Chapter 2

32

2.1. Introduction

Piperazine and piperidine derivatives are the most privileged structural motifs in

the field of nitrogen heterocyclic chemistry. They occur in several natural and synthetic

bioactive compounds. Rather they are widely used in medicine. Tens of thousands of

compounds of this series have been synthesized and studied by now; more than 300 of

them are used in medical practice as drugs. They include drugs with central and

peripheral neurotropic effects (local anesthetics, M-cholinoblockers, agonists and

antagonists of other pharmacological receptors, analgesics etc.,) agents that act on the

cardiovascular system (coronary dilative, antiarrhythmic, antihypertensive), spasmolytics,

diuretics, broncholytics, antiemetics, antinuclear drugs and many others.

Piperazine Piperidine

Both piperazine and piperidine are the two finest and classical representations of

six membered saturated nitrogen heterocycles. The name piperidine and piperazine

comes from the genus name Piper, which is the Latin word for pepper. Piperidine is a

nitrogen heterocycle with only one nitrogen atom on the six membered saturated ring,

where as piperazine is having two nitrogen atoms on the opposing side of the six

membered ring. Although they are structurally very similar, the nitrogen present on both

piperazine and piperidine makes them chemically and biologically different. The variation

of substituents on nitrogen atoms of these moieties plays an important role in selectivity

and potency against biological targets.

Chapter 2

33

The foregoing literature review gains rich knowledge on the role of piperazine and

piperidine derivatives in the field of bioorganic and medicinal chemistry.

2.1.1. Current status of piperazine and its derivatives in

the field of bioorganic and medicinal chemistry

2.1.1a. Piperazine as pharmaceutical agents:

The piperazine motif appears in many drugs encompassing a broad range of

activities (e.g. Oxatomide, Almitrine, Hydroxyzine, Buclizine, Lomerizine1). This motif

(monoaryl and diarylpiperazine) also found in drug candidates displaying anti-allergenic,2

antibacterial,3 anti-anxiety,

4 anti-emetic,

5 antimigraine

6 and platelet anti-aggregatory

activities.7 In addition, piperazine motif is present in many cardiovascular drugs

8 (e.g.,

Manidipine, Doxazosin, Trimetazidine, Flunarizine, Prazosin) and drug candidates.9-10

Piperazine and their derivatives also possess antimalarial activity,11

antioxidative activity12

and antifungal activity13

and found in many drug molecules such as Meclizine (motion

sickness drug), Cyclizine (antiemetic and antihistamine), Clozapine (antipsychotic drug),

Imatinib (leukemia drug), Befuraline (stimulant and antidepressant), Antrafenine

(analgesic), Trazodone (sedating antidepressant) and Niaprazine (sedating antihistamine)

etc.

Nitrogen in piperazine ring plays an important role in exerting biological effects.

The basicity of piperazine nitrogen plays an important role in selectivity and potency

towards the biological targets. In recent structural activity relationship study of

antimuscarinic compounds, it was shown that there was a favorable electrostatic

interaction between the protonated piperazine and the anionic region of the receptor. By

varying the substituents on the terminal piperazine nitrogen, the potency and selectivity

of the molecule toward its muscarinic receptor was greatly improved.14

Chapter 2

34

Figure 2.1: Some representative examples of piperazine derivatives being used as

pharmaceutical drugs.

Chapter 2

35

Among several piperazine derivatives known in the field of medicinal chemistry,

monoaryl and diarylpiperazine derivatives leading the piperazine family as promising

bioactive molecules. Of note, benzhydril and benzyl piperazine derivatives are emerging

as the most successful bioactive piperazine derivatives.

Benzhydrilpiperazine Benzylpiperazine

Both benzhydrilpiperazine and benzylpiperazine derivatives possess a wide range

of pharmacological properties. Chemists had wide variety of options to synthesize

biologically active derivatives of benzylpiperazine, by keeping the benzyl/substituted

benzyl group at one end of the piperazine and substituting the various substituents

including the substituents which have biological importance, at the other end of the

piperazine ring. The research which is going on the synthesis of benzyl/benzhydril

piperazine and its derivatives as which exhibit wide range of biological activities are

summarized below:

2.1.1b. Benzhydrilpiperazine:

An honored scaffold among arylpiperazine derivatives

Benzhydril group on the piperazine ring plays a paramount role in elucidating the

biological activities of these derivatives upon conjugation with other analogues.

Benzhydrilpiperazine belongs to the monoaryl piperazine family and their derivatives

possess a wide range of pharmacological properties. Hence, chemists have broad range of

pathways which paves way to synthesize biologically active derivatives of

benzhydrilpiperazine by coupling various substituents on one of the nitrogen atoms of the

piperazine moiety and without replacing the benzhydril group at the other end of the

Chapter 2

36

piperazine and they have succeeded in obtaining the compounds with marked

enhancement in the biological activity compared to the conventional drugs. Also, it is

believed that the pharmacological activities of benzhydrilpiperazine derivatives are

attributed due to the presence of two nitrogen atoms on the piperazine ring.

Moreover, monoaryl and diarylpiperazine derivatives play an important role.

Among these, benzhydrilpiperazine is a core of many bioactive compounds which exhibit

a variety of pharmacological effects which have been reported in the literature and for

instance,

Kawasaki et al.,15

have synthesized a novel series of benzhydrilpiperazine

trimethylhydroquinone derivatives 29 and found to have antioxidative and anti-allergic

effects. Among the synthesized compound, 4-[4-(4-diphenylmethyl-1-piperazinyl)-

butoxy]-2, 3, 6- trimethyl phenol exhibited good anti-oxidative and antiallergic activities.

29

where; n = 2 to10

Shanklin et al.,16

have synthesized Flunarizine 30 bearing benzhydrilpiperazine and

reported as calcium antagonist and antioxidants. Substituents on the benzene rings of the

benzhydril group had a large effect on the activity. Compounds with fluoro substituents in

the 3- and/or 4-positions of both benzene rings of benzhydril were found to be more

potent, but flunarizine has also been known to have a clinical risk of extrapyramidal side

effects caused through the binding to D2 receptor.

Chapter 2

37

30

Miyake et al.,17

have reported Tamolarizine 31, a new type of organic Ca2+

channel

blocker for its reversing effect on multidrug-resistant tumor cells. Tamolarizine

synergistically potentiated the cytotoxicity of doxorubicin for doxorubicin-resistant K562

cells at a concentration of 0.1 to10 µM. These results indicate that tamolarigine reverses

the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

31

Ito et al.18

have synthesized Lifarizine 32 bearing benzhydrilpiperazine and

imidazole skeleton which has been reported as a calcium antagonist.

Emanuel et al.,19

have synthesized Oxatomide 33 as H1-antihistamines. The

benzimidazolone skeleton is responsible for its antihistaminic activity and also the length

of alkyl chain between piperazine and benzimidazolone influenced the activity.

Chapter 2

38

Sugiyama et al.,20

have synthesized a series of o-aminophenol derivatives

possessing H1-antihistaminic activity and their effects were investigated on lipid

peroxidation in rat brain homogenates. The compound 34 with secondary amine showed

highest activity.

N N

F

F HN

HO

CH3

34

Abou-Ghabria et al.,21

have synthesized a series of polycyclic piperazinyl imides

and among the synthesized, 2-[4-[4-[bis(4-flurophenyl)methyl]-1-piperazinyl]butyl]-

4,4a,5,5a,6,6a-hexahydro-4,6-ethenocycloprop[f]isoindole-1,3(2H,3aH)-dione 35 which

demonstrated good H1-antagonist activity. They also demonstrated that substitution of a

xanthinyl moiety for the polycyclic imide group led to the identification of novel

xanthinyl-substituted piperazinyl derivatives with potent antihistamine H1 activity.

Chapter 2

39

35

Chern et al.,22

have synthesized a series of pyrazolo[3,4-d]pyrimidines 36 and

tested for their antiviral activity. SAR studies revealed that phenyl group at the N-1

position and the hydrophilic diphenylmethyl at the piperazine largely influenced the in

vitro antienteroviral activity of this new class of potent antiviral agents.

36

Hajos et al.,23

have reported substituted benzhydril-2-hydroxypropyl piperazine

derivatives 37 as cardiotonic agents.

37

where, R is hydrogen or acetyl, Ar1 and Ar2 are phenyl or substituted phenyl,

and X is selected from the group of heterocyclic compounds.

Kaneko et al.,24

have synthesized novel benzhydrilpiperazine derivatives 38 and

studied the effect of inhibiting over contraction and overextension of the myocardium

without being accompanied by a myocardium-inhibiting effect. By using the novel

Chapter 2

40

benzhydrilpiperazine derivatives as an effective ingredient, it is possible to obtain a

myocardial necrosis inhibitor which can protect against myocardial necrosis.

38

where R represents or

Baltes et al.,25

have patented the synthesis of 2-[4-(diphenylmethyl)-1-

piperazinyl]-acetic acids 39, their amides and their salts, processes for the preparation

thereof and therapeutic compositions. These compounds are found to be antiallergic,

spasmolytic and antihistaminic agents.

39

where: Y= -OH or –NH2; X and X1= H, halogen, alkoxy or

trifluoromethyl; m=1 or 2 and n=1 or 2.

Labrid et al.,26

have synthesized almitrine 40 which is a triazinylpiperazine

derivative and currently using for respiratory insufficiency.

Chapter 2

41

40

Recently, Song et al.,27

have synthesized several series of urea (A), carbamate (B),

amide (C), sulphanamide (D) and oxalamide (E) derivatives based on benzhydrilpiperazine

scaffold and tested for CB1 receptor binding affinity. The SAR studies to optimize the CB1

binding affinity led to the potent urea derivatives. After the additional SAR studies to

optimize the substituents of diphenyl rings, the combination of 2-chlorophenyl and 4-

chlorophenyl d turned out to be the most potent scaffold.

Chapter 2

42

N

HN

N

N

NHO

R4

R1

R2

R3

R1

R2

R3

N

N

O

R1

R2

R3

R5

N

N

O O-R6

N

N

S R7O

O

N

N

O

O

R8

i

ii

iii

iv

v

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

a.R1=R2=R3=Clb.R1=R3=Cl, R2=Hc.R1=R2=Cl, R2=Hd.R1=Cl, R2=R3=He.R1=Me R2=R3=Hf.R1=CF3 R2=R3=H

g.R1=R2=H,R3=Clh.R1=R3=Cl,R3=H

A

B

C

D

E

where: (i) R4-NCO, TEA, DCM, rt; (ii) R5-COOH, DCC, HOBt, DMF, rt; (iii) R6-OCOCl, TEA,

DCM, 0 0C- rt; (iv) R7-SO2Cl, TEA, DCM, rt; (v) R8-NH2, (COCl)2, TEA, DCM, 0

0C- rt.

Chapter 2

43

Yung et al.,28

have synthesized a series of hydrazones of 1-phenyl-, 1-benzyl-, and

1-benzhydril-4-aminopiperazines and evaluated for antimicrobial activity. Among the

synthesized molecules, 1-benzhydril-4-isonicotinylideneaminopiperazine 41 showed

broad spectrum of antimicrobial activity.

41

Press et al.,29

have synthesized a series of purine derivatives and evaluated for

their selective inotropic activity. Thioether-linked derivatives were superior to their

oxygen and nitrogen isosteres. Substitution of electron withdrawing groups on benzhydril

moiety of these agents increased the potency. Compound 42, carsatrin showed good

activity.

42

Chapter 2

44

2.1.1c. Benzylpiperazine:

An honored scaffold among arylpiperazine derivatives

Benzylpiperazine belongs to the arylpiperazine family and is commonly referred to

as BZP, an N-monosubstituted piperazine derivative available as either base or the

hydrochloride salt. The base form is a slightly yellowish-green liquid. The hydrochloride

salt is a white solid. BZP base is corrosive and causes burns. The salt form of BZP is an

irritant to eyes, respiratory system and skin. BZP was first synthesized in 1944 as a

potential antihelminthic (anti-parasitic) agent for use in farm animals. It was subsequently

shown to possess antidepressant activity.

Benzylpiperazine

Benzylpiperazine possess a wide range of pharmacological properties. Chemists

have wide variety of options, to synthesize biologically active derivatives of

benzylpiperazine, by keeping the benzyl/substituted benzyl group at one end of the

piperazine and substituting the various substituents including substituents which have

biological importance at the other end of the piperazine ring. Derivatives of

benzylpiperazine which exhibit wide range of biological activities are summarized below:

Bozsing et al.,30

have synthesized novel pyrimidine derivatives of benzylpiperazine

for their evaluation of 5-HT2A and 5-HT2C receptor binding affinity and found that

compounds with 5-benzylpyrimidine derivative 43 showed substantially higher affinity

compared to the other analogues in the series. The introduction of N-benzylpiperazine

moiety on the 4, 6-diamino-2-thiopyrimidine moiety results in the increase in affinity

towards 5-HT2A and 5-HT2C receptor. In continuation of this work, they have also

synthesized (3-trifluoromethylbenzyl)piperazine derivatives by varying substituents in the

position-2 of the pyrimidine benzyl group. Among the synthesized compounds, 44 is

found to be the most potent in this series and has a 15-fold 5-HT2C/2A selectivity and they

Chapter 2

45

have opined that this may be due to the presence of 2-trifluoromethyl group at the

position-2 in the pyrimidine benzyl group.

