SYNTHESIS OF NOVEL BIFUNCTIONAL BISPHOSPHONATES

     

Ercan Topçu   

 

 

 

Master’s thesis 

Department of Chemistry 

Medicinal chemistry 

475/2014

1

SYNTHESIS OF NOVEL BIFUNCTIONAL BISPHOSPHONATES

Student: Ercan Topçu (251253)

Supervisors: Elina Puljula

Sebahat Ucal

Abstract

Bisphosphonates (BPs) are synthetic molecules containing a P-C-P backbone and

are very well known class of organophosphorus compounds which have many

applications in different fields of industry. BPs have high affinity for hydroxyapatite

(HAP), which is the main mineral found in bones, and they can chelate several metal

ions, such as Mg(II), Ca(II), and Fe(II). BPs are easily taken up by the bone tissue

where they can prevent the resorption of bone. This makes them crucial to be used in

the treatment of diseases related with increased bone resorption, for instance

osteoporosis. The aim of the current research is to show an approach to chemistry

underlying the properties of novel chealating agents with BP and polyamine (PA)

moieties. In this study, two novel BP-PA conjugates were prepared and characterized by

NMR method. This thesis is a part of the investigations based on BPs and their

functions in pharmaceutical chemistry field.

Keywords : Bisphosphonate, ethenylidene bisphosphonate, polyamine, hydroxapatite,

osteoporosis, Paget’s disease, 31

P NMR, synthesis of novel chelating agents, bone

resorption, bone diseases, antiresorptive potency, osteoclast, osteocyte, osteoblast, NBP,

NNBP.

2

Abbreviations

ALN – Alendronate

ATP – Adenosine triphosphate

BP – Bisphosphonate

BMD – Bone mineral density

CLO – Clodronate

DETA – Diethylenetriamine

EDA – Ethylenediamine

ETI – Etidronate

FPP – Farnesyl pyrophosphate

FOP – Fibradysplasia ossificans progressiva

GC – Gas chromatography

GI – Gastronintestinal

HAP – Hydroxyapatite

HPLC – High performance liquid chromatography

IBA – Ibadronate

IUPAC – International union of pure and applied chemistry

MIN – Minodronate

NBP – Nitrogen containing bisphosphonate

NMR – Nuclear magnetic resonance

NNBP – Non-nitrogen containing bisphosphonate

OPG – Osteoprotogerin

PA – Polyamine

PAM – Pamidronate

PPi – Pyrophosphate

PUT – Putrescine

RANKL – Receptor activator of nuclear factor-ĸB ligand

RIS – Risodronate

SAR – Structure-activity relationship

3

SPD – Spermidine

SPECT – Single-photon emission computed chromatography

SPM – Spermine

TFA – Trifluoroacetic acid

TLC – Thin layer chromatography

TMS – Tetramethylsilane

TNFSF11 – Tumor necrosis factor ligand superfamily member 11

TRIEN – Triethylenetetraamine

TSP – Sodium 3-(trimethylsilyl)-1-propanesulfonate

ZOL – Zoledronate

4

Table of Contents

1 Bisphosphonates, their derivatives and current applications ................................................... 5

1.1 Introduction .................................................................................................................... 5

1.2 Historical aspect of development of bisphosphonates ...................................................... 6

1.3 Bisphosphonate structure and the link between bisphosphonate and pyrophosphate ......... 7

1.4 Classification of bisphosphonates ................................................................................... 9

1.5 Structure– activity relationships of bisphosphonates.......................................................10

1.6 Pharmacokinetics of bisphosphonates ............................................................................11

1.7 Use of bisphosphonates .................................................................................................14

1.7.1 Known medical applications of bisphosphonates .....................................................15

1.7.2 The effects of bisphosphonates on the treatment of bone diseases ............................19

1.8 The mechanism of action of bisphosphonates in cells .....................................................22

Effects of Bisphosphonates on Osteocytes .......................................................................22

Effects of Bisphosphonates on Osteoblasts ......................................................................23

Effects of bisphosphonates on hydroxyapatite (HAP) .......................................................24

1.9 Side effects of use of bisphosphonate-drug ....................................................................25

1.10 Future prospects on bisphosphonates ...........................................................................26

1.11 Novel bisphosphonate and polyamine conjugates .........................................................30

1.12 The aim of the study ....................................................................................................27

2 Experimental Part .................................................................................................................28

2.1. Materials and Method ...................................................................................................29

2.1.1. General ..................................................................................................................29

2.1.2. Materials................................................................................................................29

2.1.3. Prepared compounds ..............................................................................................31

2.2 Results and Discussion ..................................................................................................35

2.3 Conclusion ....................................................................................................................38

3 Acknowledgement ...............................................................................................................39

4 References ...........................................................................................................................40

5

1 Bisphosphonates, their derivatives and current

applications

1.1 Introduction

The studies in this master thesis are based on bisphosphonates (BPs). Chemically,

BPs are synthetic analogues of naturally occurring pyrophosphates (P-O-P). The

simplest type of pyrophosphate molecules naturally found widely in nature is inorganic

pyrophosphate (PPi). In the case of BP, central carbon atom replaces the oxygen atom

that has a connection to two phosphates, so that the BP structure contains a P-C-P

backbone. According to historical development of BPs, they were firstly synthesized

more than one century ago in 1800s [1]

. However, the early use of BPs was mainly as

complexing agents in textile, oil, and fertilizer industries, as corrosion inhibitors as well

as for several other industrial aims [2]

.

Utilization of BPs’ additional properties came to light subsequently in

pharmaceutical chemistry field as well. The turning point of these molecules occurred

approximately 50 years ago by using them in treatment of disorders of calcium

metabolism [3]

. Even though first BPs were synthesized over a century ago, and that the

existence of them has been known for a long time, their journey in pharmaceutical field

initiated with etidronate (ETI) which was the first BP to be used in humans for the

treatment of calcification disorders, such as myositis ossificans progressive (now known

as fibrodysplasia ossificans progressiva or FOP).

Moreover the subject of use of BPs in pharmaceutical industry is currently being

developed by scientists. Nowadays, the most interesting field for BPs use is the field of

the treatment of bone related diseases, such as osteoporosis. They can chelate several

metal ions, such as Mg(II), Ca(II), and Fe(II) [4]

. BPs have high affinity to HAP, which

is the main mineral taking place in bones [5]

. BPs are easily taken up from the

bloodstream by the bone tissue where they can prevent the resorption of bone. Therefore

BPs are used in the treatment of diseases related with increased bone resorption.

6

Applications of BPs cannot be narrowed by the fields mentioned above because the

topic of BPs and their use are significantly a wide field in chemistry. Nowadays, there

are averagely more than 20 000 publications according to a recent search on several

databases. These many different research topics about BPs have been mainly based on

their medical and non-medical applications. Hence this literature overview is intended

to feature the properties of BPs having interesting usage in many different fields of

science.

1.2 Historical aspect of development of bisphosphonates

As mentioned in introduction part before, first BPs were synthesized in 1800s and

new BPs are still being developed for several purposes. Though BP use in industry and

its importance are undeniable, this thesis will be mostly focusing on the development of

medical applications of BPs’ as the research topic is closer to that part of studies.

