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Chapter 1

Isolation of arjunolic acid from

Terminalia Arjuna plant and

synthesis of its derivatives

Chapter 1

1

1.1 Introduction

Natural products are chemical substances, which are obtained from nature and posses

biologically distinctive properties. These compounds play a vital role in the field of

medicinal chemistry. Among natural compounds, terpenoids are one of the main

groups of compounds being used either as a drug or as a drug intermediate. The term

‘terpene’ was coined after extracted from terpentine, which is a volatile liquid from

pine trees. There is a tendency to use the term ‘terpenoids’ instead of terpenes that

includes hydrocarbons and oxygen containing compounds, such as alcohols,

aldehydes or ketones. In modern definition “Terpenoids are the hydrocarbons of plant

origin of the general formula (C5H8)n as well as their oxygenated, hydrogenated and

dehydrogenated derivatives. Majority of terpenoids extracted from plant sources1,2

and very few of them also obtained from other sources like animals and microbes.3

The simpler terpenoids are volatile substances, but higher terpenoids are non-volatile

in nature.4 The international union of pure and applied chemistry (IUPAC)

recommends a systematic nomenclature of terpenoids; however, the names are not

only lengthy but also quite cumbersome. Therefore, the trivial names of

most terpenoids are used most frequently even today for naming.

1.1.1 Isoprene rule

Isoprene (methylbuta-1,3-diene, called hemiterpenes) is a hydrocarbon with the

molecular formula C5H8, which is a gaseous 1,4-diene and emitted from the leaves of

various plants during the metabolism as a byproduct. It is the most common volatile

organic compound found in nature after methane.5

Isoprene

HeadTail

Chapter 1

2

Isoprene has a “head” and a “tail”, the linking between two or more isoprene units can

occur in three ways;

1. The head of one isoprene unit can join with the head of another isoprene unit, is

called a head-to-head or 1-1 link.

2. The head of one isoprene unit can join with the tail of another isoprene unit, is

called a head-to-tail or 1-4 link.

3. The tail of one isoprene unit can join with the tail of another isoprene unit, is

called a tail-to-tail or 4-4 link.

The isoprene rule states that in most naturally occurring terpenes isoprene units are

adds through head to tail or 1,4-arrangements (Figure 1.1).

head

tail tail

tail

head

headhead

head

Figure 1.1: General representation of isoprene rule

1.1.2 Classification of terpenes

Most naturally occurring terpenoids are hydrocarbons with general formula (C5H8)n,

and the value of n is used as a basis for classification.

Chapter 1

3

Table 1.1: Classification of terpenes

Sl. No. Terpenes Isoprene units Carbon atoms

1 Monoterpenes 2 10

2 Sesquiterpenes 3 15

3 Diterpenes 4 20

4 Sesterterpenes 5 25

5 Triterpenes 6 30

6 Carotenoids 8 40

7 Rubber > 100 > 500

Examples for simple terpenes as follows;

Myrcene

CH2OH

Farnesol

OH

Vitamin A

Chapter 1

4

Carotenoids

Each class of terpenes further classified into different groups on the basis of the

number of rings present in the molecule,6,7 this as follows

Acyclic terpenoids

Monocyclic terpenoids

Bicyclic terpenoids

Tricyclic terpenoids and

Tetracyclic terpenoids

(a) Monoterpenoids are made up of two isoprene units and they are the chief

constituents of the essential oils.8

Examples:

CHOOH

Citral (Acyclic) Menthol (Monocyclic) Pinane (Bicyclic)

(b) Sesquiterpenoids are made up of three isoprene units. These are mainly obtained

from the higher boiling fraction of the essential oils

Examples:

CH2OH

Farnesol(Acyclic)

Ziniberene(Monocyclic)

Cadinene(Bicyclic)

Chapter 1

5

(c) Diterpenoids are made up of four isoprene units and most commonly found in

plants and animals.

Examples:

CH2OH

Phytol (Acyclic)

Vitamin A (Monocyclic)

OH

H2N

Abietic acid

HO

Dehydroabietylamine

O

(d) Triterpenoids are made up of five isoprene units and isolated from the liver oil of

shark. Other sources are olive oil and several other vegetable oils.9,10

Examples:

Squalene

(e) Tetraterpenoids are made up of eight isoprene units.11

Examples:

-Carotene

Chapter 1

6

ZeaxanthinHO

OH

(f) Polyterpenoids are made up of a large number of isoprene units and obtained

majorly from plant latex.

Examples:

Natural Rubber

1.1.3 Biogenetic isoprene rule

To accommodate ‘irregular’ terpenoids, ‘the biogenetic isoprene rule’ was proposed

by Ruzicka in 1953. It explains the terpenes derived from a number of biological

equivalents of isoprene units are joined in a ‘head-to-tail’ manner, but sometimes

isoprenes are on subsequent modification by enzymes to provide greater diversity in

terpenes structure.

As of now more than 25,000 terpenoids are isolated and structurally characterized and

proved that terpenoids are major constituents when compared to any other class of

naturally occurring compounds.2,12

In fact, the chemical ecology is predominantly depending on the occurrence of plant

terpenoids and hence the latter play a broad-spectrum of highly characteristic and

specific roles in the plant kingdom, such as: insect propellents13–15 and antifeedants,16

phytoalexins17–20 attractant for pheromones,21,22 defensive substances against

herbivorous animals,23–25 allelochemicals,26,27 signal molecules and in addition to

above it acts as the plant growth hormones.

Chapter 1

7

Isoprene units are also associated within the skeleton of other natural compounds for

example, indole alkaloids, quinines (vitamin K),28,29 alcohols (vitamin E) also known

as terpenols or polyprenols,30–32 vitamin A formed from β-carotene, phenols.33

Further, molecular regulation, biogenesis and role of plant terpenoids have been

significantly reported.34

Ubiquinone also called as coenzyme Q conjugated with isoprene unit and it is

responsible for biochemical conversion into varieties of compound are reported.35

1.1.4 Introduction to triterpenoids

Triterpenoids are an important class of compounds like steroids and sterols. About 20

different groups of triterpenoids are known in nature and abundantly present in plant

and animal sources. Phytosterols occur in plants,36,37 zoosterols occur in animals38,39

and mycosterols found in micro organisms.40,41 Actually triterpenoids bearing about

thirty carbon atoms in their skeleton and most naturally occurring triterpenoids are

biosynthesized from squalene.42,43

Squalene

Most steroids are obtained from squalene by various processes either by cyclistion or

loss of small molecules or ring contractions or expansion. Synthesis of cholesterol is a

suitable example.41

HO

Cholesterol

Chapter 1

8

The prostane cation is obtained from squalene synthon through cyclisation of chair-

boat-chair-boat conformations. This cation can convert to lanostrane chain, which is a

biological precursor for most of steroids.44

Further, protostane cation undergoes the cyclisation between 9th and 19th carbon to

form cycloartane skeleton. Majority of plants synthesize their triterpenoids from the

cycloartane skeleton. These triterpenes are commonly known as phytosterols.

