Developing a Rapid Test for

Designer Drug, Mephedrone Winston Churchill Fellowship Report 2014

Kathryn Kellett

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Table Of Contents

Table Of Contents ............................................................................................................. 2

1 Introduction ............................................................................................................... 4

1.1 Sensor selection .............................................................................................................. 5

1.2 Host-guest chemistry ...................................................................................................... 5

1.3 Calixarenes ..................................................................................................................... 6

1.4 Anthraquinones .............................................................................................................. 7

2 Project aim ................................................................................................................. 8

Methods .......................................................................................................................... 8

2.1 Materials and equipment ................................................................................................ 8

2.1.1 Materials and general procedures ..................................................................................... 8

2.1.2 Analytical measurements ................................................................................................... 9

2.2 Anthraquinone................................................................................................................ 9

2.2.1 Formation of 1, 8- diaminoanthraquinone......................................................................... 9

2.2.2 Formation of Di-benzyl-thiourea anthraquinone ............................................................... 9

2.2.3 Formation of Di-benzyl-thiourea anthraquinone using triethylamine ............................. 10

2.2.4 Formation of di-benzyl-thiourea anthraquinone using triethylamine in ethyl acetate ... 10

2.2.5 Formation of di-benzyl-thiourea anthraquinone in water/acetone ................................ 11

2.2.6 Formation of Di-Benzyl-Thiourea Anthraquinone using P4 Base...................................... 12

2.3 Anthracene ................................................................................................................... 12

2.3.1 Reduction of 1,8-diaminoanthraquinone ......................................................................... 12

2.3.2 Formation of di-benzyl-thiourea anthracene using triethylamine ................................... 13

2.3.3 Formation of di-benzyl-thiourea anthracene in ethanol.................................................. 13

2.4 Calixarene ..................................................................................................................... 14

2.4.1 Detertbutylation of Calixarene19 ...................................................................................... 14

2.4.2 Nitration of Calix[4]arene20 .............................................................................................. 14

2.4.3 Reduction of nitrocalix[4]arene21,22 .............................................................................. 15

2.4.4 Reduction of nitrocalix[4]arene using Pd/C22 ................................................................... 15

2.4.5 Di substitution of 4-tert-butylcalix[4]arene lower rim19 .................................................. 15

3 Results and discussion .............................................................................................. 16

3.1 Anthraquinone.............................................................................................................. 16

3.1.1 Di-thioureabenzylanthracene NMR titration studies ....................................................... 20

3.2 Calixarene ..................................................................................................................... 23

4 Conclusion ................................................................................................................ 25

5 Future work .............................................................................................................. 26

6 References ............................................................................................................... 27

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1 Itinerary

Wednesday 14th May 2014 Fly from London Heathrow to Atlanta, Georgia

Thursday 15th May 2014 Drove from Atlanta to Hattiesburg, Mississippi.

Meet with Prof. Karl Wallace at the University of

Southern Mississippi.

karl.wallace@usm.edu

Thursday 16th May 2014 Started in the Karl's lab and meet the rest of the

team I got to work with for the next 3 months.

Monday 25th August 2014 Drove back to Atlanta airport and flew home to

London.

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2 Introduction

People have been experimenting with psychoactive drugs to change their state of consciousness for

thousands of years. In more recent years there have been large influxes of novel psychoactive

substances (NPS) that are produced to circumvent controlled drug legislation, and yet still produce the

same bioactivity. Cathinones are one class of NPS that were developed to mimic the effects of

amphetamine1. Mephedrone, the most prevalent cathinone

2, first came onto the UK drug scene in

2007 as a legal replacement to amphetamine (Figure 1). Cathinones as a class were made illegal in

2011 after a number of deaths were connected to their use3. The structural similarity between these

two drugs means that a lot of the previous tests for amphetamine are now affected by false positives in

the presence of mephedrone4.

Figure 1 - Structure of Amphetamine and Mephedrone

A number of different techniques have been adopted for the detection of small molecules. The most

common include; immunoassays, ELISA, colourmetric tests and chemometric sensors. Each of these

has been utilised in the field of drug detection for common drugs of abuse5,6

. However, the speed at

which NPS’s are coming onto the market has meant that a number of these tests are no longer

sufficient. This is due to the fact that so many NPS are appearing that are very structurally similar that

previous tests that are based on structural differences in drugs are no longer adequate. Therefore new

methods of rapid drug detection are necessary that will allow for a greater degree of selectivity.

Current immunoassay tests are very selective towards the drugs that they detect, however they are

expensive, time consuming and require expertise to operate. Colourmetric tests such as the Marquis

Test are cheap and easy to use, they are however not very selective and are used to identify a number

of different drugs7. This is becoming increasing harder due to the amount of drugs present. Ideally a

new drug testing method would encompass the positive features of both of these tests. This will allow

for a rapid, cheap, selective and easy to use testing device. There is currently no test that achieves this

for mephedrone or the cathinone class.

Therefore the work that is being carried out is to develop a rapid testing device for mephedrone. This

is to be achieved using supramolecular chemistry techniques. Supramolecular chemistry utilises the

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concept of host-guest interactions. This will allow mephedrone to be detected using non-covalent

interactions such as hydrogen bonding, electrostatics and dispersion interactions. By designing a host

molecule that adopts these features in a design that is specific for mephedrone, it will allow for the

host molecule to selectively detect mephedrone.

The design of such sensors is achieved through development of a 3-point pharmacophore that is

unique for mephedrone. This was designed using computational techniques, at the University of

Hertfordshire prior to the sensor design and synthesis.