Zhao et al.,31

have synthesized 2-[4-(4-chloro-benzyl)-piperazin-1-yl]-1-(2-methyl-

2,3-dihydro-indol-1-yl)ethanone with chlorine atom at 4-position on the benzyl group 45

and 2-[4-(4-methyl-benzyl)-piperazin-1-yl]-1-(2-methyl-2,3-dihydro-indol-1-yl)ethanone

with methyl group at 4-position on the benzyl group 46 and reported as potent mixed

D2/D4 antagonists. It is believed that chlorine and methyl groups at 4-position on the

benzyl group is held responsible for the above said activity.

In continuation of the same work, the same authors also reported 32

the chiral

synthesis of indoline and piperazine containing derivatives, (R)-2-[4-(4-chloro-

benzylmethyl)piperazin-1-yl]-1-(2-methyl-2,3-dihydro-indol-1-yl)ethanone and reported

as novel class of D2/D4 antagonists. They have also studied the effect of chirality and

benzyl group substitution. Among the synthesized compounds, 47 with methyl group at 4-

Chapter 2

46

position and 48 with chlorine at 4-position and methoxy groups at 2-position showed

good activity.

Foster et al.,33

have synthesized a series of N,N-dibenzylpiperzines by introducing

substituents on the benzyl group to enhance their anticocaine activity. In this series of

compounds, 49 with chlorine atom at 3-position on the benzyl group showed greatest

sigma-2-affinity and good anticocaine activity.

49

Younes et al.,34

have synthesized simple piperazines. Amongst the synthesized

analogues, 1-benzyl-4-(2-napthyl)piperazine 50 demonstrated good affinity for sigma-1-

receptors, and weak antagonism of the locomotor effects of stimulants

(methamphetamine).

50

Bucle et al.,35

have synthesized several N-benzylpiperazino derivatives of

[1]benzopyrano[2,3-d]-1,2,3-triazol-9(1H)-one and its 5-methyl homologue and evaluated

Chapter 2

47

for their antihistamine activity on guinea pig ileum. Among the synthesized compounds,

6-[3-[4-(4-chlorobenzyl)-1-piperazinyl]propoxy][1]benzopyrano[2,3-d]-1,2,3-triazol-9(1H)-

one 51, showed most potent activity against histamine on guinea pig ileum, comparable

to that of mepyramine. In this series, chlorinated derivatives were more potent than

methyl or methoxy group containing derivatives.

51

Ohataka et al.,36

have reported the quantitative structure cerebral vasodilating

activity relationships of 1-benzyl-4-(3-hydroxy-3-phenylpropyl) piperazine derivatives. The

analyses indicate that potency depends on the number of methoxyl groups on the benzyl

moiety and also lipophilicity of substituent is to be important with respect to the activity.

Among the derivatives studied, two derivatives 52 and 53 were found to be more potent

where: 52. Y= 2,3,4-tri-OCH3

53. Y= 3,4,5-tri-OCH3

Ohataka et al.,37

have synthesized a series of ester derivatives of 1-benzyl-4-

piperazineacetic acid and evaluated as antinuclear agents. The QSAR analyses for the

esters revealed that the antinuclear activity of these compounds were considered to be

based on the cycloprotective activity, the most active and least toxic derivatives, 54 and

55 were selected for further study.

Chapter 2

48

54

N N

MeO

MeO OO

OCH3

OMe

55

Hernandez et al.,38

have synthesized two series of analogues of novel human

mitochondrial thymidine kinase inhibitors, 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine

were synthesized by replacing trimethoxy moiety by a variety of substituted amines and

carboxamides. The selectivity in all the cases against the mitochondrial enzyme was either

maintained or improved and several derivatives including benzylpiperazine moiety, 56

showed modest potent activity.

56

Dundar et al.,39

have synthesized some 1-[4-2-oxo-2,3-dihydro-1,3-(benzoxazole-

3-yl)butanoyl]-4-arylpiperazine derivatives 57 and evaluated antinociceptive activity. The

distance between the amide nitrogen of piperazine ring and the nitrogen atom located at

position 3 of 2(3H)-benzoxazole increased to four carbon atoms, antinociceptive activity

was also increased.

Chapter 2

49

57

where R = phenyl, benzyl, fluorophenyl, chlorophenyl, pyridyl.

Asaki et al.,40

have synthesized a series of 3-substituted benzamide derivatives

structurally related to STI-571 (imatinib mesylate) 58, a Bcr-Abl tyrosine kinase inhibitor

used to treat chronic myeloid leukemia (CML), and evaluated for antiproliferative activity

against the Bcr-Abl-positive leukemia cell line K562.

58

Chaudhary et al.,41

have synthesized cyano derivatives of N-alkyl and N-aryl

piperazines and screened for antibacterial and antifungal activities. All the synthesized

compounds showed the antibacterial activity against pathogenic strains of Staphylococcus

aureus, Pseudomonas aeruginosa, Streptomyces epidermidis and Escherichia coli and

antifungal activity against pathogenic strains of Aspergillus fumigates, Aspergillus flavus

and Aspergillus niger. All compounds showed mild to moderate antimicrobial activity.

However, compound, 59 showed potent antibacterial activity against pathogenic strains

used in the study.

59

Chapter 2

50

Turkmen et al.,42

have synthesized a series of sulfonamides by reacting

sulfanilamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide with X-chloroalkanoyl chlorides,

followed by replacement of the X-chlorine atom with secondary amines. Tails

incorporating heterocyclic amines belonging to the morpholine, piperidine and piperazine

ring systems have been attached to these sulfonamides, by means of an alkanoyl-

carboxamido linker containing from two to five carbon atoms. The new derivatives

prepared in this way were tested as inhibitors of three carbonic anhydrase isozymes, the

cytosolic isozymes CA I and II, and the catalytic domain of the transmembrane, tumor-

associated isozyme CA IX. Several low nanomolar CA I and CA II inhibitors were detected

both in the aromatic and heterocyclic sulfonamide series, whereas the best hCA IX

inhibitors (inhibition constants in the range of 22–35nM) all belonged to the

acetazolamide-like derivatives 60.

60

N, N-Dialkylditihiocarbamate derivatives have been well known as broad-range

fungicides. Ozkirimli et al.,43

have synthesized, the triazole derivatives of N,N-

disubstituted dithiocarbamates and evaluated for antifungal activity against M. canis, M.

gypseum, and T. rubrum. N, N-Dialkylditihiocarbamate containing benzylpiperazine 61

showed good activity.

61

Chapter 2

51

Hages et al.,44

have synthesized amide derivatives of benzylpiperazine and some

analogues of befuraline and piberaline were reported. All compounds have been tested

as antidepressive agents. According to the tests, some of the amides showed elevated

antidepressive activity.

Hashimoto et al.,45

have studied the neurotoxicity of 3,4-

methylenedioxymethatamine(MDMA) in rat brain neurotoxicity was attenuated

significantly by coadministration of several benzylpiperazines (p-nitrobenzylpiperazine, p-

chlorobenzylpiperazine and 1-piperonylpiperazine), which were weak inhibitors of [3H]6-

nitroquipazine binding to the (5-HT), 5-hydroxytryptamine transporter in rat brain. These

results suggest that benzylpiperazine may inhibit the MDMA-induced neurotoxicity by a

novel neuropharmacological effect other than 5-HT uptake inhibition.

Desai et al.,46

have synthesized a series of (4-ethylphenyl)-3,5-ditertiarybutyl-4-

hydroxybenzylamine, 1-phenyl-4-(3,5-ditertiarybutyl-4-hydroxybenzyl)piperazine, and 1-

(3,5-ditertiarybutyl-4-hydroxybenzyl)piperidine and characterized and a comparative

study of the synthesized antioxidants with the commercially available antioxidant 2,6-

ditertiarybutyl-4-methylphenol was made. The presence of phenolic and amino groups

influenced the performance of the antioxidants. The performance of the antioxidants

influenced the thermal stability of the PPCP (polypropylene copolymer).

Chapter 2

52

2.1.2. Current status of piperidine and its derivatives in the

field of bioorganic and medicinal chemistry

The piperidine and arylpiperidine scaffolds are found in many compounds that

have distinct pharmacological and therapeutic profiles. The piperidine nucleus can also be

frequently recognized in the structure of numerous naturally occurring alkaloid and

synthetic compounds with interesting biological and pharmacological properties. As a

consequence, the development of general methods for the synthesis of piperidine

derivatives have been the subject of considerable synthetic effort. Pharmacological action

of piperidine derivatives are represented below:

The piperidine motif is as wide-ranging in its therapeutic uses as it is ubiquitously

found in drugs and drug candidates. It is a key structural component of successful anti-

Parkinson’s drugs and drug candidates (e.g. Besonprodil,47

Budipine48-49

) and displays

antiviral,50

antipsychotic,51-53

metabolic,54-56

anti-inflammatory,57

and cognition disorders58

and antmicrobial activities.59-60

Piperidines offered by group of common elements of

biogenic amine regulated GPCRs, which includes the large family of serotonin, dopamine,

histamine, and glutamic acid receptors.

Chapter 2

53

Figure 2.2.: Some representative examples of arylpiperidine derivatives being used as

pharmaceutical drugs.

Among piperidine derivatives, arylpiperdine derivatives showed various kinds of

biological activities in the field of medicinal chemistry. Chemists have synthesized various

substituted arylpiperidine derivatives by varying the aryl/substitution at various positions

of piperidine ring with respect to the nitrogen atom of the piperidine ring. The biological

activity of arylpiperidine derivatives are summarized as follows:

Chapter 2

54

Sugimoto et al.,61

have synthesized indanone derivatives and studied the anti-

Ache activity, derivative, 62 with electron releasing methoxyl group at the 3-position

increased the activity by 20-fold, compared to 2- and 4 –positions.

62

Willoughby et al.,62

reported the preparation of a combinatorial library of

arylpropyl piperidine derivatives with potent CCR5 binding affinity. Compounds with

novel combinations of subunits were discovered that have high binding affinity for the

CCR5 receptor. A potent CCR5 antagonist from the library, compound 63 was found to

have potent binding and moderate antiviral activity, which would be due to the aliphatic

cyclohexyl substituent, the aryl propyl piperidine side chain and the pyrrolidine scaffold in

combination.

63

Chapter 2

55

Pissarnitski et al.,63

have synthesized a series of novel piperidine derivative as γ-

secretase inhibitors, potentially useful for the treatment of Alzheimer’s disease. Since the

introduction of a fluorine atom in the aromatic region of compound 64 and cis-

stereochemistry of the substituents at the piperidine ring was established as a

requirement for the potency.

.

64

Ting et al.,64

have synthesized a novel series of dual NK1/NK2 receptor antagonists,

based on the 2-oxo-(1, 4’-bipiperidine) template. Compound 65 with N-methylacetamide

side chain on 2-oxo-(1,4’-bipiperidine) is a potent dual NK1/NK2 antagonist and

demonstrates excellent in vivo activity and good oral plasma levels in the dog.

65

Chapter 2

56

Wang et al.,65

have synthesized a number of highly potent M2 receptor antagonists

with >100-fold selectivity against the M1 and M3 receptor subtypes. In the rat

microdialysis assay, this series of compounds showed pronounced enhancement of brain

acetylcholine release after oral administration. Among them compound 66 demonstrated

that the M2 antagonists upon oral administration, stimulated brain acetylcholine release

in functional microdialysis assay.

66

Ramalingan et al.,66

have synthesized some novel benzoxazolylethoxypiperidones

and tested for their antibacterial activity against Streptococcus faecalis, Bacillus subtilis,

Escherichia coli, Staphylococcus aureus and Pseudomonas aureuginosa and antifungal

activity against Candida albicans, Aspergillus niger, Candida-51 and Aspergillus flavus

were evaluated. Compounds 67, 68, and 69 exerted potent in vitro antibacterial activity

against Streptococcus faecalis while compounds 70 and 71 exhibited potent in vitro

antifungal activity against Candida-51.

