 (CH), 134.0 (C), 135.6 (C), 138.7 (C), 141.9 (C), 143.5 (C), 151.8 (C), 166.4

(C), 166.8 (C), 171.1 (C), 173.8 (C). MS m/z (ESI): 549.2 [(MH)+], 571.0 [M

++Na].

SYNTHESIS OF 19:

To a suspension of N-(4-aminobenzoyl)-β-alanine (5 g, 0.024 mole) in DM water (75 mL),

added concentrated hydrochloric acid (5 mL, ~36% w/w) at 20-25°C and the resulting solution

was cooled to 2 to -2°C. A solution of sodium nitrite (1.74 g, 0.025 mole) in DM water (10 mL)

was added at 2 to -2°C. The resulting diazotized solution was added to a pre-cooled solution of

4-hydroxybenzoic acid (1.65 g, 0.012 mole) in DM water (100 mL) containing sodium

carbonate (9.1 g) at 2 to -2°C and maintaining pH above 8. After completion of reaction,

reaction mass acidified, filtered, washed with DM water and dried. Thereafter, obtained product

was suspended in aqueous acetic acid (400 mL) and refluxed for 20 min. Cooled the suspension

to 25-30°C, filtered and dried to obtain impurity 8. (4.28 g, 32% yield). IR (KBr, cm-1

): 3298

(NH), 3057 (Ar-H), 2921 (aliphatic C-H), 1630 and 1604 (C=O), 1577 and 1538 (C=C

aromatic), 1430 (C-H), 1214 (C-N), 1085 (C-O), 799 and 772 (Ar-H out of plane bend). 1H

NMR (DMSO-d6, 300 MHz, δ ppm): 2.57 (m, 4H, 2CH2), 3.52 (m, 4H, 2CH2), 7.32 (d, J = 8.8

Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.95-8.15 (m, 8H), 8.24 (s, 1H), 8.72 and 8.75 (2t, J = 5.2 Hz,

2H), 11.54 (brs, 1H), 12.27 (brs, 2H). 13


(CH2), 35.7 (CH2), 114.1 (CH), 119.3 (CH), 122.2 (CH), 122.8 (CH), 128.0 (CH), 128.4 (CH),

128.5 (CH), 136.1 (C), 136.5 (C), 139.0 (C), 145.1 (C), 153.2 (C), 153.3 (C), 159.0 (C), 165.5

(C), 165.5 (C), 172.9 (C). MS m/z (ESI): 533.3 [(MH)+], 555.1 [M

++Na].

SYNTHESIS OF 20:

Suspended (10 g, 0.05 mole) N-(4-aminobenzoyl)-β-alanine in DM water (120 mL) at 20-25°C.

Added concentrated hydrochloric acid (10 mL, ~36% w/w) to the obtained suspension and



Chapter - I

cooled to 2 to -2°C. A solution of sodium nitrite (3.5 g, 0.05 mole) in DM water (20 mL) was

added at 2 to -2°C. Resulting diazotized solution was added to a solution of 4-hydroxybenzoic

acid (1.65 g, 0.01mole) in DM water (100 mL) containing sodium carbonate (18 g) at 2 to -2°C

maintaining pH above 8. After completion of reaction, temperature of reaction mass was raised

to 35-40°C and acidified,. Thereafter, precipitated product was filtered, washed and dried.

Obtained product was suspended in ethyl methyl ketone (100 mL) having 10% v/v water and

refluxed for 20min. Thereafter, suspension was cooled to 20-25°C, filtered and dried to obtain

product 9. (3.15 g, 36% yield). IR (KBr, cm-1

): 3261 (OH and NH), 1714 and 1639 (C=O), 1548

and 1494 (C=C aromatic), 1228 (aliphatic C-N), 1070 (C-O), 789 and 760 (Ar-H out of plane

bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.41 (m, 4H, 2CH2), 2.47 (m, 2H, CH2), 3.43

(m, 4H, 2CH2), 3.49 (m, 2H, CH2), 6.98 (d, J = 9.3 Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.51 (2d, J

= 8.8 Hz and 2.5 Hz, 4H), 7.87 (d, J = 8.0 Hz, 6H), 7.93 (d, J = 9.3 Hz, 1H), 8.55, 8.64 and 8.72

(3t, J = 5.2 Hz, 3H). 13


(CH2), 38.4 (CH2), 121.6 (CH), 123.8 (CH), 124.2 (CH), 124.3 (CH), 127.4 (CH), 130.4 (CH),

130.6 (CH), 134.6 (CH), 135.1 (C), 136.5 (C), 137.7 (C), 138.4 (C), 139.8 (C), 144.3 (C), 146.5

(C), 154.0 (C), 155.7 (C), 158.2 (C), 167.6 (C), 167.8 (C), 168.6 (C), 176.5 (C), 176.8 (C). MS

m/z (ESI): 724.1 [(MH)+], 746.2 [M

++Na].

SYNTHESIS OF N-acetyl-8:

Added N-(4-aminobenzoyl)-β-alanine (30 g, 0.15 mole) to acetic acid / water mixture (2:1, 90

mL), at 25-30°C. Acetic anhydride (30 g, 0.29 mole) was added to the obtained suspension

maintaining temperature 30-45°C. Stirred the reaction mass at 35-40°C for 1h.and diluted with

acetone (90 mL), Thus, precipitated product was filtered, washed with DM water followed by

acetone and dried to obtain 10. (32.46 g, 90% yield). IR (KBr, cm-1

): 3326 and 3196 (OH and

NH), 3064 (Ar-H), 2907 (aliphatic C-H), 1706, 1693 and 1626 (C=O), 1590 and 1537 (C=C

aromatic), 1429 (aliphatic C-H), 1262 (C-N), 1083 (C-O), 813 and 763 (Ar-H out of plane

bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.06 (s, 3H, CH3), 2.48 (t, 2H, CH2), 3.44 (m,



Chapter - I

2H, CH2), 7.62 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H), 8.38 (t, J = 5.2 Hz, 1H), 10.13 (s,

1H).

SYNTHESIS OF N-acetyl-2:

Added 5-aminosalicylic acid (50 g, 0.33 mole) to acetic acid / acetic anhydride mixture (1:1,

100 mL) and stirred the contents at reflux for 30min, cooled to 20-25°C. Filtered the

precipitated product, washed with DM water. Suspended the obtained product in 10% w/w

aqueous sodium hydroxide solution and stirred for 1h at 25-30°C. Adjusted the pH of solution

to 2.to precipitate the product. Filtered the product 11, washed with DM water and dried. (59.7

g, 94% yield). IR (KBr, cm-1

): 3357 and 3106 (OH and NH), 2874 (aliphatic C-H), 1685 and

1603 (C=O), 1539 and 1516 (C=C aromatic), 1420 (aliphatic C-H), 1238 (C-N), 807 and 792

(Ar-H out of plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.14 (s, 3H, CH3), 7.07 (dd, J

= 8.78 Hz and 1.92 Hz, 1H), 7.32 (d, J = 1.92 Hz, 1H), 7.74 (d, J = 8.78 Hz, 1H), 9.60 (s, 1H),

11.32 (brs, 1H).

SYNTHESIS OF 21:

To a suspension of 4-nitrobenzoyl-β-alanine, 7 (25 g, 0.11 mole) in diisopropyl ether (250 mL) ,

added thionyl chloride (15 g, 0.13 mole) and DMF (1g) at 30-35oC. The contents were heated


completion of reaction, the reaction mass was cooled to 5-10oC , and added to β-alanine, 40o

(11.21 g, 0.12 mole) in DM water (500 mL) containing sodium hydroxide (12 g) at 5-15oC


reaction. After completion of the reaction the pH of the reaction mass was adjusted to 1.8 using

aqueous hydrochloric acid at 8-12oC and thereafter, product was filtered and washed with

precooled water to obtain N-(2-(3-hydroxypropylcarbamoyl)ethyl)-4-nitrobenzamide (21).