2.1 Sensor selection

An empirical approach to the host molecule design was adopted, using binding patterns of small

molecules with large proteins to develop a pharmacophore. Inspiration was taken from how binding of

small molecules in nature occurs and these interactions were studied allowing for the host molecules

to mimic such interactions. This lead to the use of two different classes of molecules: calixarenes and

anthraquinones. Through careful design of both molecules they can be designed to incorporate the

desirable pharmacophoric features. Calixarenes are rigid, pre-organised structures that allow for

minimal re-organisation of the host molecule upon addition of the guest, whereas anthraquinones are

more flexible and therefore allow for the host molecule to adapt more to the introduction of a guest

molecule. It is important to study both flexible and rigid host molecules so as to understand the effect

this has on the binding ability as well as the selectivity of the host molecules. Thiourea groups have

been incorporated into both the sensor designs as they are known to have good hydrogen bonding

capabilities, which will be beneficial in the binding of potential host molecules.

2.2 Host-guest chemistry

In supramolecular chemistry, host-guest chemistry is the study of large ‘host’ molecules that are

capable of enclosing smaller ‘guest’ molecules via non-covalent interactions8. Host-guest chemistry

encompasses the detection of anions, cations and neutral compounds to specially designed host

molecules. This principle builds on the theory first introduced by Emil Fischer in 1894, the lock and

key mechanism. In this model only a substrate that is complimentary to the target site will bind and

produce a reaction. The binding site within the host molecule is designed to selectively attract the

guest molecule of interest. They are designed to interact using the four most common non-covalent

interactions, which are: hydrogen bonds, ionic bonds, Van der Waals and hydrophobic interactions.

The strongest interactions are hydrogen bonding at 2-30 kcal/mol while Van der Waal interactions are

the weakest at just 0.1-1 kcal/mol. When designing a host molecule it is also important to take into

account directionality for the interactions. Hydrogen bonds for example are highly directional and the

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strongest interaction occurs at 180 degrees. All of these interactions can be incorporated into the

design of a host molecule to allow it to be selective towards a target guest molecule.

2.3 Calixarenes

Calixarenes are cyclic host molecules that are synthesised by condensation of p-substituted phenol

and formaldehyde8. They form three dimensional bowl structures with hydrophobic cavities that can

encapsulate smaller molecules or ions. They come in a number of sizes that are classed by the number

of phenol groups within the ring, the most commonly studied are calix[4]arene and calix[6]arene

(Figure 2). Calixarenes can be substituted on both the ‘upper’ and ‘lower rim’8,9

. The upper rim is

defined as the wide rim, where the t-butyl groups are situated, while the hydroxyl groups are

positioned on the lower rim, or narrow rim. There is a large amount of literature surrounding the

substitution of the ‘lower rim’; as well as the ‘upper rim’ as the t-butyl groups as easily cleaved9,10

.

Figure 2 - Structures of Calix[4]arene and Cali[6]arene. The upper and lower rim in the para positions can both be

substituted

By selectively altering the upper and/or lower rim it is possible to design hosts that are capable of

binding anion, neutral and cation guest species. Agraval et al. outlined in detail the variations of

substituents that are possible on both the upper and lower rim of calixarenes11

. Upper ring substitution

is carried out by first detertbutylating the upper rim followed by a subsequent reaction. The hydrogen

bonding from the hydroxyl groups makes the compounds water insoluble, which can lead to synthetic

complications. However Shinkai et al. found that the addition of sulfonate groups on the lower rim

produces water-soluble calixarenes12

. Some of the earliest work carried out on the alterations of

calixarenes is the esterification of the lower rim hydroxyl groups. This work showed that strict

controls of the reactions conditions is necessary in order to produce the desired product11

. This work

has lead to a number of different studies looking at the effects of different substituents, their effects on

the calixarene conformations and the type of guest molecules that can be attracted, for example;

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etherification, bridged calixarenes and bis-calixarenes. The concept of dimerising calixarenes via

substitution of the lower rim has lead to the developments of selective anion sensors10

. It is the ability

to alter the cavity size of calixarenes, as well as the large number of possible substitutions, that makes

them very versatile as host-guest complexes. This makes it possible to be selective as to the size of

guest molecule that is intended to bind. This is particularly useful to allow calixarenes to be adapted

to allow for selective binding of mephedrone.

Calixarenes are pre-organised, semi-rigid structures that contain a high degree of rotation around the

methylene bond. This leads to a number of known conformations occurring; cone, partial cone, 1,3

alternate and the 1,2 alternate (Figure 3)13

.

Figure 3 – Four possible conformations that have been reported for calixarenes

It is possible to fix the conformation of the calixarenes into the more desirable cone conformation by

selective substitution of the lower rim with large functional groups such as isopropyl and benzyl

functionalities10

. The larger the number of units within the calixarenes the harder it is to fix the cone

structure due the bulkiness of the substituted lower rim. The effect of substitution on the conformation

of the calixarene is extremely important when considering the overall binding cavity of the host

molecule.

2.4 Anthraquinones

Anthraquinones are a class of molecules based on the 9,10- anthraquinone skeleton (Figure 4). They

are some of the most widely used polycyclic systems in both nature and technology due to their

unique physical properties14

. They are naturally occurring compounds that are very well studied for a

number of uses including as; dyes, pharmaceuticals and host molecules. This allows for a large variety

of derivatives to be synthesised.

8

Substituted anthraquinones are flexible with no pre-organised nature. They are synthesised via

oxidation of anthracene, typically using chromium trioxide as the oxidant15

. Literature surrounding

anthraquinone analogues shows that substitution is not limited to the 4 and 5 position. Dhananjeyan et

al. looked at the synthesis of a range of anthraquinone analogues, and their biological properties16

.

