Boronated porphyrazines as a potential boron neutron capture therapy agent.

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Boronated porphyrazines as a potential boron neutron capture therapy agent. vom Fachbereiche Biologie/Chemie Der Universität Bremen genehmigte DISSERTATION zur Erlangung des Grades eines Doktors der Naturwissenschaften - Dr. rer. Nat - vom Michal Ratajski aus Sroda Wielkopolska Bremen 2005

description

a article about bnct

Transcript of Boronated porphyrazines as a potential boron neutron capture therapy agent.

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Boronated porphyrazines as a potential boron neutron capture therapy agent.

vom Fachbereiche Biologie/Chemie Der Universität Bremen genehmigte

DISSERTATION

zur Erlangung des Grades eines Doktors der Naturwissenschaften

- Dr. rer. Nat -

vom Michal Ratajski

aus Sroda Wielkopolska

Bremen 2005

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Tag des öffentlichen Kolloquiums:

28.09.2005

Gutachter der Dissertation:

1. Gutachter: Prof. Dr. Detlef Gabel

2. Gutachter: Prof. Dr. Dieter Wöhrle

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I would like to express my sincere gratitude to the following persons: My supervisor Prof. Dr. Detlef Gabel for accepting me as a PhD-student, for the interesting theme of my PhD-work, for help, good advice and a lot of patience. Prof. Dr. Dieter Wöhrle for being my second co referent. Dr. Thomas Dülcks for help in recording and analyzing the mass spectra. Renate Alberts the secretary of working group of Prof. Gabel for help not only in the chemistry. Past and present members of working group of Prof. Detlef Gabel for nice atmosphere during the work and many interesting discussions and advice. Past and present colleges at the department of chemistry University of Bremen.

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Contest 1 INTRODUCTION............................................................................................................ 7

2 BORON NEUTRON CAPTURE THERAPY. .............................................................. 8 2.1 INTRODUCTION TO BNCT ........................................................................................... 8 2.2 DEVELOPMENT OF TUMOR-LOCALIZING COMPOUNDS................................................ 10

3 PHOTO DYNAMIC THERAPY. ................................................................................. 11 3.1 INTRODUCTION TO PDT. ........................................................................................... 11 3.2 DEVELOPING TUMOR SEEKING DRUGS FOR PDT........................................................ 12

3.2.1 Clinical used PDT drugs. .......................................................................... 12 3.2.2 PDT tumor seeking drugs as boron transporting unit in BNCT. .... 14

4 SYNTHESIS OF DODECAHYDRODODECABORATE. ........................................ 15 4.1 SYNTHESIS OF MERCAPTOUNDECAHYDRO-CLOSO-DODECABORATE. ......................... 15 4.2 CHEMISTRY OF MERCAPTOUNDECAHYDRO-CLOSO-DODECABORATE......................... 18 4.3 SYNTHESIS OF AMMONIAUNDECAHYDRO-CLOSO-DODECABORATE. .......................... 19 4.4 CHEMISTRY OF AMMONIANDECAHYDRO-CLOSO-DODECABORATE............................. 20

5 PORPHYRAZINES. ...................................................................................................... 21 5.1 STRUCTURE OF PORPHYRAZINES. .............................................................................. 21 5.2 SYNTHESIS OF THE PORPHYRAZINE............................................................................ 22

6 BORONATED PHTHALOCYANINES AND CHLORINS AS PDT AND BNCT AGENTS. ........................................................................................................................ 26

6.1 BORONATED PHTHALOCYANINES AS PDT AND BNCT AGENTS. ............................... 26 6.2 BORONATED CHLORINS AS PDT AND BNCT AGENTS. .............................................. 34

7 GOAL OF THE WORK. ............................................................................................... 37

8 SYNTHETIC STRATEGY. .......................................................................................... 38

9 RESULTS AND DISCUSSION..................................................................................... 41

9.1 PREPARATION OF CIS-1,2-DICYANO-1,2-ETHYLENEDITHIOLATE................................ 41 9.2 ATTEMPT OF THE SYNTHESIS OF THE BORONATED PORPHYRAZINE VIA SUBSTITUTION

OF PORPHYRAZINE. .................................................................................................... 42 9.3 ATTEMPT OF SYNTHESIS OF PORPHYRAZINE WITH CARBONYL LINKER....................... 45 9.4 SYNTHESIS ATTEMPT OF BORONATED PORPHYRAZINE VIA FREE ACID NITRILE

DERIVATIVE............................................................................................................... 47 9.5 SYNTHESIS ATTEMPT TO PREPARE BORON PORPHYRAZINE DERIVATIVE BY

MACROCYCLIZATION OF TWO DIFFERENT DINITRILES. ............................................... 51 9.6 BORONATED PORPHYRAZINE PREPARED VIA SCHIFF BASE FROM ALDEHYDE

DERIVATIVE OF NMT. ............................................................................................... 54 9.7 BORONATED PORPHYRAZINE PREPARED FROM THE CYCLIC DERIVATIVE OF DINITRILE.. .................................................................................................................................. 58 9.8 SYNTHESIS OF THE PORPHYRAZINE VIA THE DIHYDROXYACETONE NA MNT

DERIVATIVE............................................................................................................... 61 9.9 SYNTHESES OF PORPHYRAZINES WITH DIAMINOMALEODINITRILE (DAMN) AS A

PRECURSOR. .............................................................................................................. 62 9.10 SYNTHESES OF DAMN DERIVATIVES AS MACROCYCLIZATION PRECURSORS. ........... 64

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10 HPLC PURIFICATION OF BORONATED PORPHYRAZINE. ........................ 68

11 EXPERIMENTAL SECTION. ................................................................................. 71 11.1 GENERAL CONSIDERATION. ....................................................................................... 71 11.2 PREPARATION OF SODIUM CIS-1,2-DICYANO-1,2-ETHYLENEDITHIOLATE .................. 72 11.3 PREPARATION OF1,2-DICYANO-1,2-BIS(2-METHOXY-2-OXO-1-ETHYL)THIO ETHYLENE. .................................................................................................................................. 73 11.4 PREPARATION OF 1,2-DICYANO-1,2-BIS(3-METHOXY-3-OXO-1-PROPYL)THIO

ETHYLENE. ................................................................................................................ 74 11.5 PREPARATION OF 1,2-DICYANO-1,2-BIS(4-METHOXY-4-OXO-1-BUTYL)THIO ETHYLENE

.................................................................................................................................. 75 11.6 PREPARATION OF 2,3,7,8,12,13,17,18-OCTAKIS[4-PROPYLOXY-4-OXO-1-

BUTYL)THIO]-21H,23H-PORPHYRAZINE.................................................................... 76 11.7 PREPARATION OF 2,3,7,8,12,13,17,18-OCTAKIS[(4-HYDROXY-4-OXO-1-BUTYL)-

THIO]-21H,23H-PORPHYAZINE (HOFFMAN 2001) ..................................................... 77 11.8 PREPARATION OF 2,3,7,8,12,13,17,18-OCTAKIS[(4-(MERCAPTOUNDECAHYDRO-

CLOSO-DODECABORTYL)-4-OXO-1-BUTYL)-THIO]-21H,23H-PORPHYAZINE. ............ 78 11.9 PREPARATION OF CHLOROACETYL MERCAPTOUNDECAHYDRO-CLOSO-

DODECABORATE. ....................................................................................................... 79 11.10 PREPARATION OF 1,2-DICYANO-1,2-BIS[2(MERCAPTOUNDECAHYDRO-CLOSO-

DODECABORATYL)-2-OXO-1-ETHYL)THIO ETYLENE. ................................................. 80 11.11 PREPARATION OF 2,3,7,8,12,13,17,18-OCTAKIS{[2-(MERCAPTOUNDECAHYDRO-

CLOSO-DODECABORATYL)-2-OXO-1ETHYL]THIO}-5,10,15,20PORPHYRAZINATO]MAGNESIUM. .............................................................. 81

11.12 PREPARATION OF S,S’-BIS(4-CARBOXYPROPYL)DISULFIDE. .................................. 82 11.13 PREPARATION OF 1,2-DICYANO-1,2BIS-(4-HYDROXY-4-OXO-1-BUTYL)THIO

ETHYLENE. ............................................................................................................ 83 11.14 PREPARATION OF 1,2-DICYANO-1,2BIS(4-S-PHENYL-4-OXO-1-BUTYL)THIO

ETHYLENE. ............................................................................................................ 84 11.15 PREPARATION OF 1,2-DICYANO-1,2-BIS(4-N-PHENYL-4-OXO-1-BUTYL)THIO

ETHYLENE. ............................................................................................................ 85 11.16 PREPARATION OF {2,3,7,8,12,13,17,18-OCTAKIS[4-N-PHENYL-4-OXO-1-

BUTYL)THIO]-5,10,15,20- PORPHYRAZINATO}MAGNESIUM. ................................. 86 11.17 PREPARATION OF 1,2-DICYANO-1,2-BIS[4-(MERCAPTOUNDECAHYDRO-CLOSO-

DODECABORATYL]-4-OXO-1-BUTYL)THIO ETHYLENE TETRAMETHYLAMMONIUM SALT. ..................................................................................................................... 87

11.18 PREPARATION OF 1,2-DICYANO-1,2-BIS[4-(MERCAPTOUNDECAHYDRO-CLOSO-DODECABORATYL]-4-OXO-1-BUTYL)THIO ETHYLENE SODIUM SALT. .................... 88

11.19 PREPARATION OF {{2,3,7,8,12,13,17,18-OCTAKIS[4-(MERCAPTOUNDECAHYDRO-CLOSO-DODECABORATYL)-4-OXO-1-BUTYL]THIO}-5,10,15,20-PORPHYRAZINATO]} MAGNESIUM SODIUM SALT. ................................................................................... 89

11.20 PREPARATION OF 1,2-DICYANO-1,2-BIS(ETHOXYMETHYL)THIO ETHYLENE........... 90 11.21 PREPARATION OF 1,2-DICYANO-1,2-BIS(METHOXYMETHYL)THIO ETHYLENE. ....... 91 11.22 PREPARATION OF [2,3,7,8,12,13,17,18-OCTAKIS[(ETHOXYMETHYLO)THIO]-

5,10,15,20- PORPHYRAZINATO]MAGNESIUM. ........................................................ 92 11.23 PREPARATION OF [2,3,7,8,12,13,17,18-OCTAKIS[(METHOXYMETHYLO)THIO]-

5,10,15,20- PORPHYRAZINATO]MAGNESIUM. ........................................................ 93 11.24 PREPARATION OF 1,2-DICYANO-1,2-BIS(HYDROXYETHYL)THIO ETHYLENE........... 94 11.25 PREPARATION OF 1,2-DICYANO-1,2-BIS(HYDROXYPROPYL)THIO ETHYLENE. ........ 95

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11.27 PREPARATION OF [2,3,7,8,12,13,17,18-OCTAKIS[(2-HYDROXYPROPYL)THIO]-5,10,15,20- PORPHYRAZINATO]MAGNESIUM. ........................................................ 97

11.28 PREPARATION OF [2,3,7,8,12,13-HEXA[(2-HYDROXYETHYL)THIO]-17,18DI(4-METHOXY-4-OXO-1-BUTYL)THIO]-5,10,15,20- PORPHYRAZINATO]MAGNESIUM. .. 98

11.29 PREPARATION OF [2,3,7,8,12,13-HEXA[(2-HYDROXYPROPYL)THIO]-17,18DI[(4-METHOXY-4-OXO-1-BUTYL)THIO]-5,10,15,20- PORPHYRAZINATO]MAGNESIUM. .. 99

11.30 PREPARATION OF 1,2-DICYANO-1,2-BIS(2,2-DIETHOXYETHYL)THIO ETHYLENE.. 100 11.31 PREPARATION OF 1,2-DICYANO-1,2-BIS(2-OXOETHYL)THIO ETHYLENE. ............. 101 11.32 PREPARATION OF 1,2-DICYANO-1,2-BIS(2-IMINODODECAHYDRO-CLOSO-

DODECABORATEETHYL)THIO ETHYLENE. ............................................................ 102 11.33 PREPARATION OF 6-HYDROXY-6,7-DIHYDRO-5H-1,4-DITHIEPINE-2,3-

DICARBONITRILE. ................................................................................................ 103 11.34 PREPARATION OF 1,3-DITOSYLACETONE. ............................................................ 104 11.35 PREPARATION OF PYRIDINUM 5,6-DICYANOPYRAZINE-2-OLATE.......................... 105 11.36 PREPARATION OF ACETAMIDOACETIMIDOSUCCINONITRILE (HINKEL 1937). ....... 106 11.37 PREPARATION OF ACETAMIDOIMINOSUCINONITRILE (HINKEL 1937)................... 107 11.38 PREPARATION OF1,2-DICYANO-1-(ACETYLAMINO)-2-[(2-HYDROXY-2-OXO-

ETHEN)AMINO]ETHYLENE.................................................................................... 108 11.39 PREPARATION [OCTAKIS ACETYLOAMINOPORPHYRAZINATO] MAGNESIUM. ........ 109 11.40 PREPARATION [TETRAKIS-β-AMINO-TETRAKIS-β’-ACETAMIDO PORPHYRAZINATO]

MAGNESIUM. ....................................................................................................... 110 11.41 PREPARATION OF 1,2-DICYANO-1-AMINO-2[(4-FORMYLPHENYL)METHYLEN

AMINO]ETHYLENE. .............................................................................................. 111 11.42 PREPARATION OF 1,2-DICYANO-1-AMINO-2{[4-(UNDECAHYDRO-CLOSO-

DODECABORATE)IMINO]METHYL]PHENYLMETHYLEN AMINO}ETHYLENE. .......... 112 11.43 PREPARATION OF [TETRAKIS(β-P-FORMYL PHENYL METHIN)OCTAKIS AMINO

PORPHYRAZINATO] MAGNESIUM ......................................................................... 113 11.44 PREPARATION OF 1,2-DICYANO-1-AMINO-2-[(4-CARBOXYPHENYL)METHYLENE

AMINO] ETHYLENE. ............................................................................................. 114 11.45 PREPARATION OF [TETRAKIS(β-P-CARBOXY PHENYL METHIN)OCTAKIS AMINO

PORPHYRAZINATO] MAGNESIUM ......................................................................... 115 11.46 PREPARATION OF S-4-((E)-{[(Z)-2-AMINO-1,2-DICYANOVINYL]IMINO}METHYL)

BENZOYL MERCAPTOUNDECAHYDRO-CLOSO-DODECABORATE............................ 116 11.47 PREPARATION OF N-(-1,2-DICYANO-2-{[(4-FORMYLPHENYL)METHYLENE]AMINO}

VINYL)ACETAMIDE. ............................................................................................. 117 11.48 PREPARATION OF N-{(2Z)-1,2-DICYANO-2-[({4-[AMMONIAUNDECAHYDRO-CLOSO-

DODECABORATEIMINO)METHYL]PHENYL}METHYLENE)AMINO]VINYL}ACETAMIDE............................................................................................................................. 118

11.49 PREPARATION OF 4-[(UNDECAHYDRO-CLOSO-DODECABORATEIMINO)METHYL] BENZALDEHYDE. ................................................................................................. 119

12 SUMMARY............................................................................................................... 120

13 ABBREVIATIONS. ................................................................................................. 122

14 LITERATURE: ........................................................................................................ 124

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1 Introduction.

For a long time tumor disease has become one of the most deadly illnesses for mankind directly after heart diseases. From first cancer detection a lot of people scientists, doctors and many others have tried to find some universal treatment against tumor disease. During the years scientists have found out that cancer is not a simple problem, there are many kinds of cancer and many reasons are responsible for this terrible disease. Many different treatment methods have been developed during the last century. Some of them are strictly pharmacological like treatments that use fluorine-containing compounds and others are strictly physical using radiation to kill cancers cells. All of these methods have some advantages, and disadvantages. No universal method for all kinds of cancer has been developed until now. All methods, chemotherapy, radiation therapy and surgery can bring some positive effects in the fight against the cancer, but in many cases bring also some disorderly effects on healthy tissues without guarantee of cure. Work in the field of the right cancer treatment is still continued. Each year scientist all over the world have some new ideas, how to fight better against cancer and how to better protect people. One of the most interesting ideas in this field is BNCT (Boron Neutron Capture Therapy). This interdisciplinary method has been developed for the last sixty years. Although this kind of treatment is still in its research phase, a lot of research groups all over the world are trying to develop this method in any of its aspect (medical, physical, chemical etc.). There is some success during clinical trials on patients, but also there is a lot to do in the field of BNCT therapy. BNCT is nowadays one of the promising treatments in fight against cancer diseases. In the future it should become the most universal and effective treatment against cancer. This gives a hope to all people touched with this terrible illness for a better life and complete cure.

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2 Boron Neutron Capture Therapy (BNCT).

2.1 Introduction to BNCT

Boron Neutron Capture Therapy (called BNCT) is binary treatment, composed of a chemical factor and irradiation of this chemical agent inside of tumor tissue. BNCT based on the simple nuclear reaction between the boron isotope 10B and low energy or thermal neutrons (Barth 1992). During this reaction nuclear reaction nuclei 4He (α particle), 7Li nuclei are produced (fig. 1).

Figure 1. Nuclear reaction of 10B.

The produced particles have an energy strong enough to kill cells. The range of this radiation is short and can destroy only cells placed around of the nuclear reaction center. A sufficient quantity of 10B (chemical factor) must be localized within the cancer cell and then a beam of thermal neutrons must be delivered and absorbed by boron placed in the cells to destroy the neoplastic cells (irradiation). Such binary system has a big advantage, because each of the factors can be manipulated independently to increase the selectivity of the treatment

The primary source of the thermal and low energy neutrons for BNCT treatments is nuclear reactors. The radiation produced in the nuclear reactors consists have lower-energy and thermal neutrons, fast or high energy neutrons and gamma rays. Before irradiation it is necessary to filter the beam from unwelcome high-energy neutrons and gamma rays. Such beam of mainly of low-

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energy and thermal neutrons is useful in BNCT therapy. Nuclear reactors are not the perfect source of neutrons for this reason many of research group has been trying to find some other sources of neutrons such as accelerators (Blue 1986) or radio isotopes (Bech 1991).

Chemical agents are the second factor of this dual system therapy. 10B atoms have larger neutrons capture ability than the others endogenous nuclei (1H, 16O, 14N, 12C, 31P). A huge number of boron containing compounds has been synthesized as potential BNCT chemical agents. Such chemical agent should satisfy a few conditions. The basic condition is water solubility, because only in this form the drug could be delivered to the living tissues. The second important thing is toxicity. A potential chemical BNCT drug should be nontoxic or the toxic effect should be very low to make it useful. The next condition is selective uptake of the drug into neoplastic cells. A suitable compound should target itself to the tumor cells and should have no or low accumulation in the healthy tissues, because during the absorption of neutrons healthy tissues could be also destroyed. The drug should also leave the blood rapidly after introduction into the body. The perfect BNCT drug should consist of a ``tumor localizing unit´´ which is responsible for providing the drug in to neoplastic cells, a ``boron loaded unit´´ which is loaded with 10B and optionally a linker connecting the targeting unit with the boron load unit (fig. 2).

