DEGREE PROJECT IN CHEMICAL ENGINEERING AND TECHNOLOGY, FIRST LEVEL STOCKHOLM, SWEDEN 2019

KTH ROYAL INSTITUTE OF TECHNOLOGY KTH ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

Pre-formulation of a novel

nucleotide analogue

Mariam Rabizadegan

ii

DEGREE PROJECT Bachelor of Science in

Chemical Engineering and Technology

Title: Pre-formulation of a novel nucleotide analogue Swedish title: Preformulering av en nukleotid analog Keywords: Pre-formulation, pharmaceutical development Work place: RISE Supervisor at the work place: Ronja Widenbring Supervisor at KTH: Catharina Silfverbrand Lindh Student: Mariam Rabizadegan

Date: 2019-12-21 Examiner: Catharina Silfverbrand Lindh

iii

Sammanfattning

Studier har visat att molekylen MR1, en cGMP analog, kan vara ett lämpligt läkemedel för

neurodegenerativa ögonsjukdomar. Dessa sjukdomar leder till att synen kraftigt försämras

vilket kan leda till att patienten förlorar sin synförmåga.

I detta examensarbete har olika saltformer av MR1 syntetiserats för att frambringa molekylens

preformuleringsdata. Denna preformuleringsdata kommer sedan användas för framtida studier

inom forskningsområdet.

Olika saltformer av MR1 har syntetiserats och karakteriserats med avseende på molekylens

fysikalkemiska egenskaper såsom löslighet och karakterisering av dess fasta tillstånd.

Syntetisering av MR1 Na+ och MR1 fri form har utförts för att få fram prover till

löslighetsstudier och karakterisering av dess fasta tillstånd.

Karakteriseringen av saltformernas fasta tillstånd utfördes genom tillämpning av

röntgenmetoder, Mikroskopi, Hot Stage Controller, DSC, TGA och DVS.

Alla saltformer förutom den fria formen visade sig ha kristallina inslag. Ingen av salterna

visade någon tydlig smältpunkt efter att ha analyserats i DSC instrumentet. TGA analysen

visade 15 % viktminskning för MR1 Na+ saltet, vilket är ett relativt ovanligt resultat och bör

kontrolleras och repeteras ännu en gång. När MR1 fri form analyserades i DVS och röntgen

instrumentet för att detektera hur amorft saltet är överstämde resultaten inte med varandra.

Analysen bör därmed kontrolleras och repeteras.

Löslighetsstudierna visade att MR1 Na+ har högst löslighet i jämförelse med MR1 TEAH

+ och

MR1 fri form när salterna analyserats i olika buffertar.

iv

Abstract

Previous studies have shown that the molecule MR1, a cGMP analogue, may be a suitable

drug for neurodegenerative eye diseases. These diseases cause the eyesight to greatly

deteriorate which in turn can result in the patient becoming blind.

In this thesis, several salt forms of MR1 were synthesized and characterized to generate the

drugs physicochemical properties, such as solubility and solid-state behavior. This in order to

determine the drugs pre-formulation data.

Synthesis of MR1 Na+ and MR1 Free form were performed in order generate material for

solubility studies and solid state characterizations. Solid-state characterization was done in

terms of X-ray, Microscopy, Hot Stage Controller, DSC, TGA and DVS.

All salt forms, expect for the Free form, indicated crystallinity through X-ray analyses.

However, none of the salt forms showed any clear melting point after being evaluated by DSC.

TGA analysis of MR1 Na+ salt form indicated a huge weight decrease. A total amount of 15 %

of the sample decreased over a very short time range in just a few seconds. The result is very

extraordinary and should be controlled and analyzed once again.

Results regarding amorphicity of MR1 Free form did not completely comply with results from

DVS and X-ray. Further analyses should therefore be performed with extra material to obtain a

more consistent result.

Moreover, the solubility of the salt forms was determined and it was observed that MR1 Na

+

indicated to be most soluble in all the tested buffers, as compared to MR1 TEAH+ and MR1

Free form.

The pre-formulation data from this thesis are of importance for the work on understanding

how the drug MR1 behaves. The work carried out in this thesis will continue (beyond the

scope of this bachelor thesis) and the results herein will be used for further studies of

neurodegenerative eye diseases.

v

Acknowledgments

I cannot thank enough the staff at RISE for their knowledge and guidance.

Particular mention to my supervisor Ronja Widenbring for your constant guidance and support

throughout the whole process.

I would also like to thank my lab mentors Dileep Urimi and Oswaldo Perez for being

consistently available in the laboratory. Thank you for answering my many questions and for

all the guidance, motivation and knowledge.

Thank you to my supervisor Catharina Silfverbrand Lindh from KTH for all your support,

guidance and knowledge.

Lastly, I would like to thank my family and friends for their limitless support during these past

three years. Special thanks to my parents, Hossain and Zahra, who always believed in me and

supported my academic career.

vi

List of Abbreviations

API Active pharmaceutical ingredient

ACN Acetonitrile

DSC Differential scanning calorimetry

DVS Dynamic vapor sorption

DMSO Dimethyl sulfoxide

D2O Deuterium oxide

EtOH Ethanol

HCl Hydrochloric acid

HPLC High-performance liquid chromatography

Log P Partition constant

TCNB 1,2,4,5-tetrachloro-3-nitrobenzene

TEA Triethylamine

TEAH+ Triethylammonium

TGA Thermogravimetric analysis

MeOH Methanol

NaOMe Sodium methoxide

NMR Nuclear magnetic resonance

TGA Thermogravimetric analysis

pKa Dissociation constant

(%RH) Relative humidity

XRPD X-ray powder diffraction

ZBH Zero Background Holder

vii

Table of Contents

1. Introduction ................................................................................................... 1

2. Background .................................................................................................... 2 2.1 MR1................................................................................................................................. 2 2.2 Pre-formulation ................................................................................................................ 2 2.3 Salts................................................................................................................................. 2 2.4 Physicochemical properties ............................................................................................... 4 2.5 Analytical methods ........................................................................................................... 5

2.5.1 NMR ......................................................................................................................... 5 2.5.2 HPLC ........................................................................................................................ 5 2.5.3 DSC, TGA, X-ray and DVS ........................................................................................ 6

2.6 Acid base reactions ........................................................................................................... 6 2.6.1 Acid base reactions of MR1 ........................................................................................ 7

3. Procedure ...................................................................................................... 9 3.1 1

H Assay of TEAH+ salt of MR1 ......................................................................................... 9

3.2 MR1 Na+ salt synthesis ..................................................................................................... 9

3.3 MR1 Free acid synthesis ................................................................................................. 10 3.4 Physicochemical characterizations ................................................................................... 11

3.4.1 Standard curve .......................................................................................................... 11 3.4.2 Preparation of buffers for solubility study .................................................................. 12 3.4.3 Solubility study of the salt forms ............................................................................... 13

3.5 Solid state characterization ............................................................................................. 13 3.5.1 X-ray (XRPD) ......................................................................................................... 13 3.5.2 Microscopy ............................................................................................................. 13 3.5.3 Hot Stage Controller ................................................................................................ 13 3.5.4 DSC ........................................................................................................................ 13 3.5.5 TGA ....................................................................................................................... 14 3.5.6 DVS ........................................................................................................................ 14

4. Results ........................................................................................................ 15 4.1 Assay of TEAH

+ salt of MR1 .............................................................................................15

4.2 MR1 Na+ synthesis ...........................................................................................................15

4.3 MR1 Free acid synthesis ..................................................................................................15 4.4 Physicochemical characterization .................................................................................... 16

4.4.1 Standard curve ......................................................................................................... 16 4.4.2 Solubility of the salt forms ........................................................................................ 19