Majority of triterpenoids bears methyl groups at the 10th and 13th carbon and an alkyl

side chain at 17th carbon.

Lanostrane Cycloartane

Table 1.2: Common triterpenoids

H

COOH

HO

HOHH

OHAsiatic acid

O

O

OH

H

O

H

OHO

OHHO

OH

OHH

OH

OH

OH

Astragaloside IV

Chapter 1

9

HHH

Cucurbita 5-ene

O

O

OH

H

O

H

OHO

OHHO

OH

OHH

OH

OH

OH

Astragaloside IV

O

O

OH

H

O

H

OHO

OHHO

OH

OHH

OH

OH

OH

Astragaloside IV

O

O

HO

Diosgenin

OH

O O

O

O

OH

H

OH

H

Ganadoric acid A

O

OOO

OH

OH

HO

OH

OHHO

OH

H

OH OH

H

H

Gensenoside

Chapter 1

10

H

HOH

H-amyrin

HHO

H

COOHH

H

Betulinic acid

COOH

H

HO

H

-Boswellic acid

O

O

O

OH

H3CO

O

O

O

H

H

OHH

Oleandrin

O

HOOC

H

OH

Clestral

COOH

H

OO

O

O

HOOCHO

HO

OHHOOC

OH

OH

O

H

H

Glycyrrhizin

OH

COOH

HOH

HEnoxolone

HO

H

H H

H

Lupeol

Chapter 1

11

HO

H

HHO

OH

H COOH

HOMadecassic acid

H

HHO

H COOH

Oleanolic acid

O

O

OO

OO

O

OO

OO

HO

OHOHOH

OHOH

OH

OH

OH

OH

OHOH

OHOH

HO

OHOH

Platycodin D

O

O

HO

Pristimerin

O

NC CO2Me

HCDDO

OOOOHOHOHO HO

OH

OH

O

H

OH

OHSaikosaponin A

HO

HOH

H

H

HO Soyasapogenol B

OO

O

H

OH

H

OH O

HH

H

Withanolide A

Chapter 1

12

HO

OH

COOHSexangulic acid

H

O

COOH

Pisticialanstenoic acid

CO2Me

O

OOHHO

Scolopianate A

O

O O

H H

Dinorcucrbitane

CO2H

O O

H H

Pentanorcucurbitacins

O

OOH

H

Octanorcucurbitacins

O

HO

H

O

O

Lippiolide

O

HOOCH

COOH

3,4-Secolupane

Triterpenoids are extensively used as an ingredient in healthcare and their several

therapeutic applications are documented.45,46 The pharmacological effects and

potential remedies of triterpenoids as antioxidant,47 anti-inflammatory, anticancer,48

Chapter 1

13

antidiabetic,49,50 hypertension protection,51,52 anti-rheumatoid arthritis,53 wound

healing,54,55 brain improvement and neuroprotective effect56,57 gastric ulcer

prevention,58,59 cardioprotection,60,61 anxiolytic activity.62,63 The clinical study of

triterpenoids has shown its potential in venous hypertension-related improvements. 64

1.1.5 Introduction to Terminalia arjuna

In ancient days plant sources were widely used for the treatment of several

diseases.65–68 Even today, there is an increasing the awareness of natural products

from medicinal plants. Particularly, herbal drugs, because these are easily available,

safe, less expensive, efficient, and rarely have side effects.

World Health Organization reports revealed that the medicinal plants are the best

source to access a variety of drugs. The major constituents of medicinal plants are

small organic compounds, which provide specific physiological action on the human

body and these substances include tannins, alkaloids, carbohydrates, terpenoids,

steroids, flavonoids and phenols.

The biologically active phytocompounds are produced either by primary or secondary

metabolism of living organisms. The metabolites are chemically diverse compounds

and very difficult to understand their function. These are widely utilised in human

therapy, veterinary, agriculture, scientific research and many other. Active

metabolites, chemical constituents are extracted from the various parts of plants like

seeds, berries, leaves, bark, root or flowers. But the knowledge of the chemical

constituents of particular plants is essential for extraction/isolation of complex

chemical substances.

Terminalia arjuna is a large sized fluted tree with an average height of 30 m and 2-2.5

m diameter (Figure 1.2 and Figure 1.3). This tree belongs to Combretaceae family

Chapter 1

14

and encountered in the South Asian region. The name ‘Arjuna’ for this tree was

mentioned in the Rig Veda and Atharva Veda. ‘Arjuna’ means that ‘white’ or

‘bright’, probably denoted due to its creamy white flowers or the shining quality of its

bark.

Figure 1.2: Terminalia arjuna tree during summer season

Vernacular Names of Arjuna tree are mentioned below [www.ecoindia.com]

Kannada: Holé matti, Maddi, Matti.

Hindi: Anjan, Anjani, Arjun, Arjun, Kahua.

Sanskrit: Kakubha, Partha, Indradru, Dhavala, Devasala.

English: Arjun, White Marudah.

Telugu Vella marda, Vella matti, Yer maddi.

Tamil: Vella marda, Vella maruthu, Vella matti.

Bengali: Arjun, Arjhan.

The Arjuna tree is usually greenish leaves appearing in the summer season (February

to April). This tree is one of the most versatile medicinal plants with a wide spectrum

of biological activity.68 The extract of Terminalia arjuna is used as an antidysentric,

antipyretic, astringent, cardiotonic, lithotriptic, anticoagulant, hypolipidemi,

antimicrobial, diuretic, antihypertension and antiuremic agents. Recently in vivo

Chapter 1

14




bark.