Yield 6.8 g, (20% of theory). Chromatographic purity: 99.53 % (by HPLC, area normalization).

1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.38 (t, 2H, CH2), 2.62 (t, 2H, CH2), 3.26 (m, 2H,

CH2), 3.47 (m, 2H, CH2), 8.08 (d, J = 8.5 Hz, 2H), 8.33 (d, J = 8.5 Hz, 2H), 8.92 (m, J = 5.5 Hz,



Chapter - I

2H), 12.28 (brs, 1H). MS m/z (ESI): 308 [(M-H)-], m/z 310.1 [(M+H)

+], m/z 332.1

[(M+H)++Na].

SYNTHESIS OF 25:

To 4-nitrobenzoyl-β-alanine, 7 (260 g, 1.09 mole) suspended in ethanol (3400 mL) at 20-25oC

and stirred the contents at 20-30oC to obtain clear solution. The reaction mass was hydrogenated

at 20-35oC using 10% w/w Pd/C (18.2 g, wet) and at 15-20 Kg pressure. After completion of

reaction the reaction mass was filtered under nitrogen atmosphere and concentrated to remove

solvent. Thereafter, diluted with water (3120 mL), concentrated hydrochloric acid (260 mL) was

added at 20-25oC and stirred the reaction mass further at 5-10

oC to dissolve 8. The solids were

filtered, washed with water and dried to yield a mixture of 25 and 26. This was separated

through column chromatography. Yield 2.5 g, 25. 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.57

(t, 4H, CH2), 3.54 (m, 4H, CH2), 8.01 (d, J = 8.5 Hz, 4H), 8.06 (d, J = 8.5 Hz, 4H), 8.77 (t, J =

5.5 Hz, 2H), 12.30 (brs, 2H). MS m/z (ESI): 411.4 [(M-H)-], m/z 413 [(M+H)

+], m/z 435.1

[(M+Na)+], m/z 457.2 [(M+2Na)

+].

SYNTHESIS OF 26:

Same process as described in 25. Yield 4.3 g, 26. 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.56

(t, 4H, CH2), 3.40 (m, 4H, CH2), 6.70 (d, J = 8.5 Hz, 4H), 7.65 (d, J = 8.5 Hz, 4H), 8.17 (t, J =

5.5 Hz, 2H), 8.23 (s, 2H), 12.21 (brs, 2H). MS m/z (ESI): 413.3 [(M-H)-], m/z 415.2 [(M+H)

+],

m/z 437.3 [(M+Na)+].

SYNTHESIS OF 27:

To 4-nitrobenzoic acid, 12 (100 g, 0.6 mole) suspended in D M water (500 mL), sodium

hydroxide (24 g, 0.6 mole) was added at 20-25oC and stirred the contents at 20-30

oC to obtain

clear solution. The reaction mass was hydrogenated at 20-35oC using 10% w/w Pd/C (2 g, wet)

and at 15-20 Kg pressure. After completion of reaction the reaction mass was filtered under

nitrogen atmosphere. Thereafter, added concentrated sulfuric acid (29.35 g, 0.3 mole) to the

filtrate at 20-25oC and stirred the reaction mass further at 5-10

oC to complete the precipitation.



Chapter - I

The solids were filtered, washed with water and dried to yield 4-aminobenzoic acid, 27. Yield

70 g (85% of theory). Chromatographic purity: 99.68 % (by HPLC, by area normalization). 1H

NMR (DMSO-d6, 300 MHz, δ ppm): 5.87 (s, 2H, NH2), 6.55 (d, J = 8.5 Hz, 2H), 7.62 (d, J =

8.5 Hz, 2H), 11.96 (brs, 1H). MS m/z (ESI): 136 [(M-H)-], m/z 138 [(M+H)

+].

SYNTHESIS OF 1a-t: (General procedure for In-situ reaction)

To a suspension of 4-nitrobenzoic acid, 12 (200 g, 1.20 mole) in diisopropyl ether (2000 mL) ,

added thionyl chloride (164 g,1.38 mole) and DMF (4g) at 30-35oC. The contents were heated


completion of reaction, the reaction mass was cooled to 5-10oC , and added to amino acids,

40(a-t), (1.44 mole) in DM water (6000 mL) containing sodium hydroxide (127.8 g) at 5-15oC


reaction. After completion of the reaction the lower aqueous layer was separated containing 7(a-

t). Thereafter, the reaction mass was hydrogenated at 20-45oC using 10% Pd/C (2% w/w, based

on 12) and at 15-20 Kg/cm2

pressure. After completion of reaction the catalyst was filtered off

under nitrogen atmosphere (containing 8(a-t)), added DM water (1000 mL) and concentrated

hydrochloric (200mL, 36% w/w) to the filtrate at 20-35oC. Filtered the reaction mass and cooled

to 2 to -2oC. Added aqueous sodium nitrite solution (1.25 mole in 400mL DM water) to the

obtained filtrate maintaining temperature 2 to -2 oC. Resulting diazotized solution was added to

a solution of salicylic acid, 10 (1.44 mole in 2400 mL D M water containing sodium hydroxide

and sodium carbonate) at 2 to -2oC maintaining pH > 8. After completion of reaction, reaction

mass was heated to 60-70oC and pH adjusted to 4.2 using concentrated hydrochloric acid.

Further maintaining temperature 60-70oC for 3 hours the reaction mass was slowly cooled to

30oC, filtered, washed with water and dried to yield 1(a-t).

This crude 1(a-t), (65 g) was added to 50%w/v aqueous 1,4-dioxane (195mL) and heated the

contents to 90-95oC. After maintaining temperature 90-95

oC for 10 minutes, the suspension was

slowly cooled to 60-65oC, filtered, washed with water and dried to yield pure 1(a-t).

Chromatographic purity: >99.00 %. (by HPLC, by area normalization).



Chapter - I

The analytical data of all analogs are given in Table-1.13.

SYNTHESIS OF 32:

To salicylic acid, 10 (50 g, 0.36 mole) suspended in methanol (400 mL), DMF (1 mL, cat.) was

added at 20-25oC and then thionyl chloride (51.74 g, 0.44 mole) at 22-30

oC. The reaction mass

was heated to reflux at 60-70oC. After completion of reaction (checked by TLC), the reaction

mass was concentrated to remove solvents. Thereafter, diluted with methylene chloride (100

mL) and water (50 mL), adjusted pH to 9.8 with aqueous ammonia solution. Methylene chloride

layer was separated, washed with water (50 mL) and concentrated under reduced pressure to get

32 as an oil. Yield 50 g (91% of theory). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 3.89 (s, 3H,

CH3), 6.96 (m, J = 8.5 Hz and J = 2.5 Hz, 2H), 7.53 (m, J = 8.5 Hz and J = 2.5 Hz, 1H), 7.79

(m, J = 8.5 Hz and J = 2.5 Hz, 1H), 10.51 (s, 1H). MS m/z (ESI): 151 [(M-H)-].