Chapter 2

57

Burgey et al.,67

identified the piperidinyl-azabenzimidazolone and

phenylimidazolinone as alternative privileged structures which when incorporated into

the benzodiazepine core afford potent CGRP (Calcitonin gene-related peptide) receptor

antagonists. Among them, compound 72, in which introduction of the ring containing

nitrogen serves to improve potency by 100-fold.

R= 4-AZA

72

Dutta et al.,68

have synthesized several analogs of the potent and selective

dopamine transporter (DAT) ligand 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine and

biologically evaluated at the dopamine and serotonin transporter (SERT) sites. Several

substituents were introduced into the aromatic rings to evaluate the influences of

electronic and steric interactions in their binding to the DAT. All the novel analogs showed

preferential interaction at the DAT compared with the SERT. Different aromatic

substitutions in the phenyl ring of the N-benzyl part of the molecule played a key role in

the selectivity. In general, compounds with strong electronwithdrawing substituents were

most active and selective at the DAT. The influence of electronic factors was indicated to

Chapter 2

58

some extent by these results since the compounds containing the most electronegative

atom (F) 73 and electron-withdrawing group (NO2) 74 showed maximal preferential

interaction at the DAT. Bioisosteric replacement of one of the phenyl rings of the

diphenylmethoxy moiety by a thiophene ring was tolerated well and produced the most

potent compound 75 in the series. Furthermore, these results also may indicate the

possible existence of complimentary electropositive/electron accepting sites on the DAT

to favour the observed interaction and vice versa on SERT.

73 74 75

Sugimoto et al.,69

have synthesized a new series of 1-benzyl-4-[2-(N-benzoyl-N-

methylamino)ethyl]piperidine derivatives and evaluated as potent anti-

acetylcholinesterase (anti-AChE) activity. Introduction of a phenyl group on the nitrogen

atom of the amide moieties resulted in enhanced activity. The rigid analogue containing

isoindolone was found to exhibit potent anti-AChE activity. Furthermore, replacement of

the isoindolone with other heterobicyclic ring systems was examined. Among the

compounds prepared in these series, 1-benzyl-4-[2-[4-(benzoylamino)phthalimido]ethyl]

piperidine hydrochloride is the most potent inhibitor of AChE. Compound 76 showed a

definite selectivity to AChE.

76

Chapter 2

59

Romero et al., 70

reported a novel class of bis(heteroaryl)piperazine (BHAP)

analogs which possess the ability to inhibit NNRTI (non-nucleoside reverse transcriptase

inhibitor) resistant recombinant HIV-1 reverse transcriptase (RT) and NNRTI resistant

variants of HIV-1 has been identified via targeted screening. Further structural

modifications were required to inhibit metabolism and modulate solubility in order to

obtain compounds with the desired biological profile as well as appropriate

pharmaceutical properties. In particular, substituting an ethyl for a methyl group on the

aminopiperidine spacer enhanced the desired activities. The AAP-BHAPs with the most

suitable characteristics were compounds 77, 78, and 79.

77. R=CH3

78. R= CH2CH3 79

Contreras et al.,71

have synthesized a series of 3-amino-6-phenylpyridazines and

tested for inhibition of AChE. Among all the derivatives investigated, 3-[2-(1-

benzylpiperidin-4-yl)ethylamino]-6-phenylpyridazine 80, was found to be one of the most

potent anti-AChE inhibitors, representing a 5000-fold increase in potency compared to

weak, competitive and reversible acetylcholinesterase. Among the different analogs

tested, it is remarkable to notice that the highest potency is associated with the N-

benzylpiperidine ethyl moiety.

Chapter 2

60

80

2.1.2a. Effect of presence of benzyl group at 4th

position of the piperidine

scaffold:

In the literature, 62,68

the aryl group at 4th

position of the piperidine ring with

respect to the nitrogen atom of the piperidine ring showed broad range of activities.

Among, them 4-benzylpiperidne derivatives showed good biological activity and are

summarized as follows:

Imamura et al.,72

synthesized and reported a novel lead compound, N-{3-[4-(4-

fluorobenzoyl)piperidin-1-yl]propyl}-1-methyl-5-oxo-N-phenylpyrrolidine-3-carboxamide,

81 and identified as a CCR5 antagonist. In an effort to improve the binding affinity of the

lead molecule, a series of 5-oxopyrrolidine-3-carboxamides were synthesized.

Introduction of 3,4-dichloro substituents to the central phenyl ring 82 and 83, or

replacing the 1-methyl group of the 5-oxopyrrolidine moiety with a 1-benzyl group 84 was

found to be effective for improving CCR5 affinity.

81

NN

O

N

O

R4

Me

CH2

Cl

Cl

Chapter 2

61

82. R4= H

83. R4= F

84

Mamolo et al.,73

have synthesized 3H-1,3,4-oxadiazol-2-one derivatives and tested

for their in vitro antimycobacterial activity. Oxadiazolone derivatives, 85 in which

conjugation with 4-benzyl piperidine showed an interesting antimycobacterial activity

against the tested strain of Mycobacterium tuberculosis.

85

McCauley et. al.,74

have synthesized two classes of 5-substituted benzimidazoles

by using derivatives of 4-benzylpiperidine and identified as potent antagonists of various

receptors. Among them, 86 having piperidine class showed good activity in the

carrageenan-induced mechanical hyperalgesia assay in rats as well as excellent

pharmacokinetic behavior in dogs.

86

Borza et. al.,75

have synthesized a novel series of benzimidazole-2-carboxamide

derivatives and reported as NR2B selective NMDA receptor antagonists. The influence of

some structural elements like, H-bond donor groups placed on the benzimidazole

Chapter 2

62

skeleton and the substitution pattern of the piperidine ring, on the biological activity was

reported. The compound 87 showed excellent analgetic activity.

X =5(6)-OH

87

In continuation of the work, the above authors76

also synthesized (4-

benzylpiperidine-1-yl)-(6-hydroxy-1H-indole-2-yl)methanone derived from (E)-1-(4-

benzylpieridin-1-yl)-3-(4-hydroxy-phenyl)-propenone and identified as potent antagonist

of the NMDA receptor. Several derivatives 88 and 89 of the above compound showed

nanomolar activity both in the binding and in the functional assay.

88. Q=6- OH

89. Q=5(6)-OH

In the literature there are many more NMDA receptors are reported such as, CI-

1041 77

90, Ifenprodil (Ro-25-6981)78

91.

(CI-1041) 90 (Ifenprodil) 91

Chapter 2

63

Ting et. al.,79

have synthesized bipiperidine amide 92 and 93 which has been

identified as a CC chemokine receptor 3 (CCR3) antagonist. (enant B), which exhibits

potent receptor affinity and inhibition of both calcium flux and eosinophil chemotaxis

than the bipipiperidine amide. An incremental improvement in the biological activity was

observed with modification of the bipiperidine core, in particular, substitution was well

tolerated at the 3-position (hydroxylmethyl) of the second piperidine ring.

92

93

Wright et al.,80

have synthesized a series of bicyclic heterocyclic systems

containing p-hydrogen donor, in which phenolic moiety was replaced by heterocyclic

systems, in particular, compound 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-

dihydrobenzoimidazol-2-one 94 found to be very potent, selective NR1A/2B receptor

antagonist.

94

Chapter 2

64

Shaw et al.,81

reported the in vivo properties of a series of 2-arylindole NK1

antagonists have been improved, by modification of the amide substituent. The 1-(2-

methoxyphenyl)piperazine amide was identified as a major area of metabolism in the

lead compound 95. Replacement of this amine moiety by a 4-benzyl-4-hydroxypiperidine

resulted with reduced clearance and improved central duration of action in compound

96.

95 96

Wacker et al.,82

synthesized the CCR3 antagonist leads with IC50 values in the mM

range were converted into low nM binding compounds that displayed in vitro inhibition of

human eosinophil chemotaxis induced by human eotaxin. In particular, 4-benzylpiperidin-

1-yl-n-propylureas 97 and erythro-3-(4-benzyl-2-(α-hydroxyalkyl)piperidin-1-yl)-n-

propylureas 98 exhibited single digit nanomolar

97 98

Novak et al.,83

have synthesized N, N-disubstituted amides of long-chain fatty acids

were screened for antimicrobial activity against bacteria, yeasts, and molds. N-oleoyl-4-

benzylpiperidine exhibit a moderate spectrum of antimicrobial activity.

Zia-ur-Rehman et al.,84

Synthesised new tri-, chlorodi- and diorganotin (IV)

dithiocarboxylates of 4-benzylpiperidine-1-carbodithioate ligand (L), A subsequent

antimicrobial study indicates that the compounds are biologically active.

Chapter 2

65

2.2. Present Work

2.2.1. Design and Selection of Piperazine and Piperidine Moieties

as Heterocyclic Precursors:

As anchored earlier in the introductory part, derivatives of piperazine and

piperidine motifs are the most reoccurring structural frameworks in the field of

heterocyclic chemistry. Currently, there are avalanche of successful examples of these

derivatives as versatile drug candidates in modern drug discovery. In light of achieving

this, several researchers have shown that upon varying numerous pharmacophores to

secondary nitrogen of piperazine and piperidine with different functional groups such as

amides, sulfonamides, urea, carbamates, oxalamides etc,. This exemplifies that

modification of similar kind of insertion will fetch fruitful results in developing novel lead

molecules in the near future.

In the literature, there appear ample avenues to synthesize biologically active

piperazine derivatives by keeping various commonly encountered moieties such as

substituted derivatives of alkyl, cyclohexyl, phenyl, benzyl, benzhydril and various

heterocycles on one of the nitrogen atoms of the piperazine moiety and varying

numerous substitutions on other nitrogen atom of piperazine. Amongst, benzyl and

benzhydril moieties are versatile groups which are already in use worldwide as potent

drugs for the ailment of many devastating diseases.

Benzhydril group on the piperazine ring plays a paramount role in elucidating the

biological activities of these derivatives upon conjugation with other analogues.

Benzhydrilpiperazine belongs to the arylpiperazine family and their derivatives possess a

wide range of pharmacological properties. Hence, chemists have new horizons which

paves way to synthesize biologically active derivatives of benzhydrilpiperazine by coupling

various substituents on one of the nitrogen atoms of the piperazine moiety and without

replacing the benzhydril group at the other end of the piperazine and they have

Chapter 2

66

succeeded in obtaining the compounds with marked enhancement in the biological

activity compared to the conventional drugs. Also, it is believed that the pharmacological

activity of benzhydrilpiperazine derivatives is attributed due to the presence of two

nitrogen atoms on the piperazine ring.

In continuation of the above, there is an another class of arylpiperazine

compounds ie., benzylpiperazine moiety, which has come across in various reports

emphasizing biological importance. Here also, the presence of aromatic benzene ring on

one end and the subsequent insertion of various substituents on the other end of the

piperazine play a vital role in bringing about the marked change in the biological

properties.

Considering the importance of both benzhydril and benzyl substitutions on

piperazine skeleton, we are very much projected in evaluating the effect of these two

groups on the similar type of piperazine analogues.

On the other hand, piperidines are the class of compounds which exhibits

innumerable number of pharmacological properties which has led to its discovery in

much distinctive therapeutics. Particularly, it is very much evident from earlier research

that substitutions have been made on different position of the piperidine ring in hoping

to achieve novel molecules. Of these, when the substitution is an aryl group in particular

on the 4th

position of the piperidine ring exhibited highest biological activity. In this

connection, we have selected 4-benzyl piperidine as one more class of the heterocyclic

precursors for our study.

2.2.2. Motivation for the Conjugation of Amino Acid Residues:

Amino acids are the fundamental components of living organisms playing a crucial

role both as building blocks of proteins and as intermediates in metabolism. The diverse

functionalities of amino acid residues which can be a polar and/or non-polar, have both

hydrophilic and lipophilic regions and have a negative, positive or neutral net charge are

ought to play very unique and important role in various protein-protein, peptide-protein,

Chapter 2

67

proteins-receptors, enzyme interactions in the human body. Considering the low toxicity,

biocompatibility, likeliness and favored interaction of amino acidic residues with the

biological system, currently there is huge tendency of conjugating amino acid/peptidic

residues with small bioactive molecules in the field of biomedical research.30-42

In

particular, the scope for the conjugation of amino acids with the bioactive heterocyclic

motifs is on rise due the favorable pharmacological effects exerted by the combined

framework of heterocyclic moiety and varying side chains of amino acids on the specified

sites of biological targets. It is reported that a simple known coupling reaction between

amino acids and heterocycles has resulted in the products of biological importance and in

many cases some heterocycles linked to amino acidic residues showed enhanced

biological activity more than standard drugs. Furthermore, there are several successful

evidences available in the literature signifying the importance of amino acid conjugation

in improving the potency, selectivity, low toxicity, in vivo stability, solubility and high cell

permeability of the bioactive heterocycles.