While, Mariappan et al. reported a number of synthesised compounds that were used as host

molecules. Anthraquinone metal complexes were used for the detection of Cu2+

and Fe3+17

. Wu et al.

also looked at the detection of anions using anthraquinones that possess a single substitution on the

ring18

. In this study the host anthraquinone molecules showed good selectivity for fluoride ions as

colourmetric sensors. Both these studies used UV-Vis to quantify the interactions. There appears to be

no literature where anthraquinones are utilised for the detection of neutral small molecules, as

opposed to anions and cations. However the information from previous anthraquinone studies as host

molecules has shown that they have highly fluorescent properties. It is the highly conjugated ring

system which produces their fluorescence. This can be easily utilised and integrated into the design of

a host molecule.

3 Project aim

The aim of this project is to synthesis two potential host molecules for the detection of mephedrone;

one calixarene and anthraquinone. The host molecules will then be tested against mephedrone to

determine the binding interactions and affinity.

Methods

3.1 Materials and equipment

3.1.1 Materials and general procedures

All solvents used were obtained from Pittsburgh, Fisher Scientific. All chemical reagents and

deuterated solvents for NMR analysis were purchased from Sigma Aldrich, Montana, USA. Thin

layer chromatography (TLC) silica gel with fluorescent plates 254 nm, thickness 0.2mm were

purchased from Sigma Aldrich, Montana, USA.

Figure 4 - 9,10-Anthraquinone skeleton that all anthraquinones are based on

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3.1.2 Analytical measurements

All nuclear magnetic spectra (NMR) were recorded on a Bruker 400 MHz NMR instrument. With the

exception of the variable temperature studies, which were carried out on a Bruker 600 MHz

instrument.

3.2 Anthraquinone

3.2.1 Formation of 1, 8- diaminoanthraquinone

1,8 – dinitroanthraquinone (504 mg, 1.69 mmol), sodium sulphide (1.422 g, 6.76 mmol) and sodium

hydroxide (0.2750 g, 6.76 mmol) were combined in ethanol (15 mL) and refluxed for 6 hours. After

which the reaction was cooled in ice and left to precipitate out fully. The reaction mixture was filtered

under vacuum, and washed with excess water, yielding a deep purple precipitate (0.361 g, 90 %). 1H

NMR (CDCl3)δ: 6.75 (s, 4H, 2NH2), 6.97-6.92 (d, 2H, J=20 Hz, Ar-2CH), 7.44-7.39 (t, 2H, J=20 Hz,

Ar-2CH), 7.60-7.63 (d, 2H, J=20 Hz, Ar-2CH)

3.2.2 Formation of Di-benzyl-thiourea anthraquinone

1-8-diaminoanthraquinone (81 mg, 0.4 mmol) and benzoisothiocyanate (111 μl, 0.8 mmol) were

added to DCM (5.5 mL) and left to reflux and monitored by TLC. After 24 hours TLC still showed

significant starting material and formation of two new spots. After 24 hours the reaction appeared to

Scheme 1 - Synthesis of 1,8-diaminoanthraquinone

Scheme 2 - Formation of di-benzyl-thiourea anthraquinone

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stop progressing and the reaction was removed and left to cool. The reaction was evaporated to

dryness yielding a brown residue. The reaction mixture was separated through column

chromatography with silica gel, eluting with EtOAc/hexane 10/90, isolating starting material, by-

products and no presence of product.

3.2.3 Formation of Di-benzyl-thiourea anthraquinone using triethylamine

1-8-diaminoanthraquinone (203 mg, 0.8 mmol), benzoisothiocyanate (286 μl, 1.7 mmol) and

triethylamine (0.26 mL, 1.8 mmol) were combined in DCM (40 mL) and refluxed. Reaction was

monitored via TLC. After 24 hours the reaction appeared to stop progressing, starting material still

present along with 3 additional spots on the TLC. The reaction was filtered producing a purple solid,

which was found to be anthraquinone-starting material. Filtrate was treated with hydrochloric acid (1

M) to remove excess triethylamine, the reaction mixture was TLCed showing 3 spots remaining, one

of which corresponded to the benzothiocyanate starting material. Reaction was separated using

column chromatography with silica gel eluting with EtOAc/hexane 10/90, isolating starting material

and two additional by-products, but no product present.

3.2.4 Formation of di-benzyl-thiourea anthraquinone using triethylamine in ethyl acetate

Scheme 3 - Formation of di-benzyl-thiourea anthraquinone using triethylamine

11

1-8-diaminoanthraquinone (100 mg, 0.4 mmol), benzoisothiocyanate (111 μl, 0.9 mmol) and

triethylamine (0.26 mL, 1.8 mmol) were combined in ethyl acetate (40 mL) and refluxed. Reaction

was monitored via TLC. After 24 hours the reaction appeared to stop progressing, starting material

still present along with 3 additional spots on the TLC. The reaction was filtered producing a purple

solid, which was found to be anthraquinone-starting material. Filtrate was treated with hydrochloric

acid (1 M) to remove excess triethylamine, the reaction mixture was TLCed showing 3 spots

remaining, one of which corresponded to the benzothiocyanate starting material. Reaction was

separated using column chromatography with silica gel eluting with EtOAc/hexane 10/90, isolating

starting material and two additional by-products, but as with DCM, no product present.