SYNTHESIS OF 33:

To a suspension of 4-aminobenzoic acid, 27 (30 g, 0.22mole) in DM water (360 mL) added

concentrated hydrochloric acid (30 mL) at 20-35oC. Filtered the reaction mass and cooled to 2

to -2oC. Added aqueous sodium nitrite solution (15.86 g, 0.23mole dissolved in 60 mL D M

water), prepared separately, to the filtrate maintaining temperature 2 to -2oC. The resulting

diazotized solution was added to a solution of methyl salicylate, 32 (40 g, 0.26 mole in 630 mL

D M water containing 17.52g sodium hydroxide and 46.42 g sodium carbonate) at 2 to -2 oC

maintaining pH > 8. After completion of reaction, reaction mass was heated to 15-20oC and pH

adjusted to 6.5 to 7 using concentrated hydrochloric acid. After that, raised the temperature to

50-55oC and pH adjusted to 2. A further maintaining the reaction mass at 50-55

oC for 3 hours

and was slowly cooled to 40oC, filtered, washed with water and dried to yield 33. Yield 60.23 g

(92% of theory). Chromatographic purity: 99.21 % (by HPLC, by area normalization). 1H NMR

(DMSO-d6, 300 MHz, δ ppm): 3.94 (s, 3H, CH3), 7.22 (d, J = 8.8 Hz, 1H), 7.93 (d, J = 8.5 Hz,

2H), 8.10 (dd, J = 8.8 Hz and J = 2.5 Hz, 1H), 8.14 (d, J = 8.5 Hz, 2H), 8.34 (d, J = 2.5 Hz, 1H),

11.01 (s, 1H), 13.21 (brs, 1H). MS m/z (ESI): 299.2 [(M-H)-], m/z 300.8 [(M+H)

+].



Chapter - I

SYNTHESIS OF 37a-t: (General procedure)

Placed DCC (3.61 g, 0.017 mole), to methylene chloride (50 mL) at 20-25oC. 33 (5 g, 0.016

mole) and methyl ester hydrochloride salt of β-alanine, 40(a-t) (0.018 mole) were added to

above reaction mass, followed by cat. HOBt (0.45 g, 0.003 mole) at 20-25oC. Thereafter,

DIPEA (2.58 g, 0.02 mole) was added slowly at 20-25oC, and maintained stirring for 18hr. After

completion of reaction, cooled to 0-2oC and maintained for 1hr. Filtered out DCU and washed

with chilled methylene chloride. Obtained filtrate was washed consequently with 10% aqueous

hydrochloric acid, 10% aqueous ammonia and then with water. Methylene chloride layer was

concentrated and concentrated mass was taken in to water (20 mL). It was stirred for 2hr to get a

solid. Filtered, washed with water and dried to get the desired compound, 37(a-t) in good

quality. Yield 5.8 g (91% of theory).

The analytical data of all analogs are given in Table-1.14.

SYNTHESIS OF 1a-t: (general procedure)

To a suspension of 37(a-t) (0.013 mole) in DM water (50 mL) added 50% aqueous sodium

hydroxide solution (2.2 mL, 0.027 mole) at 20-35oC. Stirred the reaction mass at 25-35

oC to

complete the hydrolysis. After that, raised the temperature to 50-55oC and pH adjusted to 2. A

further maintaining the reaction mass at 50-55oC for 3 hours and was slowly cooled to 40

oC,

filtered, washed with water and dried to yield 1(a-t). Yield 4.28 g (92% of theory). Analytical

data given in Table-1.13.

SYNTHESIS OF 43:

Followed same operating parameters as described in synthesis of 7. 3-nitrobenzoic acid, 41 was

used as a starting material. Yield 26.3 g (92% of theory). 1H NMR (DMSO-d6, 300 MHz, δ

ppm): 2.56 (m, 2H, CH2), 3.50 (m, 2H, CH2), 7.78 (t, J = 8.5 Hz, 1H), 8.29 (m, J = 8.8 Hz and J

= 2.5 Hz, 1H), 8.38 (m, J = 8.8 Hz and J = 2.5 Hz, 1H), 8.68 (t, J = 2.5 Hz, 1H), 8.95 (t, J = 5.5

Hz, 1H), 12.25 (brs, 1H). MS m/z (ESI): 237.2 [(M-H)-], m/z 239.2 [(M+H)

+].

SYNTHESIS OF 44:



Chapter - I

Followed same operating parameters as described in synthesis of 7. 2-nitrobenzoic acid, 42 was

used as a starting material. Yield 25.1 g (88% of theory). 1H NMR (DMSO-d6, 300 MHz, δ

ppm): 2.52 (t, 2H, CH2), 3.43 (m, 2H, CH2), 7.57 (dd, J = 8.5 Hz and J = 2.5 Hz, 1H), 7.69 (ddd,

J = 8.5 Hz, J = 8.5 Hz and J = 2.5 Hz, 1H), 7.77 (ddd, J = 8.5 Hz, J = 8.5 Hz and J = 2.5 Hz,

1H), 8.03 (dd, J = 8.5 Hz and J = 2.5 Hz, 1H), 8.74 (t, J = 5.5 Hz, 1H), 12.27 (brs, 1H). MS m/z

(ESI): 237.2 [(M-H)-], m/z 239.2 [(M+H)

+].

SYNTHESIS OF 45:

Followed same operating parameters as described in synthesis of 8. 3-nitrobenzoyl-β-alanine, 43

was used as a starting material. Yield 20 g (92% of theory). 1H NMR (DMSO-d6, 300 MHz, δ

ppm): 2.46 (t, 2H, CH2), 3.40 (m, 2H, CH2), 5.60 (brs, 2H, NH2), 6.83 (dd, J = 2.5 Hz, 1H), 7.08

(dd, J = 8.8 Hz and J = 2.5 Hz, 1H), 7.15 (dd, J = 8.8 Hz and J = 2.5 Hz, 1H), 7.20 (dd, J = 8.8

Hz and J = 2.5 Hz, 1H), 8.33 (t, J = 5.5 Hz, 1H), 11.91 (brs, 1H). MS m/z (ESI): 207.2 [(M-H)-

], m/z 209.2 [(M+H)+].

SYNTHESIS OF 46:

Followed same operating parameters as described in synthesis of 8. 2-nitrobenzoyl-β-alanine, 44

was used as a starting material. Yield 18.7 g (86% of theory). 1H NMR (DMSO-d6, 300 MHz, δ

ppm): 2.55 (t, 2H, CH2), 3.48 (m, 2H, CH2), 5.20 (brs, 2H, NH2), 6.71 (dd, J = 8.8 Hz and J =

2.5 Hz, 1H), 6.84 (dd, J = 8.8 Hz and J = 2.5 Hz, 1H), 7.23 (ddd, J = 8.8 Hz and J = 2.5 Hz,

1H), 7.67 (dd, J = 8.8 Hz and J = 2.5 Hz, 1H), 8.54 (t, J = 5.5 Hz, 1H), 9.09 (s, 1H). MS m/z

(ESI): 207.2 [(M-H)-], m/z 209.2 [(M+H)

+].

SYNTHESIS OF 47:

Followed same operating parameters as described in synthesis of 1. 3-aminobenzoyl-β-alanine,

45 was used as a starting material. Yield 60 g (100% of theory). IR (KBr, cm-1

): 3279 (OH and

NH); 3062 (Ar-H); 2632 (aliphatic C-H); 1696 and 1654 (C=O); 1632 and 1539 (C=C

aromatic); 1491 and 1452 (aliphatic C-H); 1212 (C-N) 1075 (C-O), 897 and 712 (Ar-H out of

plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.56 (t, 2H, CH2), 3.50 (m, 2H, CH2), 7.18



Chapter - I

(d, J = 8.8 Hz, 1H), 7.69 (dd, J = 8.8 Hz, 1H), 7.99 and 8.02 (2dd, J = 8.8 Hz and J = 2.5 Hz,

2H), 8.12 (dd, J = 8.8 Hz and J = 2.5 Hz, 1H), 8.33 (dd, J = 2.5 Hz, 1H), 8.38 (d, J = 2.5 Hz,

1H), 8.76 (t, J = 5.5 Hz, 1H). MS m/z (ESI): 356 [(M-H)-], m/z 358 [(M+H)

+].

SYNTHESIS OF 48:


45 was used as a starting material. Yield 26.6 g (88% of theory). 1H NMR (DMSO-d6, 300

MHz, δ ppm): 2.58 (m, 2H, CH2), 3.47 (m, 2H, CH2), 6.96 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 8.8

Hz, 2H), 7.64 (dd, J = 8.8 Hz, 1H), 8.15 (dd, J = 8.8 Hz and J = 2.5 Hz, 1H), 8.18 (dd, J = 8.8

Hz and J = 2.5 Hz, 1H), 8.62 (dd, J = 2.5 Hz, 1H), 8.75 (t, J = 5.5 Hz, 1H), 12.30 (brs, 1H). MS

m/z (ESI): 314.0 [(MH)+], 336.0 [(M+Na)

+].

