dissertation to submit

1

Synthesis of a Novel

Cholecystokinin Antagonist

A Research Project presented by

Jay Vora

For the degree of MPharm

Pharmacy Department

School of Life and Health Sciences

Aston University

22/4/2013

Dr. Eric Lattmann

2

Page

Abstract 4 1.0 Introduction 5

1.1 Cholecystokinin 5

1.2 CCK Receptors 6

1.3 Uses of CCK Agonists and Antagonists 6

1.4 Previous And Current Drugs and Research 7

1.5 Aims and Objectives 8

2.0 Experimentals 9

2.1 Synthesis of 2,3-dichloro-4,4-bis(4-methylphenyl)but-2-enoic acid (bis-

arylated acid)

9

2.2 Synthesis of cyclopentyl-azanyl-2,3 dichloro, 4,4 bis (4-methylphenyl)but-

2-enoate (Amine salt)

12

2.3 Lewis-base catalysis for the formation of 2,3-dichloro-N-cyclopentyl-4,4-

bis(4-methylphenyl)but-2-enamide

14

2.4 Synthesis of 2,3-dichloro-N-cyclopentyl-4,4-bis(4-methylphenyl)but-2-

enamide (Bis-arylated amide)

14

2.5 Synthesis of 3-chloro-5-(cyclopentylamino)-4-(3-methylphenyl) cyclopent-

2-en-1-one

16

2.6 Synthesis of 2-chloro-N-cyclopentyl-3-(cyclopentylamino)-4,4-bis(4-

methylphenyl)but-2-enamide

19

3.0 Results, Analysis and discussion 22

3.1 2,3-dichloro-4,4-bis(4-methylphenyl)but-2-enoic acid (bis-arylated acid) 22

3.2 cyclopentyl-azanyl-2,3 dichloro, 4,4 bis (4-methylphenyl)but-2-enoate

(Amine salt)

28

3.3 Lewis-base catalysis for the formation of 2,3-dichloro-N-cyclopentyl-4,4-

bis(4-methylphenyl)but-2-enamide

31

3.4 2,3-dichloro-N-cyclopentyl-4,4-bis(4-methylphenyl)but-2-enamide using

ethylchloroformate (Bis-arylated amide)

34

3.5 3-chloro-5-(cyclopentylamino)-4-(3-methylphenyl) cyclopent-2-en-1-one 41

3.6 2-chloro-N-cyclopentyl-3-(cyclopentylamino)-4,4-bis(4-methylphenyl)but-

2-enamide

45

3

4.0 Pharmacokinetic properties 49

5.0 Conclusion 53

6.0 References 56

7.0 Appendix A – 1H NMR and infrared spectras 58

7.1 Appendix B – Risk assessment and COSHH forms 59

7.2 Appendix C – 1H NMR and Infrared tables used to analyse data 60

4

Synthesis of a Novel CCK Antagonist

Jay Vora and Dr. Eric Lattmann

Aston University, School of Life and Health Sciences, Pharmacy Department, Birmingham, B4 7ET.

Cholecystokinin (CCK) is a peptide hormone and a neuropeptide predominantly found in the

gastrointestinal tract and central nervous system respectively (Herranz, 2003). The therapeutic

potential of CCK agonists and antagonists is great due the abundance of CCK receptors around the

body. Over the last 25 years several compounds have been developed to selectively target CCK

receptors, but due toxicity, side-effects, inconsistent clinical trial data, very short half-lives, expiry of

patents and poor understanding of the complex physiological processes only one CCK antagonist is

currently approved by regulatory authorities. In this research project the aim was to synthesis an

amide CCK antagonist (Berna et al, 2007).

Mucochloric acid and toluene was used to synthesise 2,3-dichloro-4,4-bis(4-methylphenyl)but-2-

enoic acid, which was then reacted with cyclopentylamine using activating agents (

ethylchloroformate or DIC) and co-solvent (DMF) to synthesise cyclopentyl-azanyl-2,3 dichloro, 4,4

bis (4-methylphenyl)but-2-enoate, 2,3-dichloro-N-cyclopentyl-4,4-bis(4-methylphenyl)but-2-

enamide, 3-chloro-5-(cyclopentylamino)-4-(3-methylphenyl) cyclopent-2-en-1-one and 2-chloro-N-

cyclopentyl-3-(cyclopentylamino)-4,4-bis(4-methylphenyl)but-2-enamide. These compounds were

analysed using 1H NMR and infrared spectroscopy and qualitative analysis performed by thin layer

chromatography. Predictive software was used to assess the ‘drug-like’ properties of the new

compounds.

The key findings were the synthesis of bis-arylated acid which resulted in a crystal form due to

forming an ionic bond with the aluminium crystals, the identification and purification of amine salts

and cyclopentylamine salts which needed to me separated from the main products and finally the

synthesis of new drug compounds that showed good oral bioavailability and ‘drug-like’ behaviour.

In conclusion all compounds synthesised should go through the drug development process and a new

generation of novel drug compounds can be synthesised using the 2,3-dichloro-4,4-bis(4-

methylphenyl)but-2-enoic acid and reacting with other amines.

5

1.0 - Introduction

Drug discovery involves the targeting of an identified biological process to produce a

pharmacodynamic effect (Turner, 2007). It is important to understand the physiological

processes of a receptor and its functional units, so that a drug can be synthesised to have

complimentary functional groups to the receptor’s enabling an interaction. (Turner, 2007)

1.1 - Cholecystokinin

Cholecystokinin (CCK) formally known as pancreozymin was discovered in the 1900’s as a

33-amino acid peptide hormone found in the gastrointestinal (GI) tract (Jorpes & Mutt,

1966). It was later discovered to also act as a neuropeptide in the central nervous system

(CNS) (Ho¨kfelt et al, 1985). The human gene that encodes for CCK is located on

chromosome 3p22-p21.3, which translates the gene transcript into a 115 amino acid

peptide precursor called ‘pre-pro-CCK’ and then modified into biologically active peptides

(Deschenes et al, 1985). CCK is abundantly found in the CNS (especially in the cerebral

cortex, amygdala and hippocampus) and GI tract (mainly pancreas, gall bladder and small

intestine) (Zwanger et al, 2012) (Harrold et al 2012). The most common forms found in

humans are CCK-8S (sulphated), CCK-8NS (non-sulphated), CCK-4, CCK-33 and CCK-58. They

all share the same C-terminal pentapeptide sequence ‘Gly-Trp-Met-Phe-NH2’ which is the

biologically active component of the hormone (Radu, 2005). Another hormone, gastrin

which acts on the CCK receptors in the gut also shares this amino acid sequence as shown in

figure 1 (Harrold et al 2012).

6

Figure 1: Structures of CCK-4, CCK-8, CCK-33, CCK-58 and gastrin all show a similar C-terminal sequence (Herranz, 2003).