Before being used as inhibitors of resorption in the treatment of bone disease, BPs

were originally thought as inhibitors of calcification [6]

. In the earliest studies it was

proven that BPs could bind to HAP crystals and inhibit crystal growth as PPi also does

[7, 8]. The turning point of BPs based on biological studies was the discovery of that, BPs

inhibit bone resorption [9, 10]

.

From the first clinical use of BPs to present applications, BP studies have been in a

very long journey of science, especially in pharmaceutical chemistry field. The first

member of clinical uses of such interesting compounds, BPs, was ETI used in the

treatment of calcification disorders, such as myositis ossificans progressive [11]

. It was

also the first one to use in order to treat bone resorption when used for the patients with

Paget’s disease [12]

. As BPs have strong affinity to bone mineral, also other studies

carried out shortly after it was used in bone resorption investigations. BPs were also

started to be used as therapeutic agents in oncology during 1970s. They then became

extensively developed for the treatment and prevention of skeletal complications related

with multiple myeloma or bone metastases arising from cancers of breast, prostate, and

other organs [11]

. Nowadays the field of BPs in use is even wider than it was before.

7

Moreover there are many available resources, such as, books and articles, concerning

the chemistry, clinical application and pharmacology of BPs [11]

.

A basic understanding towards the historical development of BPs is shown below

(Figure 1).

Figure 1. The history of BPs [5]

.

1.3 Bisphosphonate structure and the link between bisphosphonate and

pyrophosphate

BPs (Figure 2) are synthetic molecules containing a P-C-P backbone and are a class

of organophosphorous compounds containing two phosphonate groups [3]

. As they have

two phosphonate (PO3) groups, they are called bisphosphonates, bis referring to the two

similar groups in the compound. They have a very similar structure to pyrophosphate

(Figure 3) which has a P-O-P structure instead. Pyrophosphates are the most soluble

ones among the phosphate groups and they are very good complexing agents for metal

ions, such as Ca2+

and Mg2+

. They have also many uses in industry [7, 13]

.

8

Figure 2. Basic structure of bisphosphonate.

Figure 3. Basic structure of pyrophosphate.

Alteration in the central atom which is between two phosphorus atoms, changes

also the name of the compound for pyrophosphate as well as its properties on intended

applications both in medicine and industry. The nomenclature is specified by presence

of the groups bound to the central pentavalent atom of phosphorus [14]

.

BP compounds have a central carbon which characterizes the whole compound

itself. The bond type between the carbon and phosphorus atoms confers upon the

chemical stability of BPs and their resistance to enzymatic or acid hydrolysis, which

actually renders the molecule resistant to biological degradation [15, 16]

. Therefore it was

thought that BPs are not metabolized in the body for a long time. This fact, was literally

consistent with the observation which they are excreted unaltered. However nowadays

some of the BPs are already known to be incorporated intracellularly into ATP

analogues [15]

.

Alongside of these bond abilities, the BP structure also has two groups

(substituents) attached to geminal carbon. These two side-chains are responsible for

most of the biological activity observed among the BPs. So basically, altering R1 and R2

groups can change on the features of BPs [17, 18]

.

9

1.4 Classification of bisphosphonates

Chemically and enzymatically stable analogues of pyrophospates, BPs, can be

separated into two types based on their structure and mechanism of action. These two

types of BPs are referred according to their mechanism of action to inhibit bone

resorption. The one having no nitrogen in its structure is called non-nitrogen-containing

BP (NNBP), and the one having at least one nitrogen atom in its R1 or R2 groups is

called nitrogen-containing BP (NBP). NNBPs are incorporated into ATP resulting into

hydrolysable adenine containing metabolite. NBPs get involved in with the mevalonate

pathway and they function by inhibiting the farnesyl pyrophosphate (FPP) synthase

enzyme being responsible for the synthesis of FPP used in protein prenylation,

cholesterol, ergosterol, heme a, ubiquinone, and dolichol production [19, 20]

. NBPs are

also involved in direct anticancer studies [21, 22, 23, 24]

.

The BP drugs currently being used in pharmaceutical field in clinics can be

classified in three generations based on their structure-activity relations. All the

variations in ability and the potency of the compounds are determined by the properties

of three-dimensional structure of these two side chains, R1 and R2, connected to the

central carbon atom [25, 26, 27]

. The first-generation, also called simple BPs, includes

NNBPs which have simple and relatively low potency R1 and R2 groups, such as ETI

and clodronate (CLO). The second-generation BPs include a primary amine group, and

have a better potency than the first-generation BPs. Alendronate (ALN), ibandronate

(IBA) and pamidronate (PAM) are the examples for the group of second-generation

BPs. ALN is about 1000-fold more potent than ETI moreover PAM is only 100-fold

more active than ETI [28]

. Affiliation of an aminoalkyl side chain at R2 increases the

antiresorptive potency, moreover, the length of carbon chain is also significant [29, 30, 31,

32]. Third-generation BPs are characterized by having a cyclic side chain and in their

structure the sp2 nitrogen is a part of a heterocyclic ring

[33]. Zoledronate (ZOL),

Minodronate (MIN) and risedronate (RIS) are in the group of third-generation of BPs.

Incorporation of a nitrogen heterocycle further enhances the potency. The most active

one among the third-generation BPs, is ZOL. Additionally, ZOL is up to 10000-fold

more potent than both CLO and ETI in some experimental systems [34]

.

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The individual profiles of specific BPs mentioned above may relate to their clinical

characteristics. According to IUPAC recommendations, BPs in acidic forms are called

with the term of “phosphonic acid” which structurally equals to (HP(O)(OH)2).

However, almost all of the BPs in clinical use are actually in their salt form. They are

therefore called by the names of their salt forms, such as CLO, ETI or ALN. In the

literature part of this thesis, the BPs in clinical use are called in the way mentioned

above.

1.5 Structure– activity relationships of bisphosphonates

Structure-activity relation (SAR) is almost always one of the key points in any kind

of research in pharmaceutical chemistry field. There are two main features playing an

important role on the ability of the BPs to inhibit bone resorption [35, 18]

. The first feature

is the high affinity to bone mineral [36, 37, 38]

and the second feature is the structure of

side chains, R1 and R2 [39]

.

The antiresorptive potency of a BP depends on the side chains, R1 and R2 groups,

attached to geminal carbon of the P-C-P moiety. The P-C-P moiety of BP plays an

important role for the strong affinity of BPs for binding to HAP. Furthermore, the

ability of BPs to inhibit bone resorption requires the structure of P-C-P acting as “bone

hook” [5]

. The compounds containing P-C-C-P and P-N-P structures are ineffective to

inhibit the resorption of bone. As discussed earlier in the literature part, altering of the

R1 and R2 substituents in BP molecule, creates a number of variations in the structure of

BP. R2 groups have their own unique effect on antiresorptive potency and this case is

thought to be mediated to the ability to affect biochemical activity [40, 41, 42]

.

The affinity of BPs for calcium is even better when there is a hydroxyl group at the

side chain of the R1 group because then chelating occurs by tridentate binding [38]

. BPs

can chelate calcium ions also by bidentate binding via the oxygen atoms.

Alteration in the structure of heterocyclic R2 group attached to P-C-P moiety, can

significantly affect the antiresorptive potency of the BP compound [43, 44]

. It is also

stated that the mechanism of action of BPs includes a stereo-specific interaction of the

11

side chains, especially on R2, in its structure [18]

. For reaching the maximum potency, the

nitrogen atom in the R2 side chain has to be both in an optimum distance with the P-C-P

moiety and in a specific spatial configuration [45]

.