4.5 Solid state characterization ............................................................................................. 22 4.5.1 X-ray (XRPD) ......................................................................................................... 22 4.5.2 Microscopy ............................................................................................................. 23 4.5.3 Hot Stage Controller ................................................................................................ 24 4.5.4 DSC ........................................................................................................................ 24 4.5.5 TGA ....................................................................................................................... 26 4.5.6 DVS ........................................................................................................................ 28

5. Discussion .................................................................................................... 30 5.1 Assay of TEAH

+ salt of MR1 ............................................................................................ 30

5.2 Na+ synthesis .................................................................................................................. 30

5.3 Free acid synthesis ......................................................................................................... 30 5.4 Physicochemical characterization .................................................................................... 31 5.5 Solid state characterization ............................................................................................. 31

viii

6. Conclusion ................................................................................................... 33

7. References ................................................................................................... 34

8. Appendix ..................................................................................................... 36 8.1 Material and chemicals ................................................................................................... 36

8.1.1 Assay of TEAH salt of MR1 ..................................................................................... 36 8.1.2 Na

+ salt synthesis ..................................................................................................... 36

8.1.3 Free acid synthesis ................................................................................................... 37 8.1.4 Standard curve ......................................................................................................... 38 8.1.5 DSC ........................................................................................................................ 38 8.1.6 TGA ....................................................................................................................... 38 8.1.7 Hot Stage Controller ................................................................................................ 39 8.1.8 Microscopy ............................................................................................................. 39 8.1.9 X-ray (XRPD) ......................................................................................................... 39 8.1.10 DVS ........................................................................................................................ 40 8.1.11 Solubility study ........................................................................................................ 40

8.2 LC analysis of solubility study for the salt forms ............................................................... 41

1

1. Introduction

The pharmaceutical enterprises are playing a major role in developing new drugs,

medical devices, and vaccines, to improve people’s living conditions. However, this

is only achievable by applying fundamental research into innovation.

Previous studies have shown that the molecule MR1, a MR1 analogue, may be a

suitable drug for neurodegenerative eye diseases such as hereditary retinal diseases.

These diseases cause the eyesight to greatly deteriorate which in turn can result in

the patient becoming blind.

No pre-formulation data is currently available for the molecule MR1. Pre-

formulation data is data regarding the drug molecule which helps us to understand

the physicochemical properties and design to be able to select a proper formulation.

The objective of this thesis is to synthesize new salt forms of the molecule MR1

and to generate the drugs pre-formulation data. This pre-formulation data will be

used for further studies of treatment of neurodegenerative eye diseases.

To solve the problem various salt forms were synthesized and characterized for

physico-chemical properties, such as solubility and solid-state behavior.

The transMed consortium combines with four non-academic and five associated

partners, one of them which is RISE Research Institutes of Sweden. The funding of

the consortium is from the European Union’s Horizon 2020 research and

innovation programme under the Marie Sklodowska-Curie grant agreement.

This thesis project is a part of the transMed consortium which aims to educate the

next generation of scientist in translational medicine, focusing on eye diseases. The

reason for educating future scientists in this field is because of a perceived gap

between basic research and successful clinical translation that delays establishment

of urgently needed therapies. [1]

The solving methods used for this thesis expect lab work were literature studies and

previous studies from transMed. The sources used for the literature study were

journals and a few books.

2

2. Background

2.1 MR1

MR1, an active pharmaceutical ingredient (API), has been developed and observed.

MR1 is a MR1 analogue. MR1 is one of the main molecules involved in the

phototransduction cascade in photoreceptors.

Scientists have discovered that the MR1 analogue can be a promising drug

candidate in the treatment of neurodegenerative eye disease of the retina. People

suffering from some of these diseases have an excessive amount of MR1 in

photoreceptor cells in the retina, which eventually leads to cell death and possibly

blindness.

Figure 1. Left: MR1. Right: the MR1 analogue MR1.

2.2 Pre-formulation

Pre-formulation is the study of a molecule’s physical, analytical, chemical, and

pharmaceutical properties. By studying these parameters one can develop a safer,

more effective, and reliable pharmaceutical formulation.

The main objective of the pre-formulation phase is to lay down a foundation for

developing the pharmaceutical drug formulation. This in such a way that the drug

can be used in the right amount, right way, and at the right target. Another

important objective during pre-formulation studies is to provide longer stability to

the drug by, for instance, protecting the drug component from environmental

conditions. [2]

2.3 Salts

Approximately half of all drug products are administrated in a salt form. [2]

Transforming a molecule into a salt form is generally used to increase the

performance of a molecule. One can improve the molecule by for example decrease

the side effects, improve the stability and modify the release dosage forms.

3

While selecting a convenient salt form, several factors need to be considered. The

primary factor that determines the convenient salt form is the type of formulation

that is to be developed. Generally, hydrochloride and sodium are the most

appropriate substances to be used to develop salt forms if the developed

formulation is an injection solution, oral solution, or tablet. Most often, an

improved solubility and better bioavailability can be found for hydrochloride and

sodium salt forms. Another important factor regarding the selection of salt form is

whether the counter ion meets the regulatory requirements and if it’s non-toxic. For

instance, use of lithium as salt form is strictly forbidden for pharmaceutical

applications. [2]

Generally, the dissolution rate of a salt form of a drug is rather different compared

to the parent compound. Potassium and sodium salts of weak organic acids and

hydrochloric salts of weak organic bases tend to dissolve more willingly compared

to their corresponding free bases or acids. [3]

4

2.4 Physicochemical properties

Determination of chemical properties of a molecule specifies the stability of that

molecule in the body and the absorption behavior. One of the most commonly used

chemical properties is the dissociation constant (pKa), partition coefficient (Log P)

and stability of the molecule under different kinds of conditions.

The dissociation constant (pKa) decides the solubility in a pH-depended

environment and the extent of ionization. Since only the unionized form can be

absorbed across any biological membrane it is necessary to determine the pKa

value of a molecule.

The pKa value gives an insight regarding the site of absorption. Depending on the

drug being acidic or basic, the pKa value changes. For instance, weakly acid drugs

have a pKa value around 4 and are mostly present in unionized form, which results

in them being best absorbed from the stomach. Basic drugs have a pKa value near 8

and are mostly present in the unionized form, which results in them being best

absorbed from the intestine. [2]

The partition coefficient (LogP) indicates the ratio of unionized drug between the

organic and aqueous phase. By studying the oil-water partition coefficient one can

gain an insight about the drugs ability to cross a cellular membrane. The balance

between the hydrophilic and lipophilic phase is one of the most significant

contributing factors for optimizing drug delivery and absorption. The amount of

drug absorbed depends highly on the lipidic nature of the biologic membrane.

Commonly, the unionized form of a molecule has greater lipophilicity, compared to

the ionized form, and therefore it has obtained much importance. [2]

If the value of LogP is 0 it specifies that the drug has equal distribution in partition

in solvent and in water. When the value of Log P is less than one it indicates that

the water solubility of the molecule is high. But if the value of Log P is greater than

one it indicates higher lipidic solubility. Several methods are available to determine

the partition coefficient of molecules. Shake flask method is a widely used

redundant that utilizes octanol-water system to decide the specific drugs

partitioning behaviors. There are many reasons why octanol is being selected as the

partitioning solvent. One reason is because octanol can copy the lipoidal behavior

as it has a nonpolar and a polar tail. [2]

Studying the solubility of the drug candidate is an important parameter during

physical characterization of a molecule. The solubility of a drug is the amount of

drug that dissolves in the solvent to generate a saturated solution at standard

pressure and temperature. Generally poor solubility often results in a failure in drug

discovery and development. For instance, the target specificity will be reduced.