1.2 - CCK Receptors

CCK binds to two types of receptors, CCK1 (formally known as CCK-A) and CCK2 (formally

known as CCK-B) (Wank, 1995). Both are specific G-protein coupled receptors which are

broadly distributed around the human body and share a 50% homology (Otsuki, 2000). CCK1

receptors are differentiated to CCK2 receptors due to their difference in affinities to CCK

analogues (Rehfeld 2012). The CCK1 receptor has a high affinity to sulphated CCK’s and

peptide analogues (Dufresne et al, 2006). The endogenous peptide CCK-8S has a ≈1000 fold

greater affinity to CCK1 compared to its non-sulphated form (CCK-8NS), but has a poor

affinity to gastrin and CCK-4 (Dufresne et al, 2006). The CCK2 receptors do not show any

selectivity towards a sulphated or non-sulphated analogue (Dufresne et al, 2006).

Upon stimulation inositol-1,4,5 triphosphate (1P3) and 1,2-diaglycerol (DAG) is formed

leading to a release of calcium ions (Ca2+) which leads to various physiological functions like

learning and memory, dopamine release, noiceception and digestion processes (enzyme

secretion, gastric acid secretion, gut motility and gall bladder contraction) (Liddle, 1997).

1.3 - Uses of CCK Agonists and Antagonists

Due to the abundance and broad physiological effects of CCK receptors in the human body,

there is a therapeutic potential by stimulating or inhibiting CCK receptors to achieve

7

pharmacological effects through the use of agonists and antagonists (Staljanssens et al,

2011). The potential therapeutic applications are summarised in table 1.

Table 1: Potential applications of CCK receptor antagonists and agonists (berna et al, 2007).

CCK receptor Antagonist CCK receptor Agonist

Treatment of pancreatitis (Acute/chronic)

Obesity therapy

Treatment of gastrointestinal motility disorders

Cholescintigraphy (analytical)

Anorexia therapy Detection of cancer

Bulimia therapy Imaging of tumours by radio-ligand binding

Treatment of cancers Delivering radiotherapy

Treatment of panic attacks Preventing gall stones

Pain management

Treating schizophrenia

Control of gastric acid secretions

Treatment of cholecstysis

1.4 - Previous and Current Drugs and Research

In the past 25 years several CCK antagonists have been developed, but only one drug is

currently available in the market (Herranz, 2003). Proglumide is an amino acid derivative

that is weak and non-selective (Herranz, 2003). It is marketed for the treatment of ulcers by

competitive antagonism of CCK-8 in the gut, reducing gastric acid secretion (Herranz, 2003).

Lead optimization was conducted to improve its selectivity, potency, affinity and

pharmacokinetic properties (Herranz, 2003). Lorglumide was developed and further

manipulated to synthesise loxiglumide and deloxiglumide (Berna et al, 2007). These

compounds had shown greater oral bioavailability, affinity, potency and selectivity to CCK

receptors (Herranz, 2003). Lorglumide showed inconsistency in clinical trials thus hindering

its progress, loxiglumide was under regulatory approval in Japan for the treatment of acute

pancreatitis, but further clinical trials were discontinued due to patent expiry (Berna et al,

2007). Deloxiglumide was discontinued due to clinical trials showing toxicity in the kidney

8

(Herranz, 2003). There have been several other developments of CCK antagonists in

producing peptide and non-peptide derivatives such as, amino acids derivatives, cyclic

nucleotides, tryptophan derivatives, peptides derivatives, benzodiazepine derivatives, indol-

2-based compounds have all been promising, but are unable to reach regulatory approval

due to toxicity, poor bioavailability or short half-lives (Berna et al, 2007) (Herranz, 2003).

1.5 - Aims and Objectives

Since amide derivates have been the closest to gaining regulatory approval, the aim is to

synthesise a novel amide CCK antagonist which has the potential to be marketed. Structural

and qualitative analysis will be performed, followed by an assessment of the

pharmacokinetic properties of the drug using predictive software.

9

2.0 - Experimentals

2.1 - Synthesis of 2,3-dichloro-4,4-bis(4-methylphenyl)but-2-enoic acid (bis-arylated acid)

Mucochloric acid (powder, 10g, 0.06mol) was dissolved in approximately 200ml of toluene

in a 500ml round-bottom flask containing a stir bar. Aluminium chloride (granules, 10g,

0.07mol) was added to the flask and a drying tube containing calcium carbonate was

attached to absorb any moisture released during the reaction. The mixture was stirred for

72 hours at room temperature. It turned from a maroon coloured solution to an orange-

yellow coloured solution within the first 24 hours. Thin layered chromatography was

performed every 24 hours to monitor the progress of the reaction.

Work-up

The system was quenched by slowly pouring the solution into a beaker containing 125g of

ice and 30mls of concentrated hydrochloric acid. This was stirred with a magnetic stirrer

until all ice dissolved and a cloudy white precipitate was formed. The mixture was left to set

for 72 hours. The precipitate was filtered using Buchner filtration apparatus and the beaker

was washed with (3x30ml) toluene. Residue was an almost clear crystal which was collected

and dried under vacuum (crop1). The filtrate was then poured into a separating funnel,

shaken vigorously and left to settle for 5 minutes. The product was extracted by collecting

the organic layer and washing the aqueous layer with toluene (3 x 50ml). The organic layers

were combined and dried using magnesium sulphate (approximately 6g) and poured into a

weighed round-bottom flask. The solvent was extracted using a rotary evaporator and

placed in a desiccator overnight. A pale-yellow ‘soft’ solid is formed.

10

Spectroscopic Analysis

1H NMR was performed at 250Mhz. Chloroform-D (CDCl3) was the solvent used to prepare

the NMR sample tube with a concentration of 10mg/ml. Infrared spectroscopy was carried

out using ID5-ATR diamond FT-infrared machine.

Qualitative Analysis

Thin layer chromatography (TLC) - stationary phase was silica plates and mobile phase was

diethyl-ether. Sample was dissolved in ethanol to make a 10mg/ml solution.

2,3-dichloro-4,4-bis(4-methylphenyl)but-2-enoic acid

Yield = Crop 1 (45.07%); Crop 2 (42.54%)

Melting point: 181-184C

Rf (diethyl ether) = 0.14

Molecular weight: 335.22

Molecular Formula: C18 H16 Cl2 O2

1H NMR (CDCL3) 250 MHz: δ (ppm) = 11.0 (1H, CO-OH); 7.3 (8H, m, Ar – H); 6.7 (1H, s, C-H);

2.3 (6H, s, CH3)

IR cm-1 = 3026cm-1 (Aromatic C-H); 2962 cm-1(sp2 C-H); 2625 cm-1 (Carboxylic acid -OH); 1782

cm-1 (Carboxylic acid C=O); 1687 cm-1 ( Aromatic C=C); 867 cm-1 ( C-Cl).