Figure 2. Model of BNCT chemical agent.

A huge number of chemical compounds have been synthesized as potential BNCT agents and work is still continued in many research groups. Each year a few new potential BNCT chemical agents are known.

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2.2 Development of tumor-localizing compounds.

The first attempt of developing tumor-localizing compounds was made by Soloway, Sweet and Brownell in the 1960s. In their research among the many compounds that seemed to have suitable properties two of them were p-carboxy-benzeneboronic acid and the sodium salt of decahydrodecaborate (fig. 3).

BHO OH

p-carboxy-benzoboronic acid decahydrodecaborate

Figure 3. Structure of p-carboxybenyzoic acid and decahydrodecaborate.

Both of the compounds were relatively non toxic and gave tumor-to-brain concentration ratios of 5-8 to 1. These compounds destroyed the tumor, but too high concentration in the blood destroyed the blood vessels as well. These experiments showed that the right BNCT agents should also appear a high tumor to blood ratio. Some number of different compounds had been analyzed and two promising sulfhydryl-containing boron anions B12H11SH-2 (fig. 4) well known as BSH and B10Cl8(SH)2

-2 were found. Since 1968 Na2B12H11SH (sodium salt of BSH) had been used by Hiroshi Hatanaka in Japan to treat patients with brain tumors. The results he obtained renewed the interest in BNCT in USA, Europe and Australia and the research work on the synthesis of new BNCT compounds were continued (Gabel 2003).

It was known that the biosynthetic pathway of melanin needs phenylalanine, Mishima used boron-containing derivatives of phenylalanine amino acid to introduce it into melanoma cells. This compound is also called BPA (fig. 4)

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BOHHO

COOH

H2N

SH

-2

BPA BSH

Figure 4. Structure of BPA and BSH.

BPA is metabolically degraded to boric acid, and then can easily diffuse out of the cell. Both boronated compounds are used nowadays in clinical trials of BNCT in Japan and Sweden, and became the main BNCT drugs.

3 Photo Dynamic Therapy (PDT).

3.1 Introduction to PDT. The tumors localizing compounds are used not only in BNCT therapy. PDT (Photo Dynamic Therapy) uses light instead of neutron radiation, and in PDT it is necessary to provide compounds into tumor cells to absorb the light. In a photochemical reaction oxygen is transferred into its singlet form. Singlet oxygen is a strong oxidizing agent, which is able to kill a living cell. In case of PDT it is also necessary to produce tumor localizing drugs (chemical factor) which could find the tumor cells, accumulate in tumor cells and absorb light from external source (physical factor) and produce active singlet oxygen to kill tumor cells. The main mechanism of PDT is very similar to BNCT.

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3.2 Developing tumor seeking drugs for PDT Many classes of photosensitizers have been developed in the field of PDT or in another words chemical agents used as chemical factor in PDT therapy until now. The main role play chemical compounds from the group of porphyrins and their derivatives. Daugherty discovered the abilities of porphyrins accidentally. He placed radiation-sensitizing agents in cell culture near the lab window and noticed significant death of cells. Daugherty isolated the agent responsible for this phenomenon and after studies it was found that it was a porphyrin (Daughtery 1978). From this time many classes of chemical agent have been developed for clinical use on the basis of porphyrins.

3.2.1 Clinical used PDT drugs. Schwartz found out that purified hematoporphyrins (fig. 5) are much less active than crude. He developed a method of activation by treatment with sulfuric acid and acetic acid mixture (Kessel 2004). As a result of these experiments he got a mixture of porphyrins (hematoporphyrin derivatives HpD). Other investigators found that HpD consist from following compounds: hematoporphyrin, hydroxyethylporphyrin, vinyl-deuteroporphyrin and protoporphyrin.

N

NH N

HN

CH3

Na+

RH3C

O

H

-O

O

H3C CH3

O

Figure 5. Structure of hematoporphyrin. Direct injection of HpD into the center of the tumor showed higher level of the porphyrin in tumor tissues than in healthy tissues (Lin 1988) (Grossweiner

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1984). HpD has already been used in clinical trials as a drug appearing reliable, active, pain-free, relatively safe and non-toxic. However the drug is not easily removable from tissues. Patients need to stay out of sunlight at least 4 weeks after treatment. The mechanism of tumor localization of HpD is not well known. Jori (Jori et al. 1984) demonstrated that the porphyrins contained in HpD show affinity for the lipoproteins in plasma and suggested that up-regulation of low-density lipoprotein receptor in neoplastic tissues could play a role (Kessel 1984), (Grossweiner 1984). The specify biological mechanism is explained by Vicente (Vicente 2002). Hematoporphyrin derivative HpD is now commercially available drug called Photofrin. Another of the most important member of the porphyrin family is Foscan (fig. 6). This drug absorbs light at 660 nm and is rapidly distributed in tissues.

N

NH N

HN

Ph

Ph

Ph

PhOH

OH

HO

HO

Figure 6. Structure of Foscan

The doses of the drug are less in comparison to Photofrin, but the cost of the drug is higher. The drug is highly efficient in converting light. Only 20 J/cm2 is required to activate the drug. This allows to reduce the time of treatment but extraordinary care must be taken not to irradiate region, which should be not irradiated. The other disadvantages are that the patient can suffer from photosensitivity even out of direct sunlight. The drug is so active that heightened sensitivity is observed even to room light (Kessel 1987) (Winkelman 1962).

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Dyes have occurred as a fast-developed family of potential PDT drugs. Many of the compounds used in ink are efficacious photosensitizers. Many dyes used clinically come from phthalocyanines (fig. 7) and their relatives as well as naphthalocyanines. Such chemical agents are active in the range of 650-850 nm and activated at energy around 100 J/cm2. Compounds from this class are mostly hydrophobic and need a delivery agent to be delivered to tumor tissues. All clinically used drugs have a structure similar to porphyrin (Allison et al. 2004).

N

NH

N

N

N

HN

N

N

Figure 7. Structure of phthalocyanine

3.2.2 PDT tumor seeking drugs as boron transporting unit in BNCT. Both kinds of therapy Photodynamic Therapy and Boron Neutron Capture Therapy have the same basis. Both therapies need tumor localizing drugs to be accumulated in neoplastic tissues. Drugs used in PDT have better selectivity and don’t need the transporting unit to be delivered to tumor tissues. In the field of BNCT many different ideas have been already tested. BNCT agents normally use some transporting unit to localize and to be accumulated in neoplastic cells. The idea has been appeared to combine both kinds of drugs used in PDT and BNCT. BNCT agent consisting of high selective transporting unit used in PDT (porphyrin family tumor localizing drugs) and connected to it a drug consisting of a large number of boron atoms like boron cluster compounds should be potential promising agents for BNCT (Morgan 1993) (Rudolph 1981) (Hawthorne 2002).

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4 Synthesis of dodecahydrododecaborate. There exist a few syntheses methods of preparation of dodecahydrododecaborate. None of them gives excellent results and high yield of product. But one of more efficient method uses sodium borohydride as starting material. Sodium borohydride is oxidized by iodine and in this process converted into sodium octahydrotriborate, which at higher temperature in a complicated process forms dodecaborate. The anion produced can be then isolated from water solution by addition of triethylammonium chloride as its bis-triethlammonium-dodecaborate salt, which is not very well soluble in water. To remove the boric acid, which is a by-product, it is necessary to use some hydrochloric acid. (fig. 8) (Komura 1987)

3 NaBH2 I2 -2NaI-2H2

NaB3H8∆H

[(C2H5)3NH] Cl[(C2H5)3NH]2 [B12H12]

Figure 8. Synthesis of dodecahydrododecaborate. Dodecahydrododecaborate is able to take part in substitution reactions. It reacts very rapidly with the halogens chlorine, bromine and iodine forming poly- and per-halogenated derivatives. The activity of halogens is ordered as it follows Cl > Br > I. However the substitution with iodine give as a main product B12H11I-2. It cannot be simply reacted with one equivalent of iodine although this procedure is satisfactory to prepared a mono substituted iodine derivative (Knoth 1964). Hydrogen atoms of the cluster might be also substituted for hydroxy, thio and amino groups. These reactions are described in the next paragraphs. 4.1 Synthesis of mercaptoundecahydro-closo-dodecaborate. Mercaptoundecahydro-closo-dodecaborate known also as BSH (fig 4) has been used as BNCT drug in Europe and Japan. This compound is relatively non toxic and has an ionic nature its sodium salt is well soluble in water. However exchanging of sodium cation to some organic cation like tetramethylammonium or tetrabutylammonium cation causes that this compound is soluble in less polar organic solvents. The sulfhydryl group of BSH has a pKa of 13,4, this could explain why BSH behaves as weak acid. In presence of base BSH can form derivatives with positive charge accumulated on the sulfur atom (B12H11S-1). This causes that BSH and its ion show nucleophilic abilities. BSH reacts easily with alkyl halides and acid chlorides (Gabel et al. 1993) which allow attaching

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BSH to many others organic systems and gives the opportunity to link it to a large number of chemical agents and increase its tumor localizing abilities. BSH in acid condition and in the presence of oxygen can form blue dimeric disulfide of BSH, which next can be oxidized by air oxygen to colorless oxidized disulfide form. To avoid this unwelcome reaction it is necessary to carry out synthesis in absence of air and low pH (fig. 9).

SH

[O]

S S

2- 4-

[O]

S S

4-O

Figure 9. BSH dimer disulfide. The preparation of BSH is possible by substitution of a hydrogen atom by a thiol group (Knoth 1964; Tolpin 1978). BSH was synthesized first by Knoth in a reaction between dodecahydrododecaborate (B12H12

-2) and hydrogen sulfide, but this method gives difficulties with isolation of BSH as a pure crystalline salt and the yield of this process was not satisfier. The reason is that during the reaction many other compounds are formed like multi-substituted thio and hydroxy derivatives and polymeric structures of dodecahydro-closo-dodecaborate (Tolpin 1978). The other synthesis method of BSH is the reaction of B12H12 with 1-methyl-2-thiopyrrolidone in the presence of concentrated hydrochloric acid, which forms its derivatives with borate, and then it is hydrolyzed in basic conditions to finally form BSH. The first step of this reaction is the protonation of boron cluster compound. This phenomenon was described by Knoth (Knoth 1964). However it is still not known what the position of these two additional protons is (fig. 10).

16

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H+ + B12H12-2 HB12H12

-

H+ + HB12H12- H2B12H12

Figure 10. Protonation of boron cluster. The protonated cluster is reactive enough to be reacted with Lewis base with the evolution of hydrogen. The attack of a Lewis base in this case 1-methyl-2-thiopyrrolidone provides two additional electrons to the skeleton of boron cluster compounds. These two electrons cause that the cage is converted from the closo form into the nido form (fig. 11).

L

-2 -1

H+

HH

N

CH3SN

CH3S

-1

-H2

Figure 11. Reaction of cluster with 1-methyl-2-thiopyrrolidone. In the nido structure two bridge hydrogen atoms are in opposite position to the bounded sulfur atom. Finally the additional hydrogen atom is removed and the nido structure comes back to closo form of the cluster with an attached 1-methyl-2-thiopyrrolidon. To form BSH it is necessary to hydrolyze the former produced compound. According to work of Knoth about the synthesis of monohydroxy derivatives (Knoth 1964) a nucleophilic attack probably takes place on carbon number 2, which is directly connected to the oxygen atom. A similar reaction might take place in the case of the mercapto derivative (fig. 12), and finally form BSH. Further protonation and further substitution is also possible which leads to multisubstituted derivatives (Tolpin 1978).

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SN

CH3 SH

OH-

-1 -2

Figure 12. Hydrolysis of substituted cluster. 4.2 Chemistry of mercaptoundecahydro-closo-dodecaborate. As it was written in former paragraphs BSH behaves as a weak acid, and the sulfur atom of the thiol group appears to be strong nucleophilic center. This can be explained by the presence of the large doubly negatively charged anion attached to the sulfhydryl group. However the chemical behavior of the BSH is very similar to the classical thiols. BSH react in presences of base with alkyl halides, and halogen derivatives of carboxylic acids. The large nucleophilicity of BSH could explain that in alkylation reaction not only mono-substituted derivatives but also di-substituted derivatives with a positive charge cumulated on sulfur atom, which makes the whole structure only singly negatively charged (Gabel 1993) (fig. 13).

SH

RX

S+

R

R-2 -1

Figure 13. Reaction of BSH with alkyl halides. For the preparation of mono-alkylated derivatives it is necessary to use special techniques. At first BSH is reacted with an excess of β-bromopropionitrile to prepare the di-substituted derivative. Strong base like tetramethylammoniumhydroxide is able to remove one of the cyanoethyl groups. Next it can be reacted with another molecule of alkyl halide and once more tetraethylammoniumhydroxide can be used to remove the second cyanoethyl group and to get only a singly substituted derivative (fig. 14) (Gabel 1993).

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SH S+

CN

CN

S

CN

S+

CN

R

S

R

Me4N+ OH-

XR

Me4N+ OH-

- C2H4CN

- C2H4CN

Figure 14. Reaction sequence leading to mono-substituted derivative of BSH. This looks completely different in case of reaction witch carboxylic acids derivatives. Each molecule of BSH can react only with one molecule of carboxylic acid derivative (fig. 15) and formation of di-substituted derivatives is not observed. The thioester is quite stable against hydrolysis, which makes the linkage between cluster and carboxyl group useful in the preparation of water stable derivatives for BNCT.

SH S

R

X

O

R

O

Figure 15. Reaction of BSH with carboxylic acid derivatives. 4.3 Synthesis of Ammoniaundecahydro-closo-dodecaborate. Hydrogen atoms can be not only substituted by a sulfhydryl group. A substitution reaction is also known, which leads to ammoniaundecahydro-closo-dodecaborate. Hydroxylamine-O-sulfonic acid has been known as agent for direct introduction of amine group into aromatic rings in the presence of aluminium chloride. In this reaction an electrophilic attack takes place by a polarized hydroxylamine-O-sulfonate aluminium chloride with the attacking

19

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species NH2+. Hertler tried to make the same type of reaction on boron cluster

compounds B10H10-2 and B12H12

-2, but presence of an acid catalyst was required and the reaction was made in water. Then the prepared compound is isolated as tetramethylammonium salt from water and in its protonated form (fig. 16) (Hertler 1964).

ONH2

SO3OH

-2δ−δ+ NH3

+

-1

Figure 16. Formation of ammoniaundecahydro-closo-dodecaborate. 4.4 Chemistry of ammoniandecahydro-closo-dodecaborate. Ammoniaundecahydro-closo-dodecaborate (BNH3) reacts like a normal amine. It is possible to substitute with alkyl halides. It can react also with carboxylic acid derivatives and it can form Schiff bases in reaction with aldehydes. BNH3 exists only in its protonated form. Therefore it is necessary first to activate the amine group by removing the proton from lone electron pair of amine group with base. The first type of reaction is a reaction with alkyl halides in basic conditions. The degree of alkylation depends of the type of alkyl used in reaction. In case of the reaction with ethyl iodide triethylamine-undecahydro-closo-dodecaborate is formed but the reaction with 2-bromopropane yields two different products, mono- and diisopropylamine is produced. So it could be made some correlation between the structure of alkyl halide and the product of the alkylation. The reaction can be controlled by steric factors, which increase in the order ethyl<benzyl<isopropyl (fig. 17) (Gabel 1997).

NH3+

NH2+

NH+

Ph

Ph

N+

Figure 17. Alkylation of BNH3

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The other route to prepare mono alkylated derivative of BNH3 is a route through the formation of Schiff base and its reduction to alkyl derivative. Here the presence of base is necessary to deprotonate the nitrogen atom that allows to react the amine group with the carbonyl group of the aldehyde. The prepared Schiff base could be reduced with NaBH4 to a corresponding mono-alkylated derivative. However only aromatic aldehydes have been reacted with BNH3. The reasons of this are not completely clear (fig. 18) (Sivaev 1999).

NH3+ R

H

O

NH+R

H

NaBH4

NH2+

R

H

Figure 18. Alternative rout of alkyl derivative formation. 5 Porphyrazines. 5.1 Structure of porphyrazines. The name porphyrazine or in other words meso-tetraaza-substituted porphyrin includes a wide class of macrocyclic porphyrin analogues or heteroanalogues. This definition includes also porphyrazine itself and its substituted derivatives signed as follows H2PA and [H2PA(β-R)8] (fig. 19).

N

NH

N

N

N

HN

N

N

N

NH

N

N

N

HN

N

N

R

R

R

R R

R

R

R

H2PA [H2PA(β-R)8]

Figure 19. Structure of non substituted and substituted porphyrazine.

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The similarity of the porphyrazines to the porphyrins causes that this class of compounds retain many of their physicochemical properties. In the UV-VIS spectra of porphyrins the band in the blue region at 390-425 nm can be observed, two strong bands characterize porphyrazines at 330-350 and at 530-620 (Rodriguez-Morgade and Stuzhin 2004). Porphyrazines as well as porphyrins has the ability to incorporate metal ions into their central core. Porphyrazines could use as materials for photovoltaic cells, photoconductors and semiconductors, photo catalyst. 5.2 Synthesis of the porphyrazine. The father of the porphyrazine chemistry was Reginald P. Linstead. He was the first to synthesize a porphyrazine and started a new research line on this field. The large number of porphyrazines is synthesized nowadays by ‘‘Linstead macrocyclization’’. The main synthetic route for porphyrazine preparation is cyclotetramerization of synthons derived from corresponding substituted unsaturated vic-dicarboxylic acid. A porphyrazine magnesium complex (MPA) is obtained in this reaction (fig. 20).

COOH

COOH

O

O

O

NH

O

O

CONH2

CONH2

CN

CN

N

NH2

OAlkAlkO

N

NH2

ClCl

NH

NH

NH

[MPA]

NH3

- H2O

AlkO- HCl

NH3

- H2O NH3

Figure 20. Synthetic routes of porphyrazine formation.

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From this scheme the most important and most useful synthetic method is the method of macrocyclization of dinitriles. This is the so called Linstead macrocyclization. First metallic magnesium is added to dry n-propyl alcohol or n-butyl alcohol; the reaction is initiated by addition of a small chip of iodine into the reaction mixture. In this step at elevated temperature a heterogeneous mixture of magnesium propyloxide in propyl alcohol is formed. The dinitrile dissolved in dry propanol is added to such mixture. The formation of pigment can be observed on the surface of magnesium alkoxide particles (Linstead 1952). The prepared material is a porphyrazine magnesium complex [MPA]. To remove the coordinated ion it is necessary to use some strong acid or acid solution and the magnesium free porphyrazine (H2PA) is formed (fig. 21). Propyl alcohol seems to be the most useful alcohol for porphyrazine preparation. In experiments with other alcohols the pigment was not formed or formed in very small yield. This phenomenon could be explained by the influence of growth of the alcohol carbon chain on the final cyclization of the ring.