3.2.5 Formation of di-benzyl-thiourea anthraquinone in water/acetone

Scheme 5 - Formation of di-benzyl-thiourea anthraquinone

1,8-diaminoanthraquinone (100 mg, 0.4 mmol), benzoisothiocyanate (111 μL, 0.9 mmol) and carbon

disulphide (0.05 mL, 0.8 mmol) were combined in Acetone/water (3:1, 20 mL) and refluxed. After

three hours a product spot was visible on the TLC as well as the anthraquinone starting material. The

reaction was monitored by TLC for 28 hours, starting material still present, but reaction has stopped

proceeding. Reaction was removed from the heat, filtered and the filtrate was evaporated to dryness.

Filtrate was analysed by NMR and was shown to be anthraquinone-starting material, filtrate was

shown to be benzothiocyanate and by-products, not product observed.

Scheme 4 - Formation of di-benzyl-thiourea anthraquinone using triethylamine in ethyl acetate

12

3.2.6 Formation of Di-Benzyl-Thiourea Anthraquinone using P4 Base

1.8-Diaminoanthraquinone (200 mg, 0.8 mmol) and phosphazene base P4 – tert butyl (1.17 g, 1.9

mmol) were stirred in ethyl acetate (35mL) and stirred at room temperature for 10 minutes.

Benzoisothiocyanate (0.25 mL, 1.9 mmol) was taken up in ethyl acetate (2 mL) and added dropwise

to the reaction mixture. The reaction was left at room temperature for 64 hours, and was monitored by

TLC. Reaction was filtered and analysed by NMR, producing starting material. No product formation

occurred.

3.3 Anthracene

3.3.1 Reduction of 1,8-diaminoanthraquinone

1,8-diaminoanthraquinone (1 g, 4.2 mmol) was dissolved up in isopropanol (50 mL) and stirred under

nitrogen for 15 minutes. Sodium borohydride (2 g, 53 mmol) was added and the reaction was refluxed

for 36 hours. The reaction mixture was added to iced water (100 mL) and a green precipitate formed,

reaction was filtered and washed with excess water. Yielding a dark green solid (0.8152 g, 93%). 1H

NMR (CDCl3)δ: 4.21 (4H, NH, s), 6.96 (2H, Ar-H, d), 7.42 (2H, Ar-H, t), 7.81 (2H, Ar-H, d), 8.22

(1H, Ar-H, s), 8.37 (1H, Ar-H, s).

Scheme 6 - Formation of di-benzyl-thiourea anthraquinone using P4 superbase

Scheme 7 - Reduction of 1,8-diaminoanthraquinone to 1,8-diaminoanthracene

13

3.3.2 Formation of di-benzyl-thiourea anthracene using triethylamine

1,8-Diaminoanthracene (700 mg, 3.4 mmol), benzoisothiocyanate (0.76 mL, 6.7 mmol) and

triethylamine (1.25 mL, 7.9 mmol) were taken up in DCM (70 mL) and refluxed for 24 hours.

Monitoring by TLC showed presence of anthracene starting material and formation of a new spot.

Reaction was removed and placed in ice, and filtered. Precipitate was washed with ethyl acetate /

hexane (1:9). TLC of precipitate showed two spots, neither corresponding to starting material. Product

was purified via column chromatography using silica gel. Both spots isolated and NMRed showing

possible multiple conformers present.

3.3.3 Formation of di-benzyl-thiourea anthracene in ethanol

1-8-Diaminoanthracene (877 mg, 4.2 mmol) and benzoisothiocyanate (1.12 mL, 8.4 mml) were taken

up in ethanol (100 mL) and refluxed for 2 hours. The reaction was cooled in ice, and filtered. The

precipitate was washed with excess water and ethanol. Product was dried under vacuum, yielding a

dark brown solid (1.04 g, 40 %). 1H NMR (Acetone-d6) δ: 4.91 (s, 4H, 2CH2), 7.19-7.75 (m,

Aromatic), 8.08-8.11 (d, 4H, J= 12 Hz, Ar-2CH-anthracene), 8.91 (s, 2H, 2NH), 9.20 (s, 2H, 2NH).

IR (FT-IR) cm-1

: 1200 (s, C=S).

Scheme 8 - Formation of di-benzyl-thiourea anthracene

Scheme 9 - Formation of di-benzyl-thiourea anthracene

14

3.4 Calixarene

3.4.1 Detertbutylation of Calixarene19

4-Tert-butylcalix[4]arene (2.0 g, 3.1 mmol), phenol (1.35 mL, 15 mmol) and anhydrous aluminium

trichloride (2.0 g, 15 mmol) were taken up in toluene (70 mL) and stirred at room temperature. The

solution went from clear to yellow after a few hours, and after stirring overnight the solution was

opaque beige in colour. Hydrochloric acid (0.2 M, 5 mL) was added dropwise. The Toluene phase

was extracted and washed with water (3 x 25 mL), the combined organic layers were combined and

evaporated yielding a beige residue. The product was precipitated with methanol in ice and filtered.

The crude white solid was recrystallized from chloroform/methanol (2:3) yielding a white crystalline

solid (0.9992 g, 76 %). 1H NMR (CDCl3)δ: 1.82 (s, 8H, CH2), 6.71-6.73 (t, 4H, J=7.8 Ar-H), 7.01 (d,

8H, Ar-H), 10.18 (s, 4H, OH).

3.4.2 Nitration of Calix[4]arene20

Acetic acid (5 mL) and nitric acid (2.2 mL, 33 mmol) were slowly combined and cooled in ice.

Calix[4]arene (250 mg, 0.6 mmol) was taken up in dichloromethane (5 mL), and cooled in ice.