11

Optimised method for Synthesis of 2,3-dichloro-4,4-bis(4-methylphenyl)but-2-enoic acid

Mucochloric acid (powder, 10g, 0.06mol) was dissolved in approximately 200ml of toluene

in a 500ml round-bottom flask containing a stir bar. Aluminium chloride (granules, 10g,

0.07mol) was added to the flask and a drying tube containing calcium carbonate was

attached. The mixture was stirred for 72 hours in room temperature. It turned from a

maroon coloured solution to an orange-yellow coloured solution within 24 hours.

Work-up

The system was quenched by slowly pouring the solution into a beaker containing 125g of

ice and 30mls of concentrated hydrochloric acid. This was stirred with a magnetic stirrer

until all ice dissolved and a cloudy white precipitate was formed. The precipitate was

filtered using Buchner filtration apparatus and the beaker was washed with (3x30ml)

toluene. Residue was collected and dried under vacuum resulting in a pure white powder.

The filtrate was then poured into a separating funnel, shaken vigorously and left to settle for

5 minutes. The product was extracted by collecting the organic layer and washing the

aqueous layer with toluene (3 x 50ml). The organic layers were combined and dried using

magnesium sulphate (approximately 6g) and poured into a weighed round-bottom flask.

The solvent was extracted using a rotary evaporator and placed in a desiccator overnight. A

pale-yellow ‘soft’ solid was formed.

12

2.2 - Synthesis of cyclopentyl-azanyl-2,3 dichloro,4,4 bis (4-methylphenyl) but-2-enoate

(Amine salt)

1g (3mmol) of bis-arylated acid was weighed into a 100ml round-bottom flask containing a

stir bar. The flask was sealed with a rubber bong and a balloon filled with argon gas was

attached. 25ml of anhydrous diethyl ether was added to the flask. The mixture was stirred

until all bis-arylated acid dissolved. Two equivalence of cyclopentylamine (0.59ml, 6mmol)

was inserted to the solution. Upon addition the mixture released heat, producing a ‘hissing’

sound. A white precipitate was formed immediately. The reaction was kept homogenous for

2 hours at room temperature.

Work up

The system was quenched by slowly pouring the solution into a beaker containing 50g of ice

and 10ml of concentrated hydrochloric acid. This was stirred with a magnetic stirrer until all

ice dissolved giving a cloudy white precipitate. The mixture was filtered using a funnel and

filter paper and the residue was collected and dried in a vacuum over night resulting in a

white powder. The filtrate was poured into a separating funnel, shaken vigorously and left

to settle for 5 minutes. The organic layer was collected and aqueous layer was washed with

diethyl ether (3 x 50ml). The organic layers were combined into a weighed round-bottom

flask. The solvent was extracted using a rotary evaporator and placed in a desiccator

overnight. A small amount of white powder was formed.


1H NMR was performed at 250Mhz. Dimethyl sulfoxide (DMSO) was the solvent used to

prepare the NMR sample tube with a concentration of 10mg/ml. Infrared spectroscopy was

performed using ID5-ATR diamond FT-infrared machine.

13


Thin layer chromatography (TLC) where the stationary phase was silica plates and mobile

phase was diethyl-ether. The sample was dissolved in ethanol to make a 10mg/ml solution.

cyclopentyl-azanyl-2,3 dichloro, 4,4 bis (4-methylphenyl)but-2-enoate (Amine salt)

Yield = 98%

Rf (Diethyl ether): Did not run

Melting point: 190C

Molecular weight: 420

Molecular formula: C23H27Cl2NO2

1H NMR (DMSO) 250 MHz: δ (ppm) = 8.1 (NH3+, s); 7.3 (8H, m, Ar-H); 6.3 (1H, s, C-H); 3.5 (1H,

s, C-H); 2.5 (6H, s, -CH3); 1.4-1.9 (8H, m, C-H);

IR cm-1= 2963 (Amine salt NH3+ broad stretch); 2900 (C-H stretch); 1627 (C=O stretch); 1627

(N-H bending) 1585 (Aromatic C=C, m)

14

2.3 - Lewis-base catalysis for the formation of 2,3-dichloro-N-cyclopentyl-4,4-bis(4-


500mg of salt was weighed into a test tube and heated up to 150C. The white powder

melted and turned into a brown solid.

500mg of salt was dissolved in 10ml toluene in a 100ml round-bottom flask and heated to

150C under reflux for four hours. This resulted in a dark brown oily substance

2.4 - Synthesis of 2,3-dichloro-N-cyclopentyl-4,4-bis(4-methylphenyl)but-2-enamide

1g (3mmol) of bis-arylated acid was weighed into a 100ml round bottom flask containing a


attached. 25ml of anhydrous diethyl ether was added to the flask. The mixture was stirred

with a magnetic stirrer until the solid was dissolved. Two equivalence (6mmol, 0.6ml) of

ethylchloroformate was added to the solution. 30 minutes later 2.2 equivalence (6.6mmol,

0.59ml) of cyclopentylamine was added. Upon addition a ‘hissing’ sound was heard and the

base of the flask warmed up due to the reaction being exothermic. A white precipitate

formed immediately. The mixture was kept homogenous for 2 hours.

Work-up


and 10mls of concentrated hydrochloric acid. This was stirred with a magnetic stirrer until

all ice dissolved and a cloudy white precipitate was formed. The mixture was filtered using a

funnel and filter paper. The residue was collected and dried in a vacuum oven set at 40C

over night resulting in a white powder. The filtrate was then poured into a separating funnel

and 50ml of diethyl ether was added. The funnel was shaken vigorously and left to settle for

5 minutes. The product was extracted by collecting the organic layer and washing the

15

aqueous layer with diethyl ether (3 x 50ml). The organic layers were combined into a


placed in a desiccator overnight, producing a light brown oily substance.

Purification

The white powder was dissolved in minimal amounts of ethanol, the un-dissolved material

was filtered and the residue dried under vacuum. The resulting solid was dissolved in

toluene and filtered, discarding the residue and extracting the solvent in a rotary evaporator

and dried in vacuum overnight. The end product is a light brown solid.



the NMR sample tube with a concentration of 10mg/ml. Infrared spectroscopy was




phase was diethyl-ether. Sample was dissolved in ethanol to make a 10mg/ml solution.

2,3-dichloro-N-cyclopentyl-4,4-bis(4-methylphenyl)but-2-enamide using ethylchloroformate (Bis-arylated

acid)

16

Yield = 1%

Rf (ether): 0.77


Molecular formula: C23H25CL2NO

1HNMR (CDCl3) 250MHz δ (ppm) = 7.1 ( 8H, m, Ar-H); 6.6 (1H D20 exchangeable, s, N-H); 6.2

(1H, s, -CH); 4.2 (1H, m, C-H); 2.3 (6H, s, -CH3); 1.3 (8H, s).

IR cm-1 = 3272 (Amide CONH stretch); 1658 (amide C=O stretch); 2849 (C-H stretch); 1635

(Aromatic C=C stretch); 814 (Aromatic C-H stretch).