CNH

H CN

MgOPrPrOH

N

N

N

N

N

N

N

NMg

N

NH

N

N

N

HN

N

NH+

MPA H2PA

Figure 21. Formation and demetalation of porphyrazine (H2PA). It is possible to substitute porphyrazine in β positions of the pyrrol rings. Mostly substitution is made on the corresponding dinitrile before cyclization and the dinitrile so prepared is further use to cyclization (Wöhrle 1996), (Hoffman 1998), (Hoffman and Barrett 2000), (Hoffman 2001), (Gül 2001). For preparation of di-substituted dinitriles two kinds of precursors are mostly used: the disodium salt of dithiolodimaleodinitrile (NaNMT) and diaminomaleodinitrile (DAMN). NaNMT is synthesized in a reaction of sodium cyanide and carbon disulfide in DMF. Nucleophilic attack of the cyanide anion on carbon disulfide leads to formation of a NCCS2Na×3DMF complex, which is later hydrolyzed to NaMNT as sodium salt (Davison 1967). Na MNT is able to react witch alkyl halides in the presence of catalytic sodium iodide in acetone to

23

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form S- substituted derivatives of NMT which could be later used for porphyrazine cyclization (fig. 22.)

S-NC

NC S- Na+

Na+

XRNaI

SNC

NC S

cyclization

NaMNT

R

R

Figure 22. Substituted MNT for cyclization. The second most useful dinitrile used in porphyrazine formation is diaminomaleonitrile. DAMN is produced in condensation of HCN. The condensation of HCN is well understood (Sanchez 1967) (Ferris 1972). The reaction proceed via the stepwise formation of dimer, trimer to form finally DAMN (fig. 23)

HCN NH CHCN NH2CH(CN)2

CNH2N

H2N CN

DAMN

Figure 23. Stepwise formation of DAMN. Mono-substituted DAMN cannot be obtained by simple reaction with alkyl halides. Such reaction leads to disubstituted derivatives. An illustration of this can be the reaction of DAMN with methyl iodide (Hoffman and Barrett 1999). During this reaction disubstituted DAMN is formed. It is possible to prepare the monosubstituted derivatives indirectly by stepwise reaction of DAMN with aldehyde first to form Schiff base, which later can be reduced to mono alkylated derivatives. However in the reaction with the aldehyde the Schiff base is formed only on one amino group. To substitute the second amino group it is necessary first to reduce the Schiff base and then to react it again with a second molecule of aldehyde and finally reduce it to the alkyl derivatives (Shepard 1974) (fig. 24).

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NH2NC

NC NH2O

R

H

NH2NC

NC N

H

R

NaBH4

NH2NC

NC NH

H

R

NH2NC

NC NHR O

R

H

NNC

NC NHR

H

RNaBH4

NHNC

NC NHR

R

Figure 24. Alkylation of DAMN. However for cyclization it is necessary to alkylate each of the nitrogen atoms of the amino groups (Hoffman and Barrett 1997). In other case if not all nitrogen position would be protected, the cyclization reaction does not work. Until now it was shown that only porphyrazines can be synthesized from one type of nitrile, but is also possible to prepare porphyrazine from two different nitriles that can react in a so called cross-cyclization. Such cyclization can lead to six different isomers (fig. 25) (Hoffman 2001).

N

NHN

NN

HNN

N

R RR

RRR

R

R

N

NHN

NN

HNN

N

R' RR

RRR

R

R'

N

NHN

NN

HNN

N

R' R'R'

RRR

R

R'

N

NHN

NN

HNN

N

R' RR

R'R'R

R

R'

N

NHN

NN

HNN

N

R' R'R'

R'R'R

R

R'

N

NHN

NN

HNN

N

R' R'R'

R'R'R'

R'

R'

Figure 25. Isomers of porphyrazine after cross-cyclization.

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However formation and distribution of isomers depends in each case on the activity of the reacting nitriles or others precursors. When one of the nitriles is much more active than other nitriles, porphyrazine richer in this nitrile would be preferred. Distribution of the isomers in reaction between nitriles of comparable activity should be as it is shown on figure 25. The only possibility to tune the distribution of formed isomers is to use appropriate stoichiometric ratio of the two nitriles. 6 Boronated phthalocyanines and chlorins as PDT and BNCT agents. 6.1 Boronated phthalocyanines as PDT and BNCT agents. The structure of the phthalocyanines is very similar to the structure of the porphyrins. The structure also consists of four pyrrol rings connected to each other with nitrogen atom instead of -CH2- in case of porphyrin. Also to each pyrrol ring s benzene ring is attached (fig 26). Phthalocyanines are synthesized from dinitrile derivative of benzene. The dinitrile units are cyclized around a central metal cation in the presence of base (fig 26).

N

N

N

N

N

N

N

N

CN

CN

M

Figure 26. Formation of the phthalocyanine. Because of this similarity to the porphyrin system it could have the same biological abilities and could also have abilities to accumulate in the tumor cells. This makes it a great chemical system, which could be used as a tumor localizing unit in both binaries therapies PDT and BNCT. In PDT therapy different phthalocyanine systems have been synthesized. In the field of the BNCT therapy phthalocyanines can be also used as provider of the boron atoms to the tumor cells. Until now preparation of a few phthalocyanines systems with

26

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boron derivatives attached to them have been tried. Kahl and coworkers made a first attempt of boronated phthalocyanines preparation. (Kahl 1996). Phthalocyanines exhibit insolubility in both water and organic solvents so the boronated phthalocyanines prepared by Kahl had groups which allowed later to dissolve the phthalocyanines in both solvents systems strong polar like water and nonpolar organic solvents. The phthalocyanine synthesized by Kahl bears carborane. In the synthesis route Kahl used as starting material 4-nitrophthalodinitrile (fig. 27) .

CN

CN

NO2

CN

CN

COOCH3H3COOC

CN

CN

COOCH3H3COOC

CN

CN

COOCH3H3COOC

CN

CN

COOCH3H3COOC

C

C

Figure 27. Synthesis route to prepare a building block for boronated phthalocyanines derivative. The nitro group of this compound was substituted by dimethyl malonate in a simple reaction. From dimethyl malonate the carboanion is generated and is able to substitute the nitro group of the starting material. The carbon atom between

27

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the two carboxylic acid is able to form again a carboanion and the prepared carboanion is able to react with alkyl bromide. In this case the formed cation was reacted with propargyl bromide. The triple bond of the propargyl chain is able to react with B10H14 to form finally the closo form of the boron cluster with two carbon atoms inside in the cluster structure. The building block so prepared was then used for cyclization. As cyclization method solid-state condensation of the boronated phthalonirile by heating and at 200°C with cobalt (II) chloride in argon atmosphere was used. The prepared phthalocyanine was not soluble in water, which is the one of the most important abilities for BNCT agents. The ester groups were hydrolyzed in basic conditions to increase the solubility in water. In this process decarboxylation of one carboxylic acid groups also occurs to give finally the water-soluble salt of the boronated phthalocyanine (fig. 28). However the toxicity and abilities as a BNCT agent of this compound have been never tested.

N

N

N

N

N

N

N

N

RR

R R

COOCH3

COOCH3

C

C

R=

N

N

N

N

N

N

N

N

RR

R R

H

COO- Na+

C

C

R=

Co Co

Figure 28. Formation of the boronated phthalocyanine salt. The next attempt of synthesis of boronated phthalocyanines was made by the Russian scientist Zakharkin (Zakharkin 2000). In this synthetic attempt

28

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Zakharkin used a similar strategy. Boronated building blocks were prepared first that later were used for cyclization reaction to obtain boronated phthalocyanine. 9-(p-hydroxyphenyl)-m-carboborane was used as an initial compound. This compound was synthesized in a four-step cycle of reaction (fig 29). The first step in this route was the formation of a Grignard reagent from p-bromoanisole by reacting it with magnesium. The obtained compound was later transferred into its zinc derivative, which reacts in presence of Pd(PPh3)4 as a catalyst with 9-iodo-m-carborane to give 9-(p-methoxyphenyl)-m-carborane. This compound was finally hydrolyzed to the phenol derivative, which was reacted with 4-nitrophthalodinitrile.

OH3C

Br

OH3C

MgBr

OH3C

C C

OH

C C

OH

C

C

CN

CN

O2NO

C

C

NC

CN

Figure 29. Formation of the boronated building block for cyclization. Such dinitrile derivative is able to be cyclized into the phthaolocyanine system. Solid-state cyclization was used similarly to the work of Kahl. The prepared phthalocyanine precursor was heated to 200°C under argon and in the presence

29

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of CoCl2. However also in this case no biological investigation has been made; toxicology, biological activity and cell uptake of the compound is not known. The first work that reported the biological investigation was made by Fabris and co workers (Fabris 2001). They were also the first to start to work with BSH as boron loaded units in their attempt in preparation of the boronated phthalocyanine. Also the synthetic method was completely different than in already presented works. The phthalocyanine was synthesized first. Then the boron loaded unit in this case BSH was attached to the prepared porphyrazine. As starting material 2-(4-carboxyphenoxy)phthalocyaninato Zn(II) was made (fig. 30).

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

O

O

OH

O

O

S

Zn Zn

Figure 30. Preparation of 2-[(4-undecahydro-closo-dodecaboromercapto carbonyl) benzoxy]Zn(II)-phthalocyanine. The carboxyl group of this compound was activated by transferring the acid group into its acid chloride derivative and reacted with BSH in anhydrous solvent in the presence of base.

30

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The compound prepared and purified was used in a series of few experiments as photosensitizer on a molecular and cellular level. In each case the starting nonboronated compound was used in the same test to compare the results to its boronated derivative. Mostly the tests were orientated to check abilities of these two compounds as potential PDT agents. However some of the biological tests gave some useful information, which can be used in investigation of the boronated derivative abilities as a BNCT agent. The first from the series of experiment is a photo bleaching experiment. Both of compounds shown in figure 30 have no significant differences in molar extinction. In both cases the UV-VIS spectra substances were irradiated by 600 – 700 nm light and in some intervals of time were measured. In both cases it appears that the absorbency decreases what means that the concentrations of the phthalocyanine and boronated phthalocyanine decrease and the conclusion is that the irradiation causes irreversible and extensive degradation of both phthalocyanines. Next very important tests, like the cell accumulation test and cell photosensitization test were made. Especially very interesting is the cell accumulation test, which would confirm abilities of the boronated phthalocyanines as a potential BNCT agent. However the investigation shows that cellular uptake of the both phthalocyanines depends on the structure. It was also found, that cellular uptake of the nonboronated derivative is higher than the molecular uptake of the boronated pthalocyanines. It could be concluded that the peripheral boron cluster caused that the phthalocyanine accumulation is a little higher than in a case of its boronated derivative. The explanation is that boron cluster attached to the phthalocyanine inhibits the process of cellular uptake of the boronated derivative. However higher accumulation of the phthalocyanine could be reached by using a higher concentration of boronated phthalocyanine in the incubation medium. Similar results were obtained from cellular photosensitization test. HT-1080 cells incubated for 24 hours with 0,5 µm both of the phthalocyanines derivatives was used as test model and then irradiated by 600-700 nm. cell survival was observed after 1 minute no residual. However the photosensitizing activity of boronated phthalocyanine derivative was still significant. To conclude it is necessary to say that this work shows that phthalocyanines are promising class of compounds to be used in both kinds of therapies PDT and BNCT. They appear to be good cell accumulated systems and good photosensitizers. Boronated derivatives of the phthalocyanine seem to be a little bit less effective as photosensitizer and has less ability in cell uptake experiments. However the results of the experiments do not exclude it as a potential PDT and BNCT agent.

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Another synthetic attempt to obtain boronated phthalocyanine was made by Wöhrle and coworkers (Wöhrle 2004). The phthalocyanines prepared by them were tetrasubstituted with carborane. Some other test of photoactivity was made in the photo oxidation experiment. Two boronated phthalocyanines macro cycle were obtained connected with four ortho-carboranes with two different oxyphenyl spacers. The synthetic route used by Wöhrle exploits as starting material 4-nitrophthalonitrile which was reacted with two different phenol derivatives p-hydroxyphenol and p-nitrophenol to finally obtain two different spacers (fig. 31).

NC

NC

NO2

HO R

O

CNNC

RR= -NO2,-OH

R= -NO2,-OH

R= -NH2

Figure 31. Precursors of the boronated phthalocynines preparation. Additionally the 4-(4-nitrophenoxy)phthalonitrile was transformed into its amino derivative. C-methylcarboraneacetic acids chlorides were synthesized to connect the phthalocyanines to the carboranes, which then were reacted with earlier produced amino and hydroxy (fig. 31) derivatives to the corresponding ester and amide derivatives. Then finally each of obtained phthalonitriles was cyclized to the phthalocyanine system to obtain five different phthalocyanines (fig. 32). Their abilities as potential PDT agent were tested in each case. As it was described earlier in paragraph 3.1 the right photosensitizer has to produce singlet oxygen, which is an extremely reactive oxidation agent. Photooxidation experiments were made to check the abilities of the obtained phthalocyanines as potential PDT agents. Dissolved photosensitizers in the methanol-chloroform mixture should produced singlet oxygen, which react with also dissolved citronellol. The mixture was flushed with oxygen and irradiated with light and consumption of oxygen as function of time was observed. These experiments

32

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showed that boronated phthalocyanines are better photosensitizers than their nonboronated derivatives. Also their photodegradation is comparable to the nonboronated derivatives. Additionally the large content of boron in the structure makes the boronated phthalocyanines a potentially suitable drug for BNCT.

N

N

N

N

N

N

N

N

R

R

R

R O NH2

R O OH

R O O

O B10H10

CH3

R O NH

O B10H10

CH3

R

Zn

Figure 32. Obtained boronated phthalocyanines. Another attempt to prepare BNCT suitable boronated phthalocyanine was reported by Soloway (Soloway 1989). This compound was prepared by the reaction of 1-(4-aminophenyl)-o-carborane with copper(II) phthalocyanine tetrasulfonylchloride followed by the hydrolysis of unreacted chlorosulfonic acid to sulfonic acid groups. Next the unsubstituted SO3H groups were converted into the sodium salt to increase the water solubility of the obtained compound (fig. 33).

33

Page 34: Boronated porphyrazines as a potential boron neutron capture therapy agent.

N

N

N

N

N

N

N

NCu

SO3NHHO3S

HO3S SO3H

C

C

Figure 33. Water soluble boronated phthalocyanine. 6.2 Boronated chlorins as PDT and BNCT agents. The next porphyrin class compounds are chlorins. The structure of the chlorins is of course very similar to the porphyrins. The structure consists of four condensed pyrrole rings connected with methylene units. The only difference in both structures is the absence of one double bond in the structure of chlorin (fig. 34).

N

HNN

NH N

HNN

NH

porhyrin chlorin Figure 34. Structure of porphyrin and chlorin.

34

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However this difference in structure has no influence on the biological activity of the chlorin and the chlorin structure compounds are also used as tumor seeking drugs and photosensitizes in PDT (Daughtery 1998, Boyle 1996, MacDonald 1999). If this class of compounds keeps the tumor seeking abilities it could be used as a boron delivery unit in boron neutron capture therapy as well as other porphyrin compounds. However until now not much has been done on this field. Preparation of a boronated chlorin has been reported by Osterloh (Osterloh 2001). The pyropheophorbide-a a derivative of chlorophyll-a was used in this work, which wasn’t prepared synthetically but was obtained by extraction from see plants (Spirulina pacifica). Chlorophyll-a was treated with sulfuric acid in methanol to obtain methylpheophorbide-a and finally to convert it into pheophorbide-a (fig. 35). This structure consists of functional ester groups and vinyl groups. The ester group can be hydrolyzed to the free carboxylic acid and then reacted with BSH to attach the boron cluster compound to the chlorin system. BSH is an ionic compound so finally the counter ion can be exchanged into sodium cation, which makes this compounds water-soluble. On the other side this kind of chlorin has also vinyl group which can be reacted with HBr and then with different alcohols to obtain chlorin ether systems with different lipophilicity.

NNH

N HN

O

O

O

Figure 35. Structure of pheophorbide-a. The suspected mechanism of tumor cell delivering is a interaction with lipoproteins of cell membranes from this reasons different liphophility of the chlorin rings can have some influence on the boronated chlorin cell uptake. Five boronated chlorins with different carbon chain length were prepared as shown in figure (fig. 36).

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NNH

N HN

O

O

SB12H11

O

R

R =

R = CH3

(CH2)2

CH3

R =(CH2)4

CH3

R =(CH2)6

CH3

R =(CH2)8

CH3 Figure 36. Cycle of the boronated chlorins. Next all of the obtained boronated chlorins were used in some biological test to check their abilities as a potential boron neutron capture therapy drugs. The first test was a cellular uptake test. V-79-chinese-hamster cells was incubated together with the different prepared chlorins in different concentration. All chlorins showed the same ability to increase the cellular uptake when the concentration is higher. Additionally it was proved that the chlorins with shorter side chain are better accumulated than chlorins with a londer side chain. V-79-chinese-hamster cells was used in the cell toxicity tests. The cells were incubed with different chlorins in different time each. The survival of the cells at the end of the incubation time. More than 50% of the cells were dead approximately after 8 hours of incubation. The general conclusion is that boronated chlorins are less toxic than its nonboronated chlorin derivatives. The in-vivo test on living animals were made also. The mice carried a tumor in the body. The different chlorins were injected into the mice and the mice were observed. The time for the compound to leave the body was estimated. The last mice was sacrificed after 72 h and it was found out that only a little bit of the agent was still left in body. More agent was found in organs like liver and spleen than in the tumor. Almost nothing of boron agents was found in blood and blood organs like the lung and the heart.

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7 Goal of the work. The goal of the work was to prepare water soluble tumor localizing BNCT agents to check their chemical, physical and biological properties such as cell uptake, and its potential abilities as selective tumor localization agents According to paragraph 2.1 the perfect BNCT agent should consist of three parts: delivery unit, boron loaded unit and optionally linker between delivery and boron loaded units. The porphyrazine derivative were chosen in this work as delivery unit. The porphyrazine system was chosen because of its similarity in chemical and physical properties to the porphyrin class, which has been already used in clinical trials as tumor localizing agent in PDT therapy (see paragraph 3.2.1). The abilities of tumor seeking boronated porphyrin derivatives have also been tested already (see paragraph 3.2.2). The boron cluster dodecahydrododecaborate (B12H12) has been chosen as boron loaded unit. This cluster is already in use in clinical trials. The substitution reaction of B12H12 to exchange one of the hydrogen atoms into thio, amino or hydroxyl group are known, which gives great possibility to attach the cluster to other chemical systems. The third reason is the ionic character of the cluster. The counter ion decides on the solubility in solvents of different polarity, which gives great opportunity to tune the solubility from completely non-polar solvents to polar solvents like water. This property is useful during the chemical synthesis and finally exchanging the counter ion to sodium makes the prepared product soluble in water, which is one of the goals of work. Although the synthesis of the porphyrazines and its derivatives is quite well known, the main problem of the work is to connect the boron cluster with the porphyrazine system. Direct connection is not possible because of the chemical character of the boron cluster and the porphyrazine system. For this reason it is necessary to use so called linker to attach the boron cluster to the porhyrazine system. This work shows a few attempts of connection cluster to porphyrin with help of linkers, and to synthesize boron cluster compounds derivatives, which could be used as precursors to synthesize boron containing porphyrazines. From chemical and biological reasons it has been tried to use different linkers between ‘‘tumor localizing unit’’ and ‘‘boron loaded unit’’. This work presents attempts to obtain different boronated porphyrazines, and different synthetic ways to prepared it. It presents also all problems occurred during synthetic work and full analytical characteristics of the obtained compounds.