Calix[4]arene solution was added dropwise into the acid solution stirring in ice. On completion of

addition reaction was bright orange. The mixture was stirred in ice for 5 hours, and then added to 25

mL of cold water. The residual solid was filtered off, washed with excess water followed by

dichloromethane, yielding a pale yellow solid. 1H NMR (DMSO-d6) δ: 8.14 (8H, s, ArH), 3.4 to 3.16

(8H, s, ArCH2Ar), 5.8 (4H, s, OH). IR (FT-IR) cm-1

: 1620 (stretch, N-O), 1300 (stretch, N-O).

Scheme 10 - De-tertbutylation of tert-butylcalix[4]arene

Scheme 11 - Nitration of Calix[4]arene

15

3.4.3 Reduction of nitrocalix[4]arene21,22

Nitrocalix[4]arene (107 mg, 0.18 mmol) and tin(ll) chloride dihydrate (0.796 g, 3.5 mmol) were added

together in ethanol (20 mL) and refluxed for 5 hours, as no starting material was present on TLC at

this point. The reaction was cooled, and potassium hydroxide (1M) was added to adjust the solution to

pH≈10. The reaction was then filtered, yielding no product. The filtrate was evaporated to dryness and

analysed, showing no presence of expected product.

3.4.4 Reduction of nitrocalix[4]arene using Pd/C22

Nitrocalix[4]arene (200 mg, 0.34 mmol), hydrazine (3.24 mL, 5.2 mmol) and Pd/C (0.19 mg, 1.76

mmol) were dissolved up in isopropyl alcohol (60 mL). The calixarene does not fully dissolve as seen

with a number of other solvents. The reaction was refluxed under nitrogen for 4.5 hours. The

calixarene is seen to be more soluble upon heating. The reaction was then cooled and evaporated to

dryness. Resultant residue was analysed and showed no presence of product but presence of

calixarene that clearly was no soluble.

3.4.5 Di substitution of 4-tert-butylcalix[4]arene lower rim19

4-Tert-butylcalix[4]arene (200 mg, 0.3 mmol) was taken up in pyridine (10 mL) and stirred in ice for

an hour. Benzoyl chloride (0.29 mL, 2.5 mmol) was added dropwise into the reaction mixture and left

to stir for 1.5 hours. The reaction mixture was poured into ice water (50 mL) and a white precipitate

immediately started to form. The precipitate was filtered off, washed with water and then

Scheme 12 - Reduction of nitrocalix[4]arene to aminocalix[4]arene

Scheme 13 - Reduction of nitrocalix[4]arene using Pd/C

Scheme 14 - Disubstituion of 4-tertbutylcalix[4]arene

16

recrystallized from chloroform/ethanol (3:5 v/v), filtered and washed with ethanol yielding a white

crystalline solid (0.0533 g, 20%). 1H NMR (CDCl3)δ: 1.04 (s, 18H, t-bu), 1.19 (s, 18H, t-bu), 3.53 –

3.57 (d, 4H, ArCH2Ar, J=14.21 Hz), 4.00-4.03 (d, J=14.17 Hz, 4H, ArCH2Ar), 5.18 (s, 2H, ArOH),

6.94 (s, 4H, Ar-H, (-benz)), 7.05 (s, 4H, Ar-H, (-OH)), 7.57 – 7.59 (t, 4H, m-Ar-H, J=7.80 Hz), 7.74-

7.78 (t, 2H, p-Ar-H, J= 7.47 Hz), 8.37-8.39 (d, 4H, o-Ar-H, J=7.08 Hz).

4 Results and discussion

4.1 Anthraquinone

The proposed reaction scheme was a three-step process (Scheme 15), starting from the commercially

available 1,8-dinitroanthraquinone. This allowed for reduction of the nitro groups to aminos followed

by the formation of the thiourea functionality. Concurrently the napthylmethylamine needs to be

converted to napthylthiocyanate to allow a coupling between the two starting materials, to form the

target compound, 1,8-dinapthylthioureaanthraquinone.

The reduction of the 1,8-dinitroanthraquinone was carried out using sodium sulphide and sodium

hydroxide in ethanol. Four equivalents of both bases were used and the reaction was refluxed for six

hours. The reaction mixture is simply filtered producing a deep purple precipitate, and was washed

with excess water to remove any remaining base. The reaction was successfully repeated with yields

of up to 90%.

As a model compound benzoisothiocyanate was originally used to couple to the 1,8-

diaminoanthraqiunone to ensure that ideal reaction conditions were achieved. This reaction was tried

a number of times using a variety of conditions. Both triethylamine and sodium hydroxide were

attempted in DCM, ethyl acetate and ethanol under reflux. Reactions were attempted from one hour to

one week all of which were monitored via TLC throughout the reaction. All of these reactions gave

starting material after the workup. The amino group on the anthraquinone appeared to be unreactive to

all of these conditions. All previous literature surrounding these reactions have been based upon

Scheme 15 - Proposed reaction scheme for the synthesis of di-napthylthiourea anthraquinone

17

anthracene moieties instead of anthraquinones therefore there is no carbonyl group positioned

between the amino groups. Most notably Kim et al. who synthesised a variety of thiourea substituted

anthracene compounds23

. It is possible that the carbonyl group is preventing amino groups from

reacting, by stabilising the structure through hydrogen bonding.