To sum up, modifications to both or either of the phosphonate groups, such as

substitution of P-C-P bonds, replacement of hydroxyl group, and the removal of one of

the phosphonate groups, reduce the antiresorptive potency [10, 39]

. The results concerning

the reduction in antiresorptive potency are not always due to the loss in affinity for bone

and it is also known that both of the phosphonate groups in the structure of BPs are

crucial for a drug to be pharmacologically active [46, 5]

. However, so far no biochemical

mechanism has been presented to explain the SAR of BPs as a whole. Therefore, there

might be a need for molecular interaction model for BPs with their pharmacological

targets.

1.6 Pharmacokinetics of bisphosphonates

The extraction of the BPs is not easy from biological fluids as all of them are

known to be very polar compounds. Moreover most of them do not include a

chromophore, and it creates a difficulty upon building a proper sensitive analytical

method [47]

. For instance, applying a simple detection method for the measurement of

BPs, such as radio-labelling, is not suitable due to the case of radiation limitations of the

compounds used in such methods. Chromatographic methods developed for direct

measurements of BPs, such as gas chromatography (GC) and high performance liquid

chromatography (HPLC), are also not sensitive enough to detect BPs in serum longer

than 24h in any case [48]

. As BPs are excreted, unaltered from the kidney, they can be

detected in urine.

One of the most well-known characteristic properties of BPs is the low oral

bioavailability that they have. The reason for the low oral bioavailability is the high

ionization at physiological pH which makes them very polar and poorly absorbed from

gastrointestinal (GI) tract [49]

. The oral bioavailability of the BPs is variable but is very

similar between the NBPs. It is also known that approximately 50% of the absorbed

12

drug is permissively taken up by the skeleton and the rest which unaltered in

metabolism yet, is excreted by excretory system in urine [48]

. Comparing the drug

concentrations in plasma or in urine after drug uptake may be helpful to figure out how

good the bioavailability of the drug is. The bioavailability of the drug can vary in both

animals and humans and this can be seen in the table below (Table 1). It is essential to

know about the amount of BP at the skeleton when taking a close look at

pharmacokinetics of BPs related with pharmacodynamics. Because BPs exert their

action at the bone surface, where they are taken up by the osteoclasts during bone

resorption [48]

.

Table 1. Bioavailability of the BP drugs in rats and humans.

Human

(%)

Rat

(%)

Clodronate 1-2 [50]

-

Etidronate 3-7 [51]

2.8-3.6 [52]

Pamidronate 0.3-0.48 [53, 54]

0.5-0.6 [55]

Alendronate 0.7 [56]

0.9 [57]

There are some parameters that effect on absorption; one of them is the

complexation with cations, such as Ca2+

and Mg2+

. The complexation between BPs and

cations might hinder the absorption properties as BPs are strong chelating agents and

they form complexes which have high molecular weight and tendency to precipitate

easily [58, 59]

. These complexes are also insoluble [49]

. Thus the complexes may widen the

paracellular tight junctions [60]

. If a higher dosage is provided, then the bioavailability of

the drug may be increased but then it might cause also some subsequent local side

effects [61]

. For instance, when the dose of ALN was increased from 2 to 40 mg orally, a

better result was obtained in the bone of rats, which reflects an increase in the extent of

drug absorption at high doses [62]

. Therefore, the oral absorption and the bioavailability

of the BPs are dose-dependent parameters.

There are two routes for drug compounds to be taken into the blood stream,

transcellular and paracellular [61]

. Transcellular transport, known as a transport in which

13

the compounds cross the epithelial barriers via biological membranes, is prevented by

the hydrophilicity of BPs. Also the charge of BPs plays a role on this prevention.

Therefore the paracellular route is the only way for BPs to be absorbed [63, 64]

. In the

paracellular transport, drug compounds reach the bloodstream via the tight junctions

between the epithelial cells. As the tight junctions are transparent to the molecules with

the molecular weight up to 150 g mol-1 [62]

the BPs having large molecular size hinder

the paracellular transport due to the selectivity controlled by the negative charge of

brush-border membrane [65]

. Hence this evidence actually shows that also

physicochemical properties of BPs are crucial.

From the bloodstream, BP is rapidly and easily taken up by the skeleton, however,

the amount of BP taken up depends on couple of parameters, such as the affinity of the

BP for bone mineral, renal function and prevalent rate of bone turnover [48]

. Saturation

in binding sites of the skeleton is virtually impossible with the amounts of BP given in

the treatment of osteoporosis and usually the capacity of the skeleton to retain is very

high [66]

.

Half-lives of BP do not actually show whether its bone uptake is well. A fully

comparable research is needed on half-lives of the BPs in order to make sense on results

as different design of studies, analytical methods and pharmacokinetics techniques

makes the comparison between studies difficult [67]

. The bioavailability of drug

compound may be observed and drug concentration in plasma may be estimated by this

way. A study on relative content of 14

C-labelled ALN and RIS in humans receiving

ALN 70 mg once-weekly or RIS 35 mg once-weekly has been performed over 10 years

ago [68]

. In this study, retention carried out at 24 h by ALN was 54.5%. This value for

RIS was 51%. There was no significant difference between them in this step. The only

difference appeared after 72 h was that less RIS than ALN had been retained by the

values of 45.5% and 51.8%. After 27 days of use, the retention values were 45.9% and

33.8% for ALN and RIS, respectively. It has also been shown that the release of BP

from bone is very slow [69]

. For instance, labeled PAM was detected in urine samples of

young patients approximately 8.5 years after halt of treatment. There have been also

several studies based on changes in bone mineral density (BMD) to the anti-fracture

efficacy of inhibitors of bone turnover and of BPs especially [70, 71]

. An example of these

studies is shown in following figure (Figure 4) [67]

. It is proposed that there are at least

14

two different processes based on the changes in BMD related with BP therapy [11]

. One

of these processes proposes that the significant increase occurring in BMD in a few

months is expected to result from filling in of “remodeling space”. The other one

proposes on that the slower increases occurring after months and years are probably

because of the increased mineralization of the existing bone being renewed slowly by

body. This bone becomes older by the time passing and thus it then becomes more

heavily mineralized. Furthermore, it is also thought that the relationship between the

increase in BMD with the various BPs and the related decreases in fractures is not very

simple.

Figure 4. Schematic presentation of bisphosphonate-induced changes in bone formation

and resorption (right panel) and in bone mineral density of the spine (LS BMD, left

panel) in osteoporosis.

1.7 Use of bisphosphonates

BPs have been used in many different fields so far, firstly as corrosion inhibitors

and complexing agents in industry [5]

. After the discovery of their abilities in

pharmaceutical fields over 40 years ago, BPs could also be applied to the treatment of

bone diseases as they have a high affinity to HAP [72]

. Their abilities in the vast field of

pharmaceutical chemistry, have been widened by several investigations on the treatment

of osteoporosis, Paget’s disease, osteolytic and metastatic bone diseases, pediatric bone

diseases and hypercalcemia related diseases [73, 74, 75, 76, 77, 78, 79, 80, 81]

. Nowadays, uses of

15

the BPs can be divided into two groups, medical applications and non-medical

applications of BPs. In this section, it will be focused on the medical applications

related with bone diseases.