Chapter 1

14




bark.















Chapter 1

15

studies showed that its leaves act as analgesic and anti-inflammatory properties.

Tumour growth in animal models is reduced with extract or isolated arjunolic acid

(commonly seen as the main bioactive) as is reduced DNA damage in response to

mutagens69–71 and these are attributed to the antioxidative capacity of arjuna which is

comparable to Vitamin C on a per weight basis. Many useful constituents are isolated

from Terminalia arjuna which includes triterpenoids for cardiovascular properties,

tannins and flavonoids for its anticancer properties, antimicrobial properties and so

on.72

Figure 1.3: The bark of terminalia arjuna is thin, shiny smooth and greenish-grey

colour.

Other possible medical applications of arjunolic acid are platelet aggregation,

coagulation, antioxidant status and gastritis with potency similar to Rantidine,

cytotoxic studies, 70,73 protective role in response to streptozotocin-induced type-2

diabetic through mitochondrial independent and dependent pathways,69 protection to

the liver and kidney likely mediated by antioxidative properties.74

Chapter 1

16

The dried bark of Terminalia arjuna has extensively used as a cardiotonic75 and for

injury or wound, emaciated condition, poison, blood disorders, obesity, urinary

disorders, and ulcer or wound. Traditionally, the drug has been administered as an

alcoholic decoction of the bark or taken with clarified butter or boiled in milk.

Major constituents of Terminalia arjuna tree are

Table 1.3: Common triterpenoids

O

O

O

HH

H

O

OH

H

Arjunolic acid (1)

O

OHH

O

O

HHO

Glu

HOArjungenin

O

OHH

O

OH

HHO

Arjunic acid

O

OH

O

O

HHO

Glu

HO

Glu

O

Arjunolitin

O

OHH

O

O

HHO

Glu

Arjunetin

O

OHH

HHO

Terminoic Acid

Chapter 1

17

O

COOH

H

OHO

Terminic acid

O

OCOOCH3

HH

H

Methyl maslinate

O

O

CH2OH

COOCH3

HH

H

Hederagenin methyl ester

OCH2OH

COOH

H

H

Hederagenin

HO

COOH

H

HO

Mislinic acid

O

OCOOH

H

HHO

Galactose

O

Arjunoside I

Chapter 1

18

Table 1.4: Common tannins

O

O

HO

HO

HOO

OO

OHHO

HO

OO

HO

HO OH

HO OO

OH

OHHO

Tetragolyl glucose

O

O

HO

HO

HOO

OO

OHHO

HO

OO

HO

HO OH

OO

OHHO

HO

OO

OH

OHHO

Pentagolyl glucose

O

O OH

OH

HO

HO

O

O

Ellagic acid

Table 1.5: Common flavonoids

O

OOH

HO

OHOH

Luteolin Leucocyanidin

O

OHOH

HO

OHOH

OH

Chapter 1

19

Kaempferol

O

OOH

OH

HO

OH

1.2 Scope of present work

From the above literature review it has been found that the Terminalia arjuna tree

posses a wide spectrum of medicinal applications. Particularly the principal

constituent, arjunolic acid is a valuable intermediate. Therefore there is a need to

improve the extraction methods to isolate the pure arjunolic acid with high yield and

also it is desirable to construct its newer derivatives with significant biologically.

1.3 Isolation and characterization of arjunolic acid

Presently there is a very limited methods are available for the isolation of free

arjunolic acid from Teminalia arjuna tree, because the bark extract contains glycoside

conjugated arjunolic acid also called saponin and low yield. Therefore, the

development of new method for the direct isolation of arjunolic acid with high yield is

desirable. Hence, herein we have chosen the wood instead of bark of Teminalia

arjuna tree. This method involves de fatting Terminalia arjuna heart wood by new

technology of supercritical fluid extraction method (SFE). Then wood residue left

after SFE was subjected to extraction with ethyl acetate followed by conventional

purification through column chromatography.

The modern extraction technique (Figure 1.4), supercritical fluids (SCFs) extraction

is highly attractive because of ‘health and safety’ and also eco-friendly in nature due

to avoiding the usage of organic solvents (DCM, Chloroform, Benzene etc.). In

Chapter 1

20

addition to this, SCFs, CO2 is widely used in industrial purification and

recrystallization operations.

Figure 1.4: Schematic diagram of supercritical fluid extraction technique

The heart wood of Terminalia arjuna was collected and cut into small pieces and

further dried in an incubator at 40 – 45 °C for 5 days. Later crushed in an electrical

grinder, then the powder was sieved through 20 mesh, 5 kg of sieved powder was

subjected to SFE (Figure 1.5) at 35-40 °C at 300 bar pressure for 24 hrs to remove the

greasy pigmented non-polar materials.

Chapter 1

21

Note: Curtesy, Sami labs ltd SFE facility, Bangalore.

Figure 1.5: Supercritical fluid extraction plant

Then the residue was extracted in methanol for 2 hrs in Soxhlet apparatus. The extract

was filtered through Whatman No. 1 filter paper and the resulting filtrate was

concentrated under reduced pressure at 40 °C in rotary evaporator. The thick gummy

residue was dissolved in 40% aqueous methanol followed by extraction with ethyl

acetate (liquid-liquid extraction), the separated ethyl acetate layer was dried over

anhydrous magnesium sulphate, filtered and evaporated the solvent under reduced

pressure at 40 °C on rota-evaporator to afford off-white crude solid.

Further the crude solid was purified by column chromatography using silica gel (60-

120 mesh). Initially, the elution was subjected in different solvent systems, starting

with 100% hexane followed by hexane:chloroform graded mixtures (95:5, 90:10,

80:20, 70:30, 60:40, 50:50) then with 100% chloroform followed by graded mixtures

of chloroform:ethyl acetate (95:5, 90:10, 80:20, 70:30, 60:40, 50:50) & finally with

100% ethyl acetate & graded mixtures of ethyl acetate:methanol (99:1, 98:2, 97:3,

96:4, 95:5). The elution was monitored by TLC and visualization by UV & vanillin–

sulphuric acid spraying reagent heated at 110 °C. Each time 10 mL eluent was

collected & combined the required eluent (TLC monitored) and concentrated under

reduced pressure to obtain pale yellow powder. This powder was crystallised to obtain

a white crystalline solid designated as compound 1. The purity of the compound 1

was assessed using HPLC technique and structural characterization was done by

spectroscopic analysis such as IR, MS and NMR. The spectral data revealed that the

compound 1 is confirmed as arjunolic acid and the structure is shown in Table 1.3.