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8 Synthetic strategy. Because it is not possible to attach boron atoms directly to the porphyrazine structure it was decided to prepare macrocyclization precursors from disodium salt of dithiolodimaleodinitrile (Na MNT) and diaminomaleonitrile (DAMN). The synthetic strategy of a boronated porphyrazine preparation from Na MNT shows figure 37 way 2. Na MNT is already known and easy to prepare. This compound could be easily reacted with alkyl halides, which gives great opportunity to attach a chemical linker to porphyrazine system. The linker could then be connected with boron cluster compounds. The boron cluster dodecahydrododecaborate with thio (BSH) and amino (BNH3) ligands was chosen as boron loaded unit. Both of these compounds react easier with activated carbonyl group. For this reason it was decided to prepare the first building blocks, which would consist of side chains with one side, attached to the dinitrile system and from the other side would have protected carboxylic acid group as its ester derivative. After cyclization such derivatives would be easy to hydrolyze to its acid form. Finally the activated carboxylic acid derivative could be reacted with BSH to obtain a boronated porphyrazine. The reaction between the BNH3 and the aliphatic carboxylic acid is not possible. If this synthetic route would for some reasons not be successful it is possible to use some other strategy to prepare boronated porphyrazines figure 37 way 1b. This alternative route could be to obtain boronated porphyrazines by cyclization of macrocyclization precursors with already attached boron cluster compound. This route gives also two different opportunities of boronated porphyrazine preparation. The boron cluster compound with the thio ligand could be used as a starting material in the first of them. The thio or amino ligand of the boron cluster compound is able to react with activated carboxylic acids. This derivative should be then attached to the dinitrile system. Selected activated carboxylic acid should have on the other end of the carbon chain a functional group, which allows them to react with Na MNT. So the best group should be halogen that could react with Na MNT, make the bond between the sulfur atom of MNT and the carbon chain of boron cluster compounds derivative. The second synthetic way shown on the figure 37 way 1a.This route could be the preparation of boronated macrocyclization precursor which uses as a starting material not the boron cluster compound but Na MNT. The ionic nature of the Na MNT allows reacting it with alkyl halides. So it would be possible to react it with halogenated carboxylic acid esters. NMT carboxylic acid ester derivatives would be obtained as a result of this reaction. This ester then could be hydrolyzed to the carboxylic acid, and finally after activation it could be reacted

38

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with BSH or BNH3 boron cluster compounds to obtain macrocyclization precursor ready for cyclization.

NC

S-NC

S- Na+

Na+

NC

SNC

SCn

Cn

O

O

OR

OR

NC

SNC

SCn

Cn

O

O

OH

OH

NC

SNC

SCn

Cn

O

O

R

R

R= -S-cluster

R

O

ORX

R= -S-cluster

way 1a way 2

SNC

SCn

Cn

O

O

OR

OR

NC

porphyrazineS

Cn

O

OR

porphyrazineS

Cn

O

OH

porphyrazineS

Cn

O

OR

R= -S-cluster

way 1b

Figure 37. General strategy of using Na NMT in syntheses. Both of the synthetic ways of this route lead to the same macrocyclization precursors. The advantage of this route is that after cyclization compound will be obtained, which already have the boron unit attached, and it allows to avoid problems with separation of the starting boron cluster and the obtained boronated porphyrazine. An acid component with different carbon chain length could be used in all proposed routes to observe difference in reactivity and physical and chemical properties of the obtained compounds in all steps.

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The boron cluster compounds used in all synthetically routes could be BSH as well as BNH3. However BNH3 can be used only in the reactions with aromatic acids and aldehydes. Different distribution of charges in both types of the clusters could also bring interesting differences between boronated porphyrazines bearing of these two types of the clusters. Reactions typical only for BNH3 cluster like reaction between its amino group and aldehyde to form a Schiff base are known. Two different synthetic routes were also selected for this solution. These routes started from Na MNT to finally get its derivative with carboxyl group able to be react with amino group of BNH3 boron cluster compound to form a Schiff base. The Schiff base could be later reduced to the amino system, which is more stable than the Schiff base.

NH2NC

NC NH2

NH2NC

NC NCH

Cn O

NH2NC

NC NCH

Cn

COOH

NH2NC

NC NCH

Cn

CR

O NH2NC

NC NCH

Cn NHR

R= -S-cluster

R= -cluster

NH2NC

NC NCH

Cn NH2

R

R= -clusterporphyrazine

NCH

Cn R

OR= -S-cluster

porphyrazineN

CHCn NH

R Figure 38. General strategy of using DAMN in syntheses.

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The synthetic strategy of preparation of a boronated porphyrazine from DAMN is shown on figure 38. Diaminomaleonitrile (DAMN) is prepared from HCN but is also commercially available. Its different chemical nature from Na MNT makes that in this case it is necessary to use a different strategy than in case of Na MNT. DAMN has two amino groups (see fig. 23). Two side chains could be attached to each of amino group. It is necessary to use a special technique through the Schiff base to obtain only mono substituted group. This technique is described in paragraph 5.2 (see also fig. 24). This technique allows also to substitute both amino groups with different side chain. In this case could be used a difunctional chain component as a linker, which allows to make reaction with amino group on DAMN on one side (aldehyde group) and with BSH or BNH3 on the other side (carboxylic acid group). This whole strategy allows to obtain a wide range of different boronated porphyrazines and gives wide range of different synthetic routes. However further investigation showed that the idea of using monoalkylated DAMN derivative was not good. All position of nitrogen atoms of the DAMN have to be substituted to use this species in the cyclization reaction. 9 Results and discussion. 9.1 Preparation of cis-1,2-dicyano-1,2-ethylenedithiolate. The most common compounds used in almost all syntheses of porphyrazines are the derivatives of cis-1,2-dicyano-1,2-ethylenedithiolate (see fig. 22). This compound is synthesized as its sodium salt (NaMNT). It has in its structure a sulfur atom with a negative charge. This makes it a strong nucleophilic agent, which can easily react with halogen derivatives of alkyls by nucleophilic attack on the α-carbon. This opens many possibilities to prepare many derivatives, which then could be later used in porphyrin synthesis. A two step synthesis of NaMNT is described by Davison and Holm (Davison 1967). The first step of the synthesis carried out in DMF. Well powdered sodium cyanide is suspended in dry DMF and carbon disulfide is added drop wise from a dropping funnel. The nucleophilic attack of cyanide anions on central carbon of carbon disulfide takes place at this step and cyanodithioformate ion is formed NCCS2Na*3HCON(CH3)2. It is necessary to provide anhydrous condition at this step to avoid hydrolysis of the formed product. It is also necessary to provide good mechanical stirring of the reaction mixture. Efficient stirring increases the yield. During the reaction the mixture rapidly darkens and the product crystallizes as a solid brown solid mass. Purification of the raw product is carried out by crystallization from isobutyl

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alcohol. The yield of this reaction is about 70 %. The brown crystals are then dissolved in water and hydrolyzed in 24 hours to the right product the second product of hydrolysis is DMF, which should be evaporated under reduced pressure together with water. At this step the raw product should be purified by crystallization. First the yellow solid substance is dissolved in hot dry ethanol and filtered to remove insoluble material. Diethyl ether is added to the cooled mixture to precipitate the product. The original literature advices to make crystallization from cold ethanol by addition of diethyl ether, but a second crystallization from hot ethanol gives better yield. The obtained substance is a yellow crystal powder, well soluble in water, methanol, and ethanol and less in acetone. The freshly crystallized product still contains a large amount of ethanol. For this reason it should be dried in high vacuum over 24 hours. When the product still contains ethanol it decomposes within a few weeks. In this case it is necessary to crystallize the partly decomposed material once more from hot ethanol and dry it carefully in high vacuum. The 1H NMR measurement was not done because of the absence of the proton in structure. The 13C NMR measurement indicates the two signals with chemical shifts in the area of the nitrile group and the double bond system carbon. Also an IR measurement indicates the typical signals for the nitrile group. 9.2 Attempt of the synthesis of the boronated porphyrazine via substitution of porphyrazine. This attempt uses as starting material Na MNT. Na MNT as described in paragraph 5.2 can react with alkyl halogens in the presence of sodium iodide as a catalyst. This allows to prepare a porphyrazine by reaction of Na MNT with ω-halogen acid esters of different chain length. Such Na MNT derivatives can be easily converted into a porphyrazine. The porphyrazine derivative with an ester side chain should be easily converted into its acid derivative and such group can be activated by converting it to the carboxyl chloride and finally reacted with BSH to obtain the boronated porphyrazine (fig 39). Dry Na MNT was reacted with methyl chloroacetate, methyl 3-chloropropionate or methyl 4-chlorobutyrate. All three reactions were carried out in similar condition. Na MNT was suspended in dry acetone. A small amount of NaI was added and the reaction mixture was stirred and refluxed in nitrogen atmosphere to avoid contact with moisture. Esters instead of acid were used o avoid reaction between acid group and the anionic thiol group of Na MNT and formation of thio esters. The reactions were carried overnight. The inorganic salt produced as second reaction product was removed by filtration and extraction with water. The yield of each reaction strictly depends on the chain length of the used ester.

42

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NC S-

S-NC

Na+

Na+

(CH2)n

O

OX CH3

NC S

SNC

(CH2)n

O

O CH3

(CH2)n

O

O CH3

N

NH

N

N

N

HN

N

N

SR

SR

SR

SRRS

RS

RS

RS

R= (CH2)n

O

O Pr

n=1,2,3X=Br, Cl

n=1,2,3

H2Pz (CH2)n

O

O Pr

KOHTHF/H2O

H2Pz (CH2)n

O

OH

SOCl2pyridineBSH

H2Pz (CH2)n

O

SB12H11 Figure 39. Synthesis scheme via acid esters derivatives. In case of the longest chain ester (Hoffmann 2001) the yield was the highest about 70 %. In case of shorter chain lengths, propionyl ester and acetyl ester the

43

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yields were smaller, about 50 %. Only the methyl chloroacetate derivative was a solid substance; the two were viscous liquids. This could be explained by their structure. The longer side chain makes these compounds unable to get its crystal form. This causes some difficulties in the further purification process. The acetyl ester derivative could be purified by simple recrystallization. Both the longer chain length derivatives are high molecular weight viscous liquids and the only way to purify them is a column chromatography. Column chromatography gives good results. Pure compounds were obtained in both cases. The structures were determined by standard analyses IR, 1H NMR, 13C NMR and mass spectroscopy. The obtained compounds have good solubility in non-polar organic solvents, are stable in air and are non-hygroscopic. However before further use in synthesis it is necessary to dry them in high vacuum and heat. The next step in this synthesis route is the cyclization to the porphyrazine. This was made according to standard procedure Linstead macrocyclization (Linstead 1952). In this synthesis it is necessary to prepare magnesium propoxide as cyclization agent. This compound is prepared by suspending metallic magnesium in propan-1-ol in the presence of a small amount of iodine. This mixture is refluxed overnight. Because magnesium propoxide is sensitive to water it is necessary to provide anhydrous conditions. For this reason the reaction requires dry propanol, dry reagents and nitrogen reaction atmosphere. NMT derivatives dissolved in dry propanol were added to such prepared magnesium propoxide suspension and the reaction mixture was refluxed 24 hours. Only the butyryl derivative yields a blue pigment. The acetyl and propionyl derivatives give no blue pigment but lead to brown mixtures of large number of compounds, probably of polymeric character. The explanation of such differences in reactivity could be sterical. The esters with shorter chain lengths are closer to the formed ring, which causes the second polymeric unit to turn to the other side and instead of the cyclization product only polymeric material is obtained (fig. 40).

CN-

C

N

Mg+

N

R R

N

RR

Figure 40. Formation of polymers during cyclization.

44

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The reaction between basic magnesium propoxide and acidic protons attached to a carbon could take place in case of the acetyl ester derivative, which leads also to other type of polymerization. However only the butyryl ester gives the blue pigment porphyrazine. Under basic conditions and in the presence of magnesium propoxide the methyl ester is converted to the n-propyl ester by transesterification. The obtained mixture was reacted with TFA to remove the magnesium cation from the core of the porphyrazine ring. The product was obtained as a mixture with few other by-products. The product was purified by column chromatography and finally by crystallization and the structure was determined by IR, 1H NMR, 13C NMR and mass spectroscopy analyses. The next step in the reaction route is the hydrolysis of the ester into its acid form. Because of the problems with solubility the reaction was made in two solvents system. Potassium hydroxide was used as hydrolyzing agent in stoichiometrical excess to the ester groups. Potassium hydroxide was dissolved in water. As a solvent for porphyrazine THF was taken, which is soluble in water. Both solutions, of potassium hydroxide in water and of the porphyrazine in THF, were mixed together and stirred over a period of few days. Then after removing of THF and neutralization of the water solution by addition of inorganic acid the product of hydrolysis precipitates. The prepared acid is ready to react with BSH to finally form the boronated porphyrazine. This reaction was made in two steps. First activation of carboxylic group by transferring it into carboxyl acid chloride and then reaction with BSH. Thionyl chloride was used to transfer the acid into the acid chloride. Porphyrazine was dissolved in pure thionyl chloride and refluxed. Then thionyl chloride was evaporated in vacuum to obtain a blue liquid residue. The obtained chloride is very sensitive to moisture. For this reason it was used for next the reaction without purification and analysis. Too long exposure to moisture in air could cause decomposition of the obtained chloride and make it completely inactive in the reaction with BSH. Excess of BSH with pyridine as base was added to the chloride dissolved in dry acetonitrile. The reaction was carried out under nitrogen for 5 days and in the absence of light to avoid formation of disulfide. After this time the acetonitrile was removed. The obtained blue residue was tried to purify by HPLC methods (described in paragraph 10.) 9.3 Attempt of synthesis of porphyrazine with carbonyl linker. It was decided to prepare porphyrazine with attached boron cluster with help of acetyl group or its longer chain homologous. It is known that BSH and BNH3 can react with carbonyl chlorides: The reactions of BSH with acetyl chloride and

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benzoyl chloride were described (Gabel 1993). In both cases the chlorides react witch BSH under mild conditions in the presence of base, in this case pyridine. The yields of these reactions are also good, about 80 % each. This reaction gives the opportunity to use the acetyl group to attach the boron cluster to another organic system in this case to the porphyrazine. Bromoacetyl bromide and chloroacetyl chloride were chosen to be used as a linker. The activated carboxyl group can easy react with the sulfur atom of BSH to give a chloro or bromo acetyl thio ester derivatives of BSH (fig 41). In case of chloroacetyl chloride this reaction is made in acetonitrile as solvent. It is also necessary to use a base. In such reaction normally pyridine is used, but in this experiment a compound was used which gives not only reaction with activated carboxyl group but where also reaction with the halogen group is possible, that leads to dialkylated derivatives of BSH (Gabel 1993). 2,6-lutidine was used in this reaction as base to avoid the alkylation reaction on the nitrogen atom. Two methyl groups shield the 2,6-lutidine nitrogen atom. The reaction mixture was also sealed with nitrogen to get rid of oxygen and to prevent disulfide formation. The reaction mixture was cooled in an ice bath to avoid too rapid reaction. In any case after addition of chloroacetyl chloride some trace of a disulfide formation was observed by the blue color of reaction mixture. The purification of the reaction product was as follows. The acetonitrile was rotary evaporated, and the residue was dried finally in high vacuum to get rid of the rest of chloroacetyl chloride. The dry residue was dissolved in the fresh portion of acetonitrile and some volume of diethyl ether was added to precipitate the boron cluster compound. The precipitate was filtered and finally recrystallized from water. This compound is able react with nucleophilic agents in this case Na MNT that has two nucleophilic sulfur atoms. DMF was chosen as reaction solvent because of different solubility of these two compounds. The reaction proceeds in presence of NaI as a catalyst in dry conditions and nitrogen atmosphere. The purification of the obtained product mixture was made similarly to acetyl derivative. The obtained orange solid substance was positively analyzed as expected product. The TMA ion was exchanged to protons by ion exchanger and addition of diluted sodium hydroxide in water finally to the sodium cation. The nitrile was reacted with magnesium propoxide to cyclize it into the boronated porphyrazine. But this reaction gave no expected product. TLC analyses showed that during this reaction the starting boronated nitrile was completely decomposed in the mixture. There was no starting material. This could be explained by the same reasons as the syntheses described in paragraph 9.2.

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SH O

X X

S

O

X

S-

NC

NC

S- Na+

Na+S

NC

NC

S S

O

S

O

Cyclization Figure 41. Synthesis schema of boronated porphyrazine preparation via acetylation of BSH. 9.4 Synthesis attempt of boronated porphyrazine via free acid nitrile derivative. Because of the problems described in a paragraphs 9.2 and 9.3 it was decided to synthesize porphyrazines only with a linker containing 4 carbon atoms in the chain. Therefore Na MNT was used as starting material, which was reacted with two equivalents of methyl 4-chlorobutanoate in the presence of catalytic amounts of NaI in dry acetone at reflux for 1 day. The obtained liquid mixture had to be purified by chromatography column. This gave the pale yellow ester in yield of about 70 % (fig 42). Next it was necessary to hydrolyze the obtained ester to get the corresponding free acid derivative.

47

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S-NC

NC S-

Na+

Na+

Cl(CH2)3 O

CH3

O SNC

NC S(CH2)3 O

CH3

O

(CH2)3 OCH3

O

H+

H2O/THF

SNC

NC S(CH2)3 OH

O

(CH2)3 OH

O

SNC

NC S

(CH2)3

O

S

(CH2)3

O

S

boronated porphyrazine

Figure 42. Synthesis schema of boronated porphyrazine via free acid nitrile derivative. The first attempt was with an aqueous solution of potassium hydroxide as base. The ester was added to the solution of potassium hydroxide and stirred over a few hours as a suspension. After the whole amount of ester was dissolved the reaction mixture was acidified with an aqueous solution of HCl to pH about 7. Crystalline material precipitated immediately which was later filtered and re-crystallized. The obtained substance after analyses appears to be not the expected dinitrile acid derivative but a completely different structure. From analyses it was found out that it is a disulfide derivative (fig 43). The base used in the experiment (aqueous solution of potassium hydroxide) was able to hydrolyze the ester groups but was also strong enough to hydrolyze both nitrile groups and form the potassium salt.