1.7.1 Known medical applications of bisphosphonates

BPs have played a significant role in medical applications based on the treatment of

various diseases since they were firstly discovered [5, 26]

. Most of the diseases treated by

BP drugs are based on variation of activity of the osteoclasts [82]

. Among the clinical

applications of BPs, the inhibition of bone resorption is the most impressive one as there

was no effective treatment existed on that previously. Therefore, BPs have been a good

option for the treatment of the diseases in which excessive osteoclast activity is a

significant parameter. Nowadays, a number of BP-drugs are pharmaceutically available

for several treatment methods concerning different bone diseases (Table 3). They are

briefly explained in this section.

Table 3. Most applicable BP-drugs in clinical use and their structures

Bisphosphonates R1 R2

Clodronate -Cl -Cl

Etidronate -OH -Me

Pamidronate -OH -CH2CH2NH2

Alendronate -OH -((CH2))3NH2

Risedronate -OH -CH2-3-pyridine

Tiludronate -H -CH2-S-phenyl-Cl

Ibandronate -OH -CH2CH2N(CH3)(pentyl)

Zoledronate -OH -CH2-imidazole

Etidronate

ETI is a salt of etidronic acid (Figure 5) which is a chelating agent. ETI was the

first BP to be used in the treatment of osteoporosis. It is an effective BP to be used in

the treatment of vertebral fractures. However there is no definite evidence for reduction

of nonvertebral fracture [11]

. ETI has a low pharmacological potency. Surprisingly, ETI

16

(Figure 6) has been shown to include a relatively rapidly reversible effect on bone

turnover. The Food and Drug Administration (FDA) approved ETI in 1977.

Figure 7. Etidronate (left) and etidronic acid (right).

Clodronate

CLO is a first-generation (non-nitrogenous) BP. Until recently, there has been no

data on that if it is effective on fractures; however, in a few countries it has been

approved for the treatment of osteoporosis [11]

. CLO (Figure 8) has a low cellular

potency and low mineral affinity.

Figure 9. Clodronate

Alendronate

ALN (Figure 7) is a well-known BP among second-generation BPs and is well

established for the treatment of osteoporosis. It has a high mineral affinity and

intermediate effects on FPPS [11, 83]

. Its effects do not appear to be as fast as in the case

of RIS although it reduces all fracture types [11]

.

17

Figure 10. Alendronate.

Pamidronate

There is not enough information about PAMs effects on fractures, however, it is

being acceptable to be used in the treatment of patients who cannot tolerate oral BPs for

osteoporosis. PAM (Figure 8) is being used to treat high blood calcium levels and some

certain bone problems such as lesions and metastases in bones [48]

. It retards the release

of calcium from bones to lower calcium levels [11]

.

Figure 11. Pamidronate.

Ibandronate

IBA (Figure 9) is meant for the treatment of osteoporosis in post-menopausal

women. It is preferred that men should get a clinical test before uptaking IBA [84]

. FDA

approved IBA as a daily treatment for post-menopausal osteoporosis.

18

Figure 12. Ibandronate.

Zoledronate

ZOL (Figure 13) is a third-generation BP developed exclusively for intravenous use

rather than oral use. ZOL retards the bone resorption. The once yearly dosing regimen

makes the chemistry in the period of clinical convenience. ZOL has an appreciable high

affinity to bone mineral [11]

. ZOL is contraindicated for the patients having the case of

pregnancy, poor renal function, hypocalcemia, paralysis and it can be helpful for

treating pain caused by bone metastases [85]

.

Figure 14. Zoledronate.

Risedronate

RIS (Figure 15) is one of the strongest inhibitors of FPPS among the BPs based on

clinical use [11]

. Although RIS has markedly lower mineral binding affinity comparing

with that ZOL and ALN have. Use of RIS is a noteworthy as it may cause esophageal

ulcers if it lodges in the esophagus. It is usually recommended that RIS should be taken

with the body upright and possibly with a glass of water [86]

.

19

Figure 16. Risedronate.

Tiludronate

TIL (Figure 17) has a wide clinical use especially for treatment of Paget’s disease.

However, it has no significant development for osteoporosis [11]

. TIL acts via the

incorporation into ATP metabolites, but not through the FPPS mechanism.

Figure 18. Tiludronate.

1.7.2 The effects of bisphosphonates on the treatment of bone diseases

Exploration of BPs as inhibitors of calcification made the scientists take some new

steps through the bone diseases [74, 87]

. BPs were first applied to the treatment of Paget’s

disease and the diagnosis of metabolic bone disease by complexation with 99m

Tc [88, 12]

.

However the BPs are nowadays most widely used for the treatment of osteoporosis

which is thought as a major health problem for humans and especially for menopausal

women [89]

.

Osteoporosis is a skeletal disease and it is characterized by reduction in bone mass

that leads to an increased fracture risk [90, 91]

. Since early 1970s, BPs have been

20

investigated for the treatment of osteoporosis [92, 93]

. BP-drugs have the licensed positive

effect in increasing BMD and on bone resorption and are taken by millions of patients

worldwide. In the late 1980s, ETI became the first BP approved for the treatment of

osteoporosis [94]

. In 1990, another successful study about use of ETI in a randomized,

double-blind, placebo-controlled study in 66 postmenopausal women over three years,

was published [95]

. The efficacy of ALE and other BPs in postmenopausal women was

proved by several studies [96, 97, 98, 99, 100]

. Clinical studies proved that all approved BPs

actually reduced the fracture risk in bone. However, some of them were more efficient

comparing with the others in preventing the fractures in different parts of body. For

instance, ALN, ZOL and RIS are more efficient than IBA in preventing hip fractures

[101].

Paget’s disease is a chronic disorder in which bone turnover of one or several bones

is increased, resulting in enlarged and weak (misshapen) bones. This type of bone

disease appears in approximately 3% of people above the age of 40 [102]

. Since the main

point of the disease is the increased bone resorption, BPs are currently being used for

the treatment of Paget’s disease [103, 104]

. So far, a number of studies have proved that

BPs in the management of patients with Paget’s disease, are efficient and the

availability of more potent, newer, BPs has improved treatment consequences [75, 105, 106,

107, 108, 109, 110, 111, 112].

The BPs tested in patients with problems based on bone metastases, have shown

reduced skeletal complications, such as reduction in osteolytic progression, fractures

and bone pain [113]

. Additionally, almost 30% of cancer patients have also

hypercalcemia during their disease. Hypercalcemia occurs in the malignant stage and is

a very serious complication. Increased osteoclast activity causes a high release of bone

calcium and it results to hypercalcemia [114]

. In the medication step of this disease,

especially ZOL, PAM and CLO showed impressive efficacy towards the treatment of

hypercalcemia of malignancy [80, 115, 116]

. Osteoclast activity plays a significant role in

bone metastases [113]

. BP-drugs are strongly recommended for the treatment of

metastatic bone disease.