Chapter 1

22

1.3.1 Characterisation of arjunolic acid

The melting point is 317 - 320 °C. (Lit. 319 – 321 °C)76; 1H-NMR (500 MHz,

DMSO-d6) δ 11.97 (s, 1H, COOH), 5.17 (m, 1H, HC=C), 4.05–4.50 (broad peak, 3H,

OH), 3.31 (d, J ¼ 10.5 Hz, 2H, OCH2), 3.17 (d, 1H, J=9.5 Hz, OCH), 3.04 (d, 1H,

J=10.5 Hz, OCH), 2.74 (dd, 1H, J1= 13.25 Hz, J2=3.75 Hz, CH), 1.98–0.74 (terpeniod

protons, 26H), 1.10 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.71 (s, 3H,

CH3).;13C-NMR (125.75 MHz, DMSO-d6): δ 179.04, 144.38, 121.95, 76.04, 75.99,

67.89, 64.40, 47.52, 47.27, 47.14, 46.48, 46.13, 45.90, 42.96, 42.20, 41.85, 41.26,

40.47, 40.39, 40.30, 40.22, 40.13, 39.96, 39.80, 39.63, 39.57, 39.46, 39.35, 37.84,

37.76, 33.78, 33.30, 32.56, 32.35, 30.86, 27.63, 26.15, 23.83, 23.78, 23.49, 23.05,

21.54, 17.93, 17.49, 17.44, 17.37, 17.22, 14.22, 14.15; IR (KBr, cm-1): 3500-3300

(br), 1660, 1686, 1647 (s); MS: m/z = 488.35 (Calculated), m/z = 487.3 [M-H]-

(Found).

1H spectrum of compound 1

Chapter 1

23

13C spectrum of compound 1

Mass spectrum of compound 1

IR spectrum of compound 1

Chapter 1

24

1.4 Synthesis of arjunolic acid derivatives

In this part, deal with the synthesis of arjunolic acid derivatives.

1.4.1 Acetylation of arjunolic acid

The purified arjunolic acid (1) was treated with acetic anhydride in presence of

pyridine to protect hydroxyl groups of arjunolic acid (Scheme 1.1).77,78

O

O

O

H

H

H

O

OH

H

AcO

AcO

AcO

O

OH

H

Ac2O

Pyridine

1 2

Scheme 1.1

1.4.2 Synthesis of acetyl 11-keto arjunolic acid (3)

The acetylated arjunolic acid was oxidized at 11th carbon using an oxidising agent

[chromium (III) trioxide] in acetic acid media to access compound 3 (Scheme 1.2).79

AcO

AcO

AcO

O

OH

H

AcO

AcO

AcO

O

OH

HO

Cr2O3

CH3COOH

2 3

Scheme 1.2

Chapter 1

25

1.4.3 Synthesis of acetylated arjunolic acid-diene (4)

Bromination of compound 2 on 11th carbon was carried out using N-bromo

succinimide in carbon tetrachloride solvent at refluxed condition to obtain compound

4. In this step, first the bromination occurs at allylic position and then it undergoes

intramolecular elimination of hydrogen bromide to form diene (4) (Scheme 1.3).80,81

AcO

AcO

AcO

O

OH

H

AcO

AcO

AcO

O

OH

H

NBS

CCl4, 60 °C

2 4

Scheme 1.3

1.4.4 Synthesis of protected arjunolic acid

The synthesis of ketal (5) from arjunolic acid (1) was performed in acetone using mild

dehydrating agent, anhydrous copper sulphate (Scheme 1.4).76

HO

HO

HO

O

OH

H

O

HO

O

O

OH

H

CuSO4

Acetone

1 5

Scheme 1.4

1.4.5 Synthesis of arjunolic glucoside

The compound 6 (arjunolic glucoside) was synthesised by reaction between

compound 2 and -bromoglucose in chloroform solvent, in presence of base (1N

sodium hydroxide) and catalytic amount of tetrabutyl ammonium bromide at refluxed

condition (Scheme 1.5).82

Chapter 1

26

CH3COO

COOHCH3COO

CH3COO

+ OOCOCH3

HH

OCOCH3

OCOCH3

H

H

OCOCH3 Br

H

a-acetobromoglucose

TBAB,CHCl3

1N NaOH,EDC, 60 °C

HO

HO

HO

OO

OH

OHOHOH

2

6

O

H

Scheme 1.5

1.4.6 Synthesis of amides of arjunolic acid

The arjunolic acid (1) was treated with various amines in DMF at 0 °C in presence of

triethylamine as a base, EDC as a coupling reagent and HOBt as an additive to get

amides 7(a-h) (Scheme 1.7).83,84 All synthesized amides are mentioned in the Table

1.6.

OH

OH

OH

O

OH

H

R-NH2+

EDC / HOBt

Et3N, DMFOH

OH

OH

O

NH

H

R

1 7 (a-h)

Scheme 1.6

Chapter 1

27

Table 1.6: Synthesis of 10,11-Dihydroxy-9-hydroxymethyl-2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydro-2H-picene-4a-carboxamides.

Entry R-NH2 7 (a-h)Reaction

time (hrs)Yield (%)

1. 7a 12 95

2.

CH3

7b 12 90

3. 7c 15 90

4.

Cl

7d 16 89

5. 7e 11 95

6. HO 7f 15 80

7.

N

NOH

OO

7g 16 85

8.

NH2

7h 12 90

Chapter 1

28

1.5 Experimental section

1.5.1 Materials and methods

All starting synthetic reagents were purchased from Sigma-Aldrich, Merck and S D

fine chemicals and used without purification. All reactions in anhydrous solvents

were performed in flame-dried glassware under an inert atmosphere of dry nitrogen.