48

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NC S

SNC

OCH3

O

OCH3

O

SOH

O

SOH

O

KOH/H2O

- CO2

Figure 43. Formation of the disulfide derivative. This could initiate the reaction of disulfide formation. After the addition of aqueous solution of acid salt was transferred to the acid and both acid groups next to the double bound could decarboxylate. However the mechanism of the reaction, which leads to the disulfide derivative was not investigated because it was not the theme of this work and it is still not known. In the next experiment instead of the potassium hydroxide was used aqueous solution of HCl. This agent cannot hydrolyze the nitrile groups and the reaction of disulfide formation is not initiated. The reaction was made in a two phase system because of the problems with solubility. The ester was dissolved in THF and an aqueous solution of HCl was added. The reaction mixture was refluxed for 10 hours. The product was extracted with methylene chloride and recrystallized, and analyzed as expected. During the cyclization reaction the ester group of the dinitrile can be converted by transesterification to the n-propyl ester. For this reason the first acid derivative was prepared by reacting it with thiophenol in the presence aniline to check if during the cyclization this acid derivatives would be stable and would not give the transesterification products. All derivatives were prepared successfully and with good yield and further cyclization reaction showed, that such derivatives are stable and do not take part in transesterification reactions. These experiments allow to assume that it is possible to make cyclization reactions with BSH derivatives. Because of problems with solubility of BSH TMA salt derivative in dry propanol it was necessary to exchange the TMA anion to sodium, which is much better soluble in propanol than its TMA salt. To exchange the cation first Dowex ion exchanger in its H+ form was used to exchange the TMA cation into H+ cation and then finally convert it with help of sodium hydroxide aqueous solution into its sodium salt but the compound seemed to be very sensible on base. During the first experiment the addition of NaOH aqueous solution was controlled by laboratory pH papers. When the pH was about 7 the solution

49

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changed the color into dark yellow. Further analysis confirms the decomposition of the compound. During the second experiment after exchanging the cation into H+ addition of the diluted NaOH solution was controlled by pH-meter with glass electrode. The pH was raised to value 6,8 to avoid basic conditions. However during evaporation of water the compound decomposed again. The probable reason of this could be not a completely reacted rest of NaOH which during the evaporation process could concentrate to make basic conditions strong enough to decompose the compound. Finally the TMA salt was exchanged on the ion exchanger as its Na+ form and sodium salt was obtained. This salt latter was used as porphyrazine precursor. The conclusion from the experiments could be that the obtained dinitrile derivative with BSH attached to its structure (fig. 42) is sensitive to basic conditions and in these conditions it is decomposed. Even a small concentration of base causes the decomposition of boronated dinitriles. Only the experiment with direct exchanging of TMA salt into its sodium salt on ion exchanger when in reaction environment there were no basic OH- ions was successful. The analysis of the obtained liquid sodium salt confirmed that the TMA cation was successfully exchanged. The 1H NMR spectrum shows no more the chemical shift typical for protons of the methyl group from the TMA cation. Also mass spectroscopy prepared by ESI ionization method shows in the negative part of spectra the boronated dinitrile derivative and positive part of spectra shows no more peak with mass 74 which belongs to TMA counter ion instead of it spectra shows only the sodium cation with mass 23. Also the IR investigation seems to confirm that the ion exchange process was successful. The IR spectra did not show any more the typical signals for ammonium. The obtained sodium salt is much better soluble in propanol than its TMA salt, which allows to make macro-cyclization reaction with dry propanol as a solvent. However it was found that the boronated dinitrile derivative is sensitive to basic conditions. The preparation method of porphyrazines uses the basic agent magnesium propoxide as cyclization agent. The decomposition of boronated dinitrile derivatives take place in presence of strong bases like NaOH. Magnesium propoxide is a much weaker organic base. For this reason it was decided to make the macro-cyclization reaction in reduced stoichiometric ratio 1:1 between the dinitrile and magnesium. The boronated dinitrile was added to the reaction mixture as solution in dry propanol. No significant changes were observed at the beginning of the reaction. The reaction mixture turned brown after few hours of refluxing in nitrogen atmosphere and further investigations showed that dinitrile structure did no longer exist. Also the boron cluster part of the compound was not found in the mixture. So in this case the reaction leading to the porphyrazine could fail for the same reasons as during the ion exchanging process. Basic conditions provided by magnesium propoxide necessary to initiate the macro-cyclization reaction could be too strong and could cause

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decomposition of the boronated dinitriles derivative, which is sensitive to basic condition. 9.5 Synthesis attempt to prepare boron porphyrazine derivative by macrocyclization of two different dinitriles. Attempts of preparation of porphyrazine from two different dinitriles are known and described in the literature (Hoffman 2001; Hoffman 2000). The main problem during so-called cross macrocyclization is to find two different dinitriles, which could react with each other to produce a porphyrazine, consisting of two dinitriles.

S

NC

NC

S

O

O

O

O

S

NC

NC

S

OH

OH

S

NC

NC

S

OH

OH

S

NC

NC

S

O

O

S

NC

NC

S

O

O

1 2

3 4

5 Figure 44. Dinitriles selected for cross cyclization. Dinitriles have different activity in reaction of macrocyclization and can form different isomers (fig. 25). Formation of porphyrazine richer in the more active component would be preferred during the reaction. For this reason it is first necessary to the find the right ratio between the two used dinitriles, which could be reacted.

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Five different dinitriles were selected. First it was tried if they could be cyclized in normal cyclization (fig. 44). Only three nitriles from this group are able to form porphyrazine product in the cyclization reaction: dinitrile number1, number 2 and number 3. In case of the two other dinitriles 4 and 5, cyclization reaction cannot be done. It was found that in this case the reactions go to a different product than porphyrazine. After reaction a mixture of many different compounds was obtained, probably products of polymerization of dinitriles. So finally it was decided to prepare porphyrazine consisting of 1 unit 2 or 3. Dinitrile number 1 was selected, because finally it would be the unit, which could be reacted with its carboxylic acid group with BSH. To avoid problems with further purification of the boronated porphyrazine it was decided to prepare the porphyrazine only with two BSH clusters. The condition of cyclization should be chosen to get finally the porphyrazine showed on the picture (fig. 45).

N

NH

N

NN

HN

N

N

RR

R

R

R R

S

S

S

S

O

O

B12H11

B12H11

Figure 45. Boronated porphyrazine obtained in cross macrocyclization. To get this isomer in the best possible yield it was necessary to make a series of experiments with different dinitrile ratios to find the optimal molar ratio between the dinitriles 1 and 2. In the first experiment both dinitriles were reacted in a molar ratio 1:1 and the molar ratio between dinitriles and metallic magnesium was 2:3. After reaction by TLC chromatography a mixture of two different porphyrazines and other by-products was found. Further investigation by H1 NMR showed that one of the product was a porphyrazine consisting only of dinitrile 2. The H1 NMR of the second compound got as 10 % of whole mixture mass shows both side ring from dinitrile 1 and 2 however the mass spectra shows no molecular ion from any possible cross isomers from

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cyclization of these two dinitriles. The probably reason of the unsuccessful experiment was the difference in activity of these two dinitriles. Only a porphyrazine with one type of the side chain was found. Formation of this isomer as only one reaction product could prove difference in activity of them. Dinitrile number 2 is probably more active and for this reason the porphyrazine with side chain of this dinitriles is formed. In the next experiment the molar ratio between dinitriles was changed to value 4:1 and the molar ratio between the dinitriles and magnesium was kept on the same level 2:3. to resolve this problem. The mixture was obtained as a result of this experiment. The simple TLC tests gave similar results as the previous experiment. The mixture consisted of two blue compounds probably porphyrazines and other by-products. Porphyrazine products have comparable RF factors as in previous experiments and other analyses H1 NMR and mass spectroscopy gave also similar results. One of component of the porphyrazine mixture is the prophyrazine with side chain only from dinitrile number 2. The second porphyrazine component got also in 10% shows in H1 NMR signals belongs to both side chain of used dinitriles, however mass spectroscopy shows no molecular ion from any of the isomers which could be formed in the cross cyclization. Other experiments in which molar ratio of the dinitriles was changed to value 1:6 and molar ratio between dinitriles and magnesium was changed to value 1:1. This brought completely the same results. This formation of only one porphyrazine compound could have sterical reason. The side chain of dinitrile 2 is shorter than dinitrile 1. This could causes that during formation of cyclic porphyrazine the bulky dinitrile 2 in this basic condition in presence of magnesium propoxide first takes part in transesterification reaction and it is to bulky to join the cyclization reaction center. The much smaller dinitrile 1 can join the reaction center faster because of its size; formation of its porphyrazine derivative is preferable. In the reaction mixture no porphyrazine made only from number 1 was found what could prove that another reason of failed cross-cyclization is a decomposition of this dinitrile in the condition used in the experiment. However another cross-cyclization experiment was made. From this sterical reasons as a second co-cyclization agent was chosen dinitrile 3 and 1. This dinitrile has the bigger side chain. In comparison with dinitrile 2 this compounds has a side chain longer by one methylene unit what makes it a little bit more bulky than dinitrile used before and this gives a hope that dinitrile with a longer side chain would eliminate the sterical effect of former macro-cyclization and finally cross-cyclization of two different dinitriles would be possible.

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In a first experiment a stoichiometric ratio of dinitriles according to expected product was used (fig. 45). The porphyrazine obtained after reaction should consist of three units of dinitrile 3 and one unit of dinitrile 1, so the ratio 3:1 between dinitriles was used and the ratio between dinitriles and magnesium was 2:3. As result of simple TLC test the reaction was obtained a mixture of three blue compounds and other by-products of the reaction. Separation of the products was carried out by chromatography column with an eluant system dichloromethan (CH2Cl2) and methanol (CH3OH) in volume ratio 4:1. The separated two products were analyzed by standard analytical methods like 1H NMR and mass spectroscopy. The blue material eluted first from chromatography column shows in 1H NMR signals typical for cyclization product consisted only from side chains of 1 and mass spectroscopy confirmed that this structure was a porphyrazine only with one sort of side chain. The second eluted compounds shows in 1H NMR signals from both of used dinitriles however the integration of the both types of signals indicates that the ratio between them is different than expected 3:1, the ratio was about 1:1. However the further analyses like mass spectroscopy show no molecular ion signal of any possible isomer of macro-cyclization. The explanation could be that the obtained sample is still a mixture of other some porphyrazine derivatives with unown structure. Finally the porphyrazine compound, which was eluted, as third was examined also with 1H NMR technique and mass spectroscopy. This test indicates the signals and molecular ion from porphyrazine derivative obtained from cyclization of dinitrile 1. As a summary it could be said that in comparison with former experiment with dinitriles with shorter side chain in case of dinitriles with longer chain in ratio 3:1 the only difference is that in this case the porphyrazine derivative with only dinitrile 1 is formed. This compound was not formed in former experiments. The conclusion could be that in these conditions cyclization of both dinitriles is possible and to obtain cross-cyclization products is only matter of the right ratio between both dinitriles and dinitriles and magnesium. However in the cycle of experiments no cross cyclization isomers were obtained. 9.6 Boronated porphyrazine prepared via Schiff base from aldehyde derivative of NMT. The reactions between aldehydes and ammoniaundecahydro-closo-dodecaborate (BNH3) with formation of Schiff base are known (Sivaev 1999). Sivaev and co-workers prepared a number of Schiff bases from BNH3 and then they reduced them to secondary amines as a new route for preparation of BNCT agents. We decided to use this synthetic route to synthesize boron containing porphyrazines with this boron cluster connected to the porphyrazine system. The synthesis was planned as in figure 44.

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S-NC

NC S- Na+

Na+

Br

O

O

SNC

NC S

O

O

O

O

Et

Et

Et

Et[H+]

SNC

NC S

O

O

SNC

NC S

O

O

NH3

2

SNC

NC S

N

N

SNC

NC S

NH

NH

cyclization to porphyrazine

Figure 46. Syntheses route through dinitrile aldehyde derivative. It was planned to make an aldehyde derivative of the dinitrile. The first step of the reaction route should be a reaction between MNT sodium salt and bromoacetaldehyde or chloroacetaldehyde. But because of the strong nucleophilic abilities and basic character of NMT sodium salt it was necessary to use a protection group to protect the aldehyde group. Protons connected to α-carbon of the aldehyde can migrate in presence of base from carbon to base and on the α−carbon the carboanion is formed. The carboanion is a very reactive chemical unit and can react with carbonyl groups of other aldehydes molecules. This reaction is called aldol condensation reaction and could lead to unwanted reaction products. It is also known that aldehydes can react with alcohols to produce so called acetals and it was decided to use an acetal derivative of chloro

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ethyl aldehyde. Reaction between Na NMT and chlorethyldiethylacetal was made in conditions typical for such experiment, with dry acetone as solvent and in presence of catalytic amount of NaI. This reaction gives two main products, mono- and di- substituted derivatives of Na MNT. After purification on chromatography column the main product was obtained with yield of about 70% as viscous liquid substance. The next step of the reaction was the deprotection of the aldehyde group. The acetal derivative was dissolved in water/ethanol mixture and the aqueous solution of the HCl was added as a catalyst of this reaction. The ethanol was evaporated and an aqueous solution of NaOH was added to neutralize the solution. In the hydrolyzation reaction the pH was raised only to value of about 6 and product was extracted from water by another organic solvent. The product was obtained as solid substance in good yield. The next step of syntheses route was a reaction with BNH3 to get a Schiff base then to reduce it to the amine system. The BNH3 is a boron cluster compound with amino ligand. The specific structure of this compound makes the amino ligand a strong nucleophilic center. The doubly negatively charged boron cage causes that electron density on the nitrogen atom of the amino groups higher and the lone electron pair of the nitrogen atom is a strong nucleophilic center. Actually the electron density is so large that the amino group of the BNH3 exists only in its protonated form as –NH3

+ group (fig. 45) and the whole boron cluster is totally only one time negatively charged.

N

HH

..e

H+

N

HH

H

+

-2 -1

-

Figure 47. Protonation of lone electron pair of BNH3 of the amino group. Such protonated amino group is completely deactivated as a nucleophilic agent. For this reason it is necessary to first activate this amino group before the reaction with BNH3 as a nucleophilic agent. It is necessary to add to the reaction environment a base to take over the proton from the deactivated amino group to activate this group. Formation of the Schiff base is a reaction in with the

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nucleophilic amino group attacks the carbonyl group of the aldehyde. In case of the reaction between the prepared aldehyde and the boron cluster BNH3 it is necessary to use a very strong base, sodium hydride NaH. It is necessary to provide anhydrous condition to work with sodium hydride because sodium hydride can react rapidly with water. The reaction was carried out in two steps. First BNH3 was dissolved in dry acetonitrile and reacted with sodium hydride. At this step some amount of hydrogen gas is evolved. After the evolution of H2 stopped in second step former prepared aldehyde was added to reaction mixture. After half an hour of stirring the reaction solvent was removed and the obtained mixture was analyzed. Only BNH3 material in its protonated form and some polymeric material were found in the mixture, but no product was found in reaction mixture. BNH3 becomes an extremely strong nucleophile after removing of the additional proton, which could react with α-carbon protons and as well as in the case of reaction with NaOH described earlier. It could form the carboanion in reaction with aldehyde. The carboanion is a very reactive chemical element and its presence in reaction environment could lead to polymerization of the second aldehyde co-reactant. So for this reason the reaction is not possible to be done. A similar reaction of formation of the Schiff base between BNH3 and an aliphatic aldehyde was never reported. Only the reaction between the BNH3 and the aromatic aldehydes are known (Sivaev 1999). It is not possible to resolve this problem by using of a protection group. For a acidity of the α-carbon’s protons have influence the carboxyl group of the aldehyde. So increasing of the carbon chain length could protect the -CH2- from influence of the carboxylic group and decries the acidity of the -CH2- enough to react it with BNH3 in its deprotonated form without formation of the carboanion and polymerization reaction. However further investigations haven’t been done and this hypothesis hasn’t been proofed. However no reaction between a aliphatic aldehyde and BNH3 has been found in the literature. The reaction between the aromatic aldehyde (Sivaev 1999) and BNH3 has been described and probably it is not possible to be done with the aliphatic systems.

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9.7 Boronated porphyrazine prepared from the cyclic derivative of dinitrile. Reactions are known in which one compound with two thio functions can react with an other bis functional compound to finally form cyclic derivatives (Corey 1971) (Musker 1983). In this reaction two thio functions react not with molecule that consist of two halogen atoms, but with different species. Corey and Musker used 2-(chloromethyl)oxirane to obtain this cyclic derivative of 1,3-propanedithiol (fig. 48).

SH

SHO

ClS

SO-NaOH

EtOH

Figure 48. Reaction of dithiol with 2-(chloromethoxy)oxirane. In this reaction two nucleophilic sulfur atoms react in the presence of NaOH with 2-(chloromethoxy)oxirane. In this reaction the oxirane ring is opened and the cyclic derivatives is produced. The same reaction should be possible if Na MNT instead of 1,3-propanedithiol is used. After reaction a cyclic seven membered ring should be produced with the hydroxyl group in the central position of the ring (fig. 49). This hydroxyl group could be later converted into a carbonyl group (Piantadosi 1976). This carbonyl group could react with the amino group of BNH3 to form the double bond between the carbon atom and the nitrogen atom, which then could be reduced to the amine system and a boronated dinitrile could be obtained. The prepared dinitriles could be used as a macro cyclization precursor and boronated porphyrazines could be obtained. However from the same reasons as in paragraph 9.6 the formation of the Schiff base is not possible. The cyclic derivative of NMT can be formed in this reaction only if one molecule of the NMT will react with only one molecule of the chloromethyloxirane. In the reaction mixture is not possible to avoid of reaction with other molecular ratio which can lead to polymerization instead of cyclization. In this reaction one molecule of MNT reacts with molecule, which has two different functional groups, chlorine atom and the second epoxide ring. A few different processes during the reaction are possible. The first of them is a nucleophilic attack of the sulfur atom on the oxirane ring. The result is that the ring is opened and Na MNT is substituted (fig. 50). In the next step of this reaction the formed derivative in presence of base in can be transferred back in

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to the oxarine ring. The attack of the second nucleophilic sulfur atom on the oxarine ring is possible at this step and cyclic derivative of is formed.

S

SNC

NC

OH

S-NC

NC S- Na+

Na+

O

Cl

S

SNC

NC

O

S

S CN

CN

HN

S

S CN

CN

H2N

cyclization to porphyrazine Figure 49. Synthesis of boronated porphyrazine via cyclic derivatives of NMT. However this process could take place not only between the one molecule of NaMNT and one molecule of 2-(chloromethoxy)oxirane. The NaMNT substituted with the oxirane ring can react as well with the another NaMNT molecule. This finally leads to the polymerization

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To avoid this phenomenon the components of the reaction was added into the reaction environment in large dilution to restrict the contact possibility of NMT molecules with more than one molecule. 2-(chloromethoxy)oxirane was also added to the solution of the Na MNT from the dropping funnel over long time. Sodium hydroxide as a base and dry ethanol as a reaction solvent were used to increase the nucleophilicity of Na MNT. To increase the speed of the reaction, it was carried out at elevated temperature. However the reaction was not successful. After the reaction no traces of starting materials, Na MNT and 2-(chloromethoxy)oxirane, were found in the mixture. The mixture consisted of many different compounds of polymeric character. The reaction was repeated at higher temperature, with faster mixing and with increasing the time of (2-chloromethyl)oxirane addition to three hours. Additionally both of the reactants were added to the reaction solvent separately. These experiments were not successful. The most probably reason is that instead of the cyclization reaction, the polymerization reaction is preferred. All transition product described former can also react with each other. This process leads to the polymeric product of the reaction

SNC

NC S

O

Cl

S CN

CN-S

SNC

NC S-

O

Cl

OH

OH-

Figure 50. Probable transition compounds in reaction of the Na NMT and (2-chloromethoxy)oxirane.