In today’s medicine amino acids continue to grow in popularity for their potential

use in drug therapy. Amino acid-based drugs are any substance that uses the different

amino acids to diagnose, prevent and treat diseases and conditions to restore or maintain

normal body functions. These have led to the discovery of numerous amino acids based

drugs of therapeutic potential, a number of which are already applied clinically. Till to

date there appears to be no reports available in the literature on amino acids conjugated

benzhydrilpiperazine. Upon triggered over this, we owed to couple various Nα-protected

Boc-L-amino acids with one of the nitrogens available on the benzhydrilpiperazine. In this

view, we would be able to elicit whether amino acid residues play a role in enhancing the

activity of the synthesized compounds after conjugation in general and also in particular

any residue/group would be held responsible for the activity.

In view of all the above facts and considering biological importance of amino acid

conjugation with the bioactive heterocycles, we are very much projected on design,

synthesis and biological evaluation of new series of amino acid conjugated

Chapter 2

68

benzhydrilpiperazine (Analogues-I), benzylpiperazine (Analogues-II) and 4-

benzylpiperidine (Analogues-III) derivatives. The representative structural frame works of

the synthesized compounds are here picturized below (Scheme 2.1 - 2.3).

The heterocyclic precursors have been either synthesized according to literature

methods or were procured commercially. The Nα-Boc protected amino acids were coupled

with aforesaid heterocyclic precursors using EDCI/HOBt as coupling agent and NMM as base.

The synthesized amino acid conjugates have been subjected to different analytical and

spectroscopic techniques in order to conformity of the structures like, elemental analysis and

1H NMR. The Boc deprotected compounds were screened for antibacterial, antifungal and

antioxidant activities. Antibacterial studies included gram +ve bacteria like Staphylococcus

aureus and various gram –ve strains like Escherichia coli, Klebesiella pneumoniae and

Pseudomonas auregenosa where as anti fungal assay employs Aspergillus niger, Aspergillus

flavus and Fusarium monoliforme. Both antibacterial and antifungal studies were carried by

agar well diffusion method. Also, the compounds have been screened for DPPH radical

scavenging effect as an antioxidant assay.

Chapter 2

69

O OH Cl N NH

b

N N

O

R

NH

O

O

+

O

HO

R

HN

O

O

d

Where R = Side chain of amino acids (Gly, Ala, Val, Leu, Phe, Glu, Lys, Arg, His, Trp, Asn, Pro)

I II III IV

V(a-l)

N N

O

R

NH2..HCl

VI(a-l)

e

a-l

a c

Reagents and conditions (a) NaBH4, methanol, rt, 5 hrs. (b) SOCl2, DCM, 0-50C, 4 hrs.

(c)Piperazine, K2CO3, DMF, 800C, 8 hrs. (d) EDCI, HOBt, NMM,

DMF, -15 0C, 24 hrs. (e) 4N HCl/dioxane

Scheme 2.1: Synthesis of amino acids conjugated bezhydrilpiperazine derivatives (Analogues-I)

Chapter 2

70

Chapter 2

71

NHNHBoc

OH

O N

NHBoc

O

N

NH2.HCl

O

b

+a

Scheme-2.3: Synthesis of amino acids conjugated 4-benzylpiperidine derivatives (Analogues-III)

R

RR

XII a-l XIII(a-l)

XIV(a-l)

Where R = Side chain of amino acids (Gly, Ala, Val, Leu, Phe, Glu, Ile, Arg, Lys, His, Trp, Pro)

Reagents and Conditions (a) EDCI, HOBt, NMM, DMF, -15 0C, 24 hrs. (b) 4N HCl /dioxane

Chapter 2

72

2.3. Experimental

2.3.1. Materials and Methods:

All the amino acids used were of L-configuration unless mentioned. All Boc-amino

acids and HOBt were purchased from Advanced Chem. Tech. (Louisville, Kentuky, USA).

EDCI and NMM were purchased from Sigma Chemicals Co. (St. Louis, MO). All the solvents

and reagents used for the synthesis and analysis were of analytical grade. TLC was carried

out on precoated silica gel plates prepared in laboratory using

chloroform/methanol/acetic acid (95:5:3, 85:15:3) as solvent systems. 4-Benzylpiperidine

(XII) was purchased from Sigma-Aldrich India. 1H NMR spectra were obtained on a 400

MHz Bruker FT-NMR Spectrometer instrument by using DMSO/CDCl3 as solvent and TMS

as an internal standard. Elemental analysis was obtained by using VARIO EL III CHNS

Elementar.

2.3.2. Synthesis

2.3.2.1. Synthesis of heterocyclic precursors:

2.3.2.1.1. Synthesis of benzhydrilpiperazine (IV):

Synthesis of benzhydrol (II) from benzophenone (I):

Benzophenone (10.92g, 60 mmol) was dissolved in 250 mL of methanol and

cooled to 0 0C. NaBH4 (2.16 g, 60 mmol) was added to the above solution at 0

0C for 1 hr

and the reaction mixture was stirred further at rt for 4 hrs. Then, methanol was distilled

under reduced pressure. The reaction mixture was diluted with water (100 mL), the

product was extracted with diethyl ether (300 mL) and the organic phase was washed

with 1N HCl, followed by a saturated NaHCO3 and finally with water. It was dried over

anhydrous sodium sulphate and evaporated under vacuum to obtain benzhydrol (II, 9.93

g, 90%), M. P. 67-68 0C (lit. 66-68

0C).

85

Chapter 2

73

Synthesis of benzhydrilchloride (III) from benzhydrol (II):

The bezhydrol (9.2 g, 50 mmol) was dissolved in DCM (50 mL) and thionyl chloride

(4.3 mL, 60 mmol) was added dropwise to the solution for 30 minutes at 0-5 0C. The

reaction mixture was stirred at room temperature for 4 hrs. The reaction mixture was

concentrated under reduced pressure to remove thionyl chloride and DCM, again and

again washed the reaction mixture (4-5 times) with 50mL of DCM; finally the DCM extract

was distilled under reduced pressure, which gives an yellow liquid, Yield (9.1 mL, 90%),

B.P. 138-139 0C (lit-139-140

0C).

85

Synthesis of benzhydrilpiperazine (IV) from benzhydrilchloride (III):

Benzhydrilchloride (8.0 mL, 45 mmol) was added dropwise to the stirred solution

of piperazine (38.74 g, 450 mmol) and potassium carbonate (12.24 g, 90 mmol) in DMF.

The reaction was heated to 80 0C for 8 hrs and monitored by TLC. Upon completion of

reaction, after filtration, the solvent was removed under vaccum. The residue was

dissolved in ethylacetate, washed with water and brine, dried over anhydrous sodium

sulphate and evaporated. The residue was chromatographed on silica gel (60-120 mesh),

chloroform: methanol (9:1) to afford 1-benzhydrilpiperazine a white powder with an yield

(9.63 g, 95%), M.P. 89-910C (lit.91-92

0C).

85

2.3.2.1.2. Synthesis of benzylpiperazine (IX):

Benzylchloride (7.45 mL, 65 mmol) was added dropwise to the stirred solution of

piperazine (55.90 g, 650 mmol) and potassium carbonate (17.68 g, 130 mmol) in DMF.

The reaction was heated to 80 0C for 8 hrs and monitored by TLC. Upon completion of

reaction, after filtration, the solvent was removed under vaccum. The residue was

dissolved in ethylacetate, washed with water and brine, dried over anhydrous sodium

sulphate and evaporated. The residue was chromatographed on silica gel (60-120 mesh),

chloroform: methanol (9:1) to afford 1-benzylpiperazine with an yield (9.88 g, 95%), B.P.

146-147 0C (lit.145-147

0C).

85

Chapter 2

74

2.3.2.2. General procedure for the coupling of Nα-Boc amino acids with

heterocycles (IV, IX and XII):

To the stirred solution of Nα-Boc-amino acid (2 mmol) and HOBt (0.31 g, 2 mmol) in

DMF (10 mL) cooled to 0 0C, added NMM (0.22 mL, 2 mmol). The mixture was further

cooled to -15 0C ± 1

0C and added EDCI (0.39 g, 2 mmol) and heterocyclic precursors (IV,

IX, XII, 2 mmol). After 20 minutes, the pH of the solution was adjusted to 8 by the

addition of NMM and the reaction mixture was stirred overnight while slowly warming to

rt. The reaction mixture was quenched with water (2 mL) and the solvent was condensed.

The residue was dissolved in chloroform (25 mL), washed with 5% NaHCO3 (3 x 20 mL),

H2O (1 x 20 mL) followed by 0.1N cold HCl (3 x 20 mL) and brine solution (3 x 20 mL), dried

over anhydrous Na2SO4. The chloroform was removed under reduced pressure to obtain

the desired products (Va-Vl, Xa-Xl and XIIIa-XIIIl). The analytical data of these compounds

are presented in Table-2.1-2.3.

2.3.2.3. Deprotection of Boc group:

The Boc group of the synthesized compounds (1 mmol) was deblocked by treating

with 4N HCl in dioxane (10 mL / g of the compound) for 1.5 hours. Excess HCl and dioxane

were removed under reduced pressure, triturated with ether, filtered, washed with ether

and dried to afford hydrochloride salts of amino acid conjugated heterocycles (VIa-VIl,

XIa-XIl and XIVa-XIVl), Yield (100%). These compounds were used for the antimicrobial

studies.

Ch

ap

ter

2

75

Ta

ble

2.1

: A

na

lyti

cal

an

d S

pe

ctro

sco

pic

Da

ta o

f th

e S

yn

the

size

d C

om

po

un

ds

(An

alo

gu

es-

I)

En

try

Sid

e c

ha

in o

f a

min

o a

cid

s

(R)

Rf

va

lue

Yie

ld

(%)

Mo

lecu

lar

form

ula

Ele

me

nta

l a

na

lysi

s (%

)

Fo

un

d

(Ca

lcu

late

d)

1H

-NM

R (

CD

Cl 3

), δ

pp

m

C

H

N

Va

H

0

.47

9

0

C2

4H

31N

3O

3

70

.41

(70

.43

)

7.6

1

(7.6

3)

10

.25

(10

.26

)

7.0

5-7

.25

(m

, 1

0H

, A

r-H

);

1.3

9 (

s, 9

H,

Bo

c);

7.9

1 (

s,

1H

, N

H-G

ly);

4.6

0 (

s, 1

H,

-

CH

-);

2.8

4

(t,

4H

, -C

H2-)

;

2.6

0 (

t, 4

H,

-CH

2-)

; 3

.9 (

s,

2H

, α

CH

2).

Vb

CH3

0

.43

9

5

C2

5H

33N

3O

3

70

.90

(70

.91

)

7.8

0

(7.8

1)

9.9

2

(9.9

2)

7.0

5-7

.25

(m

, 1

0H

, A

r-H

);

1.3

9 (

s, 9

H,

Bo

c);

7.9

3 (

s,

1H

, N

H-A

la);

4.6

2 (

s, 1

H,

-

CH

-);

2.8

8

(t,

4H

, -C

H2-)

;

2.6

2

(t,

4H

, -C

H2-)

; 4

.33

(q,

1H

, α

CH

); 1

.25

(d

, 3

H,

βC

H3).

Vc

CH3

CH3

0.4

5

93

C

27H

37N

3O

3

71

.82

(71

.83

)

8.2

0

(8.2

2)

9.2

9

(9.3

0)

7.0

5-7

.27

(m

, 1

0H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

7.9

0 (

s,

1H

, N

H);

4.6

5 (

s, 1

H,

-CH

-

Va

l);

2.8

4

(t,

4H

, -C

H2-)

;

2.6

0

(t,

4H

, -C

H2-)

; 4

.51

(d,

1H

, α

CH

-);

1.7

1

(m,

1H

, β

CH

-);

1.2

5

(d,

6H

,

γCH

3).

Ch

ap

ter

2

76

Vd

C H2

CH

CH3

CH3

0.4

5

91

C

28H

39N

3O

3

72

.24

(72

.25

)

8.4

0

(8.4

2)

9.0

2

(9.0

3)

7.0

9-7

.23

(m

, 1

0H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

7.9

0 (

s,

1H

, N

H-L

eu

); 4

.60

(s,

1H

, -

CH

-);

2.8

5

(t,

4H

, -C

H2-)

;

2.4

9 (

t, 4

H,

-CH

2-)

; 4

.50

(t,

1H

, -α

CH

-);

1.7

0

(t,

2H

,

βC

H2-)

; 1

.50

(m

, 1

H,

-γC

H-

); 1

.25

(d

, 6

H,

δC

H3).