Solubility relies on a number of kinds of properties such as temperature, pH,

molecule structure and crystal properties. The pH of a solution is a significant

factor because many drug molecules are either weak acids or bases. Some

5

techniques that can be used to improve the solubility of a molecule are particle size

reduction, chemical modification of drug and addition of a surfactant. [2,3]

Generally, lowering the pH of a weak base solution below the pKa of the weak base

causes the weak base to be ionized and to have higher solubility in the solvent. On

the other hand, when increasing the pH above the pKa of a weak acid, the weak

acid will be in an ionized state which results in a higher solubility. [3]

Commonly, a drugs solubility is analyzed by subjecting the drug to solubilization in

pH range from 1.2 to 8 using different kinds of buffers. For instance, pH 1.2

hydrochloric acid buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer and pH 7.4

phosphate buffer saline. [3]

2.5 Analytical methods

2.5.1 NMR

NMR stands for nuclear magnetic resonance. NMR spectroscopy is an analytical

method that has played a key role in almost every part of the pharmaceutical

discovery. By using NMR one can obtain a molecule’s structure and purity. NMR

can furthermore produce information regarding protonating sites, reaction

mechanisms and intermediate compound production. NMR is regularly used in

most aspects of reaction understanding, such as chemical synthesis.

In chemical synthesis, regularly a 300-500 MHz NMR instrument is used. A glass

sample tube with deuterated solvent (commonly deuterated chloroform, deuterated

water or dimethylsulfoxide) and a few milligrams of sample is prepared. The

sample is then placed in the NMR instrument for evaluation. [4,5]

2.5.2 HPLC

HPLC stands for high-performance liquid chromatography and is a commonly used

analytical technique for quantitative analysis of pharmaceuticals, polymers and

organic compounds. The HPLC system includes an autosampler, a sample injector

valve, a solvent delivery system with a pump, a high-pressure chromatography

column, and a detector.

The components in the sample are separated by distributing between the mobile and

the stationary phase. A detector observes the concentration of each separated

component in the column effluent and produces a chromatogram. [6,7]

6

2.5.3 DSC, TGA, X-ray and DVS

DSC stands for differential scanning calorimetry and indicates the relationship

between the heating or cooling of a sample and a reference. Furthermore, it

measures the differential heat flow between the sample and the reference in regards

to temperature. By using DSC, crystallization, melting points, evaporation, and

polymorph transformation can be analyzed. [8]

Crystalline materials tend to have defined arrangements of molecular chains and

sharp melting points, which can be observed in the DSC instrument. [9]

Thermogravimetric analysis (TGA) is an analytical method that indicates the

amount and rate of change in mass of a sample as a function of temperature or time.

TGA also specifies if and how various components in a material are bonded

differently. The major signals that this instrument assembles when evaluating the

sample are mass, rate of mass change, and temperature. [10]

Powder X-ray diffraction (XRPD) is a method for studying the crystalline structure

of a molecule. The method is also very useful to study modifications in the

crystalline state. Depending on the different patterns one can tell if the sample is

crystalline or amorphous. [8]

DVS stands for dynamic vapor sorption and is a gravimetric technique that

measures the kinetics and amount of solvent sorption by the sample. This is done

by varying the vapor concentration around the sample in the atmosphere and

measuring the change in mass of sample as a function of vapor and time. Generally,

water is used as the vapor but other volatile solvents such as acetone or ethanol can

also be used. In case of water the concentration of vapor is described as the percent

relative humidity (%RH), which has a maximum value of 100 % and is defined as

the ratio of adsorbate vapor pressure (P) divided by the saturated vapor pressure

(P0) of the certain adsorbate. [11]

2.6 Acid base reactions

An acid is defined as a substance that ionizes in water to produce H+, while a base

is a substance that ionizes in water to generate OH- ions. These explanations are

associated to the chemist Svante Arrhenius. Arrhenius acid and base definitions are

valuable but they are mainly describing the behavior of compound in aqueous

solution.

The chemist Johannes Brønsted-Lowry described an acid as a proton donor and a

base as a proton acceptor.

The equation below presents the ionization of the weak base ammonia (NH3).

𝑁𝐻3(𝑎𝑞) + 𝐻2𝑂 ⇆ 𝑁𝐻4+(𝑎𝑞) + 𝑂𝐻−(𝑎𝑞)

7

NH3 produces OH- in water which according to the Arrhenius definition indicates

that NH3 is a base. Ammonium is also a base in the Brønsted-Lowry sense by the

reason of it accepting a proton from the water molecule to become the ammonium

ion. [12]

Another example of an acid base reaction is when hydrochloric acid reacts with

water.

𝐻𝐶𝑙 (𝑎𝑞) + 𝐻2𝑂 ⇆ 𝐶𝑙− + 𝐻3𝑂+

According to Brønsted-Lowry definition HCl is the acid by the reason of it

donating its proton while H2O is the base because it receives a proton. [12]

2.6.1 Acid base reactions of MR1

The current synthetic method of MR1 has been developed for synthesis of the

TEAH+ salt for convenience. However, if another salt form is needed it is necessary

to perform an ion exchange after the initial synthesis.

Figure 2. Synthesis of Na salt (1) and free acid (2) of MR1.

In the first reaction, sodium methoxide (NaOMe), a strong Brønsted-Lowry base, is

utilized to neutralize the acid in the system, which is the Triethylammonium

(TEAH+) molecule.

In the second reaction, hydrochloric acid (HCl) acts as a strong Brønsted-Lowry

acid by donating a proton to the base, which is MR1 analogue.

The main purpose behind the first reaction is to remove the TEA by evaporation

and only have MR1 Na+ left. The strategy behind the first reaction is to turn TEAH

+

into neutral (non-salt) from; TEA will then be removed by evaporation.

8

The strategy behind the second reaction is to remove the TEAH+ salt by filtration

and only have the MR1 left. Filtration is being used by the reason of the molecule

being in solution. An NMR analysis will furthermore be used to control whether the

strategy worked out or not, by monitoring the ratio of TEA/TEAH+ present in the

sample compared to MR1.

9

3. Procedure

3.1 1H Assay of TEAH

+ salt of MR1

An NMR sample with 3.04 mg 99.8 % TCNB (Internal Standard), 4.93 mg of API

and 0.6 mL deuterated DMSO and 2 drops of D2O was prepared. Almost all

material dissolved. A sonicator was added to speed up the dissolution. Afterwards

proton assay experiment was performed.

3.2 MR1 Na+ salt synthesis

The general reaction for MR1 Na+ synthesis is presented in Figure 2, reaction (1).

TEAH+ API (1 g, 1.56 mmol) was weighed in a 50 mL round-bottom flask. MeOH

(30ml) was added followed by NaOMe (101 mg, 1.2 equivalents) without stirring.

The starting material was in clumps and did not dissolve, therefore stirring was

added followed by 10 mg extra NaOMe which did not make a difference. Then the

solvents were evaporated on a rotavapor. The procedure was repeated, this time

with stirring from the beginning. The mixture was heated with a heat gun to

dissolve material stuck on the walls of the flask with a condenser attached. The

mixture did not become clear and therefore, extra amount of NaOMe (16 mg) was

added, which did not cause dissolution. It was decided to continue with the

procedure by evaporating the solvents and attempting crystallization. When the

product was dry, EtOH 99.7 % (30 mL) was added while stirring and then the

mixture was heated to reflux. The solids did not dissolve, as was expected. Despite

this, the mixture was cooled down to room temperature and stirred overnight.

Later on, the solids were filtered and then washed with 99.7 % EtOH (10 mL).

Afterwards, an LC-analysis of the product was done to confirm its purity. Then the

product was put in a vacuum dryer (40 ºC). An NMR analysis was made which

showed that TEA was still present. Then the filtrate and precipitate were combined,

and the solvents evaporated to recover the API used in the previous reaction in

order to do a new reaction with it.