2.5 - Synthesis of 3-chloro-5-(cyclopentylamino)-4-(3-methylphenyl) cyclopent-2-en-1-one



attached. 25ml of anhydrous diethyl ether along with 8ml of DMF (for every 7ml of diethyl

ether added 2ml of DMF). The mixture was stirred with a magnetic stirrer until the entire

solid was dissolved. Two equivalence (12mmol, 1.2ml) of ethylchloroformate was added to

the solution. 30 minutes later 2 equivalence (12mmol, 1.2ml) of cyclopentylamine was

added to the solution. Upon addition a weak ‘hissing’ sound was heard and the base of the

flask warmed up due to the reaction being exothermic. A white-yellow precipitate was

formed and the mixture was kept homogenous by stirring with a magnetic stirrer for

24hours. The mixture turned into a light yellow solution.

Work-up



all ice dissolved. The mixture was then filtered and the filtrate poured into a separating

17

funnel where 50ml of diethyl ether was added. The funnel was shaken vigorously and left to

settle for 5 minutes. The product was extracted by collecting the organic layer and washing

the aqueous layer with diethyl ether (3 x 50ml). The organic layers were combined into a


placed in a desiccator overnight. A light yellow oily compound was produced.

Purification

Flash column chromatography was performed. The compound was dissolved in 30mls of

methanol in a 100ml round-bottom flask. Approximately four teaspoons of celite 545 was

added to the flask and gently swirled. The methanol was then evaporated using the rotary

evaporator resulting in a coarse white powder. A chromatography column with a diameter

of 40mm was filled with 40-60µm silica (approximately 15cm length of column). The

reservoir was filled with 500ml of solution containing 470ml 40:60 petroleum ether and

30ml of diethyl ether (15:1 petroleum ether : diethyl ether). A vacuum pump was attached

to the reservoir. Samples were collected in fraction sizes of 30ml and TLC (silica plates were

the stationary phase and diethyl ether as the mobile phase) was performed and compared

with the crude products TLC plate. All test tubes containing the same spot on the TLC plate

were collated and the solvent was evaporated using a rotary evaporator and then dried

under vacuum. A clear oily compound and a white ‘waxy’ compound were produced.





18




3-chloro-5-(cyclopentylamino)-4-(3-methylphenyl) cyclopent-2-en-1-one

Yield = 25%

Rf (diethyl ether): 0.87


Molecular formula: C17H20ClNO

1HNMR (CDCl3) 250MHz δ (ppm): 7.2 (4H, m, Ar-H); 6.6 (H, d, -CH); 6.1 ( 1H, m, C-H); 5.7 (H,

s, -CH); 4.2 (H, m, C-H); 3.3 (H, D20 exchangeable, NH); 2.3 (3H, m, CH3); 1.5-2.0 (8H, m, C-H)

IR cm-1 = 3273 (Amine N-H); 2957 (Aromatic C-H bonds); 2866 (sp3 C-H bonds); 1783 (C=O);

1633 (Ar C=C); 815 (C-Cl)

19

2.6 - Synthesis of 2-chloro-N-cyclopentyl-3-(cyclopentylamino)-4,4-bis(4-




attached. 25ml of anhydrous diethyl ether along with 8ml of DMF (for every 7ml of diethyl

ether added 2ml of DMF). The mixture was stirred with a magnetic stirrer until the entire

solid was dissolved. Two equivalence (12mmol, 1.86ml) of DIC was added to the solution.

30 minutes later 2 equivalence (12mmol, 1.2ml) of cyclopentylamine was added to the

solution. An orange solution was formed and stirred for 24hours. The mixture turned into a

light yellow solution.

Work-up



all ice dissolved. The mixture was then filtered and the filtrate was poured into a separating

funnel, 50ml of diethyl ether was added. The funnel was shaken vigorously and left to settle

for 5 minutes. The product was extracted by collecting the organic layer and washing the

aqueous layer with diethyl ether (3 x 50ml). The organic layers were combined into a


placed in a desiccator overnight. An orange oily compound was produced.

Purification

Flash column chromatography was performed. The compound was dissolved in 30mls of

methanol in a 100ml round-bottom flask. Approximately four teaspoons of celite 545 was

added to the flask and gently swirled. The methanol was then evaporated using the rotary

evaporator resulting in a coarse white powder. A chromatography column with a diameter

20

of 40mm was filled with 40-60µm silica (approximately 15cm length of column). The

reservoir was filled with 500ml of solution containing 470ml 40:60 petroleum ether and

30ml of diethyl ether (15:1 petroleum ether : diethyl ether). A vacuum pump was attached

to the reservoir. Samples were collected in fraction sizes of 30ml and TLC (silica plates were

the stationary phase and diethyl ether as the mobile phase) was performed and compared

with the crude products TLC plate. All test tubes containing the same spot on the TLC plate

were collated and the solvent was evaporated using a rotary evaporator and then dried

under vacuum. A light orange oily compound was formed from the top spot.








2-chloro-N-cyclopentyl-3-(cyclopentylamino)-4,4-bis(4-methylphenyl)but-2-enamide

21

Yield= 50%

Rf (ether): 0.78


Molecular formula: C28H35ClN2O

1HNMR (CDCl3) 250MHz δ (ppm) = 7.1 (8H, m, Ar-H); 6.6 (1H, s, C-H); 5.6 (1H D20

exchangeable, s, N-H); 5.4(1H D20 exchangable, s, N-H); 4.2 (1H, m, C-H); 3.8 (1H, m, C-H)

2.2(6H, s, CH3); 1.0-1.3 (16H, m, C-H).

IR cm-1 = 3304 (Amide CONH2); 2968 (Aromatic C-H); 2870 (sp3 C-H); 1710 (C=O Amides);

1651 (Alkene C=C); 1608 (Aromatic C=C).

22

3.0 – Results, Analysis and Discussion

3.1 - 2,3-dichloro-4,4-bis(4-methylphenyl)but-2-enoic acid (Bis-arylated acid) Mucochloric acid was dissolved in excess toluene, aluminium chloride was added and the

mixture was stirred for 72 hours in room temperature. Aqueous work-up was performed

and the mixture was left to settle for 72 hours. Almost clear crystals were produced, which

was separated by filtration and dried under vacuum. The aqueous phase of the filtrate was

separated from the solvent and discarded and more product was extracted by evaporating

the solvent from the organic phase resulting in a pale yellow solid. The process is

summarised in figure 3. Figure 2 shows the reaction scheme of how the main product (bis-

arylated acid) and by-product (mono-arylated furanone) were formed.

Figure 2: Reaction scheme showing how the 2,3-dichloro-4,4-bis(4-methylphenyl)but-2-enoic acid (bis-arylated acid) and 3,4-dichloro-5-(4-methylphenyl)furan-2(5H)-one (mono-arylated furanone) are formed.

23

Figure 3: A summary of the processes conducted for the synthesis the bis-arylated acid.