Ve

H2C

0.5

0

85

C

31H

37N

3O

3

74

.50

(74

.52

)

7.4

5

(7.4

6)

8.4

1

(8.4

2)

7.0

6-7

.45

(m

, 1

5H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

7.9

8 (

s,

1H

, N

H-P

he

); 4

.62

(s,

1H

,

-CH

-);

2.9

0 (

t, 4

H,

-CH

2-)

;

2.6

2 (

t, 4

H,

-CH

2-)

; 4

.58

(t,

1H

, -α

CH

-);

3.3

5

(d,

2H

,

βC

H2-)

.

Vf

H2

CC H2

CO

OH2

C

0.3

7

90

C

34H

41N

3O

5

71

.44

(71

.45

)

7.2

2

(7.2

3)

7.3

3

(7.3

5)

7.0

4-7

.37

(m

, 1

5H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

7.8

0 (

s,

1H

, N

H-G

lu);

4.6

4 (

t, 1

H,

-

CH

-);

2.8

5

(t,

4H

, -C

H2-)

;

2.5

4 (

t, 4

H,

-CH

2-)

; 4

.41

(t,

1H

, -α

CH

-);

1.8

9 (

m,

2H

, -

βC

H2-)

; 2

.16

(t,

2H

, -γ

CH

2-

); 5

.36

(s,

2H

, C

H2-P

h).

Vg

N HO

O

0.4

1

88

C

36H

46N

4O

5

70

.32

(70

.33

)

7.5

2

(7.5

4)

9.1

0

(9.1

1)

7.0

6-7

.27

(m

, 1

5H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

7.9

6 (

s,

1H

, N

H-L

ys)

; 4

.64

(s,

1H

, -

CH

-);

2.8

9

(t,

4H

, -C

H2-)

;

2.5

0(t

, 4

H,

-CH

2-)

; 4

.51

(t,

1H

, -α

CH

-);

1.5

5 (m

, 2

H,

βC

H2-)

; 1

.39

(m

, 2

H,

γCH

2-

Ch

ap

ter

2

77

);

1.4

8

(m,

2H

, δ

CH

2-)

;

2.4

2 (

t, 2

H,

εC

H2-)

; 4

.9 (

s,

2H

, C

H2),

8.0

0 (

s, 1

H,

NH

).

Vh

H2

CC H2

H2

CN H

N H

NO2

NH

0.3

5

90

C

28H

39N

7O

5

60

.73

(60

.74

)

7.0

8

(7.1

0)

17

.72

(17

.73

)

7.0

8-7

.25

(m

, 1

0H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

8.0

0 (

s,

1H

, N

H-A

rg);

4.7

3 (

s, 1

H,

-

CH

-);

2.8

8

(t,

4H

, -C

H2-)

;

2.5

9(t

, 4

H,

-CH

2-)

; 4

.54

(t,

1H

, -α

CH

-);

1.5

0 (

m,

2H

,

βC

H2-)

; 1

.30

(m

, 2

H,

γCH

2-

); 2

.59

(t,

2H

, δ

CH

2-)

; 8

.0

(s,

1H

, N

H).

Vi

NN

H2C

H2

CO

H2

C

0.4

8

90

C

36H

43N

5O

4

70

.91

(70

.93

)

7.1

0

(7.1

1)

11

.48

(11

.49

)

7.0

4-7

.35

(d,

15

H,

Ar-

H);

1.4

3 (

s, 9

H,

Bo

c);

8.0

0 (

s,

1H

, N

H-H

is);

4.7

0 (

s, 1

H,

-

CH

-);

2.9

0

(t,

4H

, -C

H2-)

;

2.5

0(t

, 4

H,

-CH

2-)

; 4

.65

(t,

1H

, -α

CH

-);

3.2

0 (

d,

2H

, -

βC

H2-)

; 6

.55

(s,

2H

,

imid

azo

le);

4

.60

(s

, 2

H,

CH

2);

5.6

1 (

s, 2

H,

CH

2).

Vj

H2C

HN

0.5

1

92

C

33H

38N

4O

3

73

.56

(73

.58

)

7.0

6

(7.0

7)

10

.37

(10

.39

)

7.0

4-7

.30

(m

, 1

4H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

7.8

0 (

s,

1H

, N

H-T

rp);

4.6

9 (

s, 1

H,

-

CH

-);

2.9

2

(t,

4H

, -C

H2-)

;

2.4

8 (

t, 4

H,

-CH

2-)

; 4

.58

(s,

1H

, -α

CH

-);

3.1

9

(d,

2H

,

βC

H2-)

; 1

0.1

2 (s

, 1

H,

NH

ind

ole

); 6

.81

(d

, 1

H,

-CH

-).

Ch

ap

ter

2

78

VK

C H2

CNH2

O

0.4

4

94

C

26H

34N

4O

4

66

.91

(66

.92

)

7.3

0

(7.3

1)

12

.00

(12

.01

)

7.0

4-7

.19

(d

, 1

0H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

7.9

8 (

s,

1H

, N

H);

4.6

7 (

s, 1

H,

-CH

-

); 2

.90

(t,

4H

, -C

H2-)

; 2

.60

(t,

4H

, -C

H2-)

; 4

.48

(t,

1H

,

-αC

H-)

; 2

.81

(d

, 2

H,

(CO

)CH

2

-);

6.0

0

(s,

2H

,

NH

2).

Vl

0.4

1

89

C

27H

35N

3O

3

72

.15

(72

.16

)

7.8

0

(7.8

1)

9.3

4

(9.3

5)

7.0

5-7

.22

(m

, 1

0H

, A

r-H

);

1.4

3 (

s, 9

H,

Bo

c);

4.6

5 (

s,

1H

, -C

H-)

; 2

.88

(t

, 4

H,

-

CH

2-)

; 2

.55

(t,

4H

, -C

H2-)

;

3.9

(t

, 1

H,

-αC

H-)

; 1

.73

(m,

2H

, -β

CH

2);

1

.48

(m

,

2H

, γC

H2);

3

.17

(t

, 2

H,

-

δC

H2);

Ch

ap

ter

2

79

Ta

ble

2.

2:

An

aly

tica

l a

nd

Sp

ect

rosc

op

ic D

ata

of

the

Syn

the

size

d C

om

po

un

ds

(An

alo

gu

es-

II)

En

try

Sid

e c

ha

in o

f a

min

o a

cid

s

(R)

Rf

va

lue

Yie

ld

(%)

Mo

lecu

lar

form

ula

Ele

me

nta

l a

na

lysi

s (%

)

Fo

un

d

(Ca

lcu

late

d)

1H

-NM

R (

CD

Cl 3

), δ

C

H

N

Xa

H

0

.44

9

4

C1

8H

27N

3O

3

64

.84

(64

.86

)

8.1

5

(8.1

6)

12

.61

(12

.62

)

7.2

-7.4

0 (

m,

5H

, A

r-H

); 1

.43

(s,

9H

, B

oc)

; 7

.80

(s,

1H

, N

H-

Gly

); 3

.8 (

s, 2

H,

-ArC

H2-)

; 2

.80

(t,

4H

, -C

H2-)

; 2

.42

(t

, 4

H,

-

CH

2-)

; 3

.85

(s,

2H

, α

CH

2).

Xb

CH3

0

.39

9

1

C1

9H

29N

3O

3

65

.67

(65

.68

)

8.4

1

(8.4

2)

12

.08

(12

.09

)

7.1

-7.3

5 (

m,

5H

, A

r-H

); 1

.43

(s,

9H

, B

oc)

; 7

.85

(s,

1H

, N

H-

Ala

);

3.8

5

(s,

2H

, -A

rCH

2-)

;

2.8

2

(t,

4H

, -C

H2-)

; 2

.40

(t

,

4H

, -C

H2-)

; 4

.40

(m

, 1

H,

αC

H);

1.1

5(d

, 3

H,

βC

H3).

Xc

CH3

CH3

0.4

1

95

C

21H

33N

3O

3

67

.20

(67

.21

)

8.7

8

(8.8

0)

11

.20

(11

.22

)

7.2

0-7

.35

(m

, 5

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 7

.80

(s,

1H

, N

H-

va

l);

3.8

5

(s,

2H

, -A

rCH

2-)

;

2.8

4

(t,

4H

, -C

H2-)

; 2

.45

(t

,

4H

, -C

H2-)

; 4

.50

(d

, 1

H,

αC

H-

); 1

.70

(m

, 1

H,

βC

H-)

; 1

.20

(d,

6H

, γC

H3).

Ch

ap

ter

2

80

Xd

C H2

CH

CH3

CH3

0.4

6

90

C

22H

35N

3O

3

67

.85

(67

.86

)

9.0

5

(9.0

6)

10

.77

(10

.79

)

7.2

-7.4

0 (

m,

5H

, A

r-H

); 1

.43

(s,

9H

, B

oc)

; 7

.90

(s,

1H

, N

H-

Leu

); 3

.80

(s,

2H

, -A

rCH

2-)

; 2

.8

(t,

4H

, -C

H2-)

; 2

.40

(t

, 4

H,

-

CH

2-)

; 4

.55

(t

, 1

H,

-αC

H-)

;

1.6

7 (

t, 2

H,

βC

H2-)

; 1

.45

(m

,

1H

, -γ

CH

-);

1.1

(d

, 6

H,

δC

H3).

Xe

H2C

0.5

2

89

C

25H

33N

3O

3

70

.90

(70

.92

)

7.7

9

(7.8

0)

9.9

2

(9.9

3)

7.1

-7.4

0 (

m,

10

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 7

.80

(s,

1H

, N

H-

Ph

e);

3

.80

(s

, 2

H,

-ArC

H2-)

;

2.9

0 (

t, 4

H,

-CH

2-)

; 2

.4 (

t, 4

H,

-CH

2-)

; 4

.75

(t,

1H

, -α

CH

-);

3.3

(d,

2H

, β

CH

2-)

Xf

H2

CC H2

CO

OH2

C

0.3

5

91

C

28H

37N

3O

5

67

.87

(67

.89

)

7.5

1

(7.5

2)

8.4

8

(8.4

9)

7.2

-7.4

(m,

10

H,

Ar-

H);

1

.43

(s,

9H

, B

oc)

; 7

.85

(s,

1H

, N

H-

Glu

);

3.8

8

(s,

2H

, -A

rCH

2-)

;

2.8

2(t

, 4

H,

-CH

2-)

; 2

.45

(t,

4H

,

-CH

2-)

; 4

.45

(s

, 1

H,

-αC

H-)

;

1.8

(m,

2H

, -

βC

H2-)

; 2

.0

(t,

2H

, -γ

CH

2-)

; 5

.20

(s,

2H

, C

H2-

Ph

).

Ch

ap

ter

2

81

Xg

N HO

O

0.3

9

90

C

30H

42N

4O

5

66

.88

(66

.89

)

7.8

5

(7.8

6)

10

.39

(10

.40

)

7.2

-7.4

0 (

m,

10

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 7

.80

(s,

1H

, N

H-

Lys)

; 3

.80

(s,

2H

, -A

rCH

2-)

;

2.8

5(t

, 4

H,

-CH

2-)

; 2

.45

(t,

4H

,

-CH

2-)

; 4

.50

(t

, 1

H,

-αC

H-)

;

1.4

5 (

m,

2H

, β

CH

2-)

; 1

.35

(m

,

2H

, γC

H2-)

; 1

.45

(m

, 2

H,

δC

H2-)

; 2

.35

(t

, 2

H,

εC

H2-)

;

7.9

5 (

s, 1

H,

NH

); 4

.85

(d

, 2

H,

O-C

H2-A

r).

Xh

H2

CC H2

H2

CN H

N H

NO2

NH

0.3

5

90

C

22H

35N

7O

5

55

.35

(55

.36

)

7.3

7

(7.3

9)

20

.55

(20

.56

)

7.2

-7.3

5 (

m,

5H

, A

r-H

); 1

.43

(s,

9H

, B

oc)

; 7

.85

(s,

1H

, N

H-

Arg

);

3.9

0

(s,

2H

, -A

rCH

2-)

;

2.8

2(t

, 4

H,

-CH

2-)

; 2

.45

(t,

4H

,

-CH

2-)

; 4

.55

(t,

1H

, -α

CH

-);

1.3

0 (

q,

2H

, β

CH

2-)

; 1

.25

(m

,

2H

, γC

H2-)

; 2

.60

(t,

2H

, δ

CH

2-

); 8

.1 (

m,

1H

, N

H-g

ua

nid

ine

).