MeOH (40 mL) was added to the reaction while stirring. This resulted in the

starting material dissolving before adding NaOMe. Then NaOMe (100 mg) was

weighed and added. After 5 min the solvents were evaporated. Afterwards, the

solids were transferred to a round-bottomed flask and 99.7 % EtOH (40 ml) was

added. Then the reaction was heated until it became a solution at 70.4 ° C and then

allowed to cool to room temperature, with stirring. After a few hours no precipitate

was observed. Therefore, a seed of MR1 Na salt was added in order to provoke a

crystallization, and then the mixture was left under stirring at room temperature for

3 days.

10

The solids were filtered and washed with EtOH 99.7 %. Then the product was

placed in a vacuum dryer. Afterwards it was analyzed on 1H NMR with deuterated

DMSO (600 µL) as solvent. An HPLC analysis was also performed by diluting a

small amount of product in EtOH 99.7 % (1 ml) in an HPLC vial.

3.3 MR1 Free acid synthesis

The general reaction for MR1 Free synthesis is presented in Figure 2, reaction (2).

MR1 TEAH+

(1 g, 1.56 mmol) and MeOH (30 mL) were stirred in a 50 mL round-

bottomed flask. Afterwards, the mixture was heated but did not turn into solution,

therefore it was left stirring for one day. Yet, when the mixture was heated once

again it became clear at 65 °C. Then TFA (0.2 g, 1.1 eq.) was pipetted into the

reaction flask, the one which has the starting material and MeOH. Followed by

turning off the heating and allowed to cool down to room temperature. While

cooling, a precipitate started to form until it was a completely white suspension

after 1 h.

The solids were filtered and then washed with MeOH (10 mL). Later on it was

placed in a vacuum dryer (40 ºC) to remove remaining solvents in the product. A

Proton NMR analysis was first made without D2O which displayed poor peak

resolution. Afterwards D2O was added in the sample which showed improved

redundant. This spectrum showed that approximately half of the TEAH+ was still

present.

The filtrates and precipitate were combined and all solvents were evaporated to

recover the API used in the previous reaction, in order to do a new reaction with it.

Afterwards, MeOH (30 ml) was added and stirred. 1 M HCL (1.8 mL, 1.1 e.q) was

then used as the acid. It was injected to the mixture after the starting material had

dissolved from heating at 70 °C. Then the heat was turned off and the mixture

allowed to cool to room temperature. After it stirred at room temperature for about

1 hour a precipitate had been formed which was filtered and washed with MeOH

(10 mL). The precipitate was then placed in the vacuum dryer (40 ºC) over the

weekend.

Afterwards it was analyzed on NMR with deuterated DMSO (600 uL) as solvent.

An HPLC analysis was also performed by diluting a small amount of product in

EtOH 99.7 % (1 ml) in an HPLC vial.

11

3.4 Physicochemical characterizations

3.4.1 Standard curve

Stock solutions

Three samples were prepared in diluted ACN.

MR1 Na+ (1.997 mg) was weighed and placed in a vial. Afterwards, 2 ml of

ACN(aq) 30 % (v/v) was added.

MR1 TEAH+ (2.007 mg) was weighed and placed in a vial. Afterwards, 2 ml of

ACN(aq) 30 % (v/v) was added.

MR1 Free form (2.015 mg) was weighed and placed a vial. Afterwards, 2 ml of

ACN(aq) 30 % (v/v) was added.

Each sample showed a clear solution after ACN was added.

Five solutions with the concentrations 20 𝜇𝑔/𝑚𝐿 to 100 𝜇𝑔/𝑚𝐿 were prepared

from each stock solution by diluting with 3:7 ACN:Water. Each concentration was

prepared in triplicates. Each solution was analyzed in the HPLC which presented

the peak areas for the analyte. A calibration curve was then generated from all data

points.

Table 1. Data for construction of standard curves.

S.No Concentration

µg/mL

Volume of

stock solution

(mL)

Volume of

diluent ACN:

Water (mL)

1 20 0.02 0.98

2 40 0.04 0.96

3 60 0.06 0.94

4 80 0.08 0.92

5 100 0.10 0.90

12

3.4.2 Preparation of buffers for solubility study

The preparation of the buffers is presented in Table 2.

Table 2. Preparation of buffers.

Buffer and

solution

Standard preparation Actual weights

taken

Observed

pH

HCl pH 1.5 Dilute 152.5 mg of 37% HCl

in 50mL of water in a

volumetric flask

146.3 mg in 50

mL water

1.55

Acetate buffer

pH 4.5

Dissolve 250 mg of sodium

acetate trihydrate and 85 mg

of acetic acid in 50mL of

water

249.55 mg +

85.90 mg in 50

mL water

4.63

Phosphate buffer

pH 6.8

Dissolve 18.75 mg of sodium

dihydrogen phosphate and

27.8 mg of disodium hydrogen

phosphate dihydrate in 50 mL

water

19.1 mg + 27.4

mg in 50 mL

water

7.23

Phosphate buffer

saline pH 7.4

Dissolve a tablet of phosphate

buffer saline in 200 ml of

water

7.52

Water pH 7 7.97

Calcium acetate

pH 7.5

Dissolve 1.58 g of calcium

acetate hydrate in 50 mL of

milli-Q water to get calcium

acetate hydration solution

7.48

13

3.4.3 Solubility study of the salt forms

Samples were prepared in vials in doublets with 0.5 ml of buffer in each vial.

Approximately 4-5 mg of each salt form was added to the buffer initially. The vials

were then observed and additional drug was added until visible particles were seen

in each vial. The vials were then kept under stirring at 500 rotations/min for 48 h to

form the saturated solutions. Then, the vials were centrifuged at 14000 ref for 15

min to remove the visible particles. Clear supernatant was collected, and the

centrifugation was repeated for 15 min at 14000 ref to make sure that the

supernatant was free of undissolved particles. After centrifugation, a clear

supernatant was collected and diluted in ACN:water (3:7) and analyzed using

HPLC to quantify the dissolved drug compound. pH values of all the solutions were

measured before and after the centrifugation.

3.5 Solid state characterization

3.5.1 X-ray (XRPD)

A small amount of each salt form was placed and spread in a uniform layer on a

Zero Background Holder (ZBH). The samples were analyzed with a 17 min scan

over 2-40 C 2.

3.5.2 Microscopy

Three microscope slides were prepared with a very small amount of each salt form.

Then the samples were placed in the Hot Stage Controller and heated from room

temperature to 300 °C, in order to observe the melting point and crystallinity.

3.5.3 Hot Stage Controller

Three microscope slides were prepared with a very small amount of each salt form.

A drop of oil and a coverslip were added to each sample. Afterwards, the sample

was placed under the microscope and slowly heated to 300 °C, in order to observe

melting points and compare the result to the DSC and TGA outcome.

3.5.4 DSC

MR1 TEAH+ (1.90 mg) was weighed and placed in a 40 µL aluminum pan. Later

on, the pan was placed in the sample holder. By using tweezer, a lid was placed

upside-down. Afterwards, a hole was made in the middle of the lid with a needle.

The lid was then laid on top of the pan. The entire sample holder and lid were

afterwards placed on the pressing tool. Then the pan and lid were sealed together by

pressing down the pressing tool. Later on, the sample was placed in the instrument

and heated from 25 °C to 350 °C with 10 K/min under nitrogen purge.

14

MR1 Na+

(4.24 mg) was weighed and placed in an aluminum pan. Then the sample

was placed in the instrument and heated from 25 °C to 350 °C with 10 K/min under

nitrogen purge.

MR1 Free form (4.15 mg) was weighed and placed in an aluminum pan. Then the

sample was placed in the instrument and heated from 25 °C to 350 °C with 10

K/min under nitrogen purge.

3.5.5 TGA

MR1 TEAH+ (3.57 mg) was weighed and placed in a 40 µL aluminum pan. Then

the sample was placed in the instrument and heated from 25 °C to 350 °C with 10

K/min under nitrogen purge

MR1 Na+ (12.23 mg) was weighed and placed in a 40 µL aluminum pan. Then the

sample was placed in the instrument and heated from 25 °C to 350 °C with 10

K/min under nitrogen purge.