24

Crop 1 (Aluminium salt crystals) – 9.01g

The formation of the crystal created a highly pure compound compared to the second crop.

The aluminium salt forms an ionic bond with oxygen which forms the crystal as shown in

figure 2. Figure 4 shows the crystals molecular structure deduced from 1H NMR spectra

(Table 2).

Figure 4: molecular structure of aluminium salt crystal

An infrared was conducted to compare the crystals from crop 1 and the pale-yellow solid

from crop 2 (shown in figure 5). Almost all peaks overlap each other meaning both crops

have similar structures, except crop 2 shows a peak at 1782cm-1 and crop 1 does not show

this peak. The 1782cm-1 peak is a C=O functional group peak and was concluded the crystal

does not contain this functional group but instead the aluminium salt as shown in figure 4.

25

Figure 5: Overlap of infrareds of crop 1 and crop 2.

Figure 6: 1H NMR Crop 1(ppm) - 11.0 (1H, -OH); 7.3 (8H, m, Ar – H); 6.7 (1H, s, C-H); 2.3 (6H, s, CH3).

26

Table 2: Analysis of 1H NMR from figure 6

δ (ppm)

No. of hydrogens X structure

11 1 -OH

7.3 8 Ar-H

6.7 1 C-H

2.3 6 CH3

Crop 2 (pale yellow solid) – 8.49g

The second crop obtained from the organic phase was an impure yield containing bis-

arylated acid and mono-arylated furanone. Thin layer chromatography (TLC) was used to

test the purity of the compounds. Crop 1 showed one spot on the TLC plate and crop 2

showed two spots (figure 7). The first (lowest) spot was the bis-arylated acid and the second

spot (upper) was the mono-arylated furanone, due to its smaller size and molecular formula

the mono-arylated furanone is more polar thus runs further up the TLC compared to the bis-

arylated acid. The 1H NMR in figure 8 shows a peak at 5.8ppm which is a 1H peak that would

be present in the mono-arylated furanone, therefore existing as an impurity.

27

Figure 7: A - pale yellow product (cop 2); B - crystal (crop 1); C – white powder from optimised method

Figure 8: 1H NMR Crop 2 (ppm) - 11.0 (1H, -OH); 7.2 (8H, m, Ar – H); 6.6 (1H, s, C-H); 2.3 (6H, s, CH3).

In order to be able to obtain a pure bis-arylated acid the product can be purified by flash

column chromatography or HPLC because there is a good separation between the two

compounds.

28

3.2 - Cyclopentyl-azanyl-2,3 dichloro,4,4 bis (4-methylphenyl)but-2-enoate (Amine salt)

Bis-arylated acid was reacted with cyclopentylamine under argon at room temperature. The

resulting reaction produced a white precipitate. Aqueous work-up, followed by filtration and

drying resulted in a pale white powder (1.02g). The reaction scheme is shown in figure 9.

TLC, 1H NMR and infrared was performed to analyse the compound formed.

Figure 9: Reaction scheme showing the formation of the amine salt (cyclopentyl-azanyl-2,3 dichloro, 4,4 bis (4-methylphenyl)but-2-enoate

By performing this experimental the difference between amine salt and amide spectras can

be understood when developing novel amide CCK antagonists. The amine salt did not run on

the TLC plates show in figure 10. DMSO was used for the 1H NMR (figure 11) because the

amine salt was insoluble in chloroform. A Notable peak was the 8.1 ppm indicating NH3+

amine salt formation. From the infrared in figure 12 it was concluded that peaks around

2963cm-1 and 1627cm-1 shows amine salt formation (NH3+ and N-H stretching). Thus the

difference between amine salt (ionic) and amide formation can now be distinguished.

29

Figure 10: TLC plate of amine salts. A, B, C all amine salts.

Figure 11: 1H NMR (ppm) -8.1 (NH3

+, s, broad); 7.3 (8H, m, Ar-H); 3.4 (1H, m, C-H); 2.3 (6H, s, CH3); 1.5-1.9

(8H, m, C-H).

30

Figure 12: Infrared of amine salt - = 2963 (Amine salt NH3+ broad stretch); 2900 (C-H stretch); 1627 (C=O

stretch); 1627 (N-H bending) 1585 (Aromatic C=C, m)

Table 3 Analysis of 1H NMR of figure 11

δ (ppm) No. of hydrogens X structure

8.1 1 NH3+

7.3 8 Ar-H

3.4 1 C-H

2.3 6 CH3

1.5-1.9 8 C-H

31

The behaviour of the salt was also observed, it is very soluble in ethanol and forms yellow

needle shaped crystals upon re-crystallisation, it is very insoluble in chloroform. This

information can be used in the purification process if salt formation is found during

synthesis of the amides.

3.3 - Lewis-base catalysis for the formation of 2,3-dichloro-N-cyclopentyl-4,4-bis(4-


The aim of the experimental was to synthesise an amide through Lewis base catalysis using

the unreacted crystal because it contained the aluminium salt. By heating a sample of the

amine salt to 150C using a hot plate resulting in a brown solid and another sample was

heated under reflux using toluene for four hours resulting in brown oil. As shown in figure

13 the amide was not formed, instead starting materials (bis-arylated acid and

cyclopentylamine) were formed. 1H NMR and infrared were carried out to analyse the

sample.

32

Figure 13: Reaction scheme of lewis base catalysis.

The 1H NMR (figure 14) and infrared (figure 15) did not show any peaks that could indicate

amide formation, instead the NMR showed similar peaks observed in figure 11 and table 3

with the addition of two peaks at 7.8ppm indicating NH2 of the cyclopentylamine, 6.6ppm

shows C-H of the bis-arylated acid and 6.3ppm points towards the C-H of mono-arylated

furanone impurity. The infrared showed no amide functional group peak. It was concluded

that heating the amine salts is a reversible process leading to the starting materials, but all

of the salt was not converted as NH3+ peaks were still observed at 8.2ppm in the NMR and

2916cm-1 in the infrared spectras. An attempt could be made to heat the amine salt in

toluene under reflux for more than 24 hours which could result in an amide formation and

pure bis-arylated acid should be used.

33

Figure 14: 1H NMR of heated amine salt

Figure 15: Infrared of heated amine salt

34

3.4 - 2,3-dichloro-N-cyclopentyl-4,4-bis(4-methylphenyl)but-2-enamide using

ethylchloroformate (Bis-arylated amide)

Bis-arylated acid was reacted with cyclopentylamine using ethylchloroformate (ECF) as the

activating agent. A white precipitate was formed almost immediately. After two hours the

reaction was quenched and aqueous work-up was carried out. The precipitate was filtered

and dried under vacuum forming a pale white solid (0.85g). Solvent extraction was used to

recover a second crop which was a light brown oily substance (50mg). The crops were then

mixed and purified using ethanol and toluene. The reaction schematic is shown in figure 16

shows the formation of the bis-arylated amide and a cyclopentylamine salt, at least two

equivalences of cyclopentylamine is required, one equivalence reacts with the chlorine and

the second equivalence reacts with the bis-arylated acid to form the amide. TLC, 1H NMR

and infrared were carried out to analyse the sample.