Figure 5 shows how it is possible for stable six membered intramolecular rings to form due to the

hydrogen bonding. This could theoretically prevent the amino groups from reacting even in the

presence of the harsh reaction conditions that have been attempted. It would also explain why only

starting material was isolated from the reaction. In order to test this theory and to be sure this reaction

will not progress, a phosphazene superbase was used. Tertbutyl-P4 is a more powerful base than most

amines with a pKBH+ value of 30.124

. 2.2 equivalents of P4 were taken up in ethyl acetate and the

reaction was refluxed for 20 hours, with monitoring by TLC. No reaction took place and just starting

material was once again isolated from the reaction mixture.

Given that the P4 reaction was not successful the anthraquinone sensor was redesigned in order to

develop a sensor that can be synthetically produced. Therefore the next step was to reduce the

anthraquinone to an anthracene moiety (Figure 6), which will free up the amino functionalities to

react and form the thiourea groups needed for the host molecule. There is already a lot of literature

surrounding the reduction of anthraquinones. The simplest reaction reported by Wong et al.25

was

carried out by refluxing sodium borohydride and 1,8-diaminoanthraquinone in isopropyl alcohol for

36 hours, yielding a brown solid with yields consistently above 80%. This is an improvement from the

published yield of just 55%.

Figure 5 - Theoretical hydrogen bonding occurring between both amino functionalities and the carbonyl

18

The di-aminoanthracene was reacted with 2.2 equivalents of benzoisothiocyanate, to see what effect

the removal of the carbonyl groups has on the reactivity. The TLC of the reaction mixture showed 3

spots including the corresponding starting material. The spots were separated using column

chromatography and analysed using 1H NMR. The first spot appeared to show formation of the

thiourea on just one amine group, producing the one arm host compound. The second spot appeared to

show possible formation of the desired di-thiourea host molecule, and was isolated as a yellow solid.

As seen in the proton NMR in Figure 7 there is a large amount of peaks within the aromatic region,

more than would be expected for this compound.

Figure 7 - Variable low temperature proton NMR for di-thioureabenzylanthracene run in acetone-d6

Figure 6 - Reduced anthraquinone to form dinitroanthracene

25°C

15°C

5°C

-5°C

-15°C

-25°C

-50°C

19

There are also four regions for amine peaks, due to the symmetry of the compound only two peaks

would be expected. This could be due to different conformers being formed, this has been found to be

true for a number of di-thiourea compounds26,27

. The conformations that have been reported are anti-

anti, syn-anti and syn-syn (Figure 8). The syn-anti conformation produces a non-symmetrical

molecule that would explain the four different amine peaks seen in the NMR. The different

conformations may also help explain why there are more peaks within the aromatic region than would

be expected. This may be due to the different conformers affecting the 9 position of the anthracene in

different ways leading to multiple peaks being seen. In order to try and freeze the conformers out to

try and see if it is possible to see just one within the proton NMR, a low temperature study was

conducted. As seen in Figure 7 this did not occur as expected, a number of peaks shifted, but the final

spectrum run at -50oC still does not represent the expected number of peaks for one conformer.

Figure 8 - Possible conformers for di-thioureabenzylanthracene

Due to the inconclusiveness of the previous reaction and the NMR study performed different reaction

conditions were attempted. Literature surrounding this reaction shows the reaction has always been

carried out in DCM. However, DCM is known to be ever so slightly acidic and this may have an

effect on how the reaction progresses. For this reason ethanol was used to try and see what effect this

may have on the reaction. The reaction was refluxed in ethanol for two hours, with the absence of

base. After two hours the reaction mixture was cooled in ice and filtered, yielding a dark brown solid

with a yield of 80%. 1H NMR for the product was as predicted showing two regions for the amines

and distinct aromatic peaks (Figure 9).

20

The reaction was repeated over 1 hour and 3 hours producing lower yields. Therefore it was

concluded that 2 hours was the ideal conditions for the reaction.

Once the model compound was synthesised using benzyl groups in place of the desired naphthyl

compounds, the focus was moved towards trying to produce the naphthyl isothiocyanate in order to be

able to produce the target host molecule, di-thiourea naphthylanthracene. Dahan et al. reported the

conversion of the amines to the corresponding isothiocyanate functionalities, however they are

directly attached to anthracene units as well as pyrene unit derivatives28

. These conditions were

attempted on the napthylmethylamine, using carbon disulphide and ethylchloroformate to form the

isothiocyanate. After work up a dark yellow solid was isolated and the proton NMR shows no

presence of amine functionalities. Analysis of the compound using FT-IR shows an absence of an

amine peak at 3400 cm-1

as seen in the starting material. A small peak can be seen at 1200 cm-1

, which

corresponds to the new carbon sulphur double bond. This reaction was not easily reproduced and did

not always yield the target compound, when successful yields of 45% were obtained.

The naphthylthiourea was reacted with the di-aminoanthracene in ethanol, as before, but no product

was isolated. The reaction was done on a small 100 mg scale, which was not attempted for the benzyl

compound. Further experiments will need to be completed on a larger scale, while closely monitoring

by TLC to see what affect this has on the reaction.

4.1.1 Di-thioureabenzylanthracene NMR titration studies

Due to the limitations of using mephedrone within the United States due to licencing requirements,

the precursor to mephedrone was used in order to carry out preliminary tests on the host molecule.

Figure 9 - 1H NMR for di-thioureabenzylanthracene (ethanol) run in acetone-d6

21

NMR titration studies allow interactions between the host and guest to be analysed by the relative

peak shifts seen in the spectrum. Acetone-d6 was used as the solvent for all titration studies, as

opposed to d6-DMSO, which it is more soluble in. This is because DMSO is a competitive binding

solvent, due to its ability to hydrogen bond29

. Therefore the guest would have to displace the DMSO

from the binding site before any interaction could occur. This can lead too smaller chemical shifts

within the NMR titration and consequently less favourable binding constants.