The progress of chemical reactions was monitored by thin-layer chromatography on

silica gel F254 plates E. Merck silica gel aluminium plates and visualized with UV

light. The following mobile phases were employed for TLC: chloroform and

methanol mixture or hexane, and ethyl acetate mixture with appropriate ratios. The

instrumental techniques used for the characterization of the newly synthesized

compounds include 1H and 13C NMR and mass spectroscopy. 1H and 13C NMR

spectra were recorded on a Fourier transform spectrophotometer in CDCl3 or

DMSO-d6 solution using tetramethylsilane (TMS) as internal standard with different

magnetic field strength. Chemical shifts were recorded in ppm relative to TMS. The

melting points were determined on Selaco melting point apparatus and are

uncorrected.

Chapter 1

29

1.5.2 Experimental procedure and Characterization data

Synthesis of 10,11-Diacetoxy-9-acetoxymethyl-2,2,6a,6b,9,12a-hexamethyl-

1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydro-2H-picene-4a-

carboxylic acid (2)

AcO

AcO

AcO

O

OH

H

2

In a two neck round bottom flask, arjunolic acid (5.0 g, 10.24 mmol) was dissolved in

acetic anhydride (25 mL), pyridine (1.65 ml, 20.48 mmol) added and reaction mixture

was heated to reflux for 12 hrs. Progress of the reaction was monitored by TLC,

which shows formation of new spots along with absence of starting material spot.

Then, the reaction mixture was quenched with ice-cold water and extracted with ethyl

acetate (3 x 100 mL). The combined organic layer was dried over sodium sulfate and

concentrated under reduced pressure to get crude product, which was purified by

column chromatography on silica gel (100-200 mesh size) with 2-3% MeOH in DCM

to obtained yield 56 % as a white solid.

mp = 320-322 °C; IR (KBr, cm-1): 3444.2, 2947.7, 1747.2, 1644.0, 1462.7, 1370.2,

1246.8, 1236.1 1044.3; 1H NMR (300 MHz, CDCl3): δ 12.03 (s, 1H), 5.14 (m, 1H),

5.03-5.10 (m, 1H), 4.83-4.95 (m, 1H), 3.82 (m, 1H), 3.51 (m, 1H), 2.72 (m, 1H), 1.75-

2.11 (m, 15H), 1.15-1.75 (m, 18H), 0.97-1.13 (m, 10H), 0.78-1.17 (m, 12H), 0.60-

0.75 (m, 3H); 13C NMR (100 MHz, CDCl3): 170.87, 170.50, 170.43, 122.09, 74.81,

69.87, 65.23, 47.63, 47.50, 46.45, 45.73, 43.47, 41.88, 41.54, 40.90, 39.27, 37.87,

Chapter 1

30

33.73, 33.73, 32.35, 32.11, 30.65, 27.51, 25.74, 23.52, 21.09, 20.89, 20.77, 17.85,

16.95, 16.81, 13.84; MS: m/z = 614.81 (Calculated), m/z = 613.3 [M-H]- (Found).

Synthesis of 10,11-Diacetoxy-9-acetoxymethyl-2,2,6a,6b,9,12a-hexamethyl-13-

oxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydro-2H-picene-

4a-carboxylic acid (3)

AcO

AcO

AcO

O

OH

HO

3

In a 100 mL two neck round bottom flask, acetyl arjunolic acid (3g, 4.87 mmol) was

stirred in glacial acetic acid (20 mL) and added chromium trioxide (1 g, 9.74 mmol) at

0 °C. The resulting mixture was allowed to stir another 12 hours at RT. Progress of

the reaction was monitored by TLC, which shows formation of new spot. The reaction

mixture was quenched with aqueous ammonium chloride solution and extracted with

ethyl acetate (3 x 100 ml). The combined organic layer was dried over sodium

sulphate and concentrated under reduced pressure to get crude material, which was

subjected to column chromatography on silica gel (100-200 mesh size) with 20-30%

EtOAc in hexane to obtained yield 80 % as a white solid.

mp = 320 °C; IR (KBr, cm-1): 3439.4, 2942.8, 1695.1, 1646.9, 1632.4, 1465.6,

1456.0, 1387.5, 1049.1; 1H NMR (400 MHz, CDCl3): δ 7.01 (m, 2H), 6.82 (d, 1H),

6.72 (t, 1H), 5.36 (br s, 1H), 3.67 (s, 2H), 2.92 (t, 2H), 2.81 (t, 2H), 1.97 (br s, 1H);

13C NMR (100 MHz, CDCl3): 138.00, 128.43, 67.41, 63.88, 45.43, 42.49, 41.38,

40.79, 37.37, 32.83, 32.10, 31.87, 30.41, 25.68, 23.37, 23.01, 22.58, 16.91, 16.75;

MS: m/z = 628.79 (Calculated), m/z = 627.8 [M-H]- (Found).

Chapter 1

31

Synthesis of 10,11-Diacetoxy-9-acetoxymethyl-2,2,6a,6b,9,12a-hexamethyl-

1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14b-hexadecahydro-2H-picene-4a-carboxylic

acid (4)

AcO

AcO

AcO

O

OH

H

4

In a 100 mL two neck round bottom flask, acetyl arjunolic acid (1g, 41.63 mmol) was

stirred in carbon tetrachloride (20 mL) and added NBS (1.1 g, 62.44 mmol) at 0°C.

The resulting mixture was refluxed for 12 hrs. Progress of the reaction was monitored

by TLC, which shows formation of new spot. The reaction mixture was quenched

with aqueous ammonium chloride solution and extracted with ethyl acetate (3 x 100

mL). The combined organic layer was dried over sodium sulphate and concentrated

under reduced pressure to get crude material, which was subjected to column

chromatography on silica gel (100-200 mesh size) with 20-30 EtOAc in hexane to

obtained yield 70 % as a half white solid.

mp = 320 °C; IR (KBr, cm-1): 3444.2, 2924.5, 1747.2, 1557.2, 1540.8, 1218.8; 1H

NMR (400 MHz, CDCl3): δ 7.01 (m, 2H), 6.82 (d, 1H), 6.72 (t, 1H), 5.36 (br s, 1H),

3.67 (s, 2H), 2.92 (t, 2H), 2.81 (t, 2H), 1.97 (br s, 1H); 13C NMR (100 MHz, CDCl3):

178.64, 170.84, 170.45, 170.40, 91.31, 74.58, 69.68, 65.12, 55.78, 52.23, 47.71,

45.60, 45.46, 43.36, 43.32, 42.43, 41.92, 39.87, 37.46, 34.09, 33.80, 33.21, 31.85,

30.56, 29.62, 29.11, 27.45, 23.54, 21.22, 21.22, 22.10, 22.05, 20.89, 20.73, 19.03,

18.42, 17.26, 13.61; MS: m/z = 612.79 (Calculated), m/z = 611.1 [M-H]- (Found).