One of the most common uses of BPs is for bone imaging with radiochemicals [5,

117]. The application of BPs as bone scanning agents may depend on their ability to be

21

combined with a gamma-emitting technetium isotope [88, 118, 119]

. This ability provides

such complexes proper locations in the sites of bone turnover [120]

. The single-photon

emission computed tomography (SPECT) is a widely used technique with gamma-

emitting BP complexes. Technetium-99m (99m

Tc) isotope is the most widely used

radionuclide with this method since its suitable nuclear properties (half-life, T1/2=

6.0067 h) and this isotope is then transformed into stable technetium-99 by nuclear

transformation [121, 120]

. These gamma-emitting BP complexes are nowadays leading the

rest used in bone imaging since they have high sensitivity, good results and low cost [122,

123].

The BPs have also antitumor [124]

, anti-inflammatory effects [125, 126]

as well as

effects on osteolytic bone disease of multiple myeloma [127, 128]

. A summary of BP’s

biomedical applications is shown in following figure (Figure 13) [72]

.

Figure 13. Biomedical applications of BPs.

22

1.8 The mechanism of action of bisphosphonates in cells

Osteoclasts are the cells that cause the resorption of bone tissue [5]

. Osteoclasts are

organized to support the massive transport of protons for the solubilization of the HAP

and the acidification of the resorption part [129, 130]

. Bone tissue is a porous structure

which contains a solid matrix, cells and a fluid phase [131]

. BPs have been used for over

50 years [37, 132]

. The studies on it have shown that BPs can affect the bone resorption

caused by osteoclasts in many ways [133, 134]

.

BPs are internalized via endocytosis by the osteoclasts during the bone resorption

[133]. Due to accumulation type of the BPs in bone, they have a close contact to

osteoclasts rather than other cells in bone [135]

. The action of vacuolar-type proton

pumps in the osteoclast ruffled membrane causes an acidification to the sub-cellular

space beneath the osteoclast during the bone resorption [136]

. This acidification initiates

the dissolution of the HAP bone mineral and it results in their accumulation and up-take

by fluid-phase endocytosis [137, 138]

. It is also known that BPs have significant effect

especially on mature osteoclasts. These mature osteoclasts are formed by the fusion of

mononuclear precursors of haematopoietic origin [5]

. By preventing and reducing the

activity of osteoclasts which cause the resorption in bone, or by increasing the apoptosis

rate of them, BPs inhibit the resorption in bone caused by osteoclasts [16]

.

1.8.1 Effects of Bisphosphonates on Osteocytes

Osteocyte (Figure 14) is a cell characteristic of mature bone tissue and that lies

within the substance of fully formed bone [139]

. It is derived from osteoprogenitors, a

fraction of which differentiate into active osteoblasts. It is also known that mature

osteoblasts may also acquire a resting phenotype becoming a lining cell [131]

. Osteocytes

are the most commonly found cells in bone [140, 139]

and the surface area within the

osteocytic network is approximately 1000-5000 m2 [141]

. This canalicular structure

allows the drugs to access [15]

.

23

Several BPs in very low concentrations can protect osteocytes against apoptosis [142,

143]. The inhibion made by BPs over the osteocyte apoptosis arises to be mediated

through the activation of extracellular signal-regulated kinases (ERKs) and opening of

connexin 43 hemichannels [144]

. BP effects on osteocystes are supported by the results of

the pharmacological studies based on transgenic animals, especially on their ability to

prevent the apoptotic effects of glucocorticoids [145]

or cyclic mechanical loading [146]

.

Experimental findings show that the potency of BPs on osteocytes doesn’t state about

their effects on osteoclasts [147]

. Hence it is also proposed that BPs having little anti-

resorptive activity might have ability to increase osteocyte survival [148]

.

Figure 14. Evolution of osteocytes [149]

.

1.8.2 Effects of Bisphosphonates on Osteoblasts

Osteoblast is a cell which bone develops from and is a mononuclear specialized

derivation of mesenchymal stem cells [150, 151]

. Osteoblast functions in groups of

connected cells and is involved in osteoclast regulation [152, 153]

. Recent studies on

osteoblasts also showed that they may play a significant role on bone disorder, such as

Paget’s disease and osteoporosis [151]

.

24

It was proven in the 1990s that the osteoblasts treated with BPs can inhibit

osteoclastogenesis [154]

. Clinical studies verified that BPs have a mitogenic effect over

the osteoblasts [155, 156]

. Other studies proposed that BPs could inhibit the expression of

receptor activator of nuclear factor-ĸB ligand (RANKL) also known as tumor necrosis

factor ligand superfamily member 11 (TNFSF11) and could also increase the expression

of osteoprotogerin (OPG) in osteoblastic cells of human [157]

. It is also known that

osteoblast effect on RANKL signaling mediates the antiresorptive effect of BPs [158]

.

Furthermore, some other studies have demonstrated that BPs with different

concentrations can decrease or increase osteoblastogenesis depending on their

concentration [156, 159, 160]

. A recent study on BP effects on osteoblasts proposed that as

the osteoblasts may be involved in bone disorders such as Paget’s disease and

osteoporosis, it is basically imaginable that BPs play a role in these diseases [151]

.

However, the observed effects of BPs mentioned above differ with the type of BP used

in the model system and the method used [161]

.

1.8.3 Effects of bisphosphonates on hydroxyapatite (HAP)

Chemical formula of HAP is Ca10(PO4)6(OH)2 [162]

. HAP is one of the most studied

biomaterials as it is structurally related with bone mineral [163]

. A close analog of this

compound, a biological apatite, constitutes the bone mineral [162]

. BPs possess high

affinity for HAP so that BPs have ability to bind to HAP [72]

. As a result of binding to

HAP, BPs are easily taken up by bone [137]

.

Long time ago it was approved that BPs have multiple effects on HAP such as,

inhibititon of crystal dissolution and inhibition of the aggregation of crystals [11, 38, 164]

.

The amount of BP attached to HAP can be predicted by a Langmuir adsorption isotherm

[11]. It was also proven that the classical adsorption isotherms are leading to binding

characteristics. Hence it is known that a saturation level on the amount of BP bound

demonstrates about the capacity of crystals for adsorbing a given BP onto their surface

[11]. Additionally, a recent chromatographic study showed that BPs differ in their

retention time for their binding ability to HAP [165]

. For instance, ALE and ZOL showed

25

significantly longer duration times than RIS. Furthermore, it is also known that small

differences on R1 and R2 groups, can enhance the binding to HAP as mentioned before

in the chapter of structure-activity relationships of BPs. Even though there is still a lack

of clear information on these biologically important issues, the findings on BPs’ ability

to bind to HAP can be used for pro-drug studies [166, 42]

.

1.9 Side effects of use of bisphosphonate-drug

BPs have some side effects which usually last in a short while. As everyone reacts

to drugs in a different way, there might be a chance to have one or more of possible side

effects. These possible side effects are listed below [167, 168]

.

Table 4. Side effects of BP use and its possible solutions.

Side Effect Simple solution

Fever or flu Taking paracetamol can help.

Hypocalcaemia(low level of calcium

concentration in blood)

Blood test based on checking the levels of

minerals first, then medication might help also.

Bone and joint pain A mild painkiller such as paracetamol may help.

Change in bowel

mechanism(constipation or diarrhoea)

Drinking fluids (6-8 glasses per day).

Feeling sick (nausea) It is a temporary feeling and lasts in a few days.

If it continues or gets worse, doctor can

prescribe an anti-sickness tablet.

Damage to kidney Regular blood test is needed in order to check

how well the kidney works.