SYNTHESIS OF 49:


46 was used as a starting material. Yield 30 g . IR (KBr, cm-1

): 2930 (Ar-H), 2804 (aliphatic C-

H), 1719 and 1694 (C=O), 1601 and 1577 (C=C aromatic), 1441 (C-H) 783 and 692 (Ar-H out

of plane bend). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 2.87 (t, 2H, CH2), 4.59 (t, 2H, CH2),

7.94 (m, J = 8.5 Hz and J = 2.5 Hz , 1H), 8.10 (m, J = 8.5 Hz and J = 2.5 Hz , 1H), 8.21 (m, J =

8.5 Hz and J = 2.5 Hz , 1H), 8.26 (m, J = 8.5 Hz and J = 2.5 Hz , 1H), 12.46 (brs, 1H). MS m/z

(ESI): 218 [(M-H)-], m/z 220 [(M+H)

+], 242.2 [(M+Na)

+].



Chapter - I

Table-1.13

S. No. COMPOUND 7 1

1 a 3.97 (d, J = 6 Hz, 2H), 8.12 (d, J = 9 Hz, 2H), 8.35 (d,

J = 9 Hz, 2H), 9.23 (t, J = 6 Hz, 1H), 12.70 (brs, 1H).

MS m/z (ESI): 223 [(M-H)-], Calculated; m/z 224.

3.98 (d, J = 3 Hz, 2H), 7.11 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 9 Hz,

2H), 8.08 (d, J = 9 Hz, 2H), 8.12 (dd, J = 3 Hz and J = 9 Hz, 1H),

8.38 (d, J = 3 Hz, 1H), 9.05 (t, J = 6 Hz, 1H), 12.18 (brs, 1H).

MS m/z (ESI): 342.1 [(M-H)-], Calculated; m/z 343.

2 b 1.42 (d, J = 9 Hz, 3H), 4.44 (m, J = 9 Hz and J = 6 Hz,

1H), 8.12 (d, J = 9 Hz, 2H), 8.32 (d, J = 9 Hz, 2H),

9.05 (d, J = 6 Hz, 1H), 12.69 (brs, 1H).

MS m/z (ESI): 237 [(M-H)-], 239.1 [(M+H)

+],


1.43 (d, J = 6 Hz, 3H), 4.46 (m, J = 9 Hz and J = 6 Hz, 1H), 7.19 (d, J

= 9 Hz, 1H), 7.96 (d, J = 9 Hz, 2H), 8.11 (d, J = 9 Hz, 2H), 8.14 (d, J

= 3 Hz and J = 9 Hz, 1H), 8.39 (d, J = 3 Hz, 1H), 8.88 (d, J = 9 Hz,

1H), 12.28 (brs, 2H).

MS m/z (ESI): 356.1 [(M-H)-], 358.2 [(M+H)

+], Calculated; m/z 357.

3 c 1.00 (dd, J = 6 Hz, 6H), 2.20 (m, J = 6 Hz, 1H), 4.32

(t, J = 6 Hz and J = 9 Hz , 1H), 8.11 (d, J = 9 Hz, 2H),

8.33 (d, J = 9 Hz, 2H), 8.84 (d, J = 6 Hz, 1H), 12.76

(brs, 1H).

MS m/z (ESI): 265 [(M-H)-], 267 [(M+H)

+],


1.00 (dd, J = 6 Hz and J = 9 Hz, 6H), 2.22 (m, J = 6 Hz and J = 9 Hz,

1H), 4.32 (t, J = 6 Hz and J = 9 Hz , 1H), 7.18 (dd, J = 3 Hz and J = 9

Hz, 1H), 7.95 (d, J = 9 Hz, 2H), 8.09 (d, J = 9 Hz, 2H), 8.14 (dd, J =

3 Hz and J = 9 Hz, 1H), 8.39 (d, J = 3 Hz, 1H), 8.64 (d, J = 6 Hz,

1H), 12.70 (brs, 1H).

MS m/z (ESI): 384 [(M-H)-], 386 [(M+H)


4 d 1.00 (dd, J = 6 Hz, 6H), 2.20 (m, J = 6 Hz, 1H), 4.32

(t, J = 6 Hz and J = 9 Hz , 1H), 8.11 (d, J = 9 Hz, 2H),

8.33 (d, J = 9 Hz, 2H), 8.84 (d, J = 6 Hz, 1H), 12.76

(brs, 1H).

MS m/z (ESI): 265 [(M-H)-], 267 [(M+H)

+],


0.99 (dd, J = 6.6 Hz, 6H), 2.23 (m, J = 6.6 Hz, 1H), 4.32 (t, J = 7.2

Hz and J = 7.8 Hz , 1H), 7.16 (d, J = 9 Hz, 1H), 7.94 (d, J = 8.4 Hz,

2H), 8.10 (d, J = 8.4 Hz, 2H), 8.13 (dd, J = 2.4 Hz and J = 8.4 Hz,

1H), 8.38 (d, J = 2.4 Hz, 1H), 8.64 (d, J = 8.1 Hz, 1H), 12.68 (brs,

1H).

MS m/z (ESI): 384 [(M-H)-], 386 [(M+H)


5 e 1.06 (s, 9H), 4.39 (d, J = 9 Hz, 1H), 8.08 (d, J = 9 Hz , 1.07 (s, 9H), 4.40 (d, J = 9 Hz, 1H), 7.17 (d, J = 8.7 Hz , 1H), 7.81 (d,



Chapter - I

2H), 8.32 (d, J = 9 Hz, 2H), 8.66 (d, J = 9 Hz, 1H),

12.83 (brs, 1H).

MS m/z (ESI): 279.1 [(M-H)-], 281.2 [(M+H)

+],


J = 8.4 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H), 8.13 (dd, J = 2.4 Hz and J =

6.6 Hz, 1H), 8.38 (d, J = 2.4 Hz, 1H), 12.72 (brs, 1H).

MS m/z (ESI): 398.1 [(M-H)-], 400.2 [(M+H)


6 f 3.05 (dd, J = 10.8 Hz and J = 3 Hz, 1H), 3.23 (dd, J =

4.2 Hz and J = 9.6 Hz, 1H), 4.64 (m, J = 1.5 Hz, J =

2.4 Hz and J = 4.5 Hz , 1H), 7.18 (m, J = 6.9 Hz and J

= 6.6 Hz , 1H), 7.29 (m, J = 6.9 Hz, J = 2.4 Hz and J =

7.8 Hz, 4H), 8.01 (d, J = 8.7 Hz, 2H), 8.30 (d, J = 8.7

Hz, 2H), 9.10 (d, J = 8.1 Hz, 1H), 12.85 (brs, 1H).

MS m/z (ESI): 313 [(M-H)-], 315 [(M+H)

+],


3.11 (dd, J = 10.8 Hz and J = 3 Hz, 1H), 3.23 (dd, J = 4.2 Hz and J =

9.6 Hz, 1H), 4.67 (m, J = 4.5 Hz, J = 6.3 Hz and J = 6.9 Hz , 1H),

7.17 (d, J = 9 Hz, 1H), 7.20 (m, J = 7.2 Hz, 1H), 7.30 (m, J = 7.5 Hz

and J = 7.8 Hz, 4H), 7.92 (d, J = 7.8 Hz, 2H), 8.00 (d, J = 8.4 Hz,

2H), 8.11 (dd, J = 8.7 Hz and J = 0.9 Hz , 1H), 8.37 (t, J = 0.9 Hz and

J = 1.5 Hz, 1H), 8.93 (d, J = 7.8 Hz, 1H), 12.26 (brs, 1H).