MR1 Free form (2.63 mg) was weighed and placed in a 40 µL aluminum pan. Then

the sample was placed in the instrument and heated from 25 °C to 350 °C with 10

K/min under nitrogen purge.

3.5.6 DVS

MR1 Free form (12.60 mg) was placed in a 40 µL aluminum pan and then put in

the instrument for evaluation.

15

4. Results

4.1 Assay of TEAH+ salt of MR1

The spectrum showed broad peaks and wrong phase correction. However, when 2

drops of D2O was added to the NMR sample the peaks became sharper, and the

instrument phase-corrected successfully as shown in Figure 3. NMR gave an assay

of 104 %.

The peak at 8.4 ppm belongs to the internal standard. The other peaks in 7-9 ppm

correspond to protons in the aromatic rings of the MR1 molecule, while the peaks

in 4-6 ppm correspond to protons in the sugar ring. Finally, the peaks at 3 ppm and

1 ppm belong to the triethylammonium ion.

Figure 3. NMR Assay of TEAH

+ salt of MR1.

4.2 MR1 Na+ synthesis

After filtrating, the solids had a white color and a smooth appearance. Later on it

was placed in a vacuum drier for one day to remove remaining solvents in the

product. Product weighed 0.59 g giving a yield of 67.4 %.

4.3 MR1 Free acid synthesis

After filtrating the solids had an off-white color and a smooth appearance. Later on

it was placed in a vacuum drier (40 ºC) for one day to remove remaining solvents in

16

the product. The product weighed 0.70 g. NMR showed half of the TEAH+ still

present.

When HCl was used as the acid the product was, after vacuum drying, still a white

solid and weighed 0.45 g giving a yield of 58.2 %. 1H NMR showed no TEAH

+

present in the compound. 31

P NMR showed no phosphorus signals.

After repeating the NMR experiment with more sample and 2 drops of D2O the

phosphorus NMR shows one peak at 56.9 ppm. The peaks corresponding to the

TEAH+

(Figure 3) have mostly disappeared, except for the 0.04 integral at TEAH+

methyl signal (triplet at 1.18 ppm), which indicated that approximately 0.4 %

TEAH+ is present (Figure 4).

HPLC showed a single peak at the retention time 3.2 min. Both the retention time

and UV absorption spectrum were the same as standard samples of the starting

material (TEAH+ salt) and the sodium salt.

Figure 4. NMR of MR1 Free acid.

4.4 Physicochemical characterization

4.4.1 Standard curve

The mean peak area of MR1 Na+, which was measured from the concentration

20𝜇𝑔/𝑚𝐿 to 100𝜇𝑔/𝑚𝐿, is presented in Table 3.

17

Table 3. Mean area of MR1 Na+.

S.No Concentration µg/mL Area 1 Area 2 Area 3 Mean area

1 20 5.267 5.320 5.267 5.285

2 40 10.848 10.848 10.896 10.864

3 60 16.329 16.438 16.288 16.352

4 80 21.871 21.932 22.732 22.178

5 100 27.631 25.168 27.603 26.801

The mean area values from Table 3 were afterwards used to create a standard curve

which is presented in Figure 5.

Figure 5. Standard curve of MR1 Na

+.

The mean peak area of MR1 TEAH+, which was measured from the concentrations

20𝜇𝑔/𝑚𝐿 to 100𝜇𝑔/𝑚𝐿, is presented in Table 4.

y = 0.2717x - 0.0081 R² = 0.9988

0,000

5,000

10,000

15,000

20,000

25,000

30,000

0 20 40 60 80 100 120

MR

1 N

a+ p

eak a

rea

Concentration (µg/mL)

Standard curve: MR1 Na+

18

Table 4. Mean area of MR1 TEAH

+.

S.No Concentration µg/mL Area 1 Area 2 Area 3 Mean area

1 20 5.043 4.932 5.004 4.993

2 40 10.099 9.998 10.084 10.060

3 60 15.362 15.383 15.362 15.369

4 80 20.609 20.283 20.288 20.393

5 100 25.347 25.666 25.565 25.526

The mean area values from Table 4 were afterwards used to create a standard curve

which is presented in Figure 6.

Figure 6. Standard curve of MR1 TEAH

+.

The mean peak area of MR1 Free form, which was measured from the

concentrations 20𝜇𝑔/𝑚𝐿 to 100𝜇𝑔/𝑚𝐿, is presented in Table 5.

Table 5. Mean area of MR1 Free form.

S.No Concentration µg/mL Area 1 Area 2 Area 3 Mean area

1 20 4.562 4.568 4.698 4.609

2 40 9.975 9.899 9.886 9.920

3 60 15.337 15.435 15.565 15.446

4 80 20.764 20.807 20.891 20.821

5 100 26.400 25.911 26.071 26.127

y = 0.257x - 0.1516 R² = 0.9999

0,000

5,000

10,000

15,000

20,000

25,000

30,000

0 20 40 60 80 100 120

MR

1 T

EA

H+ p

eak a

rea

Concentration (µg/mL)

Standard curve: MR1 TEAH+

19

The mean area values from Table 5 were afterwards used to create a standard curve

which is presented in Figure 7.

Figure 7. Standard curve of MR1 Free form.

4.4.2 Solubility of the salt forms

Following tables indicates the amount of API added to each of buffers until visible

particles were seen.

y = 0.2697x - 0.7966 R² = 1

0,000

5,000

10,000

15,000

20,000

25,000

30,000

0 20 40 60 80 100 120

MR

1 F

ree f

orm

p

eak a

rea

Concentration (µg/mL)

Standard curve: MR1 Free form

20

Table 6. Amount of API added to each buffer sample before particles dissolved, and the

resulting pH of the sample after 24h of stirring.

S.No Buffer

Amount of

MR1 Na+

added (mg)

Amount of

MR1 TEAH+

added (mg)

Amount of

MR1 Free

form added

(mg)

1 HCl 3.86 3.51 3.61

2 HCl 2.67 3.11 3.74

3 Acetate

buffer 34.09

3.55 15.69

4 Acetate

buffer 36.16

3.77 15.47

5 Phosphate

buffer 25.83

4.28 3.46

6 Phosphate

buffer 25.76

4.42 2.82

7 Phosphate

buffer saline 8.90

3.35 5.90

8 Phosphate

buffer saline 11.39

3.99 5.41

9 Water 29.29 3.82 2.30

10 Water 33.69 3.74 3.01

11 Calcium

acetate 2.13

3.63 1.25

12 Calcium

acetate 1.45

4.00 1.59

21

Table 7. pH value of salt forms before and after centrifugation.

TEAH

+ salt of

MR1

Free form of

MR1 Na

+ salt of MR1

Buffer

pH

(Theoreti

cal)

pH

(Observe

d)

pH

before

centrifu

gation

pH

After

centrifu

gation

pH

before

centrifu

gation

pH

After

centrifu

gation

pH

before

centrifu

gation

pH

After

centrifu

gation

HCl 1.5 1.55 1.51 1.53 1.47 1.53 1.56 1.62

HCl 1.53 1.41 1.48

Acetate buffer 4.5 4.63 4.84 4.84 2.64 2.68 4.77 4.92

Acetate buffer 4.83 2.65 4.75

Phosphate

buffer 6.8 7.23 6.99 6.99 2.73 2.77 6.81 6.72

Phospate

buffer 7.03 2.77 6.7

Phosphate

buffer saline 7.4 7.47 7.36 7.37 2.43 2.43 7.29

Phosphate

buffer saline 7.38 2.44 7.23

Calcium

acetate 7.5 7.48 7.35 7.3 6.53 6.55 7.37 7.41

Calcium

acetate 7.32 6.43 7.31

Water 7 7.97 4.65 5.05 2.72 2.78 5.16 5.52

Water 4.65 2.68 4.78

Each sample with drug was placed in two vials. The mean area of the solubility for

each of the buffers is presented below.