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9.8 Synthesis of the porphyrazine via the dihydroxyacetone Na MNT derivative. Because of the unsuccessful experiment described in paragraph 9.7 in preparation of cyclic seven-membered ring compound as a precursor of boronated porphyrazine formation it was decided to prepare a seven-member ring in a different synthetic way.

O

OHHOCH3S

O

O

Cl

O

OOTsTs

S-NC

NC S- Na+

Na+

S

SNC

NC

O

S

SNC

NC

N

cluster

S

SNC

NC

NH

cluster

cyclization Figure 51. Synthetic route through the tosylated dihydroxyacetone. As starting material dihydroxyacetone was chosen. This symmetric diol compound should be able to react with tosyl chloride to form its ditosylated derivative. This activated diol would be able to react with nucleophilic agent on its α carbon to the carboxylic group. Na NMT is a nucleophilic agent that can react with the tosylated dihydroxyacetone to finally form seven-membered cyclic NMT derivatives with carbonyl group in the central position of the ring.

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The reaction of carboxyl group with the amino group of the ammoniaundecahydro-closo-dodecaborate is known to form the Schiff base, which later can be reduced to the amino derivative (Sivaev 1999). However this reaction is known only with aromatic systems. This dinitrile derivative could be cyclized to the porphyrazine system (fig. 51). The first step of reaction was made according to the literature reaction of 1,3-propandiol with tosylchloride (Graeme 1984). The diol is added to a cooled solution of tosyl chloride in dry pyridine. After two hours the whole reaction mixture is poured into ice water to precipitate the product and then to isolate the tosylated diol from the precipitate (Hoffman 1995). The same procedure was used in the reaction of dihydroxyacetone with tosylchloride. Tosylchloride was dissolved in dry pyridine. To the mixture dihydroxyacetone was added drop wise in solution. The whole mixture was stirred at about 0°C for three hours. After this time the mixture was poured over ice water to precipitate the product. At this step the white transparent mixture turns into a brown red solution. Further analytical investigation proves that the obtained brown red mixture contains mostly pyridine and some other hard to analyze polymeric compounds. The next trial was made in dry dichloromethane as reaction solvent. Excess of the tosyl chloride and amount of pyridine necessary only for reaction with tosyl chloride was dissolved in dry dichloromethane. Next the dihydroxyacetone was added dropwise to the reaction mixture. Also in this case no tosylation product was obtained. In further experiments instead of pyridine other organic amines were used like tri-ethylamine diethyl amine and 1,6-lutidine. However the product of tosylation was not obtained. The conclusion is that α protons are to sensitive for basic agents but without basic agent the tosylation reaction cannot be made. 9.9 Syntheses of porphyrazines with diaminomaleodinitrile (DAMN) as a precursor. Diaminomaleodinitrile (DAMN) is a molecule similar to Na NMT. It consists of two cyano groups, which later would be functional group to form the tetra cycle pyrrole system of porphyrazine. Some porphyrazines have already been made (Hoffman 1997) (Hoffman 2000). The main difference is that instead of the two thio groups it has two amino groups. The macrocyclization process is the as in the case of Na MNT but because of the different chemical character of the amino group it is necessary to take a completely different strategy to attach side chain to this molecule. Each amino group of the DAMN can make two bonds with others chemical systems. To introduce to the molecule only one side chain on each nitrogen atom it is necessary to use a special technique (see paragraph 5.2 (fig. 24). This technique allows also to introduce to the DAMN molecule two completely different side chain (see fig. 52). It was decided to attach the boron cluster compound to the dinitrile structure through a benzene ring. The

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benzene ring selected to this synthetic attempt should allow to be reacted with DAMN and with one of the chosen boron cluster compounds. For this reason two functional benzyl ring derivatives were selected. First is 4-carboxybenzaldehyde. This compound has two different functional groups. One of them, the aldehyde should react with one amino group of the DAMN structure to form a Schiff base, which could be later reduced to the more stable amino structure. On the other side the carboxyl acid group through its acid chloride derivative can be reacted with boron cluster compound with thio or amino ligand to finally form a boronated macro-cyclization precursor. A group of few difunctional compounds was selected to make a linker between DAMN and boron cluster compound.

NH2NC

NC NH2

RO

H

NNC

NC NH2

R

H

NHNC

NC NH2

R

H

NHNC

NC NH

R

R'

R'= RX

RO

X

O

R

Figure 52. Introduction of two side chains into DAMN structure technique. However no success was achieved with DAMN using this synthetic method. The most probable reason is that the mono alkylated amino of DAMN derivative is not able to cyclize. In the literature were described cyclization of DAMN derivative with disubstituted amino group (Hoffman 1997).

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9.10 Syntheses of DAMN derivatives as macrocyclization precursors. It is necessary to use a linker to connect DAMN with boron cluster compound because direct connection between DAMN and any of boron cluster compounds is not possible. Such linker should be a difunctional compound. To attach it to the amino group of DAMN the best possibilities gives an aldehyde function which reacts with the amino group of DAMN to form a Schiff base which can be reduced to the amino derivatives of the DAMN. A few difunctional aldehyde derivatives of different chemical structure were selected. The aldehyde group was chosen as a second function, which allows to attach boron cluster compound with amino ligand by formation of the Schiff base (Sivaev 1999) and as well with carboxyl group, which allows to attach a boron cluster compound with thio ligand. For this purposes the following compounds were chosen: glyoxylic acid, terephthaloaldehyde, terephthalic acid. Here also no success was achieved, because monosubstituted derivatives of DAMN were used to the cyclization reaction. Until mow only the aimno disubstituted derivatives of DAMN were cyclized. The obtained compounds should be first reduced to singel bond C-N, then the amino group of DAMN should be substtituted with a methyl group to obtain disubstituted derivative of DAMN and cyclized. Glyoxylic acid was chosen as a first linker to connect DAMN with boron cluster compounds. This simple difunctional compound consists of an aldehyde group and a carboxylic group. The aldehyde group can easily react with the amino group of the DAMN to form a Schiff base, which could be later reduced to the amine. From the other side glyoxylic acid has a carboxylic group which is able to react with the amino or the thio derivative of the B12H11 boron cluster compound to form finally the boronated building block for cyclization to the porphyrazine system. The reaction was made in methanol as solvent. Pyridine was added to the reaction mixture to activate the amino group of the DAMN compound. The reaction yielded white a crystal product which was analyzed as a completely type of structure than planned. The obtained product appears to be a heterocyclic ring system (fig. 53)

NH2NC

NH2NC

O

O

OH

base

NC

NC N

N O

Figure 53. Formation of the hetero cyclic derivatives of the DAMN. It seems to be that not only the aldehyde group is able to react with the amino group to form the Schiff base but the second functional group, the carboxylic

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acid group is able to react with the second amino group of DAMN to finally form a cyclic structure as derivative of DAMN. To avoid formation of the cyclic derivative it is necessary to protect one of the DAMN’s amino group against reaction with the second carboxylic group of glyoxylic acid. It is known in literature that DAMN can be acetylated in two ways (Hinkel 1973). According to work of Hinkel and co-workers the amino groups of DAMN can be monoacetylated or diacetylated. This acetylation reaction can be easily controlled by condition used in the synthesis. Only the monoacetylated derivative is formed at slightly elevated temperature and with acetic anhydride as a co-reactant in solution. The increased temperature and use of acetic anhydride without any solvent in short reaction time leads to the di-acetylated derivative of the DAMN. The monoacetylated derivative of DAMN was made in milder conditions. This still contains the free amino group, which can be reacted with the aldehyde group of the glyoxylic acid to form the Schiff base. This was made in methanolic solution with base as a catalyst. The mixture was obtained after reaction. TLC tests proved that this mixture consists of two different compounds. After isolation process a white crystallic substance was obtained. However instrumental analysis did not confirm the expected substances. 1H and 13C NMR spectra confirm the structure but mass spectrum showed no molecular ion but some other ions. However the right structure of the obtained compound was not found. Until now only aliphatic systems were used as a linker between DAMN and boron clusters but also aromatic system could be used as linker to form boronated dinitrile precursors for cyclization. For this purposes the two following difunctional systems were chosen. To anchor the aromatic system on amino group of DAMN it is necessary to have an aromatic system with the aldehyde group. The aldehyde group described already in former paragraphs reacts easily with the amino group of DAMN to form a Schiff base. For this reason one of the function of the aromatic system has to be aldehyde function. An aldehyde or carboxylic acid function can be chosen as a second function. This second function connects the boron cluster compounds with the rest of the cyclization precursor. There were two different boron clusters available with –NH3 and –SH ligands. To attach the boron cluster with the –SH ligand to the rest of the system itr is necessary to react it with activated carboxylic acid group. The carboxylic acid group has first to be activated by transferring it into its chloride derivative by reaction with some chlorinating agents like oxalyl chloride or thionyl chloride. Such activated carbonyl group is reactive enough to form a bond with the thiol group of BSH or the amino group of BNH3. However in both cases it is necessary to use some amine as a catalyst of the process. BSH cluster forms thioester bond between a carboxyl group of the dinitrile system

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and thiol group of the BSH as second product of this reaction is obtained base hydrochloride. Similar situation is with the reaction between activated carboxylic acid and BNH3. As it was described in paragraph 4.4. the amino group of the BNH3 boron cluster exists in its protonated form. The lone electron pair of amino group can attack the carbonyl group, but in case of BNH3 this lone electron pair is deactivated by coordination bond with a proton. For this reason it’s necessary to use some base to remove the proton from nitrogen atom and activate the amino group again to make an active reactant in reaction with carboxylic acid chloride. A difunctional derivative of the benzene with aldehyde and carboxylic acid groups was selected for this reason. In case of the BNH3 boron cluster reaction between the amino group and the aldehyde group with formation of Schiff base is also possible. In this case it possible to use a benzene derivative with two aldehyde functions as a linker. Of course there are many different isomers of such difunctional benzene derivatives. It was decided in each case to use para isomers. In difunctional para isomers these two functional groups are at the largest possible distance to each other. In case of other isomers these distances are much shorter and make the cyclization precursor bulky, which could later disturb the cyclization process as described in paragraph 9.2 (fig. 40). Reaction between the DAMN and corresponding difunctional benzene derivatives was made to check if the Schiff base formation reaction is possible with DAMN (fig. 54). Cyclization was tried with both DAMN derivatives. However none of them formed a porphyrazine system, only brown polymeric material. The reason of polymers formation instead of the porphyrazine system could be that the second amino group of DAMN is not substituted.

NH2NC

NC NH2

NH2NC

NC NH2

O

H O

H

O

H O

OH

NNC

NC NH2

O

H

NNC

NC NH2

O

OH

Figure 54. Formation of the aromatic derivative of the DAMN.

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The amino group could react with magnesium propoxide, which is used as co-reactant in synthesis of porphyrazine. From this reason it was decided to protect the second free amino group of the DAMN. The acetyl group was selected as a protection group. It was tried to acetylate the free amino group of the mono substituted DAMN derivative. However this reaction did not work for unknown reasons. Because of this problem it was decided to first prepare the mono acetylated DAMN derivative and then make a reaction with an aldehyde to prepare a Schiff base. Monoacetylation of the DAMN was carried out in mild condition with 1,4-dioxane as a reaction solvent at 50°C. The monoacetylated DAMN derivative after purification was obtained in good yield. This derivative was then used for reaction with an aldehyde (fig. 55).

NC

NH N

CN

O

NC

NH N

CN

O

CHO

Figure 55. Formation of the acetylated derivative of the Schiff base. The reaction was made in the same conditions as before. As reaction solvent methanol was used and the mixture was stirred at room temperature in two hours. Then the reaction solvent was evaporated and the residue was dissolved in acetone and some activated carbon was added to decolorize the mixture. Then the reaction mixture was filtered to get rid of the activated carbon. Next acetone was evaporated and the obtained solid residue was re-crystallized from hot carbon tetrachloride. The obtained white crystalline material was analyzed by 1H NMR, 13C NMR, IR and mass spectroscopy. The analyses fully confirmed the structure of the compounds (fig.55). So this compound was transformed to the porphyrazine system, but unfortunately this reaction failed. The probably reason of the negative results is the same as during the experiment described in paragraph 9.2 (fig. 40). The aromatic ring is a too bulky group to allow cyclization of the dinitrile system to the porphyrazine system. The same situation was with synthesis with acid derivative. Its second amino group was protected with acetyl group and transferred it in porphyrazine derivative. In this case this attempt also failed. Additionally terephthalaldehyde was reacted with the BNH3 to form Schiff base, which can later be reduced to amino system and such derivative is still able to react with amino group of the DAMN (fig. 56).

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NH3

CHO

O H

NH

CHO

NH2H2N

CN

CN

HN

1. condensation2. reduction

1. condensation2. reduction

Figure 56. Formation of the linker between boron cluster and DAMN with terephthalaldehyde. 10 HPLC purification of boronated porphyrazine. The reaction mixture obtained in paragraph 9.2 could not be purified by the standard methods. TLC test showed that mixture after reaction contains of not reacted BSH as its TMA salt and very polar blue rest. TLC test showed also that the blue residue has a different polarity than the starting porphyrazine acid derivative. Some problems with isolation of potential reaction product appeared at this step. The mixture was soluble in organic solvents such as acetonitrile, DMF, DMSO. Also water appears to be a good solvent for this mixture. However a suitable crystallization solvent system was not found. Even exchange of the counter ion to TBA gave no results in the purification of the obtained mixture. It was decided to make an attempt of purification by column chromatography. However it was not possible to move the blue component of the mixture absorbed on silica gel. Only the excess of BSH was found in the eluant. No solvent could remove the rest of the absorbed material, even attempt of desorption in polar solvents like methanol, water and solvents like DMSO and DMF gave no results. Finally it was decided to purify the mixture on silica gel with reversed phase RP-18 with the HPLC system to separate the mixture of the obtained compounds. First the experiments on the analytical column were

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carried out to find the right condition for separation. The UV spectrum of mixture was recorded to find right observation wavelength of the porphyrazine for HPLC experiments. The obtained UV spectra showed that maximum absorbance is at 640 nm and this wavelength was chosen as observation wavelength during the analytical HPLC experiments.

Figure 57. Chromatogram of reaction mixture with graduated eluant system at 640 nm wavelength. The next step was to find a right eluant to separate the mixture. The measurement was carried out on HPLC system Hitachi L6200 that allows to make experiments with gradient of eluant mixture. Water and acetonitrile were chosen as an eluants system, TBA hydrogen sulfate 10 mmol cation was added to the eluant ph=5,5. The eluant program was as follows: at the beginning 100 % of pure acetonitrile for 10 minutes, then the amount of water in eluant was increased after ten minutes to finally reach 100 % after 1 hour. The chromatogram made at wavelength 640 nm shows two main peaks with retention time rt1=24,8 min. and rt2=31,3 min (fig. 57). It was latter calculated, that substance corresponding to the peak with retention time rt1 flows out from column with eluant ratio acetonitrile : H2O 53:47, and the substance corresponding to peak with retention time rt2 flows out with eluant ratio acetonitrile : H2O 75:25. Because the measurements were made at wavelength 640 nm both peaks should correspond to porphyrazine derivatives. Finally it was decided to use as separation eluant acetonitrile : H2O 52:48 mixture. One more measurement at 220 nm was made at this eluant ratio to observe, if there is still unreacted BSH which should be eluted with retention time rtBSH=1,5 min. (the

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value was taken from earlier HPLC experiment) and experimentally it was proven that BSH is still present in the reaction mixture at is eluted from column with retention time rt=1,54.

Figure 58. Chromatogram from separation experiment eluant system MeCN : MeOH 52:48 at 640 nm wavelength. The next experiment with the same eluant system but at 640 nm shows that the next two peaks are eluted with much longer retention time which allows to separate this two substances from unreacted BSH. Column with gel RP-18 eluant system MeCN:H2O 52:48 was used for separation. Two fractions with retention time rt1=14,9 min and rt2=54,4 min were obtained from this experiment (fig. 58). The both fraction were collected directly after analystator to the different glasses. These two main fractions were then later separated from TMA sulfate salt and analyzed by IR spectroscopy. Both of the spectra show the peak typical for boron cluster compounds at 2500 cm-1 which proves that both compounds are boron containing compounds. Additionally spectra from peak number one show peak at 1660 cm-1 typical for carbonyl group what could prove that the obtained compound has a thio ester bond which corresponds to planned structure. However mass spectroscopy detects no product in both sample.

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11 Experimental section. 11.1 General consideration. All reagents were obtained from Aldrich Chemical Co., Acros Chemicals and Roth chemicals and used without further purification. Dry solvents ethanol, propanol, butanol, acetonitile, tetrahydrofuran, dichloromethane were first distilled and then dried with molecular sieves 4A and 3A. All solvents used in column chromatography were first distilled. Na MNT was prepared by a modification of the literature method (Davision 1967). For column chromatography silica gel Normasil 60A 40-63 µm produced by VWR company was used. All TLC experiments were made on TLC plates Polygram Sil G/UV254 produced by Macherey-Nagel company. All 1H NMR and 13C NMR spectra were recorded on a Bruker DPX 200 spectrometer. IR spectra were recorded on a Bio Rad FTS 155 spectrometer. All EI spectra were recorded on a MAT 8200 and MAT 95. All ESI spectra were recorded on an Esquire spectrometer. Analytical and preparative HPLC experiments were made on a Merck-Hitachi L-6200 system with a column filled with reversed phase gel RP-18 produced by Merck Hilbar pre packed column diameter 250-25. The flow rate was 3.

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11.2 Preparation of sodium cis-1,2-dicyano-1,2-ethylenedithiolate (Davison 1967).