Figure 16: Reaction scheme on formation of bis-arylated amide

TLC (figure 17) showed impurities were present in the crops. When compared to the amine

salt the lowest spots were of the same Rf value and the top spot was the bis-arylated amide

formed. The middle spot was the impurity of a reaction between mono-arylated furanone

and the cyclopentylamine. 1H NMR (figure 18) and infrared (figure 19) show the amine salt is

present due to the peaks at 8.2ppm and 2963cm-1 (NH3+), a peak at 5.8ppm is the mono-

35

arylated furanone impurity. Peaks at 4.2ppm in the NMR and the high activity it 2963cm-1

region suggest that bis-arylated amide is present.

Figure 17: TLC of crops 1 and 2 (D) and amine salt (E)

Figure 18: 1H NMR of crude crops 1 and 2

36

Figure 19: Infrared of crude crops 1 and 2

To purify the crude product it was initially dissolved in minimal quantities of ethanol to

dissolve the amine salts. The solution was filtered and the residue was dried, then dissolved

into toluene and filtered again. The cyclopentylamine salt is insoluble in toluene and after

filtration and drying a silver coloured solid is formed, the 1H NMR in figure 21 is of the

cyclopentylamine salt. The filtrate contains the pure bis-arylated amide which is recovered

by evaporating the solvent, resulting in a light brown solid. A flow chart to the purification

process is shown in figure 20. A pure compound was obtained (10mg). 1H NMR shown in

figure 22 and is of the pure bis-arylated amide, table 4 explains what each peak indicates.

From the infrared spectra (figure 23) the peak at 3272cm-1 indicates the amide (CONH2)

functional group of the bis-arylated amide, which was previously not seen because the

amine salts would affect the spectra due to its ionic nature.

37

Figure 20: Flow chart of purification process for bis-arylated amide

38

Figure 21: 1H NMR of cyclopentylamine salt δ (ppm) – 8.0 (NH3

+); 3.5 (1H, m, C-H); 3.3 (H20 peak), 2.5 (DMSO

peak); 1.5-2.0 (8H, m, C-H)

Figure 22: 1H NMR of pure bis-arylated amide δ (ppm) = 7.1 ( 8H, m, Ar-H); 6.6 (1H D20 exchangeable, s, N-

H); 6.2 (1H, s, -CH); 4.2 (1H, m, NH-H); 2.3 (6H, s, -CH3); 1.3 (8H, s).

39

Table 4: Analysis of 1H NMR of figure 20

δ (ppm) No. of hydrogens X structure

7.1 8 Ar-H

6.6 1 N-H

6.2 1 C-H

4.2 1 C-H

2.3 6 CH3

1.3 8 C-H

40

Figure 23: Infrared of pure bis-arylated amide cm-1

– 3272 (amide C0NH stretch); ); 1658 (amide C=O stretch); 2849 (C-H stretch); 1635 (Ar C=C stretch); 814 (Ar C-H stretch).

The yield of the bis-arylated amide is very poor, this is due to the instant amine salt

formation, the mono-arylated furanone impurity also reacts with the cyclopentylamine and

the chloride ions from the ECF react with cyclopentylamine to form a salt. These unwanted

side reactions reduce the yield of the bis-arylated amide. A better synthesis method is

required to produce a better yield. The use of a pure bis-arylated acid instead, a co-solvent

could be added to prevent amine formation and a range of activating agents can be used to

assess which produces the best yield. The addition of triethylamine may prevent the

cyclopentylamine salt formation leading to a better yield. Flash column chromatography

could be used for the purification, due to the amide moving further up in the TLC plates.

41

3.5 - 3-chloro-5-(cyclopentylamino)-4-(3-methylphenyl) cyclopent-2-en-1-one

Bis-arylated acid was reacted with cyclopentylamine using ethylchloroformate (ECF) as the

activating agent and dimethylformamide (DMF) as a co-solvent to prevent amine salt

formation. As a result, less white precipitate was formed during the reaction. After two

hours the reaction was quenched and aqueous work-up was carried out, no residue was

found on the filter paper indicating no amine salt formation. Upon solvent extraction a

crude oil was produced weighing 2g. TLC plates (figure 24) showed two products existed in

the oil and flash column chromatography (figure 25) was used to separate them, thus

purifying the oil. This resulted in recovering unreacted bis-arylated acid as a clear oil and

waxy compound, 3-chloro-5-(cyclopentylamino)-4-(3-methylphenyl)cyclopent-2-en-1-one

(489mg). The compound was formed as a result of the impurity mono-arylated furanone

which was found in the pale-yellow solid when synthesising the bis-arylated acid. The mono-

arylated furanone reacted with the cyclopentylamine to form 3-chloro-5-

(cyclopentylamino)-4-(3-methylphenyl) cyclopent-2-en-1-one. TLC, 1H NMR and infrared was

used to analyse both products

42

Figure 24: TLC plate of – A - crude oil from synthesis of 3-chloro-5-(cyclopentylamine)-4-(3-methylphenyl) cyclopent-2-en-1-one; B- crude oil from synthesis of 2-chloro-N-cyclopentyl-3-(cyclopentylamine)-4,4-bis(4-methylphenyl)but-2-enamide; C – pure bis-arylated amide

Figure 25: TLC plates as a result of flash column chromatography

The unreacted bis-arylated acid was the bottom spot on the TLC in figure 25. The peaks at

1.3ppm and 4.2ppm in the 1H NMR of figure 26 is of diethyl ether which had not been fully

evaporated and thus detected as an impurity in the NMR. In order to achieve a better yield

more ECF and cyclopentylamine should be used thus less bis-arylated acid will be recovered.

If pure bis-arylated acid is used, bis-arylated amide is more likely to be synthesised.

The analysis of 1H NMR of figure 27 is shown in table 5. The infrared for 3-chloro-5-

(cyclopentylamino)-4-(3-methylphenyl)cyclopent-2-en-1-one is shown in figure 28, the most

notable functional group is the 3273cm-1 indicating an amine N-H.