Table 8. Solubility of the salt forms.

Buffer

Solubility of

MR1 TEAH+

Solubility of

MR1 Free form

Solubility of MR1

Na+

Solubility

(mg/mL)

Solubility

(mg/mL)

Solubility

(mg/mL)

HCl 0.095 0.25 1.59

Acetate buffer 2.69 22.35 27.98

Phosphate buffer 5.72 3.56 37.04

Phosphate buffer

saline 0.40 3.03 Not measured

Calcium acetate 0.06 0.15 1.12

Water 1.17 3.95 37.4

22

Figure 8. Solubility result of the salt forms.

4.5 Solid state characterization

4.5.1 X-ray (XRPD)

All the salt forms indicated crystallinity. However, the Free form shows peak

broadening and some baseline shift which may indicate presence of amorphous

material.

05

10152025303540

MR1 TEAH+ MR1 Free form MR1 Na+

So

lub

ilit

y (

mg

/ml)

Salt form

Solubility study of the salt forms

HCl Acetate buffer

Phosphate buffer Phosphate buffer saline

Calcium acetate Water

23

Figure 9. X-ray analysis of the salt forms and the Free form.

4.5.2 Microscopy

The Free form showed large agglomerates which made it difficult to observe any

individual crystal habit. However, Na+ and TEAH

+ salts showed needle sharp

crystals. The following figures below present the results of the three salt forms.

Figure 10. Microscopic picture of MR1 Free form.

24

Figure 11. Microscopic picture of MR1 Na+.

Figure 12. Microscopic picture of MR1 TEAH+.

4.5.3 Hot Stage Controller

MR1 Free form did not show any clear melting point and crystallinity. No clear

shapes were observed. The result therefore corresponds to the DSC outcome.

MR1 Na+

showed crystals but no melting point. At the temperature 230°C the

crystals became black, most likely because of decomposition. The result therefore

corresponds to the DSC outcome.

MR1 TEAH+ appeared to have many crystals. During the temperature range 200-

210 °C the crystals became black most likely because of decomposition. This also

corresponds to the DSC result which showed strong exothermic event at the same

temperature interval, which in turn also can indicate a degradation of the molecule.

4.5.4 DSC

The following figures below present the change in heat flow over temperature

during the analysis. Endotherms are going down in the diagram while exotherms

point upwards.

25

Figure 13. DSC result of MR1 Free form.

Figure 14. DSC result of MR1 Na

+.

26

Figure 15. DSC result of MR1 TEAH

+.

As seen from the figures above none of the forms showed any clear melting point,

since none of the peaks are going down. However, there are strong exothermic

events in all three DSC-curves which indicate a degradation of the molecule.

4.5.5 TGA

TGA analysis was performed to observe the change in mass over temperature

during the experiment.

The analysis shows that for the Free form (Figure 16) there is a weight loss of 7.9

% from 25 °C to 145 °C. In the interval 150-253 °C there is a weight loss of 15.1

%.

The Na+

sample (Figure 17) showed a weight loss of 8.3 % from 25 °C to 170 °C.

In the temperature interval 183-240 °C a total weight loss of 14.7 % was seen. The

very sharp change at 215 °C is a very extraordinary result.

Finally, as seen in Figure 18 TEAH+ showed a weight loss of 3.1 % from 25 °C to

183 °C. In the temperature interval 190-322 °C there is a weight loss of 17.8 %.

27

Figure 16. TGA result of MR1 Free form.

Figure 17. TGA result of MR1 Na

+.

28

Figure 18. TGA result of MR1 TEAH

+.

4.5.6 DVS

The following figures below present the DVS result of the salt forms. Figure 19

present the change in mass (%) and relative humidity of the sample as a function of

time, while Figure 20 show the change in mass (%) and as a function of relative

humidity.

Figure 19. DVS result for MR1 Free form

29

Figure 20. DVS result for MR1 Free form.

Analysis shows that the Free form (Figure 19) has no phase transition by the reason

of the curves on the left and right side is identical. The molecule also has no

amorphous features.

However, when the change in mass was 4 % a hydrate was formed (Figure 20).

This was revealed because a sharp step between the two red dots occurred on the

left side of the figure. From 20-70 % P/P0 the sample was absorbing water and

therefore can have a hygroscopic behavior.

30

5. Discussion

5.1 Assay of TEAH+ salt of MR1

NMR gave an assay of 104 %. One of the reasons can be that not all internal

standard did dissolve. NMR only detects material that has dissolved, which leads to

a disagreement between analytical reading and calculations.

There is an assumption that the ratio between the MR1 anion and TEAH+

should be

1:1. If this assumption is wrong and the ratio is different, the molar mass will also

be different which in turn causes either a higher or lower assay than expected.

5.2 Na+ synthesis

NaOMe was added to neutralize the acid. In previous experiments done by

Oswaldo Perez NaOMe has caused dissolution when it has been added to the

suspension. However, this time the starting material did not dissolve. Therefore,

stirring was applied to increase the speed of the process and dissolution.

One reason for the compound not dissolving could have to do with it simply having

a low solubility. In order to tackle this, a few approaches were tested. Since

solubility is related to temperature, the easiest and first test was to heat the mixture.

When a heat gun was applied to the compound for several minutes, it caused the

solvent to start boiling. However, it did not become a clear solution.

In addition, extra NaOMe was added in order to rule out the possibility of not

having enough reagent to provoke a reaction. No dissolution was observed. An

NMR analysis was performed in order to observe whether TEA was still present,

which it was. That indicate that the experiment failed. In the next attempt were

more MeOH was used, the starting material finally dissolved. The whole procedure

was done again, and NMR analysis showed that no TEA was present.

5.3 Free acid synthesis

NMR showed that half of the TEAH+ still was present when TFA was used as the

acid. It is possible that is was because TFA has about the same pKa as the free acid

form of MR1, so only 50 % of the starting API was protonated to free form. Therefore, it was suspected that, by using a stronger acid, TEAH

+ would not be

present. Indeed, when HCL was used instead, NMR showed that no TEAH+ was

present in the compound.

31

5.4 Physicochemical characterization

The R2 value of the standard curve for each salt form indicated that the quality was

reliable and could be used for further solubility studies.

Generally, salts have higher solubility and are therefore easily dissolving in water.

Sodium salt of weak organic acids dissolves more willingly compared to their

particular free acids or bases.

When MR1 Free form was used, the pH of acetate buffer, phosphate buffer,

phosphate buffer saline, and water, decreased significantly compared to the

theoretical pH values. One reason for this could be because MR1 Free form lacks

counter ions.

The solubility for all salt forms increased from the hydrochloric acid buffer to the

phosphate buffer. One reason can be because of the molecules property. Since MR1

is an acid it has low solubility in buffers with lower pH and high solubility in

buffers with higher pH.

However, MR1 Na+

was more soluble in water compared to MR1 Free form and

MR1 TEAH+. This could be since salts generally have a higher solubility in water.

MR1 Free form and MR1 TEAH+ indicated a solubility decrease when being in the

phosphate buffer saline as compared to the phosphate buffer, which was quite

surprising since the theoretical pH values for phosphate buffer and phosphate buffer

saline are quite close to each other. There may be several reasons for this, but one

reason can be because of the molecules property. The result might be unusual and

should be controlled and repeated once again.

Phosphate buffer saline became a gel when MR1 Na+ was added, which made it

difficult to separate the particles from the gel. The solubility was therefore not

measured for the MR1 Na+

salt form.