Osteonecrosis of the jaw If BP is taken for longer than a year, a dental

check-up is needed before treatment starts.

According to a recent study, among the side effects of BP use, the most frequent

one was GI discomfort appearing 19.8% of the patients on medication [169]

while other

studies found it as 25% [170]

and 20.8% [171]

, respectively. In another study it was also

26

reported that the patient who suffered from GI side effects had poor acquiescence to the

treatment progress [172]

.

As the tolerance of any patient for any drug is different, side effect topic is very

wide and might not be understood or generalized easily.

1.10 Future prospects on bisphosphonates

Even though there have been many discussions on the treatment way of bone

diseases with BPs, the clinical use of BP in this matter is being very well studied [5]

.

Additionally, there are also remarkably successful experimental studies and

observations about mortality reducing ability and anti-cancer effects of BP drugs [173, 174]

as well as other effects of them as mentioned before in previous chapters. But almost all

of these studies have also their own issues such as, the optimal duration of therapy,

gender, oral versus intravenous therapy, which means that there might be a need of

further studies on such issues.

The idea that BPs might be used as carriers to deliver some other pharmacological

agents to the bone was achieved before [175]

. It has also given a future prospect based on

that to release different active drugs via BP conjugates to the target organs or tissues in

human body.

In addition to current uses of BP, a recent study has proven that BPs have shown a

positive effect to inhibit arterial calcification [176]

. However there is still a further

research needed on recruiting these promising studies.

Beside all of these, there are still a lot of studies to be done on BP effects on the

treatment methods of several diseases as well as endless future prospects in which the

BPs can be used. In this case, it might be thought that knowing more about the

mechanism of action of BP drugs in human body, may give more opportunities to

scientists to build up brand-new ideas to overcome some of major diseases for humans

[177]. In the following figure, future prospects of BP are summarized

[5] (Figure 15).

27

Figure 15. Future ideas for BPs.

1.11 The aim of the study

The literature part of the thesis is intended to explain about general knowledge and

aspects related with the main properties, medical applications, the mechanism of action

and future prospects of BPs.

The purpose of present study is based on the synthesis of novel bifunctional BPs

and their conjugation with PAs. By this investigation, synthetic pathway and potential

problems of the synthesis of (2-((2-((2-aminoethyl)amino)ethyl)amino)ethane-1,1-

diyl)bis(phosphonicacid) and (2-((2-aminoethyl)amino)ethane-1,1-

diyl)bis(phosphonicacid) were learnt. This study also aims to give an aspect on the

synthesis of these BP-PA conjugates and the difficulties observed during this project.

28

2 Experimental Part

2.1 Novel bisphosphonate and polyamine conjugates

PAs are a class of the compounds containing two or more primary amino groups

[178]. They exist in all eukaryotic cells with their concentrations in mM range.

Biochemically the term polyamines means the naturally occurring PAs, spermidine

(SPD), putrescine (PUT) and spermine (SPM) which are essential for growth [179]

. They

are found in vegetables and fruits, fermented food products (yoghurt, beer), foods of

animal origin (milk, fish, eggs and meat).

PAs have also several synthetic derivatives, such as triethylenetetraamine (TRIEN)

[180]. TRIEN (Figure 16) is a selective chelator for Cu

2+ and also for other ions. It is used

as an alternative treatment in Wilson’s disease, an autosomal recessive disorder of

copper metabolism, where the excessive copper accumulation leads to several

neurological and mental problems.

Figure 16. Triethylenetetraamine.

Although there have been separately many publications on both PAs and BPs, there

has been only some studies based on BP-PA (Figure 17) conjugates [181]

. BP-PA

conjugates could possibly be used as selective chelating agents. In medicine, they could

potentially be used as imaging agents when combined with radioisotopes, such as 99m

Tc.

These new conjugates might have also interesting metal chelating abilities [4]

.

29

Figure 17. The basic structure of novel polyamine bisphosphonate conjugate.

2.2 Materials and Method

2.2.1 General

The main method used for observation and analysis of both the reaction and the

compounds obtained, was 1H,

31P and

13C NMR spectroscopy. The NMR

measurements were carried out on a Bruker Avance 500 DRX instrument with working

frequency of 500.1 Hz, 202.4 Hz, and 125.7 Hz for 1H,

31P and

13C nuclei respectively.

NMR spectra were obtained with internal standards, tetramethylsilane (TMS) in CDCl3

and sodium 3-(trimethylsilyl)-1-propanesulfonate (TSP) in D2O. When required and

possible, thin layer chromatography (TLC) method was used for the purity evaluation

and observation with KMnO4 and Ninhydrin. All the compounds involved in this

research, are named based on the software of Chembiodraw 13.

2.2.2 Materials

Both solvents and dry solvents in reactions were used as they are supplied.

Tetraisopropyl-methylenebisphosphonate (98%) was used as prepared before.

Tetraisopropyl-ethenylidenebisphosphonate was prepared according to known methods

published by Degenhardt et al. [182]

and the purity was not less than 99% obtained by

NMR. Paraformaldehyde (95%), di-tert-butyldicarbonate (98%) and

Ethyltrifluoroacetate (99%) were supplied by Fluka, diethylamine (99,5%) and

diethylenetriamine (99%) were supplied by Sigma-Aldrich, ethylenediamine (98%),

30

magnesium sulfate and potassium carbonate were supplied by J.T Baker.

Toluenesulfonic acid (99%) was supplied by Merck. Tert-butyl (2-aminoethyl)

carbamate (3b) was prepared according to the literature in the methods published by J.

Chadwick et al. [183]

and tert-butyl(2-aminoethyl)(2-((tert-butoxycarbonyl)amino)ethyl)

carbamate (4b) was prepared according to known methods published by M. Carland et

al. [184]

. For the methodical column chromatography, Silica gel 60 (supplied by Merck)

was used. TLC was accomplished using Merck TLC aluminium plates (Silica gel 60

F254, 20x20) and observation by staining on them was enhanced with iodine and

potassium permanganate.

31

2.2.3 Prepared compounds

The general aspect of the research carried out in this thesis is shown in following scheme (Scheme 1).

R1 (iso-propyl group) = C3H7

Scheme 1. The pathway of reactions in current study.

32

Tetraisopropyl ethenylidenebisphosphonate (2)

Paraformaldehyde (6.78 g, 225 mmol) and diethylamine (3.35 g, 45 mmol) were

combined in 100 ml of dry methanol and the mixture was warmed until it got clear.

Then heat was removed and afterwards compound 1 (12.3 g, 35 mmol) was added into

the mixture. Then the mixture was refluxed for 6 days. Afterwards it was concentrated

under reduced pressure. 20 ml of dry toluene was then added and the mixture was

concentrated. This step was repeated twice in total to ensure complete removal of

methanol from expected intermediate (CH3OCH2CH(PO3i-Pr2)2) which was obtained as

a clear liquid. This intermediate was dissolved in 100 ml of toluene and afterwards

catalytic amount of toluenesulfonic acid (monohydrate) was added. In a Dean-Stark

trap, methanol was removed by refluxing over night. After refluxing, the mixture was

concentrated under reduced pressure. Compound 2 was obtained as a clear liquid.