7 g 6.03 (dd, J = 7.5 Hz, 1H), 7.40 (m, J = 4.5 Hz and J =

2.7 Hz, 2H), 7.55 (m, J = 4.5 Hz, J = 2.4 Hz and J =

2.7 Hz , 2H), 8.14 (d, J = 8.7 Hz, 2H), 8.33 (d, J = 9

Hz, 2H), 9.54 (d, J = 7.5 Hz, 1H), 13.18 (brs, 1H).

MS m/z (ESI): 333 [(M-H)-], Calculated; m/z 334.5.

6.07 (d, J = 6 Hz, 1H), 7.17 (d, J = 9 Hz, 1H), 7.41 (m, J = 6 Hz and J

= 3 Hz, 2H), 7.58 (m, J = 9 Hz and J = 3 Hz , 2H), 7.95 (d, J = 9 Hz,

2H), 8.12 (d, J = 9 Hz, 2H), 8.14 (d, J = 9 Hz and J = 3 Hz, 1H), 8.39

(d, J = 3 Hz, 1H), 9.36 (d, J = 6 Hz, 1H), 13.18 (brs, 2H).

MS m/z (ESI): 452.4 [(M-H)-], Calculated; m/z 453.5.

8 h 3.08 (m, J = 9.9 Hz and J = 3.9 Hz, 2H), 4.53 (m, J =

4.5 Hz and J = 4.2 Hz, 1H), 8.12 (d, J = 8.7 Hz, 2H),

8.35 (d, J = 8.7 Hz, 2H), 9.04 (d, J = 7.8 Hz, 1H).

MS m/z (ESI): 269.1 [(M-H)-], 271.2 [(M+H)

+],


3.12 (m, J = 9 Hz and J = 3.9 Hz, 2H), 4.54 (m, J = 5 Hz and J = 3

Hz, 1H), 7.19 (d, J = 9 Hz, 1H), 7.98 (d, J = 9 Hz, 2H), 8.14 (d, J = 9

Hz, 2H), 8.16 (dd, J = 9 Hz and J = 2.5 Hz, 1H), 8.40 (d, J = 2.5 Hz,

1H), 9.06 (d, J = 9 Hz, 1H).


9 i 3.82 (d, J = 6 Hz, 2H), 4.51 (m, J = 6 Hz, J = 9 Hz and

J = 3 Hz, 1H), 8.14 (d, J = 9 Hz, 2H), 8.34 (d, J = 9

Hz, 2H), 8.87 (d, J = 9 Hz, 1H), 12.84 (brs, 1H).

MS m/z (ESI): 253 [(M-H)-], 255.1 [(M+H)

+],

3.84 (d, J = 6 Hz, 2H), 4.52 (m, J = 6 Hz, J = 9 Hz and J = 3 Hz, 1H),

7.19 (d, J = 9 Hz, 1H), 7.97 (d, J = 9 Hz, 2H), 8.12 (d, J = 9 Hz, 2H),

8.15 (dd, J = 9 Hz and J = 2.5 Hz, 1H), 8.39 (d, J = 2.5 Hz, 1H), 8.73

(d, J = 9 Hz, 1H), 12.22 (brs, 1H).



Chapter - I

Calculated; m/z 254. MS m/z (ESI): 372 [(M-H)-], Calculated; m/z 373.

10 j 2.46 (dd, J = 3.9 Hz, 1H), 2.77 (dd, J = 6 Hz, and J =

9.9 Hz, 1H), 4.54 (m, J = 3.9 Hz, J = 6 Hz and J = 7.5

Hz, 1H), 8.09 (d, J = 8.7 Hz, 2H), 8.33 (d, J = 9 Hz,

2H), 8.82 (d, J = 7.5 Hz, 1H).

MS m/z (ESI): 281.1 [(M-H)-], 283.1 [(M+H)

+],


2.74 (dd, J = 8.4 Hz and J = 8.1 Hz, 1H), 2.89 (dd, J = 5.7 Hz, and J =

10.8 Hz, 1H), 4.79 (m, J = 8.1 Hz and J = 5.7 Hz, 1H), 7.18 (d, J =

8.7 Hz, 1H), 7.96 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.13

(dd, J = 2.4 Hz and J = 6.3 Hz , 1H), 8.39 (d, J = 2.4 Hz, 1H), 8.96 (d,

J = 7.8 Hz, 1H), 11.13 (brs, 1H), 12.76 (brs, 2H).


11 k 2.63 (m, J = 3 Hz, J = 6 Hz, J = 9 Hz and J = 12 Hz,

2H), 4.74 (m, J = 6 Hz and J = 9 Hz, 1H), 6.97 and

7.42 (d, J = 135 Hz, 2H), 8.08 (d, J = 9 Hz, 2H), 8.34

(d, J = 9 Hz, 2H), 9.05 (d, J = 6 Hz, 1H), 12.85 (brs,

1H).

MS m/z (ESI): 280.1 [(M-H)-], 282.2 [(M+H)

+],


2.69 (m, J = 6 Hz, J = 9 Hz and J = 12 Hz, 2H), 4.77 (m, J = 6 Hz and

J = 9 Hz, 1H), 6.99 and 7.49 (d, J = 150 Hz, 2H), 7.16 (d, J = 9 Hz,

1H), 7.95 (d, J = 9 Hz, 2H), 8.07 (d, J = 9 Hz, 2H), 8.14 (dd, J = 9 Hz

and J = 2.5 Hz, 1H), 8.38 (t, J = 2.5 Hz, 1H), 8.90 (d, J = 9 Hz, 1H),

12.50 (brs, 1H).


12 l 1.85 (m, J = 6 Hz, 1H), 1.89 (m, J = 9 Hz, 1H), 1.94

(m, J = 3 Hz, 1H), 2.29 (m, J = 6 Hz and J = 9 Hz ,

1H), 3.46 (m, J = 3 Hz and J = 6 Hz , 1H), 3.61 (m, J =

9 Hz , 1H), 4.44 (m, J = 3 Hz and J = 6 Hz , 1H), 7.77

(d, J = 9 Hz, 2H), 8.30 (d, J = 9 Hz, 2H), 12.70 (brs,

1H).

MS m/z (ESI): 263.1 [(M-H)-], 265.1 [(M+H)

+],


1.91 (m, J = 6 Hz and J = 9 Hz, 3H), 2.30 (m, J = 6 Hz and J = 9 Hz,

1H), 3.58 (m, J = 3 Hz, J = 6 Hz and J = 9 Hz, 2H), 4.44 (m, J = 6 Hz

and J = 3 Hz , 1H), 7.17 (d, J = 9 Hz, 1H), 7.73 (d, J = 9 Hz , 2H),

7.93 (d, J = 9 Hz, 2H), 8.11 (dd, J = 3 Hz, 1H), 8.37 (d, J = 1.5 Hz,

1H), 12.60 (brs, 1H).


13 m 1.99 (m, J = 3 Hz and J = 6 Hz, 1H), 2.26 (m, J = 9 Hz

and J = 12 Hz, 1H), 3.25 (m, J = 12 Hz, 1H), 3.70 (m,

J = 3 Hz, J = 6 Hz and J = 24 Hz , 1H), 4.31 (m, 1H),

4.54 (m, J = 9 Hz , 1H), 7.80 (d, J = 9 Hz, 2H), 8.32

(d, J = 9 Hz, 2H).

1.99 (m, J = 3 Hz and J = 6 Hz, 1H), 2.25 (m, J = 9 Hz and J = 12 Hz,

1H), 3.34 (m, J = 9 Hz, 1H), 3.79 (m, J = 3 Hz and J = 6 Hz , 1H),

4.31 (m, J = 6 Hz, 1H), 4.54 (m, J = 9 Hz , 1H), 7.18 (d, J = 9 Hz,

1H), 7.74 (d, J = 9 Hz, 2H), 7.96 (d, J = 9 Hz, 2H), 8.12 (dd, J = 3 Hz

and J = 9 Hz, 1H), 8.38 (dd, J = 3 Hz, 1H), 12.10 (brs,1H).