Chapter 1

32

Synthesis of protected arjunolic acid:

O

HO

O

O

OH

H

5

To the solution of arjunolic acid (1g, 2.04 mmol) in acetone (25 mL), copper (II)

sulfate (0.65 g, 4.08 mmol) was added and then the reaction mixture was refluxed for

1.5 hrs. The progress of the reaction was monitored by TLC, which shows formation

of new spot. The solvent was removed under reduced pressure the reaction mixture

was quenched with water and extracted with ethyl acetate (3 x 100 mL). The

combined organic layer was dried over sodium sulphate and concentrated under

reduced pressure to get crude material, which was subjected to column

chromatography on silica gel (100-200 mesh size) with 2-3% MeOH in DCM to

obtained the compound 5 white solid (400 mg, 80%).

mp = 320 °C; IR (KBr, cm-1): 3210.5, 3020.8, 2800.6, 1800.6, 1400.6, 1382.6,

1162.3, 1030.7; 1H NMR (400 MHz, CDCl3): δ 7.01 (m, 2H), 6.82 (d, 1H), 6.72 (t,

1H), 5.36 (br s, 1H), 3.67 (s, 2H), 2.92 (t, 2H), 2.81 (t, 2H), 1.97 (br s, 1H); 13C NMR

(100 MHz, CDCl3): 96.14, 94.05, 91.02, 86.27, 72.66, 60.26, 51.40, 47.58, 46.40,

45.69, 45.61, 40.98, 39.34, 38.08, 36.90, 33.75, 33.05, 32.19, 32.03, 30.67, 29.73,

27.60, 25.94, 23.53, 22.86, 21.16, 13.4; MS: m/z = 528.76 (Calculated), m/z = 527.2

[M-H]- (Found).

Chapter 1

33

Synthesis of 10,11-Dihydroxy-9-hydroxymethyl-2,2,6a,6b,9,12a-hexamethyl-


carboxylic acid 3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl ester

(6)

HO

HO

HO

O O

OH

OHOHOH

O

H

6

1:1 equivalent arjunolic acid and acetobromo glucoside was taken in chloroform

solvent and added TBAB to this solution, then heated the reaction mixture at 60 °C

for about 4 hrs. The reaction mixture was allowed to stir for overnight at room

temperature. The reaction progress was monitored by TLC, after completion of the

reaction solvent was removed under reduced pressure and then the reaction mixture

was dissolved in ethylene dichloride and treated with 1N sodium hydroxide solution.

The reaction was stirred about 1 hr at room temperature. Finally the reaction mixture

was washed with water (5 mL x 2), brain solution (5 mL x 2) and then dried organic

layer with sodium sulfate and concentrated under reduced pressure to access the crude

product. The product was purified by column chromatography on silica gel (100-200

mesh size) with 2-3% MeOH in DCM to obtained compound 6.

mp = 197-199 °C; IR (KBr, cm-1): 3471.2, 2946.7, 1751.0, 1694.2, 1644.0, 1372.1,

1239.0, 1034.6; 1H NMR (400 MHz, DMSO-d6): δ 7.01 (m, 2H), 6.82 (d, 1H), 6.72

(t, 1H), 5.36 (br s, 1H), 3.67 (s, 2H), 2.92 (t, 2H), 2.81 (t, 2H), 1.97 (br s, 1H); 13C

NMR (100 MHz, CDCl3): 179.3, 150.2, 102.7, 80.2, 74.2, 71.0, 68.8, 68.2, 50.4, 47.6,

46.3, 45.3, 41.6, 38.0, 35.6, 33.4, 30.0, 29.9, 29.1, 25.2, 24.6, 23.0, 19.7, 19.1, 12.0;

MS: m/z = 650.84 (Calculated), m/z = 614.81 [M+H]+ (Found).

Chapter 1

34

Arjuno-amide derivatives

Dissolve the 1 equivalent of arjunolic acid in DMF and cooled the solution to 0 oC. To

this solution the coupling reagent, EDC.HCl (1.2 equiv) followed by HOBt (1.2

equiv) was added. The reaction mixture was neutralised with triethyl amine (1.1

equiv) and allowed to stir for 30 min. Further, aromatic amine (1 equiv) and triethyl

amine (1.1 equiv) in DMF were added to the reaction mixture at 0 oC. The reaction

was continued at room temperature for 12-20 hrs and the progress of reaction was

monitored using TLC. After the completion of reaction, the reaction mixture was

washed with water (5 mL x 2), dilute HCl (5 mL x 2), NaHCO3 (5 mL x 2), and brain

solution (5 mL x 2) and then the product was extracted with ethyl acetate. The

combined organic layer (ethyl acetate) was dried over anhydrous sodium sulfate and

concentrated under reduced pressure to get crude product. All products were purified

by column chromatography on silica gel (60-120 mesh size) with 2-3% MeOH in

DCM.

10,11-Dihydroxy-9-hydroxymethyl-2,2,6a,6b,9,12a-hexamethyl


carboxylic acid phenylamide (7a)

HO

HO

HO

O

NH

H

7a

Obtained Yield 95%; mp = 126-128 °C; IR (KBr, cm-1): 3416.3, 2948.6, 2857.0,

1810.8, 1713.4, 1618.9, 1462.7, 1446.4, 1388.5, 1280.5, 1154.2; 1H NMR (400

MHz, CDCl3): δ 7.64 (d, 2H), 7.24 (m, 2H), 7.01 (m, 1H), 5.29 (t, 1H), 3.74-3.68 (d,

Chapter 1

35

2H), 3.46-3.40 (m, 2H), 2.90 (m, 1H), 2.20 (d, 1H), 2.11-1.97 (m, 1H), 1.77 -1.62 (m,

1H), 1.62-1.50 (m, 5H), 1.45-1.29 (m, 9H), 1.29-1.00 (m, 11H), 1.00-0.80 (m, 11H),

0.80-0.60 (m, 5H), 0.60-0.45 (m, 3H); 13C NMR (100 MHz, CDCl3): 185.80, 180.04,

145.38, 122.95, 145.80, 133.7, 129.8, 126.4, 76.80, 75.99, 67.89, 64.40, 48.62, 47.27,

47.14, 46.75, 46.26, 46.90, 43.56, 43.48, 41.85, 41.39, 40.55, 40.39, 40.30, 41.21,

40.13, 40.11, 39.80, 39.63, 39.57, 39.46, 39.35, 37.84, 37.76, 33.78, 33.30, 32.56,

32.35, 30.86, 27.63, 26.15, 24.83, 23.78, 23.49, 23.05, 21.54, 17.93, 17.49, 17.44,

17.37, 17.12, 14.36, 14.21; MS: m/z = 563.81 (Calculated), m/z = 564.42 [M+H]+

(Found).