NaCN+

CS2

DMF

NCCS2Na*3HCON(CH3)2

NC S-

S-NC Na+

Na+

6DMF 2S

NCCS2Na*3HCON(CH3)2

H2O

Procedure: Dry, powdered sodium cyanide 29g (0.60 mol) and 180 ml of dimethylformamide (DMF) were placed in a 1L three necked round bottom round-bottom flask equipped with reflux condenser, dropping funnel and Hirshberg stirrer. The flask was placed in a water bath to provide external cooling. Carbon disulfide 36,2 ml was added dropwise to the stirred suspension over 10 minutes period. A red brown solid substance is produced at this step. The mixture was vigorously stirred for 30 min. Then stirring was stopped and 500 ml. of isobutyl alcohol was added to the mixture and heated. The solution was filtered hot to remove of unreacted material. The filtrate was cooled on ice salt bath. The product crystallized and was collected by filtration and washed with ethyl ether until the washing were pale yellow. This product was dissolved in 1000 ml of water. After 24 hours the yellow solution was filtered to remove sulfur, and the filtrate was evaporated to dryness by rotary evaporator. The obtained solid was dissolved in the minimum of boiling ethanol and filtered to remove insoluble material. The filtrate was cooled down and anhydrous diethyl ether was added (250ml). The product crystallized on cooling in ice bath. Crystals were filtered and once more recrystallized from ethanol. Yield: 26g; 48% (lit. 48%) 13C NMR (D2O): δ 122,7 (C=C); δ 126,3 (-CN) IR: 2206 (-CN); 1676 (C=C); 1627; 1506; 1371; 1302; 1246; 1044; 770; 569; 454 MS (ESI): [A]-2=70, [K]+= 23 Melting point: >250°C (lit. no data)

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11.3 Preparation of1,2-dicyano-1,2-bis(2-methoxy-2-oxo-1-ethyl)thio ethylene.

NC S-

S-NC Na+

Na+O

OBr CH3

NaIMethanol

NC

SNC

O

OS CH3

O

O CH3

Procedure: A two necked round bottom flask equipped with reflux condenser was filled with 70 ml of dry methanol, 3g (16 mmol) sodium cis-1,2-dicyano-1,2-ethylenedithiolate, 2,8g (1,75 ml, 35 mmol) methyl bromoacetate and 0,3g (2 mmol) sodium iodide. The mixture was refluxed in nitrogen atmosphere overnight. Then the solvent was evaporated and the residue was dissolved in 40 ml of CH2Cl2 and washed with a large amount of water to remove water soluble material. Next the CH2Cl2 solution was dried with anhydrous sodium sulfate, filtered and the solvent was evaporated. The residue was dissolved in a minimum amount of CH2Cl2. N-hexane was added to the solution to crystallize the product. The product crystallized on cold as white needles in a few hours. The product was filtered and dried for few hours in high vacuum. Yield: 2,1 g; 46% 1H NMR (CDCl3): δ 3,83 (s, 3H, -CH3); δ 3,93 (s, 2H, -CH2-) 13C NMR (CDCl3): δ 36,2 (-CH2-); δ 53,8 (-CH3); δ 112,0 (C=C); δ 122,0 (-CN); δ 167,7 (-CO-) IR: 3014 (C-H); 2931(C-H); 2215 (-CN); 1732 ( -CO-); 1509 (C=C); 1433; 1383; 1314; 1156; 986; 881; 767; 689; 572; 500 MS (EI, 70 eV): [M+] 286 Melting point: 65°C

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11.4 Preparation of 1,2-dicyano-1,2-bis(3-methoxy-3-oxo-1-propyl)thio ethylene.

NC S-

S-NC Na+

Na+

NaIAceton

NC

NC

S(CH2)2

O

O

S(CH2)2

O

OBr

(CH2)2

O

O

Procedure: A two necked round bottom flask equipped with a reflux condenser was filled with 70 ml of dry acetone, 3g (16 mmol) sodium cis-1,2-dicyano-1,2-ethylenedithiolate, 3,75g (2,4ml, 32 mmol) methyl 3-bromopropionate and 0,3g (2 mmol) sodium iodide. The mixture was refluxed in nitrogen atmosphere overnight. The reaction mixture was filtered to remove inorganic salt. The filtrate was rotary evaporated. The residue, yellow oil was dissolved in 40 ml of CH2Cl2 and washed with the large amount of water to remove water soluble material. Next the solution was dried with anhydrous sodium sulfate, filtered and CH2Cl2 was rotary evaporated. The obtained oil was separated by column chromatography (eluant CH2Cl2: MeOHl 9:1). Pale yellow oil it was obtained. Yield: 2,78 g; 55% 1H NMR (CDCl3): δ 2,78 (t J=7 Hz, 2H, -CH2-CO-); δ 3,39 (t J=7 Hz, 2H, -S-CH2-); δ 3,74 (s, 3H, -CH3) 13C NMR (CDCl3): δ 30,3 (-S-CH2-); δ 34,7 (-CH2-CO-); δ 52,6 (-CH3); δ 112,3 (C=C); δ 121,6 (-CN); δ 171,2 (-CO-) IR: 3004; 2956; 2213 (-CN); 1747 (-CO-); 1503; 1438; 1367; 1175; 1017; 979; 848; 824; 671; 508 MS (EI 70 eV): [M+] 314

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11.5 Preparation of 1,2-dicyano-1,2-bis(4-methoxy-4-oxo-1-butyl)thio ethylene (Hoffman 2001).

NC S-

S-NC Na+

Na+

NaIAceton

NC

NC

S(CH2)3

O

O

S(CH2)3

O

OCl

(CH2)3

O

O

Procedure: A two necked round bottom flask equipped with reflux condenser was filled with 70 ml of dry acetone, 3g (16 mmol) sodium cis-1,2-dicyano-1,2-ethylenedithiolate, 4,3g (3,8ml, 32 mmol) methyl 4-chlorobutanoate and 0,3g (2 mmol) sodium iodide. The mixture was refluxed in nitrogen atmosphere overnight. The reaction mixture was filtered to remove inorganic salt. The filtrate was rotary evaporated. The residue, yellow oil was dissolved in 40 ml of CH2Cl2 and washed with large amount of water to remove water soluble material. Next the solution was dried with anhydrous sodium sulfate, filtered and CH2Cl2 was rotary evaporated. The obtained oil was separated by column chromatography (eluant CH2Cl2). Pale yellow oil was obtained. Yield: 3,9 g; 72% (lit 65%) 1H NMR (CDCl3): δ 2,08 (q, J=7Hz, 4H,-CH2-CH2-CH2-); δ 2,49 (t, J=7Hz, 4H,-CH2-CO-); δ 3,20 (t, J=7Hz, 4H, -S-CH2-); δ 3,70 (s, 6H, -CH3) 13C NMR (CDCl3): δ 25,5 (-CH2-CH2-CH2-); δ 32,5 (-CH2-CO-); δ 34,5 (-S-CH2-); δ 52,2 (-CH3); δ 112,3 (C=C); δ 121,6 (-CN); δ 172,9 (-CO-) IR (KBr): 3001 (C-H); 2785; 2215 (-CN); 1735 (-CO-); 1529; 1263; 1008; 754; 688; 628; 528 MS (EI 70 eV): [M+] 342

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11.6 Preparation of 2,3,7,8,12,13,17,18-octakis[4-propyloxy-4-oxo-1-butyl)thio]-21H,23H-porphyrazine.

N

NH

N

N

N

HN

N

N

R

R

R

RR

R

R

R

PrOMgPrOH

R= SO

Pr

O

NC

NC

S(CH2)3

O

O

S(CH2)3

O

O

Procedure: 0,56 g (23 mmol) of magnesium turnings and 0,1g of iodine were placed in dry propanol and heated under reflux in nitrogen atmosphere overnight. 2g (5,8 mmol) of 1,2-dicyano-1,2-bis(4-methoxy-4-oxo-1-butyl)thio ethylene was dissolved in 20 ml of dry propanol and the mixture was added to the suspension and refluxed for 8 hours. The mixture was cooled down and the solvent was rotary evaporated. The residue was dissolved in 40 ml of CH2Cl2 and 10 ml of trifluoracetic acid was added and stirred for 1 hour. The mixture was neutralized with 1% aqueous solution of NaHCO3 and washed with a large amount of water, dried with anhydrous Na2SO4 and rotary evaporated. The residue was chromatographed on silica gel (eluant AcOEt:CH2Cl2 = 1:19). The purified porphyrazine was crystallized from CH2Cl2-MeOH system. Yield: 0,69g; 30% 1H NMR (CDCl3): δ -1,11 (br, 2H, -NH-); δ 0,85 (t, J=7,5Hz, 24H, -CH3); δ 1,58 (m, J=7,2Hz, 16H,-CH2-CH3); δ 2,23 (m, J=7,1Hz, 16H-CH2-CH2-CH3); δ 2,69 (t, J=7,3Hz, 16H, -CH2-CO-); δ 3,96 (t, J=6,6Hz, 16H, -S-CH2-); δ 4,16 (t, J=7,0Hz, 16Hz, -COO-CH2-) 13C NMR (CDCl3): δ 10,7(-CH3); δ 22,3 (-CH2-CH2-CH3); δ 26,1 (-CH2-CH2-CH2-); δ 33,4 (-S-CH2-); δ 34,7 (-CH2-CO-); δ 66,5 (-COO-CH2-); δ 78,1 (ring); δ 140,8 (ring); δ 173,4 (-CO-) IR: 2967(C-H); 1728 (-CO-); 1396; 1316; 1210; 1173; 1025; 996; 782; 742; 708;684;543 MS (ESI) [M-H]- = 1593; [M+Na]+ = 1617

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11.7 Preparation of 2,3,7,8,12,13,17,18-octakis[(4-hydroxy-4-oxo-1-butyl)-thio]-21H,23H-porphyazine (Hoffman 2001)

N

NH

N

N

N

HN

N

N

R

R

R

RR

R

R

R

R= SO

Pr

O

N

NH

N

N

N

HN

N

N

R

R

R

RR

R

R

R

R= SOH

O

LiOHTHF/H2O

Procedure: 0,24g (10 mmol) lithium hydroxide was dissolved in 15 ml of water and this solution was added to 0,5g (0,3 mmol) of 2,3,7,8,12,13,17,18-octakis[4-propyloxy-4-oxo-1-butyl)thio]-21H,23H-porphyrazine dissolved in 15 of THF. The mixture was stirred for 4 days until the hydrolyzed compounds was completely portioned into lower aqueous layer. Then the aqueous layer was washed with CH2Cl2 and acidified with diluted HCl to precipitate the product. The product was isolated by filtration and washed with water to remove the HCl. Yield: 0,37g; 96% (lit:100%) 1H NMR (DMSO): δ -1,83 (s, 2H, -NH-); δ 1,98 (m, J=6,84Hz, 16H, -CH2-CH2-CH2-); δ 2.53 (t, J=7,09Hz, 16H, -CH2-CO-); δ 4,03 (t, J=6,85Hz, 16H, -S-CH2-); δ 12,11 (br, 16H, -OH) 13C NMR (DMSO ): δ 26,1 (-CH2-CH2-CH2-); δ 33,3 (-S-CH2-); δ 34,6 (CH2-CO-); δ 140,4(ring); δ 153,0(ring); δ 174,7 (-CO-) IR (KBr): 2928 (C-H); 1696 (-CO-); 1426; 1309; 1182; 996; 943; 867; 791; 682 MS: (ESI) [M-2H+Cu]-: 1320.9 Melting point: > 250°C

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11.8 Preparation of 2,3,7,8,12,13,17,18-octakis[(4-(mercaptoundecahydro-closo-dodecabortyl)-4-oxo-1-butyl)-thio]-21H,23H-porphyazine.

N

NH

N

N

N

HN

N

N

R

R

R

RR

R

R

R

R= SOH

O

N

NH

N

N

N

HN

N

N

R

R

R

RR

R

R

R

R= SS

O

B12H11

1. (COCl)22. BSH

Procedure: 0,2g (0,16 mmol) of 2,3,7,8,12,13,17,18-octakis[(4-hydroxy-4-oxo-1-butyl)-thio]-21H,23H-porphyazine was dissolved in 10 ml of thionylchloride and refluxed over a night. Then the rest of the thionylchloride was evaporated and dried in the high vacuum, separately 0,6g (2 mmol) of mercaptoundecahydro-clos-dodecaborate TMA salt was placed in 10 ml of dry acetonitrile and 0,2g (2 mmol) of 2,6-lutidine was added and stirred for 1 hour. Then the acetonitrile solution was added to the former prepared chloride and stirred in the absence of light for 7 days period of time. Next the reaction solvent was evaporated and the residue was washed with water. The obtained blue solid was separated on HPLC (water:acetonitrile 48:52). Yield: purification failed.

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11.9 Preparation of chloroacetyl mercaptoundecahydro-closo-dodecaborate.

SH O

Cl Cl

S

O

Cl2,6-lutidineacetonitrile

-2

-2

Procedure: 0,5g (1,5 mmol) of mercaptoundecahydro-closo-dodecaborate TMA salt was dissolved in 20 ml of dry acetonitrile and to solution 0,16g (0,17ml, 1,5 mmol) 2,6-lutidine was added, for 20 minutes, then the nitrogen was sealed through the flask to get rid of the oxygen from the reaction mixture. Next mixture was cooled down with ice-cooling and 1,65g (1,17ml, 12,4 mmol) of chloroacetyl chloride dissolved in 10 ml of dry acetonitrile was added from dropping funnel in 20 min period of time. The ice bath was removed and the mixture was stirred in next 2 hours, then solvent was evaporated in high vacuum in room temperature and the residue was dissolved in fresh 10 ml of acetonitrile 25 ml of diethyl ether was added to precipitate the product, which was filtered and washed with diethyl ether. The obtained precipitate was crystallized from water, filtered and dried on air. Yield: 0,52g (85%) 1H NMR (DMSO): δ 0,3-1,8 (br, 11H, cluster); δ 3,06 (s, 24H N(CH3)2); δ 4,75 (s, 2H, -CH2-) 13C NMR (DMSO): δ 43,9 (-CH2-); δ 56,2 (N(CH3)4); δ 191,2 (-CO-) 11B NMR (DMSO): δ -9,61 (1B); δ -14,19 (10B); δ -15,63 (1B) IR (KBr): 3031 (C-H); 2491 (B-H); 1657 (-CO-); 1468; 1284; 1049; 949; 843; 815; 728 MS (ESI): [A2-+K+]-=324, [A2-+3K+]+=472, [K+]=74 Melting point: > 250°C

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11.10 Preparation of 1,2-dicyano-1,2-bis[2(mercaptoundecahydro-closo-dodecaboratyl)-2-oxo-1-ethyl)thio etylene.

S

O

Cl NC S-

S-NC

Na+

Na+

NC S

SNC

S

O

S

O

NaIDMF

-2 -4

Procedure: 20 ml of dry DMF was placed in a two necked flask. Next 0,5g (1,25 mmol) of chloroacetyl mercaptoundecahydro-closo-dodecaborate TMA salt and 0,11g (0,6 mmol) of sodium cis-1,2-dicyano-1,2-ethylenedithiolate and 0,1g (0,67 mmol) of NaI were added. The mixture was heated at 80°C under nitrogen overnight. DMF was evaporated in high vacuum and the residue was dissolved in 10 ml of acetonitrile. Then 25 ml of diethyl ether was added to precipitate the product. The brown-orange precipitate was filtered and crystallized from water, filtered, washed with diethyl ether and dried on air. Yield: 0,82g (66%) 1H NMR (DMSO): δ 0,2-1,8 (br, 24 H, cluster); δ 3,07 (s, 24H, N(CH3)2); δ 4,64 (s, 2H, -CH2-) 13C NMR (DMSO): δ 41,1 (-CH2-); δ 56,2 (N(CH3)2); δ 121,5 (C=C); δ 123,6 (-CN); δ 194,3 (-CO-) 10B NMR (DMSO): δ -9,45 (1B), δ -14,23 (10B), δ -15,87 (1B) IR: 3031; 2491 (B-H); 2216 (-CN); 1637 (-CO-); 1484; 1258; 1175; 1048; 966; 948; 843; 813; 721 MS (ESI): [A-+2K+]-=359, [K+]=74 Melting point: > 250°C

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11.11 Preparation of 2,3,7,8,12,13,17,18-octakis{[2-(mercaptoundecahydro-closo-dodecaboratyl)-2-oxo-1ethyl]thio}-5,10,15,20porphyrazinato]magnesium.

NC S

SNC

S

O

S

O

PorphyrazineMgOBuBuOH

-4

Procedure: 0,22g (0,16 mmol) of magnesium turnings was placed in a two necked flask, next 10 ml. of dry propanol and 0,001g (0,01 mmol) of iodide were added. The mixture was refluxed under nitrogen overnight. Then mixture was cooled down and 0,5g (0,63 mmol) of 1,2-dicyano-1,2bis[2(mercaptoundecahydro-closo-dodecaboratyl)-2-oxo-1-ethyl)thio etylene dissolved in dry butanol was added to the reaction mixture. The reaction mixture was refluxed for 24 hours. Then the reaction mixture was cooled down. A porphyrazine system was not found. Yield: reaction failed.

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11.12 Preparation of S,S’-bis(4-carboxypropyl)disulfide.

NC

NC

S(CH2)3

O

O

S(CH2)3

O

O

S

S

COOH

COOH

KOHH2O/EtOH

Procedure: 1g (3 mmol) of 1,2-dicyano-1,2bis-(4-methoxy-4-oxo-1-butyl)thio ethylene was dissolved in 30 ml. of ethanol. 0,67g (12 mmol) of KOH dissolved in 30 ml of water was added to the reaction mixture. The mixture was refluxed for two hours, then cooled down and a 5% solution of HCl was added to the reaction mixture until the pH of the solution reached 1. The product precipitated immediately as pale yellow crystals, which were filtered and recrystallized from acetone. Yield: 0,5g (72%) 1H NMR (DMSO): δ 1,81 (m, J=7,2, 4H, -CH2-CH2-CH2-); δ 2,30 (t, J=7.3, 4H, -CH2-CO-); δ 2,69 (t, J=7,3, 4H, -S-CH2-) 13C NMR (DMSO): δ 24,8 (-CH2-CH2-CH2-); δ 32,9 (-CH2-CO-); δ 37,7 (-S-CH2-); δ 174,8 (-CO-) IR (KBr): 3112; 2956 (C-H); 1718 (-CO-); 1450; 1421; 1315; 1247; 1230; 1198; 1030; 899; 776; 677;466 MS (EI 70eV): [M+]238 MS (ESI): [M-H+]-=237 Melting point: 114°C

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11.13 Preparation of 1,2-dicyano-1,2bis-(4-hydroxy-4-oxo-1-butyl)thio ethylene.

NC

NC

S(CH2)3

O

O

S(CH2)3

O

O

NC

NC

S(CH2)3

O

OH

S(CH2)3

O

OH

HClH2O/THF

Procedure: 2g (6 mmol) of 1,2-dicyano-1,2bis-(4-methoxy-4-oxo-1-butyl)thio ethylene was dissolved in 25 ml. of THF and 20 ml of 5% HCl was added to the reaction mixture. The mixture was refluxed for 7 hours and then THF was evaporated. The aqueous solution was extracted 3 times with 30 ml of ethyl acetate. The ethyl acetate solution was dried with anhydrous sodium sulfate, filtered and rotary evaporated. The pale yellow solid residue was crystallized from butanol-hexane system on cold. Product was filtered, washed with hexane and dried on air. Yield: 1,35g (74%) 1H NMR (MeOD): δ 1,99 (m, J=7.09, 4H, -CH2-CH2-CH2-); δ 2,45 (t, J=7.33, 4H, -CH2-CO-); δ 3,22 (t, J=7.09, 4H, -S-CH2-) 13C NMR (MeOD): δ 25,4 (-CH2-CH2-CH2-); δ 32,0 (-S-CH2-); δ 34,1 (-CH2-CO-); δ 112,2 (C=C); δ 121,8 (-CN); δ 175,0 (-CO-) IR (KBr): 3036; 2937 (C-H); 2209 (-CN); 1700 (-CO-); 1499; 1430; 1238; 1181; 1149; 936; 793; 669; 631; 539; 507 MS (ESI): [M-H+]-=313 Melting point: 98°C

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11.14 Preparation of 1,2-dicyano-1,2bis(4-S-phenyl-4-oxo-1-butyl)thio ethylene.