Chapter - I

MS m/z (ESI): 279.1 [(M-H)-], 281.2 [(M+H)

+],



14 n 1.97 (m, J = 3 Hz and J = 6 Hz, 1H), 2.12 (m, J = 6 Hz

and J = 9 Hz, 1H), 2.39 (m, J = 6 Hz and J = 9 Hz,

2H), 4.44 (m, J = 3 Hz, 1H), 8.11 (d, J = 9 Hz , 2H),

8.33 (d, J = 9 Hz , 2H), 9.00 (d, J = 6 Hz, 1H), 12.59

(brs, 1H).

MS m/z (ESI): 295.1 [(M-H)-], 297.2 [(M+H)

+],


2.00 (m, J = 5.4 Hz and J = 9.3 Hz, 1H), 2.12 (m, J = 6 Hz and J =

7.8 Hz, 1H), 2.40 (t, J = 7.2 Hz, 2H), 4.44 (m, J = 3 Hz and J = 9.6

Hz, 1H), 7.17 (d, J = 9 Hz, 1H), 7.95 (d, J = 9 Hz, 2H), 8.10 (d, J = 9

Hz, 2H), 8.13 (dd, J = 9 Hz and J = 2.4 Hz, 1H), 8.38 (d, J = 2.4 Hz ,

1H), 8.83 (d, J = 7.5 Hz, 1H), 12.20 (brs, 1H).


15 o 2.54 (t, 2H), 3.48 (m, 2H), 8.06 (d, J = 8.5 Hz, 2H),

8.32 (d, J = 8.5 Hz, 2H), 8.89 (t, J = 5.5 Hz, 1H),

12.28 (brs, 1H).


2.54 (t, 2H), 3.50 (m, 2H), 6.95 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 8.5

Hz, 2H), 8.02 (d, J = 8.5 Hz, 2H), 7.95 (dd, J = 8.8 Hz and 2.5 Hz,

1H), 8.34 (d, J = 2.5 Hz, 1H), 8.68 (t, J = 5.5 Hz, 1H), 12.12 (brs,

1H).


16 p 1.37-1.50 (m, J = 9 Hz and J = 12 Hz, 10H), 2.31 (s,

2H), 3.44 (d, J = 6 Hz, 2H), 8.07 (d, J = 9 Hz, 2H),

8.32 (d, J = 9 Hz , 2H), 8.58 (t, J = 6 Hz , 1H), 12.11

(brs, 1H).

MS m/z (ESI): 319.1 [(M-H)-], 321.2 [(M+H)

+],


1.37-1.50 (m, J = 9 Hz and J = 7.2 Hz, 10H), 2.33 (s, 2H), 3.44 (d, J =

5.7 Hz, 2H), 7.08 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 8.03

(d, J = 8.7 Hz, 2H), 8.06 (d, J = 8.4 Hz, 1H), 8.37 (d, J = 2.4 Hz ,

1H), 8.43 (t, J = 5.7 Hz , 1H), 11.94 (brs, 1H).


17 q 0.87 (dd, J = 2.7 Hz and J = 6.6 Hz, 6H), 1.15 (m, J =

6.6 Hz and J = 6.9 Hz, 2H), 1.70 (m, J = 6 Hz, 1H),

2.16 (m, J = 12.9 Hz, 1H), 2.11 (m, J = 6.6 Hz, 1H),

2.32 (m, J = 7.8 Hz , 1H), 3.33 (m, J = 7.8 Hz, 1H),

3.38 (m, J = 7.8 Hz, 1H), 8.07 (d, J = 8.7 Hz, 2H), 8.33

(d, J = 8.7 Hz, 2H), 8.81 (t, J = 5.7 Hz, 1H), 12.12 (brs,

1H).

0.89 (dd, J = 2.7 Hz and J = 6.6 Hz, 6H), 1.16 (m, J = 6 Hz and J = 9

Hz, 2H), 1.71 (m, J = 6 Hz, 1H), 2.10 (m, J = 6 Hz, 1H), 2.15 (m, J =

9 Hz and J = 3 Hz, 1H), 2.33 (m, J = 9 Hz and J = 3 Hz , 1H), 3.17

(m, J = 6 Hz, 1H), 3.34 (m, J = 6 Hz, 1H), 7.19 (d, J = 8.7 Hz, 1H),

7.93 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 8.12 (dd, J = 9 Hz

and J = 3 Hz , 1H), 8.38 (d, J = 3 Hz, 1H), 8.66 (t, J = 6 Hz and J = 3

Hz, 1H), 12.03 (brs, 1H).



Chapter - I

MS m/z (ESI): 307.2 [(M-H)-], 308.9 [(M+H)

+],



18 r 0.87 (dd, J = 2.7 Hz and J = 6.6 Hz, 6H), 1.15 (m, J =

6.6 Hz and J = 6.9 Hz, 2H), 1.70 (m, J = 6 Hz, 1H),

2.16 (m, J = 12.9 Hz, 1H), 2.11 (m, J = 6.6 Hz, 1H),

2.32 (m, J = 7.8 Hz , 1H), 3.33 (m, J = 7.8 Hz, 1H),

3.38 (m, J = 7.8 Hz, 1H), 8.07 (d, J = 8.7 Hz, 2H), 8.33

(d, J = 8.7 Hz, 2H), 8.81 (t, J = 5.7 Hz, 1H), 12.12

(brs, 1H).

MS m/z (ESI): 307.2 [(M-H)-], 308.9 [(M+H)

+],


0.89 (dd, J = 2.7 Hz and J = 6.6 Hz, 6H), 1.16 (m, J = 6 Hz and J = 9

Hz, 2H), 1.71 (m, J = 6 Hz, 1H), 2.10 (m, J = 6 Hz, 1H), 2.15 (m, J =

9 Hz and J = 3 Hz, 1H), 2.33 (m, J = 9 Hz and J = 3 Hz , 1H), 3.17

(m, J = 6 Hz, 1H), 3.34 (m, J = 6 Hz, 1H), 7.19 (d, J = 8.7 Hz, 1H),

7.93 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 8.12 (dd, J = 9 Hz

and J = 3 Hz , 1H), 8.38 (d, J = 3 Hz, 1H), 8.66 (t, J = 6 Hz and J = 3

Hz, 1H), 12.03 (brs, 1H).


19 s 0.87 (m, J = 2.7 Hz, J = 3.9 Hz, J = 5.1 Hz, J = 6.6 Hz

and J = 7.5 Hz, 6H), 1.18 (m, 1H), 1.40 (m, 1H), 1.48

(m, J = 2.7 Hz and J = 3.9 Hz, 1H), 2.21 (m, 1H), 2.23

(m, J = 3.3 Hz, J = 4.5 Hz, J = 5.1 Hz and J = 8.1 Hz ,

2H), 3.13 (m, 1H), 3.38 (m, 1H), 8.07 (d, J = 8.7 Hz,

2H), 8.33 (d, J = 8.7 Hz, 2H), 8.83 (t, J = 5.1 Hz, 1H),

12.13 (brs, 1H).

MS m/z (ESI): 307.2 [(M-H)-], 308.9 [(M+H)

+],


0.87 (m, J = 2.7 Hz, J = 3.9 Hz, J = 5.1 Hz, J = 6.6 Hz and J = 7.5

Hz, 6H), 1.20 (m, 1H), 1.35 (m, 1H), 1.48 (m, J = 2.7 Hz and J = 3.9

Hz, 1H), 2.15 (m, 1H), 2.22 (m, J = 3.3 Hz, J = 4.5 Hz, J = 5.1 Hz

and J = 8.1 Hz , 2H), 3.18 (m, 1H), 3.36 (m, 1H), 7.18 (d, J = 8.7 Hz,

1H), 7.94 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 8.13 (dd, J =

8.7 Hz and J = 2.4 Hz , 1H), 8.37 (d, J = 2.4 Hz, 1H),8.61 (d, J = 5.1

Hz, 1H), 12.10 (brs, 1H).