The three peaks that are predicted to shift upon addition of the guest molecule are the two amine

peaks at 9.2 and 8.9 ppm as well as the C9 position on the anthracene at 7.6 ppm. The guest was

added from 0 equivalents to 5 equivalents of the host molecule and the peak shifts can be observed in

Figure 10. The other peaks within the spectrum do not show any sign of shifting; this shows that there

is little, if no interaction between the host and guest molecule aromatic functionalities allowing for pi-

stacking interactions. What cannot be observed is the binding plateauing out at higher equivalents.

This suggests that the host-guest binding is not first order.

Figure 9 - 1H NMR titration study using host molecule, di-thioureabenzylanthracene against guest molecule, 4-

methylpropiophenone in acetone-d6

The next step will be to test the host molecule against mephedrone hydrochloride in order to

understand how the amine group affects binding and whether more intense binding interactions can be

observed.

22

To ensure that the interactions seen in the NMR titration study using the mephedrone precursor are

valid, a control study was carried out. For the control test tetrabutylammonium chloride (TBACl) was

used. TBACl will test the host molecules affinity for chloride anions. The same equivalents were used

as carried out for the mephedrone precursor titration to allow for a direct comparison. The results

from the titration showed an interesting effect on the host molecule spectrum. At just 0.25 equivalents

of the TBACl there was a significant shift seen in all three of the peaks of interest, by 1 equivalent the

peaks stop shifting and were not affected by the further addition of TBACl. This suggests a first

order-binding constant, which was not seen for the mephedrone precursor. The significant shift seen

between 0 and 0.25 equivalents makes it hard to calculate any conclusive binding constant, therefore a

further titration study was carried out with more points been 0 and 1 equivalents (Figure 11). Even

with these further data points there is still a very large shift between 0 and 0.1 equivalents. The shift

of 1.5 ppm seen for 0.1 equivalents of TBACl is larger then the shift seen after the addition of 5

equivalents of the mephedrone precursor.

Figure 10 - 1H NMR titration study using host molecule, di-thioureabenzylanthracene against guest molecule, TBACl in

acetone-d6

Given the effect that chloride has on the host molecule further anions were tested to see if they also

show good binding affinities. Full NMR titration studies for the TBA salts of bromine, iodine and

nitrate were carried out. Anions of varying sizes were used to understand how this affects binding.

23

Figure 12 shows the different binding isotherms for the anions, chlorine shows the best binding as

expected due to its smaller size. The nitrate anion, being the largest of the anions shows weakest

binding, there is still however a noticeable shift in the NMR peaks for all of the anions. Further

titrations will need to be carried out on larger anions such as fluoride, acetate and sulphate. This will

help to establish the size limit for the cavity when binding anions.

Figure 11 - Binding isotherms for nitrate, bromide, chloride and iodide anions in acetone-d6

This clearly presents a new challenge, not only because mephedrone is found as a hydrochloride salt,

but also because various anions may be found within a street sample of mephedrone. However, as the

sensor still binds both the mephedrone precursor and the anion it may be possible to use the sensor as

a joint anion and cation sensor. This would be a very unique sensor concept that has not been

implemented for drug detection before. Further conclusions cannot be drawn until the host molecule is

tested against mephedrone and mephedrone hydrochloride. It will also be important to see how the

host molecule interacts with the free base of mephedrone with the absence of chloride to truly

understand the interactions that are occurring.

4.2 Calixarene

The aim was to synthesis 1,3-dithioureanapthylcalixarene, and then use the understanding gained

from this to develop a number of derivatives based on differing number of substitutions on the

calixarene bowl. Scheme 16 shows the proposed synthetic route for 1,3-dithioureanapthylcalixarene.

8.8

8.9

9

9.1

9.2

9.3

9.4

9.5

9.6

9.7

9.8

9.9

0 0.02 0.04 0.06 0.08 0.1

NM

R s

hif

t (p

pm

)

Anion Concentration (mg/ml)

[Cl-]

[Br-]

[I-]

[NO3-]

24

Scheme 16 - Proposed reaction procedure for the synthesis of di-thioureanapthylcalix[4]arene

The first step involves detertbutylation of the upper rim of the starting tertbutyl-calix[4]arene. This is

a well established method reported in a number of literature sources9,12,19

. This was carried out in

toluene via retro-Friedel-Crafts detertbutylation; five equivalents of aluminium chloride were used.

The reaction was left at room temperature overnight, before being worked up following a procedure

set out by Elçin et al19

. The most recent NMR predictions for calixarenes carried out by Magrans et

al.30

showed that the detertbutylated calixarene could not exclusively adopt the cone conformation.

However NMR analysis of compound (2) shows the Ar-CH2-Ar chemical shifts at 3.57 and 3.88 ppm,

which would suggest a cone conformation, with di-substitution of the upper rim given the two

chemical environments present. This may be due to the rapid exchange between the cone and 1,3

alternate conformation in solution.

Compound (1) was then nitrated using acetic acid and nitric acid in DCM. To prevent over nitration of

the upper rim, the reaction was run using nitric acid as the limiting reagent and was reacted at low

temperature. DCM was used due to the insolubility of compound 1 in aqueous media, however the

starting material was still not completely soluble within the reaction mixture. After reacting for 2

hours the reaction was worked up as outlined in Kumar et al.20

. Crude 1H NMR showed presence of

starting material as well as product, washing with DCM and ethanol produced the pure product with

di-substitution of the upper calixarene rim. IR data corroborates these findings showing IR stretches at

1620 and 1300 cm-1

indicative of N-O stretch of the nitro groups.