Xi

NN

H2C

H2

CO

H2

C

0.4

4

91

C

30H

39N

5O

4

67

.50

(67

.53

)

7.3

6

(7.3

7)

13

.10

(13

.11

)

7.2

-7.3

5(m

, 1

0H

, A

r-H

); 1

.45

(s,

9H

, B

oc)

; 7

.90

(s,

1H

, N

H-

His

);

3.8

5

(s,

2H

, -A

rCH

2-)

;

2.8

5

(t,

4H

, -C

H2-)

; 2

.50

(t

,

4H

, -C

H2-)

; 4

.60

(t,

1H

, -α

CH

-

); 3

.12

(d,

2H

, -β

CH

2-)

; 6

.50

(s,

2H

, im

ida

zole

);

4.5

5(s

, 2

H,

CH

2);

5.6

0 (

s, 2

H,

CH

2).

Ch

ap

ter

2

82

Xj

H2C

HN

0.5

0

94

C

27H

34N

4O

3

70

.11

(70

.12

)

7.3

6

(7.3

7)

12

.12

(12

.14

)

7.2

-7.3

0 (

m,

9H

, A

r-H

); 1

.45

(s,

9H

, B

oc)

; 7

.80

(s,

1H

, N

H-

Trp

);

3.8

0

(s,

2H

, -A

rCH

2-)

;

2.8

8

(t,

4H

, -C

H2-)

; 2

.45

(t

,

4H

, -C

H2-)

; 4

.55

(s,

1H

, -α

CH

-

); 3

.20

(d

, 2

H,

βC

H2-)

; 1

0.1

0

(d,

1H

, N

H o

f in

do

le);

6.8

0 (

d,

1H

, -C

H-)

.

Xk

0.4

2

91

C

22H

35N

3O

3

67

.84

(67

.86

)

9.0

5

(9.0

6)

10

.79

(10

.80

)

7.2

-7.5

0 (

m,

5H

, A

r-H

); 1

.43

(s,

9H

, B

oc)

; 7

.85

(s,

1H

, N

H-

Ile

);

3.8

2

(d,

2H

, -A

rCH

2-)

;

2.8

4 (

t, 4

H,

-CH

2-)

; 2

.40

(t

,

4H

, -C

H2-)

; 4

.50

(d

, 1

H,

-αC

H-

); 2

.60

(m

, 1

H,

-βC

H-)

; 2

.0 (

m,

2H

, -γ

CH

2 -

); 1

.0(t

, 6

H,

CH

3).

Xl

0.4

3

90

C

21H

31N

3O

3

67

.52

(67

.53

)

8.3

5

(8.3

7)

11

.23

(11

.24

)

7.1

0-7

.50

(m

, 5

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 3

.85

(s,

2H

, -

ArC

H2-)

; 2

.85

(t

, 4

H,

-CH

2-)

;

2.4

0

(t,

4H

, -C

H2-)

; 3

.90

(t

,

1H

, -α

CH

-);

1.8

0

(m,

2H

, -

βC

H2);

1

.47

(m

, 2

H,

γ-C

H2);

3.1

5 (

t, 2

H,

δ-C

H2).

Ch

ap

ter

2

83

Ta

ble

2.3

: A

na

lyti

cal

an

d S

pe

ctro

sco

pic

Da

ta o

f th

e S

yn

the

size

d C

om

po

un

ds

(An

ao

lgu

es-

III)

En

try

S

ide

ch

ain

of

am

ino

aci

ds

(R)

Rf

Va

lue

Mo

lecu

lar

form

ula

Yie

ld

(%)

Ele

me

nta

l a

na

lysi

s (%

)

Fo

un

d

(Ca

lcu

late

d)

1H

-NM

R (

CD

Cl 3

), δ

pp

m

C

H

N

XII

Ia

H

0.4

9

C1

9H

28N

2O

3

93

6

8.6

3

(68

.65

)

8.4

8

(8.4

9)

8.4

1

(8.4

3)

7.2

0-7

.40

(m

, 5

H,

Ar-

H);

1.4

1(s

, 9

H,

Bo

c);

2.4

(d

, 2

H,

-ArC

H2-)

; 2

.65

(t,

4H

, -C

H2-)

;

1.7

0

(m,

4H

, -C

H2-)

;

1.9

(m,1

H,-

CH

-

pip

eri

din

e);

7.8

5

(s,1

H,N

H-

Gly

); 3

.85

(s,

2H

, -C

H2)

XII

Ib

CH3

0

.45

C

20H

30N

2O

3

90

6

9.3

2

(69

.33

)

8.7

1

(8.7

3)

8.0

9

(8.0

9)

7.2

0-7

.40

(m

, 5

H,

Ar-

H);

1.4

3(s

, 9

H,

Bo

c);

7.8

(s,

1H

,

NH

-Ala

); 2

.45

(d,

2H

,-A

rCH

2-

); 2

.65

(t,

4H

, -C

H2-)

; 1

.7 (

q,

4H

, -C

H2-)

; 1

.91

(m,

1H

,-C

H-

pip

eri

din

e);

4

.5 (

q,

1H

, α

-

CH

-);1

.10

(d,

3H

, C

H3)

XII

Ic

CH3

CH3

0.4

7

C2

2H

34N

2O

3

85

7

0.5

4

(70

.55

)

9.1

3

(9.1

5)

7.4

6

(7.4

8)

7.1

0-7

.40

(m

, 5

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 8

.00

(s

,

1H

, N

H-V

al)

; 2

.45

(d

, 2

H,

-

ArC

H2-)

; 2

.65

(t,

4H

, -C

H2-)

;

1.7

0

(m,

4H

, -C

H2-)

; 1

.90

(m,

1H

, -C

H-p

ipe

rid

ine

); 4

.5

(q,

1H

, α

-CH

-);

1.7

0 (

m,

1H

,

Ch

ap

ter

2

84

βC

H-)

; 1

.2 (

d,

6H

, γC

H3).

XII

Id

C H2

CH

CH3

CH3

0.4

5

C2

3H

38N

2O

3

92

7

1.0

9

(71

.10

)

9.3

3

(9.3

4)

7.2

0

(7.2

1)

7.1

5-7

.40

(m

, 5

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 7

.85

(s

,

1H

, N

H-L

eu

); 2

.4 (d

, 2

H,

-

ArC

H2-)

; 2

.65

(t,

4H

, -C

H2-)

;

1.7

2 (

q,

4H

, -C

H2-)

;1.9

2 (

m,

1H

, -C

H-p

ipe

rid

ine

); 4

.45

(t,

1H

, -α

CH

-);

1.7

0

(t,

2H

,

βC

H2-)

; 1

.45

(m

, 1

H,

-γC

H-)

;

1.2

(d,

6H

, δ

CH

3).

XII

Ie

H2C

0.5

4

C2

6H

34N

2O

3

90

7

3.9

0

(73

.91

)

8.1

0

(8.1

1)

6.6

2

(6.6

3)

7.1

-7.3

0

(m,

10

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 7

.90

(s

,

1H

, N

H-P

he

); 2

.49

(d

, 2

H,

-

ArC

H2-)

; 2

.7 (

t, 4

H,

-CH

2-)

;

1.8

0 (

q,

4H

, -C

H2-)

; 1

.90

(m

,

1H

, -C

H-p

ipe

rid

ine

); 4

.55

(t,

1H

, -α

CH

-);3

.1

(d,

2H

,

βC

H2-)

.

XII

If

H2

CC H2

CO

OH2

C

0.4

0

C2

9H

38N

2O

5

95

7

0.4

1

(70

.42

)

7.7

5

(7.7

6)

5.6

5

(5.6

6)

7.2

-7.4

0

(m,

10

H,

Ar-

H);

1.4

3 (

s, 9

H,

Bo

c);

8.0

(s,

1H

,

NH

-Glu

);

2.4

0

(d,

2H

, -

ArC

H2-)

; 2

.69

(t,

4H

, -C

H2-)

;

1.7

5(q

, 4

H,

-CH

2-)

; 1

.95

(m

,

1H

, -C

H2-)

; 4

.35

(t,

1H

, -

αC

H-)

; 1

.7 (

m,

2H

, -

βC

H2-)

;

1.9

9 (

t, 2

H,

-γC

H2-)

; 5

.15

(s,

2H

, C

H2-P

h).

Ch

ap

ter

2

85

XII

Ig

N HO

O

0.5

1

C3

1H

43N

3O

5

90

6

9.2

3

(69

.25

)

8.0

5

(8.0

6)

7.8

2

(7.8

3)

7.2

5-7

.45

(m,

10

H,

Ar-

H);

1.4

5(s

, 9

H,

Bo

c);

7.9

2(s

, 1

H,

NH

-Lys)

; 2

.42

(d

, 2

H,

-

ArC

H2-)

; 2

.60

(t,

4H

, -C

H2-)

;

1.9

1 (

m,

1H

, -C

H-)

; 1

.80

(q

,

4H

, -C

H2-)

;

4.4

0

(t,

1H

, -

αC

H-)

; 1

.59

(m

, 2

H,

βC

H2-)

;

1.3

5

(m,

2H

, γC

H2-)

;1.4

5

(m,

2H

, δ

CH

2-)

; 2

.10

(t,

2H

,

εC

H2-)

; 4

.85

(s,

2H

, -O

-CH

2-

Ar)

; 8

.0 (

s, 1

H,

NH

).

XII

Ih

H2

CC H2

H2

CN H

N H

NO2

NH

0.4

0

C2

3H

36N

6O

5

92

5

7.9

8

(57

.99

)

7.6

0

(7.6

1)

17

.62

(17

.63

)

7.3

0-7

.45

(m

, 5

H,

Ar-

H);

1.4

5

(s,

9H

, B

oc)

; 7

.90

(s

,

1H

, N

H-A

rg);

2.4

5 (

d,

2H

, -

ArC

H2-)

; 2

.60

(t,

4H

, -C

H2-)

;

1.9

0 (

m,

1H

, -C

H-)

; 1

.7 (

q,

4H

, -C

H2-)

;

4.5

0

(t,

1H

, -

αC

H-)

; 1

.30

(m,

2H

, β

CH

2-)

;

1.2

0 (

m,

2H

, γC

H2-)

; 2

.55

(t,

2H

, δ

CH

2-)

; 8

.05

(m

, 1

H,

gu

an

idin

e).

XII

Ii

NN

H2C

H2

CO

H2

C

0.4

3

C3

1H

40N

4O

4

89

6

9.8

9

(69

.90

)

7.5

5

(7.5

7)

10

.52

(10

.53

)

7.2

5-7

.45

(m,

10

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 7

.90

(s

,

1H

, N

H-H

is);

2.4

9 (

d,

2H

, -

ArC

H2-)

; 2

.75

(t,

4H

, -C

H2-)

;

2.0

0 (

m,

1H

, -C

H-)

; 1

.77

(q

,

4H

, -C

H2-)

; 4

.58

(t

, 1

H,

-

αC

H-)

; 3

.15

(d

, 2

H,

-βC

H2-)

;

6.4

8

(s,

2H

, im

ida

zole

);

4.5

3

(s,

2H

, C

H2);

5

.55

(s

,

Ch

ap

ter

2

86

2H

, C

H2).

XII

Ij

H2C

HN

0.4

9

C2

8H

35N

3O

3

90

7

2.8

5

(72

.86

)

7.6

3

(7.6

4)

9.0

9

(9.1

0)

7.2

0-7

.45

(m

, 9

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 7

.90

(s

,

1H

, N

H-T

rp);

2.4

5 (

d,

2H

, -

ArC

H2-)

; 2

.77

(t,

4H

, -C

H2-)

;

1.9

9 (

m,

1H

, -C

H-)

; 1

.80

(q

,

4H

, -C

H2);

4

.50

(t

, 1

H,

-

αC

H-)

; 3

.12

(d

, 2

H,

βC

H2-)

;

10

.00

(d

, 1

H,

NH

-in

do

le);

6.7

0(s

, 1

H,

-CH

-).

XII

Ik

0.4

1

C2

3H

36N

2O

3

90

7

1.0

9

(71

.10

)

9.3

3

(9.3

4)

7.1

9

(7.2

1)

7.2

0-7

.40

(m

, 5

H,

Ar-

H);

1.4

3

(s,

9H

, B

oc)

; 7

.90

(s

,

1H

, N

H-I

le);

2

.50

(d,

2H

, -

CH

2-)

; 2

.72

(q

, 4

H,

-CH

2-)

;

1.9

5 (

m,

1H

, -C

H-)

; 1

.70

(q

,

4H

, -C

H2-)

; 4

.45

(d,

1H

, -

αC

H-)

; 2

.5(m

, 1

H,

βC

H-)

;

1.9

0 (

m,

2H

, γC

H2-)

; 1

.0 (

d,

6H

, δ

CH

3).