20 t 0.88 (t, J = 9 Hz, 3H), 1.28 (m, J = 12 Hz, 6H), 2.09

(m, 1H), 2.17 (m, J = 9 Hz and J = 15 Hz, 1H), 2.31

(m, J = 3 Hz and J = 12 Hz, 1H), 3.20 (m, J = 6 Hz,

1H), 3.35 (m, J = 6 Hz, 1H), 8.07 (d, J = 8 Hz, 2H),

8.33 (d, J = 8 Hz, 2H), 8.80 (t, J = 6 Hz, 1H), 12.24

(brs, 1H).

MS m/z (ESI): 307.2 [(M-H)-], 308.9 [(M+H)

+],


0.88 (t, J = 6.6 Hz, 3H), 1.30 (m, J = 12 Hz, 6H), 2.09 (m, 1H), 2.18

(m, J = 7.2 Hz and J = 15 Hz, 1H), 2.34 (m, J = 5.4 Hz and J = 9.6

Hz, 1H), 3.19 (m, J = 5.1 Hz, 1H), 3.36 (m, J = 6 Hz, 1H), 7.16 (d, J

= 9 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 8.12

(dd, J = 5.7 Hz and J = 2.4 Hz, 1H), 8.37 (d, J = 2.4 Hz, 1H), 8.63 (t,

J = 5.5 Hz, 1H), 12.21 (brs, 1H).




Chapter - I

Table-1.14

S. No. COMPOUND 37

1 a 3.68 (s, 3H), 3.94 (s, 3H), 4.05 (d, J = 6 Hz, 2H), 7.21 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 9 Hz, 2H), 8.08 (d, J = 9 Hz, 2H), 8.12 (dd,

J = 3 Hz and J = 9 Hz, 1H), 8.35 (d, J = 3 Hz, 1H), 9.15 (t, J = 6 Hz, 1H), 11.06 (brs, 1H).


2 b 1.44 (d, J = 6 Hz, 3H), 3.67 (s, 3H), 3.94 (s, 3H), 4.53 (m, J = 9 Hz and J = 6 Hz, 1H), 7.21 (d, J = 9 Hz, 1H), 7.96 (d, J = 9 Hz,

2H), 8.11 (d, J = 9 Hz, 2H), 8.14 (d, J = 3 Hz and J = 9 Hz, 1H), 8.36 (d, J = 3 Hz, 1H), 8.98 (d, J = 9 Hz, 1H), 11.05 (brs, 2H).

MS m/z (ESI): 384.1 [(M-H)-], 386 [(M+H)


3 c 1.00 (dd, J = 6 Hz and J = 9 Hz, 6H), 2.22 (m, J = 6 Hz and J = 9 Hz, 1H), 3.68 (s,3H), 3.94 (s, 3H), 4.33 (t, J = 6 Hz and J = 9 Hz

, 1H), 7.21 (dd, J = 3 Hz and J = 9 Hz, 1H), 7.95 (d, J = 9 Hz, 2H), 8.10 (d, J = 9 Hz, 2H), 8.14 (dd, J = 3 Hz and J = 9 Hz, 1H),

8.35 (d, J = 3 Hz, 1H), 8.80 (d, J = 6 Hz, 1H), 11.06 (brs, 1H).


4 d 0.99 (dd, J = 6.6 Hz, 6H), 2.23 (m, J = 6.6 Hz, 1H), 3.68 (s,3H), 3.94 (s, 3H), 4.33 (t, J = 7.2 Hz and J = 7.8 Hz , 1H), 7.16 (d, J =

9 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H), 8.13 (dd, J = 2.4 Hz and J = 8.4 Hz, 1H), 8.35 (d, J = 2.4 Hz, 1H),

8.80 (d, J = 8.1 Hz, 1H), 11.06 (brs, 1H).


5 e 1.05 (s, 9H), 3.68 (s, 3H), 3.94 (s, 3H), 4.46 (d, J = 6 Hz, 1H), 7.22 (d, J = 8.7 Hz , 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4

Hz, 2H), 8.13 (dd, J = 2.4 Hz and J = 6.6 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H), 8.57 (d, J = 5.4 Hz, 1H), 11.06 (brs, 1H).


6 f 3.17 (m, J = 10.8 Hz and J = 3 Hz, 2H), 3.66 (s, 3H), 3.94 (s, 3H), 4.70 (m, J = 6.3 Hz and J = 9.1 Hz , 1H), 7.20 (d, J = 9 Hz,

1H), 7.29 (m, J = 7.5 Hz and J = 7.8 Hz, 5H), 7.92 (d, J = 8.5 Hz, 2H), 8.00 (d, J = 8.4 Hz, 2H), 8.11 (dd, J = 8.7 Hz and J = 0.9

Hz , 1H), 8.33 (t, J = 0.9 Hz and J = 1.5 Hz, 1H), 9.06 (d, J = 7.8 Hz, 1H), 10.94 (brs, 1H).



Chapter - I


7 g 3.73 (s, 3H), 3.94 (s, 3H), 6.11 (d, J = 6 Hz, 1H), 7.19 (d, J = 9 Hz, 1H), 7.42 (m, J = 6 Hz and J = 3 Hz, 2H), 7.53 (m, J = 9 Hz

and J = 3 Hz , 2H), 7.95 (d, J = 9 Hz, 2H), 8.12 (d, J = 9 Hz, 2H), 8.14 (d, J = 9 Hz and J = 3 Hz, 1H), 8.34 (d, J = 3 Hz, 1H), 9.49

(d, J = 6 Hz, 1H), 10.56 (brs, 1H).

MS m/z (ESI): 480.4 [(M-H)-], Calculated; m/z 481.5.

8 j 2.86 (dd, J = 8.4 Hz and J = 8.1 Hz, 1H), 2.98 (dd, J = 5.7 Hz, and J = 10.8 Hz, 1H), 3.64 (s, 3H), 3.67 (s, 3H), 3.94 (s, 3H), 4.88

(m, J = 8.1 Hz and J = 5.7 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.14 (dd, J = 2.4

Hz and J = 6.3 Hz , 1H), 8.34 (d, J = 2.4 Hz, 1H), 9.12 (d, J = 7.8 Hz, 1H), 11.04 (brs, 1H).


9 k 2.69 (m, J = 6 Hz, J = 9 Hz and J = 12 Hz, 2H), 3.67 (s,3H), 3.96 (s,3H), 4.89 (m, J = 6 Hz and J = 9 Hz, 1H), 6.99 and 7.49 (d, J

= 150 Hz, 2H), 7.18 (d, J = 9 Hz, 1H), 7.95 (d, J = 9 Hz, 2H), 8.07 (d, J = 9 Hz, 2H), 8.14 (dd, J = 9 Hz and J = 2.5 Hz, 1H), 8.38

(t, J = 2.5 Hz, 1H), 8.85 (d, J = 9 Hz, 1H), 12.50 (brs, 1H).


10 l 1.92 (m, J = 6 Hz and J = 9 Hz, 3H), 2.30 (m, J = 6 Hz and J = 9 Hz, 1H), 3.58 (m, J = 3 Hz, J = 6 Hz and J = 9 Hz, 2H), 3.70 (s,

3H), 3.94 (s, 3H), 4.52 (m, J = 6 Hz and J = 3 Hz , 1H), 7.20 (d, J = 9 Hz, 1H), 7.73 (d, J = 9 Hz , 2H), 7.93 (d, J = 9 Hz, 2H), 8.10

(dd, J = 3 Hz, 1H), 8.33 (d, J = 1.5 Hz, 1H), 11.03 (brs, 1H).