5.5 Solid state characterization

XRPD indicated that all salt forms mainly are crystalline but may have some

presence of amorphous material in the Free form. The Free form was partly

amorphous because it had broad peaks and some baseline increase. To receive a

clearer understanding regarding the sample, a DVS analysis was performed, which

did not identify any amorphous features in the molecule. The results did therefore

not resemble completely to each other. Further analyses should be performed with

extra material, because if there were any contaminants left in the sample it would

certainly have an impact on the crystals and the out coming result. Purer material

should therefore be produced and analyzed in order to minimize the risk of

receiving a contaminated sample.

When analyzing the Free form in the DVS instrument, no phase transitions were

present by the reason of the curves on the left and right were almost identical.

32

However, it was a very small difference between the first and the second cycle. The

second cycle did not go all the way down to 0, it stopped at 1500 min. One reason

for this can be that a kinetic effect has occurred. Another reason can be because the

first cycle has a longer drying time. When the change in mass was 4 % a hydrate

(0.03 moles) was formed. From 20-70 % P/P0 water was absorbed by the structure

and the molecule could therefore be hygroscopic.

MR1 Free form did not show any melting point after being heated to 350 °C in the

DSC instrument. To confirm the DSC result the sample was analyzed in the Hot

Stage Controller and microscope, which displayed no melting point or crystallinity.

The DSC outcome and the Hot Stage Controller did therefore correspond to each

other. The lack of melting point is due to thermal instability of the molecule which

is confirmed by the exotherms in the DSC as well as the large weight loss

according to TGA. This is not unusual for organic substance by the reason that

many of them decomposes far before melting.

MR1 Na+

and MR1 TEAH+ did not show any melting point after being heated to

350 °C in the DSC instrument. To confirm the DSC result the samples were

analyzed in the Hot Stage Controller and microscope, which displayed that the

crystals became black, most likely because of decomposition. This also corresponds

to the DSC results which showed strong exothermic events at the same temperature

interval, which in turn can indicate a degradation of the molecule.

However, crystalline material and salt forms generally should have a sharp melting

point. The results from the DSC and the Hot Stage Controller did therefore not

correspond with each other. There may be several reasons for why the DSC

instrument did not display any melting point. One reason can be that the sample

breaks down when being heated which resulted in it losing solvent and in turn

losing its crystallinity.

To receive a clearer understanding of what occurred with the sample during heating

the TGA result was analyzed. The TGA analysis of MR1 Na+ salt form indicated a

huge weight decrease. 15 % of the sample was decreased over a very short time

range in just a few seconds. One reason for the sample showing such a huge weight

decrease can be because a large amount of solvent leavening due to decomposition

when the sample is being heated. The result is very extraordinary and should be

controlled and analyzed once again.

33

6. Conclusion

During this thesis, several salt forms of MR1 has been synthesized and

characterized to generate the drugs physicochemical properties, such as solubility

and solid-state behavior. This in order to determine the drugs pre-formulation data.

Synthesis of MR1 Na+ and MR1 Free form has been done in order to generate

material for solubility study and solid-state characterization. Solid-state

characterization was done in terms of X-ray, Microscopy, Hot Stage Controller,

DSC, TGA and DVS.

All salt forms expect for the Free form indicated crystallinity when being analyzed

in the X-ray instrument. However, none of the salt forms showed any clear melting

point after being evaluated in the DSC instrument.

The TGA analysis of MR1 Na+ salt form indicated a huge weight decrease. A total

amount of 15 % of the sample was decreased over a very short time range in just a

few seconds. The result is very extraordinary and should be controlled and analyzed

once again.

The results regarding the amorphicity for MR1 Free form did not completely

resemble with the results from DVS and X-ray. Further analyses should therefore

be performed with extra material to obtain a more consistent result.

Moreover, the solubility of the salt forms was determined and it was observed that

MR1 Na+ indicated to be most soluble in all the tested buffers compared to MR1

TEAH+ and MR1 Free form. This could be since salts generally have a higher

solubility.

The pre-formulation data generated in this thesis are of importance for the work on

understanding how the drug MR1 behaves. The work carried out in this thesis will

continue (beyond the scope of this bachelor thesis) and the results herein will be

used for further studies of neurodegenerative eye diseases.

34

7. References

[1] transMed. transMed – Educating the next generation of scientists in translational

medicine: Focus on eye diseases. URL: http://www.transmed-itn.eu/ [Accessed

27th September 2019].

[2] Patel P. Preformulation Studies: An Integral Part Of Formulation Design.

IntechOpen; 2019 URL: https://www.intechopen.com/online-first/preformulation-

studies-an-integral-part-of-formulation-design [Accessed 28th September 2019].

[3] Acharya P, Shetty S, Fernandes C, Suares D, Maheshwari R, Tekade R.

DOSANGE FORM DESIGN CONSIDERATIONS. London; ACADEMIC PRESS;

2018.

URL:https://www.researchgate.net/profile/Piyoosh_Sharma2/publication/32681985

0_Dissolution_Profile_Consideration_in_Pharmaceutical_Product_Development/li

nks/5c39c85692851c22a36f5c5d/Dissolution-Profile-Consideration-in-

Pharmaceutical-Product-Development.pdf#page=532 [Accessed 3th October

2019].

[4] Bernstein M, Stefinovic M, Sleigh C. Optimising reaction performance in the

pharmaceutical industry by monitoring with NMR. MAGNETIC RESONANCE IN

CHEMISTRY. 2007; 45: 564–571. URL:

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mrc.2007 [Accessed 26th

September 2019].

[5] Gauglitz G, Vo-Dinh T. Handbook of Spectroscopy. Wiley-VCH; 2003.

URL: https://onlinelibrary-wiley-com.focus.lib.kth.se/doi/pdf/10.1002/3527602305.

[Accessed 26th September 2019].

[6] Harris D, A. Lucy C. Quantitative Chemical Analysis. 9 ed. New York: W.H.

Freeman and Company; 2015.

[7] Ahuja S, Dong MW. Handbook of pharmaceutical analysis by HPLC. 1st ed.

[internet] Amsterdam; San Diago: Elsevier Academic Pres As; 2005. URL:

https://app.knovel.com/web/toc.v/cid:kpSSTVHPA2/viewerType:toc//root_slug:vie

werType%3Atoc/url_

[Accessed 27th September 2019].

[8] Gombás Á, Szabó-Révész P, Kata M, Regdon Jr G, Erõs I. QUANTITATIVE

DETERMINATION OF CRYSTALLINITY OF α-LACTOSE MONOHYDRATE

BY DSC. Journal of Thermal Analysis and Calorimetry. 2002; 68(2): 503-510.

URL: https://link.springer.com/content/pdf/10.1023/A:1016039819247.pdf

[Accessed 4th October 2019].

[9] Arif P M, Bhodje Gowd E, Kalarikkal N, Thomas S. Crystallization in

Multiphase Polymer Systems. 1 ed. ELSEVIER; 2018.URL:

35

https://www.sciencedirect.com/book/9780128094532/crystallization-in-multiphase-

polymer-systems#book-info

[Accessed 8th October 2019].

[10] Xu W, Li S, Whitley N, Pan, W. Fundamentals of TGA and SDT. Thermal

analysis. Fundamentals and applications to material characterization. 2005. p: 1-7.

URL:

https://ruc.udc.es/dspace/bitstream/handle/2183/11485/CC80%20art%201.pdf?sequ

ence=1

[Accessed 10th October 2019].

[11] Sheokand S, Modi S. R, Bansal A. K. Dynamic Vapor Sorption as a Tool for

Characterization and Quantification of Amorphous Content in Predominantly

Crystalline Materials. Journal of pharmaceutical sciences. 2014; 103(11):3364-

3376.