XII

Il

0.4

5

C2

2H

32N

2O

3

90

7

0.9

2

(70

.94

)

8.6

4

(8.6

6)

7.5

0

(7.5

2)

7.2

-7.5

(m

, 5

H,

Ar-

H);

1.4

5

(s,

9H

, B

oc)

; 2

.49

(d

, 2

H,

-

ArC

H2-)

; 2

.70

(t,

4H

, -C

H2-)

;

1.9

0 (

m,

1H

, -C

H-)

; 1

.75

(m

,

4H

,-C

H2-)

; 3

.85

(t,

1H

, -

αC

H-)

; 1

.67

(m

,2H

, -β

CH

2);

1.4

5 (

m,

2H

, γC

H2);

3.1

(t,

2H

, -δ

CH

2);

Chapter 2

87

2.3.4. Biological Studies

2.3.4.1 Antibacterial assay

The antibacterial assay was carried out against gram +ve and gram -ve bacteria by

following the procedure of Perez et al.,86

with slight modifications.

2.3.4.1a Bacterial cultures used:

The following pure cultures of bacteria were obtained from Department of

Pathology and Microbiology, JSS Medical College, Mysore, India.

• Klebesilla pneumoniae (gram –ve)

• Pseudomonas auregenosa (gram –ve)

• Escherichia coli (gram –ve)

• Staphyllococcus aureus (gram +ve)

2.3.4.1b. Media preparation:

Nutrient broth media was used for antibacterial screening and the composition is

as follows: Peptic digest of animal (5 g), sodium chloride (5 g), beef extract (1.5 g) and

yeast extract (1.5 g). The fresh media was prepared by dissolving 13.0 g in one litre

distilled water. The pH of the media at 25

0C was 7.4 ± 0.2 and it was sterilized at 121

0C

(15 lbs pressure) for 15 minutes.

2.3.4.1c. Preparation of inoculums:

One day prior to the testing, inoculation of the bacterial fresh cultures were

cultivated separately into 10 mL of sterile distilled water and kept at 37 0C overnight.

Chapter 2

88

2.3.4.1d. Quantification of bacterial colonies:

After cultivation, the bacteria were harvested and their density was determined

by measuring A600 and referring to previously determined standards. The final density of

bacteria was adjusted to 5 x 106 CFU/mL.

2.3.4.1e. Preparation of samples of synthesized compounds

All the Boc deblocked compounds and standard Strepomycin were dissolved in

presterilized distilled water to get the respective stock solution of concentration 200

μg/mL. For each antibacterial assay, 50 μL of the stock solution was added to the well

made at the centre of the petriplate (10 μg/well). The distilled water was taken as a

negative control where as Strepomycin was used as positive control.

2.3.4.1f. General method for antibacterial assay:

In vitro antibacterial assays were performed against Staphylococcus aureus,

Escherichia coli, Klebesiella pnemoniae and Pseudomonas auregenosa by using agar well

diffusion method.86

The bacterial strains were cultivated in Muller-Hinton broth and the

inocculum concentration was adjusted by the method of mid-logarithmic phase (OD

600=0.5). The molten media was prepared by adding Muller-Hinton agar in sterile distilled

water and autoclaved for 1 hr. The autoclaved molten media was poured into pre-

sterilized 90 mm petriplate and allowed to solidify. Then, the media was scooped out at

the center by using 8 mm sterilized cup-borer and inocculum were spread over the media

and 50 μL of stock solution of compounds (10 µg/well) was added to the well made in the

petriplate and kept for 3-4 days at 37 0C. All the synthesized compounds were tested in

triplicate, Streptomycin was used as positive control and water as negative control. The

zone of inhibition was measured in mm and presented in Table-2.4, 2.6, 2.8 and Figure-

2.3, 2.5, 2.7 respectively.

Chapter 2

89

2.3.4.2. Antifungal activity:

The antifungal activities of the synthesized compounds were evaluated by

following the procedure of Singh et al., 87

with slight modifications.

2.3.4.2a. Fungal cultures used:

The pure cultures of fungi were obtained from Department of Studies in

Microbiology, University of Mysore, Mysore, India. The fungal strains used were,

• Aspergillus niger

• Aspergillus flavus

• Fusarium monoliforme

2.3.4.2b. Media preparation:

Potato dextrose agar (PDA) media was used for the antifungal screening. The

molten media was prepared by dissolving 39 g of PDA in one litre distilled water. The pH

of the media was adjusted to 7.2 and sterilized at 121 0C (15 lbs pressure) for 15 minutes.

One day prior to the testing, inoculation of the fungi were made separately into 25

ml of sterile distilled water and kept at rt.

2.3.4.2c. Preparation of inoculums:

The fresh spores were harvested in sterilized normal saline (0.9 % NaCl in distilled

water containing a drop of Tween-80). Spore suspension was spread on PDA media (20

mL) in a sterile petriplate and incubated at 25 0C until sufficient sporulation occurs (3-6

days).

2.3.4.2d. Quantification of fungal spores:

Approximate quantity of fungal spores was taken in 20 mL of culture broth with

0.02 % Tween-80. Suspension was filtered through double-layered muslin cloth.

There are two types of squares in Haemocytometer viz., big squares and small

squares. The spore count was taken in big squares and spore load or inoculums size per

ml was calculated using the formula,

Chapter 2

90

Inoculum size/ ml = N x10, 000 or N x 104

Where, N = Mean number of chambers counted in one big square.

(Factor 10, 000, because, each big square has a volume of 1mm x 1mm x 0.1 mm or 1/10

cm x 1/ 10 cm x 1/ 100 cm = 1/ 10, 000 cm3 or 10

- 4 cm

3)

Thus, inoculums size for

A. niger = 6.8 x 104/ ml

A. flavus = 4.6 x 104/ ml

F. monoliforme = 5.2 x 104 / ml

2.3.4.2e. Preparation of samples of synthesized compounds

All the Boc deblocked compounds and standard Bavistin were dissolved in

presterilized distilled water to get the respective stock solution of concentration 10

μg/mL. For each antifungal assay, 0.4 mL of this stock solution was taken and spreaded

over the media of petriplate uniformely. The distilled water was taken as a negative

control and Bavistin is used as positive control.

2.3.4.2f. General method of antifungal assay:

In vitro antifungal assays were performed against Aspergillus niger, Aspergillus

flavus and Fusarium monoliforme by using agar well diffusion method. The fungal cultures

were raised by growing on PDA media of pH 7.4 for six days at 25 0C. The spores were

harvested in sterilized normal saline (0.9 % NaCl in distilled water) and its concentration

was adjusted to 1 x 106/ml with a Haemocytometer. The autoclaved molten media (20mL)

was poured in to each 90 mm sterilized petriplate and allowed to solidify. To study the

growth response of fungi species, 0.4 mL of the synthesized compounds (10 µg/mL) was

poured in to each plate and spreaded uniformly over the agar media. A volume of 10 µl

spore suspension was poured in to the small depression made at the center of the plate

and kept for 6 days at 25 0C. After six days of incubation, the plates were observed and

compared with their respective controls. The control plates contained only distilled water

for which fungal growth is taken as 100% growth (no inhibition). The fungicidal activity of

Chapter 2

91

the synthesized compounds (Analogues - I, II, III) was assessed by comparing the zone of

fungal growth in treated plates with that of control plates in mm and the results are

presented in (Table - 2.5, 2.7, 2.9 and Figure - 2.4, 2.6 and 2.8) respectively.

2.3.4.3. Evaluation of antioxidant activity by using DPPH

(1, 1-Diphenyl-2-picrylhydrazyl) radical scavenging method:

The DPPH radical scavenging effect was carried out according to the method

employed by Blois.88

Compounds of different concentrations (Analogues I, II, III) were

dissolved in DMF and prepared in distilled ethanol, 1mL of each compound solutions having

different concentrations (10 µM, 50 µM, 100 µM, 150 µM and 250 µM) were taken in

different test tubes, 4 mL of a 0.1 mM ethanol solution of DPPH was added and shaken

vigorously. A DPPH blank was prepared (without compound) using 1 mL of DMF along with 4

mL of ethanol for the baseline correction. The tubes were then incubated in dark room at rt

for 20 min. The change (decrease) in the absorbance at 517 nm was measured using a UV-

Visible Spectrophotometer. Percent decrease in the absorbance was recorded for each

concentration and calculated on the basis of the observed decrease in absorbance of the

radical. BHA and AA were used as reference standards. The radical scavenging activity was

expressed as the inhibition percentage and was calculated using the formula:

Radical scavenging activity (%) = [(A0 – A1) / A0 ×100]

Where A0 = Absorbance of the control (blank, without compound) and

A1 = Absorbance of the compound.

IC50 (concentration required for 50 % inhibition) value of the compound can be

calculated by plotting graph, taking concentration of compounds on X-axis and % of

radical scavenging activity on Y-axis. IC50 values of analogues-I, II and III are presented in

(Table-2.10 - 2.12) respectively.

Chapter 2

92

Tables and Figures

Table - 2.4: Antibacterial activity of Analogues – I:

Compoundsa

Inhibitory Zone (diameter) mmb

Staphylococcus

aureus

Escherichia

coli

Klebesiella

pneumoniae

Pseudomonas

auregenosa

IV 04 03 03 04

VIa 08 08 05 06

VIb 07 08 06 07

VIc 09 08 07 08

VId 09 08 09 10

Vie 13 13 12 11

VIf 10 09 09 08

VIg 07 07 06 07

VIh 07 06 05 06

Vii 10 09 07 09

VIj 12 12 11 14

VIk 05 04 05 06

VIl 07 06 07 08

Streptomycin 12 12 10 11

a Concentration of compounds and reference drug: 10 µg/well.

b Values are mean of three determinations, the ranges of which are less than 5% of the

mean in all cases.

Chapter 2

93

Table - 2.5: Antifungal activity of Analogues – I:

Compoundsa

Inhibitory Zone (diameter) mmb

Aspergillus

niger

Aspergillus

flavus

Fusarium

monoliforme

IV 02 04 05

VIa 05 04 05

VIb 06 05 04

VIc 02 07 05

VId 06 06 05

Vie 06 07 05

VIf 07 05 07

VIg 05 03 03

VIh 05 06 04

Vii 07 08 07

VIj 05 07 06

VIk 04 05 06

VIl 06 05 06

Bavistin 09 10 09

a Concentration of compounds and reference drug: 10 µg/mL

b Values are mean of three determinations, the ranges of which are less than 5% of the

mean in all cases.

Chapter 2

94

Table - 2.6: Antibacterial activity of Analogues – II:

Compoundsa

Inhibitory Zone (diameter) mmb

Staphylococcus

aureus

Escherichia

coli

Klebesiella

pneumoniae

Pseudomonas

auregenosa

IX 03 02 02 03

XIa 04 06 04 03

XIb 05 06 05 06

XIc 07 06 05 04

XId 08 07 06 08

XIe 09 11 09 11

XIf 04 07 04 05

XIg 06 08 05 06

XIh 04 06 06 07

XIi 08 09 08 11

XIj 10 09 12 12

XIk 07 05 05 06

XIl 08 09 07 06

Streptomycin 12 12 10 11

a Concentration of compounds and reference drug: 10 µg/well.

b Values are mean of three determinations, the ranges of which are less than 5% of the

mean in all cases.

Chapter 2

95

Table - 2.7: Antifungal activity of activity of Analogues – II:

Compoundsa

Inhibitory Zone (diameter) mmb

Aspergillus

niger

Aspergillus

flavus

Fusarium

monoliforme

IX 02 03 04

XIa 02 03 03

XIb 02 04 04

XIc 04 06 05

XId 07 06 06

XIe 10 09 08

XIf 07 09 09

XIg 06 07 08

XIh 05 03 05

XIi 05 06 04

XIj 09 10 08

XIk 02 03 04

XIl 05 04 06

Bavistin 09 10 09

a Concentration of compounds and reference drug: 10 µg/mL

b Values are mean of three determinations, the ranges of which are less than 5% of the

mean in all cases.

Chapter 2

96

Table - 2.8: Antibacterial activity of Analogues – III:

Compoundsa

Inhibitory Zone (diameter) mmb

Staphylococcus

aureus

Escherichia

coli

Klebesiella

pneumoniae

Pseudomonas

auregenosa

XII 01 02 02 02

XIVa 03 04 04 03

XIVb 04 04 03 03

XIVc 05 04 05 04

XIVd 06 05 06 05

XIVe 08 07 06 07

XIVf 04 06 06 04

XIVg 06 06 05 06

XIVh 04 06 05 05

XIVi 08 06 07 08

XIVj 08 08 07 07

XIVk 06 05 06 05

XIVl 06 05 05 06

Streptomycin 12 12 10 11

a Concentration of compounds and reference drug: 10 µg/well.

b Values are mean of three determinations, the ranges of which are less than 5% of the

mean in all cases.