43

Figure 26: 1H NMR of clear oil (bis-arylated acid)

Figure 27: 1H NMR of white waxy 3-chloro-5-(cyclopentylamine)-4-(3-methylphenyl) cyclopent-2-en-1-one. δ

(ppm): 7.2 (4H, m, Ar-H); 6.6 (H, d, -CH); 6.1 ( 1H, m, C-H); 5.7 (H, s, -CH); 4.2 (H, m, C-H); 3.3 (H, D20 exchangeable, NH); 2.3 (3H, m, CH3); 1.5-2.0 (8H, m, C-H)

44

Table 5: Analysis of 1H NMR of figure 26

δ (ppm) No. of hydrogens X Structure

7.2 4 Ar-H

6.6 1 C-H

6.1 1 C-H

5.7 1 C-H

4.2 1 C-H

3.3 1 N-H

2.3 3 CH3

1.5-2.0 8 C-H

45

Figure 28: infrared of waxy 3-chloro-5-(cyclopentylamine)-4-(3-methylphenyl) cyclopent-2-en-1-one cm-1

– 3273 (Amine N-H); 2957 (Aromatic C-H bonds); 2866 (sp

3 C-H bonds); 1783 (C=O); 1633 (Ar C=C); 815 (C-Cl).

3.6 - 2-chloro-N-cyclopentyl-3-(cyclopentylamino)-4,4-bis(4-methylphenyl)but-2-enamide

Bis-arylated acid was reacted with cyclopentylamine using diisopropylcarbodiimide (DIC) as

the activating agent and dimethylformamide (DMF) as a co-solvent to prevent amine salt

formation. As a result no precipitate was formation. After two hours the reaction was

quenched and aqueous work-up was carried out, no residue was found on the filter paper

indicating no amine salt formation. Solvent extraction produced a crude oil weighing 2g. TLC

plates (figure 24, spot B) showed four products in the oil. The top spot was main product

and bottom three spots were impurities in very minimal quantities. Flash column

chromatography was conducted to separate the three products, giving a light orange oil as

the main product with a 50% yield. A 1H NMR (figure 31) is analysed in table 6 showing

which hydrogen atoms the peaks represent.

46

Figure 29: TLC plates as a result of flash column chromatography

The reaction scheme in figure 30 shows the formation of 2-chloro-N-cyclopentyl-3-

(cyclopentylamino)-4,4-bis(4-methylphenyl)but-2-enamide. Initially a bis-arylated amide is

formed as an intermediate form which is attacked by another cyclopentylamine to form 2-

chloro-N-cyclopentyl-3-(cyclopentylamino)-4,4-bis(4-methylphenyl)but-2-enamide. This

occurs because the cyclopentylamine is in excess. If one equivalence is used instead of two

equivalences a bis-arylated amide can be formed. Due to equilibrium balance the

cyclopentylamine will not displace the chlorine but instead attack another bis-arylated acid.

47

Figure 30: Reaction scheme for the formation of 2-chloro-N-cyclopentyl-3-(cyclopentylamine)-4,4-bis(4-methylphenyl)but-2-enamide

Figure 31: 1H NMR of 2-chloro-N-cyclopentyl-3-(cyclopentylamine)-4,4-bis(4-methylphenyl)but-2-enamide δ

(ppm) = 7.1 (8H, m, Ar-H); 6.6 (1H, s, C-H); 5.6 (1H D20 exchangeable, s, N-H); 5.4(1H D20 exchangable, s, N-H); 4.2 (1H, m, C-H); 3.8 (1H, m, C-H) 2.2(6H, s, CH3); 1.0-1.3 (16H, m, C-H)

48

Table 6: Analysis of 1H NMR in figure 30

δ (ppm) No. of hydrogens X Structure

7.1 8 Ar-H

6.6 1 C-H

5.6 1 N-H

5.4 1 N-H

4.2 1 C-H

3.8 1 C-H

2.2 6 CH3

1.0-1.3 16 C-H

49

4.0 – Pharmacokinetic Properties

Before a compound goes into clinical trials, it undergoes a development phase which

involves testing the compounds pharmacokinetic properties such as absorption,

distribution, metabolism and elimination (Turner, 2007). This could be a time consuming

and costly process. In order to increase the chances of success a software prediction engine

http://ilab.cds.rsc.org/ (ACD/I-Lab) was used to predict the possible outcomes if

pharmacokinetic tests were carried out. By doing this labour intensive work on compounds

that would have no potential for marketing is avoided and potential problems that could

slow down the development process are detected. The software assesses the compounds

functional groups, physicochemical properties and compares to existing compounds in its

database to predict the probable properties.

The oral route is the most common and convenient method for drug delivery, it contains

several barriers that could prevent drug absorption such as metabolism by enzymes

(CYP3A4), bacterial degradation, P-glycoprotein efflux and the intestinal wall may act as a

barrier (Perrie, 2010). This limits the amount of drug that can enter the blood (oral

bioavailability), preventing the achievement of therapeutic levels (Jambhekar & Philip,

2009). The major physicochemical factors that affect oral bioavailability of a drug are

solubility and membrane permeability (Aulton 2007). For a drug to be absorbed it must be in

solution (Aulton, 2007). Dissolution is the process of how drug particles dissolve (Aulton,

2007). The solubility of a drug is a parameter of dissolution rate, thus the more soluble the

drug the better its rate of dissolution (Aulton, 2007). Table 7 shows that all the drug

compounds are insoluble in pure water, this will greatly affect the dissolution rate but can

be resolved by dissolving the drug in ethanol which will greatly increase the water solubility

(Aulton, 2007).

http://ilab.cds.rsc.org/

50

Once a drug is dissolved in solution it must pass through the intestinal epithelium so that it

can enter the blood stream (Jambhekar & Philip, 2009). A drug can cross the epithelial

barrier by transcellular, carrier-mediated, paracellular and transcytosis routes (Jambhekar &

Philip, 2009). The most common route for many drugs is passive diffusion through the

transcellular pathway as drugs can pass through the lipid membrane from a region of high

concentration in the lumen to a region of low concentration in the blood (Jambhekar &

Philip, 2009). Since all the drug compounds predicted log P is greater than 2 meaning that

the compounds are lipophilic in nature and with a molecular weight of <500Da they can

easily pass through the intestinal barrier, into the blood stream by passive diffusion through

the transcellular route.

Some drugs fail to pass the development phase due poor oral stability (Herranz, 2003). They

are degraded in stomach either by enzymes or the acidic pH, thus altering the chemical

structure resulting in loss of biological activity. All compounds shown in table 7 are stable in

pH<2, enabling them to enter the small intestine for absorption with their chemical

structures intact.

First pass metabolism effect reduces the net absorption rate, if elimination rate is greater

than rate of absorption therapeutic serum levels cannot be achieved (Jambhekar & Philip,

2009). All compounds showed no significant first pass metabolism effect.

P-glycoprotein (P-gp) efflux affects the net absorption (Perrie, 2010). A drug can enter back

into the lumen by binding P-gp receptors resulting in less drug concentration in blood serum

(Perrie, 2010). Only 3-chloro-5-(cyclopentylamine)-4-(3-methylphenyl) cyclopent-2-en-1-one

showed some P-gp efflux, but is not significant enough to affect the net blood

concentrations.