10,11-Dihydroxy-9-hydroxymethyl-2,2,6a,6b,9,12a-hexamethyl-


carboxylic acid o-tolylamide (7b)

HO

HO

HO

O

NH

H

7b

Obtained Yield 90 %; mp = 125-127 °C; IR (KBr, cm-1): 3419.2, 2942.8, 1808.9,

1695.1, 1462.7, 1387.5, 1280.5, 1049.1; 1HNMR (400MHz, CDCl3): δ 8.04 (d, 1H),

7.52-7.48 (m, 1H), 7.40-7.33 (m, 2H), 5.38 (t, 1H), 3.74-3.68 (d, 2H), 3.46-3.40 (m,

2H), 2.90 (m, 1H), 2.45 (s, 3H), 2.20 (d, 1H), 2.11-1.97 (m, 1H), 1.77 -1.62 (m, 1H),

1.62-1.50 (m, 5H), 1.45-1.29 (m, 9H), 1.29-1.00 (m, 11H), 1.00-0.80 (m, 11H), 0.80-

0.60 (m, 5H), 0.60-0.45 (m, 3H); 13C NMR (100 MHz, CDCl3): 150.34, 146.23,

133.24, 120.65, 119.34, 115.4, 106.20, 94.36, 80.34, 78.09, 64.86, 53.35, 48.87,

Chapter 1

36

45.88, 42.92, 40.09, 36.85, 35.68, 31.06, 30.0, 29.55, 26.56, 20.09, 19.05, 18.80,

15.06, 11.10; MS: m/z = 577.84 (Calculated), m/z = 578.39 [M+H]+ (Found).



carboxylic acid naphthalen-1-ylamide (7c)

HO

HO

HO

O

NH

H

7c


1810.8, 1694.2, 1660.4, 1462.7, 1388.5, 1280.5; 1H NMR (400MHz, CDCl3): δ 7.61

(d, 2H), 7.30 (m, 2H), 7.16 (d, 1H), 7.15 (m, 1H), 6.55 (d, 1H), 5.26 (t, 1H), 3.74-3.68

(d, 2H), 3.46-3.40 (m, 2H), 2.90 (m, 1H), 2.45 (s, 3H), 2.20 (d, 1H), 2.11-1.97 (m,

3H), 1.77 -1.62 (m, 1H), 1.62-1.50 (m, 5H), 1.45-1.29 (m, 9H), 1.29-1.00 (m, 11H),

1.00-0.80 (m, 11H), 0.80-0.60 (m, 5H), 0.60-0.45 (m, 3H); 13C NMR (100 MHz,

CDCl3): 161.35, 145.03, 140.05, 135.06, 132.86, 129.86, 129.09, 126.20, 120.84,

109.89, 98.82, 95.30, 90.30, 90.08, 85.06, 825.91, 79.72, 76.34, 71.19, 65.06, 52.01,

50.19, 48.03, 46.13, 30.12, 26.07, 22.14, 19.64, 16.13, 12.10; MS: m/z = 613.87

(Calculated), m/z = 614.81 [M+H]+ (Found).



carboxylic acid (3-chloro-phenyl)-amide (7d)

Chapter 1

37

HO

HO

HO

O

NH

H

Cl7d


1810.8, 1730.8, 1696.1, 1456.0, 1379.8; 1H NMR (400 MHz, CDCl3): δ 8.06-8.01 (d,

1H), 7.52-7.48 (m, 1H), 7.40-7.38 (m, 2H), 5.36 (t, 1H), 3.74-3.68 (d, 2H), 3.49-3.47

(m, 2H), 2.96 (m, 1H), 2.20 (d, 1H), 2.16-1.96 (m, 1H), 1.77-1.62(m, 1H), 1.62-1.50

(m, 5H), 1.45-1.29 (m, 9H), 1.29-1.00 (m, 11H), 1.00-0.80 (m, 11H), 0.80-0.60 (m,

5H), 0.60-0.45 (m, 3H); 13C NMR (100 MHz, CDCl3): 147.36, 143.20, 130.26,

117.63, 116.43, 112.6, 103.30, 91.33, 77.64, 75.13, 61.73, 61.73, 50.33, 45.88, 43.88,

43.89, 39.91, 37.13, 33.88, 32.17, 28.06, 26.14, 20.13, 19.26, 16.44, 11.14; MS: m/z

= 598.26 (Calculated), m/z = 599.3 [M+H]+ (Found).



carboxylic acid p-tolylamide (7e)

HO

HO

HO

O

NH

H

7e

Obtained Yield, 95 %; mp = 132-134 °C; IR (KBr, cm-1): 3420.1, 2945.7, 2880.2,

1810.8, 1732.7, 1716.3, 1462.7, 1387.5; 1H NMR (400 MHz, CDCl3): δ 7.52-7.48 (d,

1H), 7.09-7.04 (d, 1H), 5.32 (t, 1H), 3.74-3.68 (d, 2H), 3.46-3.40 (m, 2H), 2.90 (m,

1H), 2.45 (s, 3H), 2.20 (d, 1H), 2.11-1.97 (m, 1H), 1.77 -1.62 (m, 1H), 1.62-1.50 (m,

Chapter 1

38

5H), 1.45-1.29 (m, 9H), 1.29-1.00 (m, 11H), 1.00-0.80 (m, 11H), 0.80-0.60 (m, 5H),