URL:https://onlinelibrary.wiley.com/doi/pdf/10.1002/jps.24160?casa_token=1z-

KN8jqcXwAAAAA:W1JKZci3Myb60TDxUBE2ZZeTr_b2sHroDs_f_c63rJ2FCw

T4Vt6BPve1QzcxoIKRkKvnHBSbAOKrQl6d

[Accessed 6th November 2019].

[12] Burge J. Chemistry. 4 ed. New York:McGraw-Hill Education; 2016.

36

8. Appendix

8.1 Material and chemicals

8.1.1 Assay of TEAH salt of MR1

Materials

Nuclear Magnetic Resonance (NMR spectrometer)

Ultrasonicator

NMR tube

Digital milligram balance

Spatula

Chemicals

Triethylammonium salt of API (MR1)

1,2,4,5-tetrachloro-3-nitrobenzene

Dimethyl sulfoxide-d6

Deuterium oxide

8.1.2 Na+

salt synthesis

Materials

Nuclear Magnetic Resonance (NMR spectrometer)

Ultrasonicator

NMR tube

50 ml round-bottom flask

30 ml graduated cylinder

Beaker

Heating plate and magnetic stirrer

Digital milligram balance

Vacuum dryer

Heat gun

Condenser

Magnetic stirrer

Sintered glass

Spatula

Glass vial

HPLC

37

Chemicals

Triethylammonium salt of API (MR1)

1,2,4,5-tetrachloro-3-nitrobenzene

Dimethyl sulfoxide

Deuterium oxide

Methanol

Sodium methoxide

Ethanol

8.1.3 Free acid synthesis

Materials

50 ml round bottomed flask

Magnetic stirrer

Hot plate and magnetic stirrer

Glass pipette

Evaporator flask 100 ml

Condenser

Balance

Sintered glass

Vacuum dryer

Nuclear Magnetic Resonance (NMR spectrometer)

NMR tube

Spatula

HPLC

Beaker

Graduated cylinder

Plastic syringe

Chemicals

Methanol

Trifluoroacetic acid

MR1 TEAH salt (starting material)

Hydrochloric acid

Dimethyl sulfoxide

Deuterium oxide

Ethanol

38

8.1.4 Standard curve

Materials

Vial

HPLC

Spatula

Balance

Micropipette

Chemicals

MR1 TEAH+ salt

MR1 Na+ salt

MR1 Free form

ACN:water

8.1.5 DSC

Materials

Differential Scanning Calorimeter (DSC)

Sample holder

Tweezer

Lid

Pressing tool

Aluminum pan

Balance

Spatula

Needle

Chemicals

MR1 TEAH+ salt

MR1 Na+ salt

MR1 Free form

8.1.6 TGA

Thermogravimetric analysis (TGA)

Balance

Tweezer

39

Aluminum pan

Spatula

Chemicals

MR1 TEAH+ salt

MR1 Na+

MR1 Free form

8.1.7 Hot Stage Controller

Materials

Hot Stage Controller

Microscope slides

Spatula

Chemicals

MR1 TEAH+ salt

MR1 Na+

MR1 Free form

8.1.8 Microscopy

Materials

Microscope

Microscope slides

Oil

Coverslip

Spatula

Chemicals

MR1 TEAH+ salt

MR1 Na+

MR1 Free form

8.1.9 X-ray (XRPD)

Materials

40

X-ray power diffraction (XRPD)

Spatula

Zero Background Holder (ZBH)

Chemicals

MR1 TEAH+ salt

MR1 Na+

MR1 Free form

8.1.10 DVS

Materials

DVS instrument

Spatula

Aluminum pan

Chemicals

MR1 TEAH+ salt

MR1 Na+

MR1 Free form

8.1.11 Solubility study

Materials

Vials

Spatula

Pipette

Balance

Duran flask

Round bottomed flask

Centrifugator

Digital pH meter

Chemicals

MR1 TEAH+ salt

MR1 Na+ salt

MR1 Free form

41

Hydrochloric acid

Acetic acid

Sodium acetate trihydrate

Sodium dihydrogen phosphate

Disodium hydrogen phosphate dihydrate

Phosphate buffer saline

Water

Calcium acetate

8.2 LC analysis of solubility study for the salt forms

Table 9. LC analysis of solubility for MR1 TEAH+ salt

Solubility of MR1 TEAH+ salt

S.

No Sample Y X

Dilution

Factor Concentration Solubility (mg/mL)

1 HCl pH 1.5 1 0,179 1,286381323 80 102,9105058 0,10

2 HCl pH 1.5 2 0,152 1,181322957 80 94,50583658 0,09

3 Acetate buffer

pH 4.5 1 8,614 34,107393 80 2728,59144 2,73

4 Acetate buffer

pH 4.5 2 8,327 32,99066148 80 2639,252918 2,64

5

Phosphate

buffer pH 6.8

1

18,624 73,05680934 80 5844,544747 5,84

6

Phosphate

buffer pH 6.8

2

17,802 69,85836576 80 5588,669261 5,59

7 PBS pH 7.4 1 1,114 4,924513619 80 393,9610895 0,39

8 PBS pH 7.4 2 1,149 5,060700389 80 404,8560311 0,40

9 Calcium

acetate 1 0,051 0,788326848 80 63,06614786 0,06

10 Calcium

acetate 2 0,049 0,780544747 80 62,44357977 0,06

11 Water 1 2,007 8,39922179 80 671,9377432 0,67

12 Water 2 5,172 20,71439689 80 1657,151751 1,66

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Table 10. LC analysis of solubility for MR1 Free form.

Solubility of MR1 Free form

S.

No Sample Y X

Dilution

Factor Concentration Solubility (mg/mL)

1 HCl pH 1.5 1 0,791 5,886540601 40 235,461624 0,24

2 HCl pH 1.5 2 0,945 6,457545421 40 258,3018168 0,26

3 Acetate buffer

pH 4.5 1 29,27 111,4816463 200 22296,32925 22,30

4 Acetate buffer

pH 4.5 2 29,407 111,9896181 200 22397,92362 22,40

5 Phosphate

buffer pH 6.8 1 23,622 90,5398591 40 3621,594364 3,62

6 Phosphate

buffer pH 6.8 2 22,834 87,61809418 40 3504,723767 3,50

7 PBS pH 7.4 1 9,624 38,63774564 80 3091,019651 3,09

8 PBS pH 7.4 2 9,228 37,16944753 80 2973,555803 2,97

9 Calcium acetate

1 0,215 3,75083426 40 150,0333704 0,15

10 Calcium acetate

2 0,212 3,73971079 40 149,5884316 0,15

11 Water 1 8,434 34,22543567 40 1369,017427 1,37

12 Water 2 8,746 35,3822766 40 1415,291064 1,42

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Table 11. LC analysis of solubility for MR1 Na+.

Solubility of MR1 Na+ salt

S. No Sample Y X Dilution

Factor Concentration Solubility (mg/mL)

1 HCl pH 1.5 1 5,389 20,449089 80 1635,927117 1,64

2 HCl pH 1.5 2 5,009 19,091461 80 1527,316899 1,53

3 Acetate buffer pH

4.5 1 7,508 28,01965 1000 28019,64987 28,02

4 Acetate buffer pH

4.5 2 7,484 27,933905 1000 27933,90497 27,93

5 Phosphate buffer

pH 6.8 1 9,647 35,661665 1000 35661,66488 35,66

6 Phosphate buffer

pH 6.8 2 10,417 38,412647 1000 38412,64737 38,41

7 PBS pH 7.4 1 1,1957842 0 0,00

8 PBS pH 7.4 2 1,1957842 0 0,00

9 Calcium acetate 1 0,069 1,4423008 80 115,3840657 0,12

10 Calcium acetate 2 0,069 1,4423008 80 115,3840657 0,12

11 Water 1 8,092 30,106109 1000 30106,10932 30,11

12 Water 2 12,187 44,736334 1000 44736,33441 44,74

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