In order to introduce the thiourea functionalities the nitro groups need to be reduced to amines. A few

different reaction conditions were attempted for the reduction. There is currently no literature

associated with this reaction using calixarenes; therefore known reduction conditions for different

compounds were attempted. The first attempt was using tin (II) chloride, in ethanol. The reaction

25

mixture was refluxed for 5 hours, until no starting material was present on the TLC. The reaction was

worked up, but the NMR showed no presence of product. Another well-known procedure is palladium

on carbon. This was carried out in isopropyl alcohol and refluxed. The starting calixarene did not

appear to dissolve up in these reaction conditions, therefore starting material was isolated upon work

up. The reaction conditions used for the reduction of the nitro-anthraquinones was attempted, using a

basic solution of ethanol and four equivalents of sodium sulphide. The nitro-calixarene was not

soluble within this reaction mixture even upon reflux. Therefore a small amount of DMSO was added,

in order to be able to take the starting compound up in solution. This allowed the reaction to proceed,

however the work up with DMSO meant that it was not possible to get the compound out of solution.

Evaporation of the reaction mixture, led to a mixture of starting materials and the 1H NMR was not

conclusive. It was concluded that DMSO allows for the starting material to dissolve, however

isolation of any desired product is not possible in this solvent system. The solubility of compound 2

was tested in a range of organic solvents, however DMSO is the only solvent in which it appeared to

show any level of solubility. This is a characteristic of the calixarenes that has been noted in literature

before31

. Substitution of the lower rim of the calixarenes aids the solubility of these compounds both

in aqueous and organic media, by disrupting the hydrogen bonding between the lower rim hydroxyl

groups. In order to further study the calixarenes as host molecules, substitution on the lower rim to

increase solubility was carried out.

Selective substitution of two hydroxyl groups for benzoyl groups was carried out as laid out in Elçin

et al19

. Substituting two positions with benzoyl groups, improved the solubility of the calixarene,

specifically in nitric acid to allow for nitration to occur, it also allowed for detertbutylation to occur

on just two positions on the upper rim. Detertbutylation was carried out as previously shown, however

the presence of the benzoyl groups prevented removal of the tertbutyl groups on those units. This

should allow for the final product to remain di-substituted. The proton NMR shifts corresponds to that

seen in the original paper. This reaction was repeated successfully multiple times, consistently

produced 20% yields of the desired product, this is significantly lower then the 65% reported in the

literature. The nitration step as carried out previously was repeated on the benzoyl calixarene

derivative and no product was seen. The reaction was done on a 100 mg scale, due to the low yielding

first step; therefore it is possible that the scale was too small to allow for successful isolation of the

desired product. This reaction will need to be repeated on a larger scale to understand how the

reaction works and how to successfully work it up. Once this is carried out further experiments can be

carried out on the upper rim in order to produce the desired calixarene host molecule.

5 Conclusion

The original target host molecule, 1,8-dinapthylthiourea anthraquinone was not successfully

synthesised. This was concluded to be due to the strength of the hydrogen bonding between the

26

carbonyl and amine functionalities, making the compound too stable to undergo reactions to form the

thiourea. However, by reducing the anthraquinone to an anthracene moiety 1,8-dibenzylthiourea

anthracene was successfully synthesised using a new 3-step reaction procedure in 80% yields. This

novel host molecule has shown some affinity towards the mephedrone precursor in proton NMR

titration studies. As well as this a number of anions guests were tested using full titration studies and

the host molecule has shown very strong binding interactions in particular with chlorine anions. From

the data collected large anions such as nitrate and iodide have also shown strong interactions.

The starting tetra-tertbutylcalix[4]arene was successfully detertbutylated with 76% yields, which is an

improvement of the 68% that was recorded in the literature. Di-substitution of the calix[4]arene was

carried out using harsh acidic conditions and was characterised using both NMR and IR spectroscopy

techniques. Reduction of the nitro groups was not successful due to the insolubility of the

nitrocalix[4]arene compounds in both aqueous and organic media. In order to overcome this, the

lower rim of the tetra-tertbutylcalix[4]arene was di-substituted with benzoyl groups prior to

detertbutylation. This was successfully carried out with yields of 20%. Further nitration was not

successful, due to poor yields of the previous steps. Further work will be carried out to increase the

yields and additional attempts of the nitration reaction will be carried out.

6 Future work

The anthracene host molecule will be tested against mephedrone hydrochloride in order to gain data

on the binding interactions that occur. So as to understand the binding between mephedrone and the

host molecule without the presence of chloride anions, the mephedrone free base will also need to be

tested. This is to ensure that all interactions that are seen are due to just the mephedrone and not the

presence of the hydrochloride salt. Since the mephedrone freebase is not very stable for long periods

of time, mephedrone with a different counter ion will also be tested. Tetraphenylborate is a very large

anion and may be able to replace the hydrochloride salt to stabilise the mephedrone without

interacting with host molecule, as it should be too large to interact. To ensure this is true, a control

study will be carried out using tetrabutylammonium tetraphenylborate. As well as this further reaction

will be carried out in order to try and synthesise the original host molecule containing the naphthyl

groups as opposed to the benzyl groups.

Further work will be carried out with regards to the calixarenes. Additional nitration reactions will be

attempted on the more soluble lower rim substituted derivative. This will hopefully allow for thiourea

functionalities to be added to the calixarene, and then this molecule can also be tested against

mephedrone. This will allow for conclusions to be drawn between the flexible anthracene host

molecule and the pre-organised calixarenes.

27

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