Column chromatography was carried out on the compound (silica gel, 1:1

acetone/hexane). Compound 2, yield 3,461 g : 1H NMR (CDCl3) δ 7.0- 6.8 (m, 2H,

H2C), 4.9-4.6 (m, 4H, OCH), 1.6-1.2 (m, 24H, CH3); 13

C NMR (CDCl3) δ 147.3 (s, 1C,

=CH2), 135.1 (t, 1C, PCP, 1JCP=168.34), 71.62- 71.29 (m, 4C, OCH), 24.415-23.85 (m,

8C, CH3); 31

P NMR (CDCl3) δ12.22.

Tert-butyl(2-aminoethyl)carbamate (3b)

A solution of di-tert-butyl dicarbonate (1.4 g, 6 mmol) in anhydrous 1,4-dioxane (50

ml) was added over 3 h to a stirring solution of ethylenediamine (2,12 g, 35 mmol) in

1,4-dioxane (50 ml). After stirring overnight the solvent was removed under reduced

pressure. Afterwards 70 ml of water was added to the residue and the insoluble bis-

substituted product was collected by filtration. The filtrate was extracted with CH2Cl2

(5x20 ml). The organic extracts were dried over Na2SO4, filtered and concentrated

under reduced pressure to give compound 3b. The compound was synthesized with a

different starting material, ethylenediamine was used instead of 1,4-diaminobutane

mentioned in original paper. Compound 3b (purity 74%) yield 0.43 g : 1H NMR

(CDCl3) δ 4.9 (s, 1H, NHBOC), 3.3-3.1 (m, 2H, CH2), 2.9-2.7 (m, 2H, CH2), 1.6-1.4

(m, 9H, CH3); 13

C NMR (CDCl3) δ 156.6 (s, 1C, C=O), 79.44 (s, 1C, CH), 42.22 (s,

2C, CH2), 28.7 (s, 3C, CH3).

33

Tert-butyl(2-((2,2-bis(diisopropoxyphosphoryl)ethyl)amino)ethyl)carbamate (3c)

In order to combine protected amine and bisphosphonate groups, compound 3b (1.0 eq.

, 44 mg, 0.085 mmol) and compound 2 (1.0 eq. , 100 mg, 0.28 mmol) in 25 ml of

acetonitrile, were taken into a flask. The mixture was refluxed for 2h. According to

spectra result, 73 mg of compound 3b was added in order to react with unreacted

compound 2. Then the mixture was refluxed for 1h and concentrated under reduced

pressure to give 3c. Compound 3c (purity 90%) yield 197 mg : 1H NMR (CDCl3) δ

4.86-4.7 (m, 4H, CH), 3.24-3.14 (m, 2H, CH2), 3.1 (td, 2H, 3JHP=17.55 Hz,

3JHH=5.5 Hz,

CH2), 2.7 (m, 2H, CH2), 2.44 (tt, 1H, 2JHP=23.76 Hz,

3JHH=5.5 Hz, PCH), 1.5-1.25 (m,

33H, BOC + i-Pr groups); 13

C NMR (CDCl3) δ 156.4 (s, 1C, C=O), 78.8 (s, 1C, OC),

72-70.7 (m, 4C, OCH), 48.32 (s, 2C, CH2), 45.33 (s, 1C, CH2), 39.3 (t, 1C, J=134.6,

PCP), 28.5 (s, 3C, CH3), 24.6-23.8 (m, 8C, CH3); 31

P NMR δ 21.94.

(2-((2-aminoethyl)amino)ethane-1,1-diyl)bis(phosphonicacid) (3d)

Compound 3c (150 mg, 0.29 mmol) in 25 ml of HCl (4M) was refluxed for 1h and then

stirred for 20 minutes. Then the mixture was concentrated under reduced pressure to

give compound 3d. Compound 3d (purity 67%) yield 99 mg : 1H NMR (D2O) δ 3.65-

3.4 (m, 6H, CH2), 2.85-2,7 (m, 1H, CH); 13

C NMR (D2O) δ 45.66 (s, 1C, CH2) 44.76

(s, 1C, CH2), 41.93 (t, 1C, J=123.72, CH), 35.92 (s, 1C, CH2); 31

P NMR δ 16.82.

Tert-butyl(2-aminoethyl)(2-((tert-butoxycarbonyl)amino)ethyl)carbamate (4b)

Diethyltriamine (1 g, 9.69 mmol) was dissolved in 40 ml of methanol. Reaction mixture

in a flask was placed into a liquid nitrogen and methanol bath at -78°C. Then ethyl

trifluoroacetate (1.37 g, 1.0 eq.) mixed with 10 ml of methanol, was added into the

mixture. The mixture was kept in the bath for 1h and then taken to an ice bath at 0°C.

Afterwards, di-tert-butyl dicarbonate (8.46 g, 4.0 eq.) mixed with 6 ml of methanol was

added into the mixture and the mixture was kept at 25°C over night. Then the mixture

was concentrated under reduced pressure to give 4b. Compound 4b, yield 1.872 g : 1H

NMR (CDCl3) δ 5.9-5.6 (m, 1H, NH), 3.35-3.18 (m, 8H, CH2), 1.56-1.33 (m, 18H,

34

NHBOC, NBOC); 13

C NMR (CDCl3) δ 156.4 (s, 2C, C=O), 79.54 (s, 2C, C(CH3)),

51.35-41.12 (m, 8C, CH2), 28.8 (s, 6C, CH3)

Tert-butyl(2-((2,2-bis(diisopropoxyphosphoryl)ethyl)amino)ethyl)(2-((tert-

butoxycarbonyl)amino)ethy)carbamate (4c)

Compound 2 (100 mg, 0.28 mmol) and compound 4b (85 mg, 0.28 mmol) were taken

into a flask and then 25 ml of acetonitrile was added. Then the mixture was refluxed for

1h and then concentrated under reduced pressure. Compound 4c, yield 178 mg : 1H

NMR (CDCl3) δ 4.85-4.7 (m, 4H, CH) 3.4-3.2 (m, 8H, CH2), 3.12 (td, 2H, 3JHH= 5.39,

3JHP= 16.32, CH2), 2.7-2.6 (broad-s, 1H, NH), 2.58-2.37 (m, 1H, CH), 1.5-1.39 (m, 24H,

CH3), 1.38-1.27 (m, 18H, NHBOC, NBOC); 13

C NMR (CDCl3) δ 156.5 (s, 2C, C=O),

48.8-38.05 (m, 5C, CH2), 29.4 (m, 6C, CH3), 25.26-23.53 (m, 1C, CH); 31

P NMR δ

21.79.

(2-((2-((2-aminoethyl)amino)ethyl)amino)ethane-1,1-diyl)bis(phosphonic acid) (4d)

Compound 4c (178 mg, 0.27 mmol) in 25 ml of HCl (4M) was refluxed for 1h. Then the

mixture was concentrated under reduced pressure to give 4d. Compound 4d (purity

63%) yield 107 mg : 1H NMR (D2O) δ 3.5-3.2 (m, 8H, CH2), 2.7-2.56 (m, 2H, CH2),

2.55-2.42 (m, 1H, CH); 13

C NMR (D2O) δ 46.15-43.86 (m, 5C, CH2), 36.94 (t, 1C,

1JCP= 121.33, PCP);

31P NMR δ 16.08.