11 m 2.03 (m, J = 3 Hz and J = 6 Hz, 1H), 2.23 (m, J = 9 Hz and J = 12 Hz, 1H), 3.69 (s,3H), 3.80 (m, J = 3 Hz and J = 6 Hz , 1H), 3.94

(s, 3H), 4.32 (m, J = 6 Hz, 1H), 4.61 (m, J = 9 Hz , 1H), 5.16 (d, J = 3 Hz , 1H), 7.21 (d, J = 9 Hz, 1H), 7.74 (d, J = 9 Hz, 2H),

7.96 (d, J = 9 Hz, 2H), 8.12 (dd, J = 3 Hz and J = 9 Hz, 1H), 8.34 (dd, J = 3 Hz, 1H), 11.05 (brs,1H).


12 n 2.05 (m, J = 5.4 Hz and J = 9.3 Hz, 1H), 2.14 (m, J = 6 Hz and J = 7.8 Hz, 1H), 2.47 (t, J = 7.2 Hz, 2H), 3.60 (s, 3H), 3.67 (s, 3H),

3.94 (s, 3H), 4.50 (m, J = 3 Hz and J = 9.6 Hz, 1H), 7.20 (d, J = 9 Hz, 1H), 7.97 (d, J = 9 Hz, 2H), 8.09 (d, J = 9 Hz, 2H), 8.14

(dd, J = 9 Hz and J = 2.4 Hz, 1H), 8.36 (d, J = 2.4 Hz , 1H), 8.95 (d, J = 6 Hz, 1H), 11.06 (brs, 1H).



Chapter - I


13 o 2.63 (t, J = 6 Hz, 2H), 3.52 (m, J = 6 Hz, 2H), 3.62 (s, 3H), 3.94 (s, 3H), 7.20 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 8.03

(d, J = 8.5 Hz, 2H), 8.12 (dd, J = 8.8 Hz and 2.5 Hz, 1H), 8.33 (d, J = 2.5 Hz, 1H), 8.74 (t, J = 5.5 Hz, 1H), 11.01 (brs, 1H).


14 p 1.37-1.50 (m, 10H), 2.40 (s, 2H), 3.42 (d, J = 5.7 Hz, 2H), 3.58 (s, 3H), 3.94 (s, 3H), 7.20 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 8.4 Hz,

2H), 8.03 (d, J = 8.7 Hz, 2H), 8.12 (d, J = 8.4 Hz, 1H), 8.33 (d, J = 2.4 Hz , 1H), 8.37 (t, J = 5.7 Hz , 1H), 11.01 (brs, 1H).


15 q 0.87 (dd, J = 2.7 Hz and J = 6.6 Hz, 6H), 1.16 (m, J = 6 Hz and J = 9 Hz, 2H), 1.68 (m, J = 6 Hz, 1H), 2.21 (m, J = 6 Hz, 2H),

2.42 (m, J = 9 Hz and J = 3 Hz, 1H), 3.17 (m, J = 6 Hz, 1H), 3.34 (m, J = 6 Hz, 1H), 3.54 (s, 3H), 3.94 (s, 3H), 7.21 (d, J = 8.7 Hz,

1H), 7.93 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 8.12 (dd, J = 9 Hz and J = 3 Hz , 1H), 8.34 (d, J = 3 Hz, 1H), 8.66 (t, J = 6

Hz and J = 3 Hz, 1H), 11.05 (brs, 1H).


16 r 0.88 (dd, J = 2.7 Hz and J = 6.6 Hz, 6H), 1.16 (m, J = 6 Hz and J = 9 Hz, 2H), 1.69 (m, J = 6 Hz, 1H), 2.24 (m, J = 6 Hz, 2H),

2.40 (m, J = 9 Hz and J = 3 Hz, 1H), 3.18 (m, J = 6 Hz, 1H), 3.34 (m, J = 6 Hz, 1H), 3.56 (s, 3H), 3.94 (s, 3H), 7.20 (d, J = 8.7 Hz,

1H), 7.94 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 8.11 (dd, J = 9 Hz and J = 3 Hz , 1H), 8.32 (d, J = 3 Hz, 1H), 8.66 (t, J = 6

Hz and J = 3 Hz, 1H), 11.06 (brs, 1H).


17 s 0.87 (m, J = 2.7 Hz, J = 3.9 Hz, J = 5.1 Hz, J = 6.6 Hz and J = 7.5 Hz, 6H), 1.17 (m, 1H), 1.35 (m, 1H), 1.48 (m, J = 2.7 Hz and J

= 3.9 Hz, 1H), 2.26 (m, 1H), 2.32 (m, J = 3.3 Hz, J = 4.5 Hz, J = 5.1 Hz and J = 8.1 Hz , 2H), 3.18 (m, 1H), 3.36 (m, 1H), 3.52 (s,

3H), 3.94 (s, 3H), 7.21 (d, J = 8.7 Hz, 1H), 7.93 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.7 Hz, 2H), 8.13 (dd, J = 8.7 Hz and J = 2.4 Hz ,

1H), 8.34 (d, J = 2.4 Hz, 1H),8.62 (d, J = 5.1 Hz, 1H), 11.06 (brs, 1H).


18 t 0.88 (t, J = 6.6 Hz, 3H), 1.30 (m, J = 12 Hz, 6H), 2.14 (m, 1H), 2.25 (m, J = 7.2 Hz and J = 15 Hz, 1H), 2.43 (m, J = 5.4 Hz and J

= 9.6 Hz, 1H), 3.19 (m, J = 5.1 Hz, 1H), 3.34 (m, J = 6 Hz, 1H), 3.57 (s, 3H), 3.95 (s, 3H), 7.20 (d, J = 9 Hz, 1H), 7.90 (d, J = 8.4

Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.12 (dd, J = 5.7 Hz and J = 2.4 Hz, 1H), 8.32 (d, J = 2.4 Hz, 1H), 8.65 (t, J = 5.5 Hz, 1H),



Chapter - I

11.07 (brs, 1H).




Chapter - I

SPECTRA:

…..IR SPECTRUM OF COMPOUND 7

…..1H NMR SPECTRUM OF COMPOUND 7 IN DMSO-d6

…..MASS SPECTRUM OF COMPOUND 7



Chapter - I






Chapter - I

…..IR, 1H NMR,

13C NMR AND MASS SPECTRUM OF COMPOUND 1



Chapter - I



…..13

C NMR SPECTRUM OF COMPOUND 13 IN DMSO-d6



Chapter - I


…..13





Chapter - I

…..IR, 1H NMR,




Chapter - I

…..IR, 1H NMR AND

13C NMR SPECTRUM OF COMPOUND 17



Chapter - I

….. 1H NMR,




Chapter - I

…..IR, 1H NMR,




Chapter - I

…..IR, 1H NMR AND

13C NMR SPECTRUM OF COMPOUND 20



Chapter - I

…..IR, 1H NMR AND

MASS SPECTRUM OF COMPOUND N-Ac-8



Chapter - I

…..IR, 1H NMR AND

MASS SPECTRUM OF COMPOUND N-Ac-2



Chapter - I

….. 1H NMR SPECTRUM OF COMPOUND 25

….. MASS SPECTRUM OF COMPOUND 25



Chapter - I





Chapter - I


….. 1H NMR AND MASS SPECTRUM OF COMPOUND 33



Chapter - I





Chapter - I

….. 1H NMR AND MASS SPECTRUM OF COMPOUND 43 AND 44



Chapter - I

…..IR, 1H NMR AND MASS SPECTRUM OF COMPOUND 47



Chapter - I

…..IR, 1H NMR AND MASS SPECTRUM OF COMPOUND 49



Chapter - I

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Chapter - I

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