Chapter 2

97

Table - 2.9: Antifungal activity of activity of Analogues – III:

Compoundsa

Inhibitory Zone (diameter) mmb

Aspergillus

niger

Aspergillus

flavus

Fusarium

monoliforme

XII 02 02 02

XIVa 02 02 03

XIVb 03 03 03

XIVc 04 04 03

XIVd 04 05 03

XIVe 04 06 05

XIVf 05 04 04

XIVg 04 04 05

XIVh 04 04 03

XIVi 05 05 06

XIVj 05 06 06

XIVk 05 05 04

XIVl 05 04 04

Bavistin 09 10 09

a Concentration of compounds and reference drug: 10 µg/mL

b Values are mean of three determinations, the ranges of which are less than 5% of the

mean in all cases.

Chapter 2

98

Table - 2.10: Antioxidant activity of Analogues – I:

Entry IC50 in µM

IV >250±0.11

VIa >250±0.11

VIb >250±0.13

VIc >250±0.15

VId >250±0.12

VIe >250±0.14

VIf >250±0.10

VIg >250±0.11

VIh >250±0.15

VIi >250±0.15

VIj 42.5±0.13

VIk >250±0.12

AA 15±0.10

BHA 15±0.12

Values represent mean ± SD (n=3).

IC50: Concentration required for 50% reduction of 0.1 mM DPPH radical.

Chapter 2

99

Table - 2.11: Antioxidant activity of Analogues –II:

Entry IC50 in µM

IX >250±0.10

XIa >250±0.15

XIb >250±0.15

XIc >250±0.10

XId >250±0.10

XIe >250±0.12

XIf >250±0.11

XIg >250±0.11

XIh >250±0.11

XIi >250±0.15

XIj 45±0.14

XIk >250±0.11

AA 15±0.12

BHA 15±0.11

Values represent mean ± SD (n=3).

IC50: Concentration required for 50% reduction of 0.1 mM DPPH radical.

Chapter 2

100

Table - 2.12: Antioxidant activity of Analogues –III:

Entry IC50 in µM

XII >250±0.10

XIVa >250±0.12

XIVb >250±0.12

XIVc >250±0.10

XIVd >250±0.11

XIVe >250±0.13

XIVf >250±0.12

XIVg >250±0.15

XIVh >250±0.12

XIVi >250±0.12

XIVj 49±0.14

XIVk >250±0.10

AA 15±0.11

BHA 15±0.14

Values represent mean ± SD (n=3).

IC50: Concentration required for 50% reduction of 0.1 mM DPPH radical.

Chapter 2

101

Figure 2.3: Antibacterial activity of Analogues-I

Figure 2.4: Antifungal activity of Analogues-I

Figure

Figure

0

2

4

6

8

10

12

14

Zo

ne

of

Inh

ibit

ion

(m

m)

S.aureus

0

2

4

6

8

10

12

14

Zo

ne

of

Inh

ibit

ion

(m

m)

A. niger

102

Figure 2.5: Antibacterial activity of Analogues-II

Figure 2.6: Antifungal activity of Analogues-II

Compounds

E. coli K. pneumoniae

Compounds

A. niger A. flavus F. monoliforme

Chapter 2

II

II

P. auregenosa

F. monoliforme

Figure

Figure

0

2

4

6

8

10

12

14

Zo

ne

of

Inh

ibit

ion

(m

m)

S.aureus

0

2

4

6

8

10

12

14

Zo

ne

of

Inh

ibit

ion

(m

m)

A. niger

103

Figure 2.7: Antibacterial activity of Analogues-III

Figure 2.8: Antifungal activity of Analogues-III

Compounds

E.coli K.pnemoniae

Compounds

A. niger A. flavus F. monoliforme

Chapter 2

III

III

P.auregenosa

F. monoliforme

Ch

ap

ter

2

10

4

Fig

ure

-2.9

. 1H

NM

R S

pe

ctra

of

Bo

c-A

la-B

HP

(V

b,

An

alo

gu

es-

I)

Ch

ap

ter

2

10

5

Fig

ure

-2.1

0.

1H

NM

R S

pe

ctra

of

Bo

c-Le

u-B

HP

(V

d,

An

alo

gu

es-

I)

Ch

ap

ter

2

10

6

Fig

ure

-2.1

1.

1H

NM

R S

pe

ctra

of

Bo

c-P

ro-B

ZP

(X

e,

An

alo

gu

es-

II)

Ch

ap

ter

2

10

7

Fig

ure

-2.1

2.

1H

NM

R S

pe

ctra

of

Bo

c-P

he

-BZ

P (

Xl,

An

alo

gu

es-

II)

Ch

ap

ter

2

10

8

Fig

ure

-2.1

3.

1H

NM

R S

pe

ctra

of

Bo

c-A

la-B

P (

XII

Ib,

An

alo

gu

es-

III)

Ch

ap

ter

2

10

9

Fig

ure

-2.1

4.

1H

NM

R S

pe

ctra

of

Bo

c-V

al-

BP

(X

IIIc

, A

na

log

ue

s-II

)

Chapter 2

110

2.4. Results and Discussion

We have synthesized a new class of amino acids conjugated benzhydrilpiperazine

(analogues-I, Va-Vl), benzylpiperazine (analogues-II, Xa-Xl) and 4-benzylpiperidine

(analogues-III, XIIIa-XIIIl) by coupling benzhydrilpiperazine, benzylpiperazine and

4-benzylpiperidine respectively with N-protected Boc-amino acids (a-l) using EDCI/HOBt

as coupling agent and NMM as a base. The product obtained was gummy and

characterized by TLC, elemental analysis and 1H NMR. The Boc deprotected synthesized

analogues were used for both antimicrobial and antioxidant activities.

Antibacterial activity:

Structural activity relationship of analogues-I, II and III

All the Boc deprotected analogues I, II and III were tested against strains of gram +ve

and gram -ve bacteria such as Staphylococcus aureus, Klebesiella pneumoniae,

Pseudomonas auregenosa and Escherichia coli. Streptomycin was used as positive control

and water as a negative control. The concentration used for both test compounds and

that of standard remains the same. Among all the synthesized analogues, analogues-I

showed highest activity in comparison to the other two analogues. In analogues-I,

benzhydrilpiperazine conjugated phenylalanine VIe and tryptophan VIj showed equally

good antibacterial activity as that of conventional antimicrobial drugs, where as

benzhydrilpiperazine conjugated valine VIc, leucine VId and histidine VIi showed

moderate activity when compared to the standard drug. The observed enhancement in

antibacterial activity of the above said synthesized analogues may be due to the presence

of both the heterocyclic moiety and amino acid functionalities in the synthesized

compounds. The following factors may be held responsible for the enhancement of

antibacterial activity, viz., (i) the hydrophobicity of amino acid side chains (ii) the presence

of aromatic group in phenylalanine (iii) the presence of heterocyclic indole ring in

tryptophan and (iv) the presence of imidazole ring in the side chain of histidine. The

Chapter 2

111

presence of these helps the molecule to interact/penetrate more with cell membrane of

the microorganisms thereby inactivating them.

On the other hand the two phenyl rings of benzhydril group and the basicity of the

two nitrogen atoms may also influence the activity. The remaining compounds which are

aliphatic in nature showed slightly increasing activity from glycine (having only one

hydrogen atom), alanine (with one methyl group), valine (presence of two methyl groups)

to leucine (with more number of carbon atoms comparatively) analogues. Thus, it can be

summarized that as the number of alkyl groups increased the activity also increased. Even

though, benzhydrilpiperazine which taken in isolation was inactive or weakly active

towards these bacterial strains, upon conjugation with various amino acids, there is a

marked increase in the activity.

Where as the benzylpiperazine derivatives (analogues-II) showed less activity over

the benzhydrilpiperazine derivatives (analogues-I) which may be due the lack of one

phenyl ring in the benzylpiperazine moiety (analogues II). Since, there is one additional

phenyl ring in the benzhydril part, this may be held responsible for the difference in

activity between the two analogues. In analogues-II also phenylalanine XIe, histidine XIi

and tryptophan XIj conjugates showed highest activity but lower to conventional

antibiotics, and the remaining showed moderate activity.

4-Benzylpiperidine derivatives (analogues-III) showed less activity among the three

analogues, here the absence of one nitrogen atom in piperidine ring compared to the

piperazine ring may held responsible for the less activity. In analogues-III, phenylalanine

XIVe, histidine XIVi and tryptophan XIVj conjugates showed highest activity, but lower

than other two analogues-I, II and conventional antibiotics and the remaining showed

moderate activity.

Antifungal activity:

All the synthesized analogues were tested against fungal strains such as Aspergillus

niger, Aspergillus flavus and Fusarium monoliforme. Among all the synthesized analogues,

analogues-II, showed highest antifungal activity. Among the analogues-I, phenylalanine

Chapter 2

112

analogues VIe, histidine analogues VIi and tryptophan analogues VIj showed better

activity over the other compounds, but lower to conventional standard drug bavistin. The

other compounds in all the three analogues showed mild to moderate antifungal activity.

Here also the factors explained under antibacterial activity equally holds good.

Thus, the compounds containing Trp, Phe and His residues showed enhancement

towards both antibacterial and antifungal activities. This may be attributed due to the

presence of aromatic systems both in the amino acid residue as well as in the heterocyclic

system. The presence of aromaticity in both the moieties enhanced the antimicrobial

properties of the compounds synthesized. But this is not the case with other molecules

where in the amino acid residues lacks the aromatic ring system (with only aliphatic side

chains) and presence of aromaticity in heterocyles only reveals the activity towards good

to moderate properties. Hence, the molecules exhibiting enhanced property could be

regarded as lead molecules in the series.

Benzylpiperazine derivatives (analogues-II) showed less activity over the analogues-I.

In contrast, phenylalanine (XIe) and histidine (XIi) conjugated benzylpiperazine

derivatives showed marginal increased activity to their respective benzhydrilpiperazine

counterparts (VIe and VIi).

Where as the analogues-III (4-benzylpiperidine derivatives) showed less activity than

the other two analogues, here it could be due to the absence of the basic nitrogen atoms

as in the case of the piperazine ring, and here the piperidine ring lack one nitrogen atom

may influence the activity moderately. In analogues-III, phenylalanine XIVe, histidine XIVi

and tryptophan XIVj conjugates showed highest activity, but lower to other two

analogues - I, II and conventional antibiotics and the remaining compounds in the series

showed moderate activity.

Overall the results of antibacterial and antifungal activities showed the importance of

amino acids conjugation with the heterocycles.

Chapter 2

113

Antioxidant activity

All the synthesized analogues I, II, III were evaluated for antioxidant by using

DPPH radical scavenging method. In all the three analogues, analogues-I showed better

activity by a small margin, but analogues III showed less activity which may be due to lack

of one nitrogen in piperidine ring compared to piperazine motif. From the literature, it is

revealed that the compounds carrying the phenolic hydroxyl group (-OH) and the

aromatic secondary -NH have shown potent antioxidant activity. The lone pair of

electrons on the hydroxyl and -NH involved in electron transfer reaction with DPPH

radical. In agreement with this, in all the three analogues synthesized, tryptophan

conjugate showed potent activity. The observed activity may be due to the presence of

secondary -NH in indole ring of tryptophan but showed less activity compared to standard

drug AA and BHA. The remaining compounds in all the three analogues showed poor/no

activity ie., >250µM. Among tryptophan derivatives of three analogues (VIj, XIj and XIVj),

benzhydrilpiperazine analogues showed highest activity, followed by benzylpiperazine

and 4-benzylpiperidine analogues. Benzhydrilpiperazine, benzylpiperazine and 4-

benzylpiperidine taken in isolation showed no activity (>250 µM).

In all the three synthesized analogues, results show the importance of amino acids

substituents in DPPH free radical effect.

Chapter 2

114

2.5. Conclusion

In an effort to discover a new heterocyclic conjugated amino acid analogue as

novel bioactive molecule, we found that phenylalanine, tryptophan, histidine and proline

analogues showed good antimicrobial as well as antioxidant activities, among the other

tested amino acid conjugates. In comparison to the three amino acid conjugated

analogues, benzhydrilpiperazine conjugated amino acid analogues showed the highest

antimicrobial and antioxidant activities which may be due to the effect of two nitrogen

atoms of benzhydrilpiperazine moiety which is absent in other two analogues.

On the whole, all the amino acid conjugates of benzhydrilpiperazine,

benzylpiperazine and 4-benzylpiperidine have showed enhanced antibacterial, antifungal

and antioxidant activities when compared to the respective parent compounds. The

enhancement of biological activities might be due to the combined effect of both

heterocyclic skeleton and amino acid residues. The obtained results indicate that further

study on conjugation of these heterocycles with the peptides of varying chain

length/composition might be of interest for the identification of this new class of

antimicrobial and antioxidant agents.

Chapter 2

115

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