51

The volume of distribution (Vd) gives an idea of how the drug distributes itself within the

body (Aulton, 2007). All compounds showed a Vd of <10L, implying there is high protein

binding and the drug is mainly found in the blood serum (Jambhekar & Philip, 2009). This

could be beneficial as small amounts of drug are required to achieve serum concentrations

and due to high protein binding the half-life tends to be longer, thus a lower dosing

frequency (Jambhekar & Philip, 2009). A disadvantage to a low Vd value is that, if the

targeted receptor is deep within cells it is unlikely that enough drug would reach its target

(Jambhekar & Philip, 2009). This can be avoided by formulating the drug within micelles,

liposomes or nano-particles that have receptor targeting abilities.

Table 7: Prediction of pharmacokinetic properties using ACD/I-Lab

Compoun

d name

Solubility in pure water (Sw) (µg/ml)

Log P Probability of

F (oral)>30%

Stability pH < 2

First pass metabolism

P-glycoprotein efflux

Volume of distribution (Vd) (L/Kg)

Bis-arylated acid 0.46 5.40 0.805 Good stability

No significant

none 0.31

Amine salt 0.29 5.91 0.811 Good stability

No significant

none 5.58

Bis-arylated amide 0.12 6.75 0.719 Good stability

No significant

none 4.44

3-chloro-5-(cyclopentylamine)-4-(3-methylphenyl) cyclopent-2-en-1-one

180 2.87 0.724 Good stability

No significant

low 3.18


0.23 6.62 0.888 Good stability

No significant

none 4.71

52

The ‘biopharmaceutical classification scheme’ is also used to predict the bioavailability of a

drug by placing it in four classes as shown in table 8 (Aulton, 2007). All drug compounds fall

into class II which is accepted as having a good oral bioavailability.

Table 8: Biopharmaceutical classification scheme (Jambhekar & Philip, 2009)

High Permeability Low Permeability

High Solubility Class I Class III

Low Solubility Class II Class IV

Lipinski rule of five is used to assess compounds ‘drug-like’ properties, it uses the main

physicochemical properties to predict if a drug compound has a good chance of absorption

(Kerns & Di, 2008). The rule allows one property to be out of its range, in this case all

compounds except 3-chloro-5-(cyclopentylamine)-4-(3-methylphenyl) cyclopent-2-en-1-one

have >5 Log p value, but all other values fell within the rules. 3-chloro-5-(cyclopentylamine)-

4-(3-methylphenyl) cyclopent-2-en-1-one had all values within the range of the rules. This

means all the compounds synthesised show drug like properties and further development is

highly recommended (Kerns & Di, 2008).

53

Table 9: Lipinksi rule of five

Compound Name Log P

< 5

< 5 H-bond

donors

<10 H-bond

acceptor

MW < 500

Bis-arylated acid 5.40 1 1 335.22

Amine salt 5.91 3 1 420

Bis-arylated amide 6.75 1 1 402

3-chloro-5-

(cyclopentylamine)-4-(3-

methylphenyl) cyclopent-

2-en-1-one

2.87 1 1 289

2-chloro-N-cyclopentyl-3-

(cyclopentylamino)-4,4-

bis(4-methylphenyl)but-2-

enamide

6.62 2 1 451

To increase reliability, a variety of predictive software’s (commercial and non-commercial)

can be used followed by in-vitro testing.

54

5.0 – Conclusion

The aim the project was to synthesise a novel amide CCK antagonist. Using mucochloric acid

and toluene 2,3-dichloro-4,4-bis(4-methylphenyl)but-2-enoic acid was synthesised, which

was then reacted with cyclopentylamine to synthesise,

cyclopentyl-azanyl-2,3 dichloro, 4,4 bis (4-methylphenyl)but-2-enoate – the amine

was synthesised to understand the difference between amine salt and amide

formation. The benefit in recognizing when an amine salt was formed during amide

synthesis will make future purification processes simpler.

2,3-dichloro-N-cyclopentyl-4,4-bis(4-methylphenyl)but-2-enamide – the yield of the

bis-arylated was very poor and further studies need to be done. A range of

activating agents can be used to optimize amide formation.

3-chloro-5-(cyclopentylamino)-4-(3-methylphenyl) cyclopent-2-en-1-one – the

reaction of the mono-arylated furanone resulted in a very drug-like compound.


– the yield of the enamide was very good and the use of this method to synthesis a

variety of amides is a good future implication

All compounds were purified and analysed using 1H NMR, infrared and TLC. Predictive

software, biopharmaceutical classification system and lipinski’s rule of five prove the

compounds have drug like properties and are eligible for drug development phases. The

compounds should undergo in-vitro opiate assay using guinea-pig ileum, Hallin & Xu (1996)

found CCK antagonists to be highly effective in pain management. Grabowska et al (2008)

had found that drugs targeting the CCK receptors can have anti-cancer effects, especially in

55

the gastrointestinal therefore the effect of the drug compounds should also be tested on

cancer cell lines.

A new generation of compounds using different amines, can be reacted with the bis-

arylated acid to generate a library of novel compounds which can be optimized for drug

development.

56

6.0 – References

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Dufresne, M. Seva, C. Fourmy, D (2006), Cholecystokinin and Gastrin receptors.

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Grabowska, A. Morris, T. McKeinze, A. Kumari, R. Hamano, H. Emori, Y. Yoshinga, K. Waston,

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Hallin, Z & Xu, X. (1996), Role of CCK in noiceception, neuropathic pain and opiate

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Harrold, J. Dovey, T. Blundell, J. Halford, J (2012), CNS regulation of

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Herranz, R (2003), Cholecystokinin Antagonists: Pharmacological and Therapeutical

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Ho¨kfelt T, Skirboll L, Everitt B, Meister B, Brownstein M, Jacobs T, Faden A, Kuga S,

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Jambhekar,S. & Breen, P. (2009), Basic Pharmacokinetics. London, Pharmaceutical Press,

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Liddle, R (1997), Cholecystokinin Cells. Annual Review Physiology, 59, 221-242.

Otsuki, M (2000), Pathophysiological role of cholecystokinin in humans. Journal of

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Pharmaceutical Press, 59-68.

Radu, D (2005), Aspects of psychopharmacology of cholecystokinin, Edita Norstedt Tryckeri

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Rehfeld, J. Agersnap, M (2012), Unsulphated cholecystokinin: An overlooked

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Staljanssens, D. Azari, E. Christiaens, O. Beaufays, J. Lins, L. Camp, J. Smagghe, G (2011), The

CCK (-like) receptor in the animal kingdom: Functions, evolution and structure. Peptides, 32,

607-619.

Turner, J. (2007). Drug Discovery. In: New Drug Development: design, methodology and

analysis, John Wiley & Son Inc, New Jersey, 32-35.

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Zwanger, P. Domschke, K. Bradwejn, J (2012), Neuronal Network of Panic Disorder: The role

of the neuropeptide cholecystokinin. Depression and Anxiety, 29, 762-774.

59

7.0 - Appendix A – 1H NMR and Infrared spectras

60

7.1 – Appendix B – Risk Assessment and COSHH forms

61

7.2 - Appendix C – 1H NMR and Infrared tables used to analyse data

dissertation to submit

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