35

2.3 Results and Discussion

The studies on preparing the BP-PA conjugates desired (Scheme 1) was initiated

by the easiest starting point, ethenylidenebisphosphonate (2). This starting point allows

Michael addition to be carried out on the double bond in structure of 2 for conjugation

reaction between PA and BP [181, 182]

.

The first step through the current study was taken via the purification of compound

1 by vacuum distillation. In an uneventful manner, expected fraction (the purest of 1)

was collected with the purity of 96.7% at the temperature range of 60-110°C (1 mmHg).

Following step was to convert the BP tetraester, 1, into 2 by the known method [182]

.

Compound 2 was obtained without difficulties but due to the impurities not desired,

column chromatography method was applied for purifying 2. Afterwards, the fractions

which have the purities over 95% were collected to be used for further steps.

In the next step Boc protected PA, 3b, was prepared by the known method [183]

.

Ethylenediamine (EDA), 3a, was used as a starting material instead of 1,4-

diaminobutane mentioned in the method. In this step 3b was successfully synthesized.

The only problem in the process was about separation of organic extracts with CH2Cl2.

Sometime also water phase was analyzed and extraction process was done for the

second time over it in order to obtain a better yield. The development in extraction

process might give desired organic phase immediately at once, but then also the

conditions should be optimum for it.

Afterwards compound 3b was conjugated to 2 by using Michael type addition. The

optimum solvent used in this reaction was acetonitrile as it showed the best activity and

provided the best result and environment among the solvents used (Table 5) for the

purity of 3c. Typical problem faced in this step was the case when all of 2 was reacted,

there was still unreacted 3b. This unreacted PA problem was solved by adding a

calculated amount of 2 according to 1H NMR spectra corresponding to the result of the

reaction. Eventually the intermediate compound, 3c, was successfully synthesized after

refluxing in acetonitrile.

36

Table 5. The optimization of solvents used in conjugation of 2 and 3b and the purities

of 3c compounds obtained.

Solvent Name Purity observed by 31

P NMR (%)

Acetonitrile 80

DCM 38

Diethyl ether 66

Ethyl acetate 64

Toluene 69

For obtaining one of the final products desired, 3d, both Boc protective groups and

isopropyl groups of 3c were removed with acid catalysis hydrolysis. The acidic

environment of mixture caused the reaction to go reverse. The amine group attached to

the central carbon via a CH2 bridge detached due to the acid catalysis hydrolysis used

for the cleavage of Boc and isopropyl groups. Thus compound 2 was unintentionally

obtained after the cleavage of amine group. Then 2 reacted with water in acidic

conditions and instead of the amine group desired, the OH group attaching to the side

chain was observed. Therefore in 31

P NMR spectra there were obvious three different

peaks corresponding to 2, 3d and the compound (2- hydroxyethene-1,1-

diyl)bis(phosphonic acid) (Figure 18) which has OH group in its side chain. The

existence of OH group in side chain was also proven by general 1H NMR spectra table.

A purification method, crystallization with ethanol and water, on 3d slightly worked out

and it developed the purity of the compound but was not as efficient as expected.

Eventually 3d was successfully synthesized with some undesired impurities.

Compound 3c was also treated with TFA in order to remove Boc and isopropyl

groups. But this attempt couldn’t be carried out to remove them out of 3c. It was

observed clearly by 31

P NMR that the reaction go reverse and there was still the peak

corresponding to 2 and not to 3d. According to findings, it can be proposed that if

optimum conditions for the cleavage with HCl or TFA are provided, then removal of

both Boc and isopropyl groups might be smoothly carried out without such problems.

37

Another Boc protected PA in the reaction pathway, 4b, was prepared from

diethylenetriamine (DETA), 4a, by the known method [184]

. As a result 4b was

successfully synthesized. The only problem faced in this step was about the third Boc

group not desired to attach to DETA (Scheme 2) after removal of trifluoroacetamide

protection at one of the amine sides of DETA. This problem was also proven by 1H

NMR spectra as there was the peak corresponding to three Boc groups. The case further

reactions were carried out successfully, showed that there was more 4b than the

compound containing three Boc protective groups and this problem didn’t cause any

important further issue for the pathway.

Scheme 2. Trifluoroacetamide attaching to 4a, Boc protective groups attaching to 4a

with trifluoroacetamide, an undesired compound containing three Boc groups (III) and

the synthesis of 4b(IV).

In the next step 4b was conjugated to 2 by using Micheal type addition. Acetonitrile

was the most efficient solvent among the solvents used for this conjugation reaction to

be carried out, toluene, DCM, acetonitrile, respectively. The intermediate, 4c, was easily

obtained after refluxing in acetonitrile without any problem.

In the last step acid catalysis hydrolysis was carried out on 4c in order to remove

both Boc protective and isopropyl groups for obtaining the end product, 4d. Compound

4c was treated with 4M HCl. As it was also observed in the cleavage of Boc and

isopropyl groups of 3d, acidic environment caused the reaction go reverse in the case of

4c. In the 1H and

31P spectra of 4d there were peaks corresponding to the structures of

4d, 2 and the compound undesired (Figure 18) containing OH group in its side chain.

Eventually 4d was obtained but with some impurities. By the crystallization with water

and ethanol, the purity of 4d was developed to 63%.

38

Figure 18. An undesired impurity occured during the cleavage of Boc and isopropyl

groups of the compounds, 3c and 4c.

All the prepared compounds in this study were identified by using 1H,

13C and

31P

NMR spectroscopy methods of analysis. 2D 1H-

1H COSY (homonuclear correlated

spectroscopy), 2D 13

C-1H HSQC (heteronuclear single quantum correlation

spectroscopy) were used to identify the assignments of the signals on NMR spectra

when needed.

2.4 Conclusion

A method to synthesize (2-((2-((2-aminoethyl)amino)ethyl)amino)ethane-1,1-

diyl)bis(phosphonicacid) and (2-((2-aminoethyl)amino)ethane-1,1-

diyl)bis(phosphonicacid) was developed starting from tetraisopropyl

ethenylidenebisphosphonate.

The yields mentioned in experimental part show that the reaction conditions

are sub-optimal. Since the final compounds are not stable enough in acidic environment,

they might not be used as imaging agents in medicine when combined with

radioisotopes. But if it was possible to purify them and they were stable enough, then

they might have interesting metal and selective chelating abilities as well as the

tendency to be imaging agents.

39

3 Acknowledgement

First of all I would like to express my special and deepest appreciation to the

Master’s Degree Programme for Research Chemists for giving me this great

opportunity. By this education, I strongly believe in that I improved my knowledge and

skills.

A special thanks to Professor Jouko Vepsäläinen for accepting me to his research

team and providing me such an interesting research project.

I would especially like to thank my supervisors, MSc Elina Puljula and MSc

Sebahat Ucal for their endless support, inspirational suggestions and good friendships

during my thesis. I also would like to thank to my friends, Aino and Maritta, for their

valuable help at laboratory. My dear friend, Rinez Thapa, is also greatly appreciated for

joyful lunches that we had at every single restaurant in Kuopio. I owe many thanks to

all of you.

I know that the words are useless to express on how grateful I am to my dear

family for their precious support for anything I do in my life but especially for my

education.

At the end, I am also thankful to all of my friends who supported and encouraged

me to strive towards my goal during this abroad adventure.

Thank You!

Ercan Topçu

40

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