0.60-0.45 (m, 3H); 13C NMR (100 MHz, CDCl3): 144.25, 143.21, 130.20, 117.63,

116.43, 112.06, 103.30 90.18, 88.38, 76.34, 72.74, 64.13, 59.84, 50.10, 49.19, 40.06,

39.06, 33.34, 29.19, 27.94, 25.64, 22.45, 19.19, 46.64, 12.13, 11.17; MS: m/z =

577.84 (Calculated), m/z = 578.56 [M+H]+ (Found).



carboxylic acid [2-(4-hydroxy-phenyl)-ethyl]-amide (7f)

HO

HO

HO

O

HN

HOH

7f

Obtained Yield 80%; mp = 120-122 °C; IR (KBr, cm-1): 3399.9, 2926.4, 2875.3

1715.4, 1633.4, 1515.8, 1456.0, 1378.9, 1364.4, 1268.0, 1244.8; 1H NMR (400 MHz,

CDCl3): δ 7.62-7.58 (d, 1H), 7.29-7.14 (d, 1H), 5.26 (t, 1H), 4.52 (d, 2H), 3.74-3.68

(d, 2H), 3.46-3.42 (m, 2H), 2.92 (m, 1H), 2.81 (d, 2H), 2.45 (s, 3H), 2.28 (d, 1H),

2.11-1.97 (m, 1H), 1.77 -1.62 (m, 1H), 1.62-1.50 (m, 5H), 1.45-1.29 (m, 9H), 1.26-

1.06 (m, 11H), 1.03-0.81 (m, 11H), 0.81-0.62 (m, 5H), 0.61-0.45(m, 3H); 13C NMR

(100 MHz, CDCl3): 147.16, 144.21, 131.13, 126.26, 121.12, 103.13, 96.93, 89.14,

77.70, 68.34, 60.73, 54.01, 50.44, 49.13, 46.34, 46.34, 39.90, 36.04, 32.66, 29.16,

22.10, 16.29, 14.66, 12.12, 11.64; MS: m/z = 577.84 (Calculated), m/z = 578.73

[M+H]+ (Found).

Chapter 1

39

4-{[(10,11-Dihydroxy-9-hydroxymethyl-2,2,6a,6b,9,12a-hexamethyl-


carbonyl)-amino]-hydroxyimino-methyl}-4-phenyl-piperidine-1-carboxylic acid

tert-butyl ester (7g)

HO

HO

HO

OH

N

NHN

HO

O

O

7g

Obtained yield 85%; mp = 170-172 °C; IR (KBr, cm-1): 3399.9, 2942.8, 2877.3,

1808.9, 1694.2, 16635.3, 1455.0, 1433.8, 1388.5, 1366.3, 1303.6, 1280.5, 1250.6,

1162.9; 1H NMR (400 MHz, CDCl3): δ 7.18 (d, 2H), 7.08 (m, 2H), 7.01 (m, 1H), 5.48

(t, 1H), 3.74-3.68 (d, 2H), 3.46-3.40 (m, 2H), 2.90 (m, 1H), 2.82-2.72 (m, 4H), 2.45

(s, 3H), 2.20 (d, 1H); 2.11-1.97 (m, 5H), 1.77 - 1.62 (m, 1H), 1.62-1.50 (m, 5H), 1.49

(s, 1H), 1.45-1.29 (m, 9H), 1.29-1.00 (m, 11H), 1.00-0.80 (m, 20H), 0.80-0.60 (m,

5H) 0.60-0.45 (m, 3H); 13C NMR (100 MHz, CDCl3): 189.10, 164.06, 160.3, 138.08,

128.03, 126.06, 124.09, 98.31, 91.31, 74.58, 68.68, 65.46, 54.76, 52.26, 47.88, 45.71,

43.36, 42.34, 41.33, 39.81, 37.36, 34.19, 33.06, 31.85, 30.16, 29.01, 27.39, 23.32,

22.10, 20.89, 19.03, 17.17, 11.01; MS (m/z): 790.08 (Calculated), m/z = 791.03

[M+H]+ (Found).



carboxylic acid (2-amino-phenyl)-amide (7i)

Chapter 1

40

HO

HO

HO

OH

7h

HN

H2N

Obtained yield 90%; mp= 175-177 °C; IR (KBr, cm-1): 3412.4, 2943.8, 28880.2,

1810.8, 1693.2, 1667.2, 1455.0, 1388.5, 1303.6, 1279.5, 1267.0, 1240.0, 1085.7; 1H

NMR (400 MHz, CDCl3): δ 7.76 (d, 2H), 7.20 (m, 2H), 7.01 (m, 1H), 5.29 (t, 1H),

5.01 (s, 1H), 3.74-3.68 (d, 2H), 3.46-3.40 (m, 2H), 2.90 (m, 1H), 2.20 (d, 1H); 2.11-

1.97 (m, 1H), 1.77 -1.62 (m, 1H), 1.62-1.50 (m, 5H), 1.48 (s, 1H), 1.45-1.29 (m, 9H),

1.29-1.00 (m, 11H), 1.00-0.80 (m, 11H), 0.80-0.60 (m, 5H) 0.60-0.45 (m, 3H); 13C

NMR (100 MHz, CDCl3): 142.13, 144.32, 132.45, 118.64, 117.64, 113.5, 107.10,

95.38, 81.44, 79.26, 65.99, 54.55, 49.86, 46.89, 43.99, 41.09, 38.09, 37.10, 29.06,

28.66, 23.56, 20.56, 19.08, 18.10, 16.10, 12.27, 11.12; MS (m/z): 578.41 (Calculated),

m/z = 573.29 [M+H]+ (Found).

Chapter 1

41

1.6 Conclusion

In conclusion we isolated the arjunolic acid for the first time from the heart wood of

Terminilia Arjuna using modern supercritical fluid extraction technique in good yield.

Further we have synthesized the derivatives of arjunolic acid and their structure was

analysed using various spectral techniques such as IR, NMR and Mass.

Chapter 1

42

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APPENDICES

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Chapter 1

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Chapter 1

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Chapter 1

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Chapter 1

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Chapter 1

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1H spectrum of compound 7a

IR spectrum of compound 7a

Mass spectrum of compound 7a

Chapter 1

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1H spectrum of compound 7b

IR spectrum of compound 7b

Mass spectrum of compound 7b

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