NC

NC

S(CH2)3

O

OH

S(CH2)3

O

OH

1.(COCl)22.thiophenol

NC

NC

S(CH2)3

O

S

S(CH2)3

O

S

Procedure: 0,5 g (1,6 mmol) of 1,2-dicyano-1,2-bis(4-methoxy-4-oxo-1-butyl)thio ethylene was dissolved in 25 ml of dry CH2Cl2 and 5ml. of oxalyl chloride was added to the mixture, then mixture was refluxed for 4 hours. Next solvent and excess of oxalyl chloride were evaporated and the residue was dried in high vacuum for 1 hour. Separately in 20 ml. of dry CH2Cl2 was dissolved 0,36g (3,27 mmol) of thiophenol and 0,36g ( 3,27mmol) of 2,6-lutidine. This mixture was added to the former prepared chloride and the mixture was stirred overnight. After this the solvent was removed by evaporation and the residue was dissolved in 25 ml. of ethyl acetate and washed three times with 25 ml. of water. Next solution was dried with anhydrous sodium sulfate, filtered and ethyl acetate was removed. The obtained liquid residue was chromatographed on silica gel (eluant CH2Cl2). Yield: 0,57g (73%) 1H NMR (CDCl3): δ 2,09 (m, J=7,2Hz, 4H, -CH2-CH2-CH2-); δ 2.45 (t, J=7,4Hz, 4H,-CH2-CO-); δ 3,22 (t, J=7,4Hz, 4H, -S-CH2-); δ 4,87 (br, 10H, ring) 13C NMR (CDCl3): δ 25,9 (-CH2-CH2-CH2-); δ 34,3 (-S-CH2-); δ 42,1 (-CH1-CO-); δ 113,2 (C=C); δ 122,1 (-S-Car-); δ 128,0 (-CN); δ 130,2 (Car); δ 130,4 (Car); δ 135,2 (Car); δ 196,7 (-CO-) IR (KBr): 3033 (C-H); 3015 (C-H); 2223 (-CN); 1692 (-CO-); 1605 (C=C); 1527; 1467; 1447; 1174; 754; 695; 505 MS (EI 70eV): [M+] 498

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11.15 Preparation of 1,2-dicyano-1,2-bis(4-N-phenyl-4-oxo-1-butyl)thio ethylene.

NC

NC

S(CH2)3

O

OH

S(CH2)3

O

OH

1.(COCl)22.aniline

NC

NC

S(CH2)3

O

NH

S(CH2)3

O

NH

Procedure: 0,5g (1,6 mmol) of 1,2-dicyano-1,2-bis-(4-methoxy-4-oxo-1-butyl)thio ethylene was dissolved in 25ml. dry CH2Cl2 and 5 ml. of oxalyl chloride was added to the mixture then the mixture was refluxed for 4 hours. solvent and excess of oxalyl chloride were evaporated and dried in high vacuum for 1 hour. Separately in 20 ml. of dry CH2Cl2 were dissolved 0,3g (3,22 mmol) of aniline and 0,345g (3,22 mmol)of 2,6-lutidine. Such prepared mixture was added to the former prepared chloride and the mixture was stirred overnight. After this the solvent was removed by rotary evaporation and residue was dissolved in 25 ml. of ethyl acetate and washed three times with 25 ml. of water. The solution was dried with anhydrous sodium sulfate, filtered and ethyl acetate was removed. The obtained liquid was chromatographed on silica gel (eluant CH2Cl2:MeOH 9:1). The crystal product was obtained. Yield: 0,56g (78%) 1H NMR (CDCl3): δ 1,91 (br, 2H, -NH-); δ 2,16 (m, J=7,09Hz, 4H, -CH2-CH2-CH2-); δ 2,53 (t, J=7,09Hz, 4H, -CH2-CO-); δ 3,26 (t, J=6,84Hz, -S-CH2-); δ 7,12 (t, J=7,33Hz, 2H, ar); δ 7,31 (t, J=7,58Hz, 4H, ar); δ 7,50 (d, J=7,82Hz, 4H, ar) 13C NMR (CDCl3): δ 26,0 (-CH2-CH2-CH2-); δ 34,9 (-S-CH2-); δ 35,4 (-CH2-CO-); δ 113,2 (Car); δ 119,9 (C=C); δ 121,9 (-CN); δ 123,9 (Car); δ 129,5 (Car); δ 140,0 (Car); δ 170,8 (-CO-) IR (KBr): 3359; 3300; 2210 (-CN); 1677 (-CO-); 1601; 1538; 1495; 1441; 1418; 1328; 1308; 1246; 1128; 969; 754; 695; 502 MS (EI 70 eV): [M+] 464 Melting point: 102°C

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11.16 Preparation of {2,3,7,8,12,13,17,18-octakis[4-N-phenyl-4-oxo-1-butyl)thio]-5,10,15,20- porphyrazinato}magnesium.

NC

NC

S(CH2)3

O

NH

S(CH2)3

O

NHN

N

N

N

N

N

N

N

R

R

R

RR

R

R

R

MgPrOMgPrOH

S(CH2)3

O

NHR=

Procedure: 0,062g (2,58 mmol) of magnesium turnings together with 0,02g (0,15 mmol) of iodine were placed in two necked 50ml flask filed with 10 ml. of dry propanol. The mixture was refluxed overnight under nitrogen. The mixture was cooled down and 0,3 (0,64 mmol) of 1,2-dicyano-1,2-bis(4-N-phenyl-4-oxo-1-butyl)thio ethylene was added dissolved in 10 of dry propanol and refluxed under nitrogen for 8 hours. The reaction mixture was cooled down and filtered. The filtrate was rotary evaporated and the residue was chromatographed on silica gel (eluant CH2Cl2:MeOH 9:1). Yield: 0,1g (31%) 1H NMR (CDCl3): δ 1,87 (br, 8H, -NH-); δ 2,24 (m, J=7,09Hz, 16H, -CH2-CH2-CH2-); δ 2,47 (t, J=7,09Hz, 16H, -CH2-CO-); δ 3,51 (t, J=6,84Hz, 16H, -S-CH2-); δ 7,12 (t, J=7,33Hz, 8H, ar); δ 7,40 (t, J=7,58Hz, 16H, ar); δ 7,52 (d, J=7,82Hz, 16H, ar) 13C NMR (CDCl3): δ 25,4 (-CH2-CH2-CH2-) ; δ32,2 (-S-CH2-); δ 34,3 (-CH2-CO-); δ 78,1 (ring); δ 119,9 (Car); δ 123,8(Car); δ 129,4 (Car); δ 140,0 (Car); δ 140,8 (ring); δ 170,8 (-CO-); IR (KBr): 3367; 3305; 1683 (-CO-); 1634; 1553; 1434; 1441; 1445; 1364; 1246; 1023; 987; 652; 693; 521 MS (ESI): [M+H+]= 1881

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11.17 Preparation of 1,2-dicyano-1,2-bis[4-(mercaptoundecahydro-closo-dodecaboratyl]-4-oxo-1-butyl)thio ethylene tetramethylammonium salt.

NC

NC

S(CH2)3

O

OH

S(CH2)3

O

OH

1. (COCl)22. BSH

NC

NC

S(CH2)3

O

S

S(CH2)3

O

S

B12H11

B12H11

TMA+

-4

Procedure: 0,5g (15 mmol) of 1,2-dicyano-1,2-bis(4-hydroxy-4-oxo-1-butyl)thio ethylene was dissolved in 25 ml of the dry CH2Cl2 and 5ml. of oxalyl chloride was added. The mixture was refluxed in 4 hours period of time. The reaction solvent and the rest of oxalyl chloride were rotary evaporated and dried in high vacuum for 1 hour. Separately 1,18g (3,65 mmol) of mercaptoundecahydro-closo-dodecaborate TMA salt and 0,39g (0,3 65mmol) of dry pyridine dissolved in 30 ml of dry acetonitrile was stirred under nitrogen and added to the former prepared chloride. The reaction mixture was stirred overnight in absence of light. 5ml of water was added to the reaction mixture and the solvent was evaporated. The residue was dissolved in fresh 10 ml. of acetonitrile. 40 ml of diethyl ether was added to precipitate the product. The product was filtered and crystallized from water, filtered and dried on air. Yield: 1,13g (81%) 1H NMR (DMSO): δ 0,2-1,1 (br, 22H, cluster); δ 1,76 (m, J=7,2, 4H, -CH2-CH2-CH2-); δ 2,86 (t, J=7,3, 4H, -CH2-CO-); δ 3,07 (s, 48H, N(CH3)2); δ 3,2 (t, J=7.3, 4H, -S-CH2-) 13C NMR (DMSO): δ 26,5 (-CH2-CH2-CH2-); δ 34,7 (-S-CH2-); δ 43,5 (-CH2-CO-); δ 55,3 (N(CH3)2); δ 113,2 (C=C); δ 121,4 (-CN), δ 205,6 (-CO-) 11B NMR (DMSO): δ -4,7 (1 B); δ -6,7 (11 B) IR (KBr): 3601; 3031; 2487 (B-H); 2213 (-CN); 1655 (-CO-); 1458; 1449; 1418; 1287; 1175; 1048; 948; 843; 813; 784; 720; 510 MS (ESI): [A+3K]-=848, [A+5K]+ Melting point: >250°C

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11.18 Preparation of 1,2-dicyano-1,2-bis[4-(mercaptoundecahydro-closo-dodecaboratyl]-4-oxo-1-butyl)thio ethylene sodium salt.

NC

NC

S(CH2)3

O

S

S(CH2)3

O

S

B12H11

B12H11

4TMA+

NC

NC

S(CH2)3

O

S

S(CH2)3

O

S

B12H11

B12H11

4Na+-4 -4

Procedure: The ion exchanger Amberlite in its Na+ form was washed with water:acetonitrile 3:2. 1,13g of (2Z)-2,3-bis(4-(mercaptoundecahydro-closo-dodecaborate butanoate)thio) but-2-enedinitrile tetramethylammonium salt dissolved in this solution was put through the column. The fractions collected were controlled by reaction with aqueous solution of 0,25% PdCl2 to check in which fraction the boronated compound is. The solvent of the boron contained fraction was rotary evaporated and a yellow liquid was obtained: Yield: 0,87g (96%) 1H NMR (D2O): δ 0,2-1,1 (br, 22H, cluster); δ 1,65 (m, J=7,2, 4H, -CH2-CH2-CH2-); δ 2,73 (t, J=7,3, 4H, -CH2-CO-); δ 3,02 (t, J=7,3, 4H, -S-CH2-) 13C NMR (D2O): δ 26,5(-CH2-CH2-CH2-); δ 34,7 (-S-CH2-); δ 43,5 (-CH2-CO-); δ 113,2 (C=C); δ 121,4 (-CN); δ 205,6 (-CO) 11B NMR (D2O): δ -4,7 (1B); δ -6,7(11B) IR (KBr): 2946; 2932; 2445 (B-H); 2234 (-CN); 1701 (-CO-); 1432; 143ß; 1167; 1067; 965; 834; 765; 510 MS (ESI): [A-4+2Na++K+]-=711

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11.19 Preparation of {{2,3,7,8,12,13,17,18-octakis[4-(mercaptoundecahydro-closo-dodecaboratyl)-4-oxo-1-butyl]thio}-5,10,15,20-porphyrazinato]} magnesium sodium salt.

N

N

N

N

N

N

N

N

R

R

R

RR

R

R

R

Mg

S(CH2)3

O

SHR=

NC

NC

S(CH2)3

O

S

S(CH2)3

O

S

B12H11

B12H11

4Na+

B12H11

Na+

PrOMgPrOH

Procedure: 0,066g (2,75 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 20 ml of dry propanol were placed in a two necked flask and refluxed under nitrogen overnight. The reaction mixture was cooled down and 0,5g (0,7 mmol) of (2Z)-2,3-bis(4-(mercaptoundecahydro-closo-dodecaborate butanoate)thio) but-2-enedinitrile sodium salt dissolved in 20 ml of dry propanol was added and the mixture was refluxed for 24 hours. A porphyrazine system was not found. Yield: Reaction failed.

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11.20 Preparation of 1,2-dicyano-1,2-bis(ethoxymethyl)thio ethylene.

NC S-

S-NC

Na+

Na+

NC S

SNC

O

O

NaI, BrCH2OEtacetone

Procedure: 2g (10 mmol) of Na MNT, 0,1g (0,7 mmol) of anhydrous sodium iodide, 3,25g (23,5 mmol) of bromomethyl ethyl ether and 30 ml of dry acetone were placed in a round bottom flask and stirred for 6 hours. The reaction mixture was filtered to remove formed inorganic salt, the solvent was evaporated and the residue was dissolved in 40 ml of CH2Cl2 and washed three times with 40 ml of distilled water, dried with anhydrous sodium sulfate, filtered and rotary evaporated. The residue was chromatographed on silica gel (eluant CH2Cl2). A yellow liquid was obtained. Yield: 1,77g (64%) 1H NMR (CDCl3): δ 1,25 (t, J=7,16, 6H, -CH3); δ 3,65 (q, J=7,16, 4H, -O-CH2-); δ 5,21 (s, 4H, -S-CH2-O-) 13C NMR (CDCl3): δ 15,0 (-CH3); δ 66,0 (-O-CH2-CH3); δ 75,5 (-S-CH2-O-); δ 112,4 (C=C); δ 122,7 (-CN) IR (KBr): 2980; 2900; 2202 (-CN); 1495; 1429; 1311; 1265; 1178; 1076; 855; 805; 645; 486 MS (EI 70 eV): [M+] 258

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11.21 Preparation of 1,2-dicyano-1,2-bis(methoxymethyl)thio ethylene.

NC S-

S-NC

Na+

Na+

NC S

SNC

O

O

NaI, BrCH2OMeacetone

Procedure: 2g (10 mmol) of Na MNT, 0,1g (0,7 mmol) of anhydrous sodium iodide, 2.9 g (1,9ml, 23 mmol) of bromomethyl methyl ether and 30 ml of acetone were stirred for 5 hours period of time. THE mixture was filtered to remove inorganic salt and rotary evaporated. The residue was dissolved in 40 ml of CH2Cl2 and washed three times with water. The organic layer was dried with anhydrous sodium sulfate, filtered and CH2Cl2 was rotary evaporated. The residue was chromatographed on silica gel (eluant CH2Cl2:n-hexane 9:1). A yellow solid was obtained. Yield: 1,7g (70%) 1H NMR (CDCl3): δ 3,31 (s, 6H, -CH3); δ 5,15 (s, 4H, -CH2-) 13C NMR (CDCl3): δ 53,3 (-CH3); δ 72,6 (-CH2-); δ 118,3 (C=C); δ 121,5 (-CN) IR (KBr): 2993 (-CH); 2981 (C-H); 2231 (-CN); 1623 (C=C); 1163; 1504; 1401; 1306; 1185; 766; 600 MS (EI 70eV): [M+]230 Melting point: 21°C

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11.22 Preparation of [2,3,7,8,12,13,17,18-octakis[(ethoxymethylo)thio]-5,10,15,20- porphyrazinato]magnesium.

NC S

SNC

O

O N

N

N

N

N

N

N

N

R

R

R

RR

R

R

R

Mg

R= S O

PrOMgPrOH

Procedure: 0,37g (15 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 25 ml of dry propanol was placed in round bottom flask and refluxed overnight under nitrogen. The reaction mixture was cooled down and 1g (3,8 mmol) of 1,2-dicyano-1,2-bis(ethoxymethyl)thio ethylene dissolved in 20 ml of dry propanol was added to the reaction mixture and refluxed under nitrogen in next 24 hours. A porphyrazine system was not found. Yield: Reaction failed.

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11.23 Preparation of [2,3,7,8,12,13,17,18-octakis[(methoxymethylo)thio]-5,10,15,20- porphyrazinato]magnesium.

NC S

SNC

O

O N

N

N

N

N

N

N

N

R

R

R

RR

R

R

R

Mg

R= S O

PrOMgPrOH

Procedure: 0,41g (17 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 25 ml of dry propanol were placed in a round bottomed flask and refluxed overnight under nitrogen. The reaction mixture was cooled down and 1g (4,3 mmol) of 1,2-dicyano-1,2-bis(methoxymethyl)thio ethylene dissolved in 20 ml of dry propanol was added to the reaction mixture and refluxed under nitrogen for 24 hours. A porphyrazine was not found. Yield: Reaction failed.

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11.24 Preparation of 1,2-dicyano-1,2-bis(hydroxyethyl)thio ethylene.

NC S-

S-NC

Na+

Na+

NC S

SNC

OH

OH

NaI, Br(CH2)2OHacetone

Procedure 3g (13 mmol) of Na MNT, 0,1 (0,7 mmol) of anhydrous sodium iodide and 30 ml of dry acetone were placed in a round bottom flask and cooled down in ice salt bath. From the dropping funnel 4,4g (2,6 ml, 35,5 mmol) of 2-bromoethanol dissolved in 10ml of dry acetone was added over 20 min period of time. Then the ice bath was removed and the reaction mixture was stirred overnight. The mixture was filtered from formed inorganic salt and acetone was removed on the rotary evaporator. The residue was dissolved in 40 ml of CH2Cl2 and washed five times with water to remove water soluble material. Then CH2Cl2 was dried with sodium sulfate, filtered and evaporated on rotary evaporator. The solid residue was crystallized from CH2Cl2 n-hexane system as white small crystals, which were filtered and dried on air. Yield: 2,7g (73%) 1H NMR (CDCl3): δ 3,33 (t, J=5,88Hz, 4H, -S-CH2-); δ 3,93 (t, J=5,88Hz, 4H,-CH2-OH) 13C NMR (CDCl3): δ 38,3 (-S-CH2-); δ 60,7 (-CH2-OH); δ 113,4 (C=C); δ 121,8 (-CN) IR (KBr): 3362 (-OH); 2933; 2879; 2206 (-CN); 1492; 1402; 1291; 1185; 1154; 1061; 1015; 999; 859; 844; 823; 565; 503; 485 MS (EI 70 eV): [M+] 230 Melting point: 132°C

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11.25 Preparation of 1,2-dicyano-1,2-bis(hydroxypropyl)thio ethylene.

NC S-

S-NC

Na+

Na+

NC S

SNC

OH

OH

NaI, Cl(CH2)3OHacetone

Procedure: 3g (13mmol)of Na MNT, 0,1g (0,7 mmol) of anhydrous sodium iodide, 3,3g (2,5 ml, 35 mmol) of 3-chloro-1-propanol and 30 ml of dry acetone were placed in a round bottomed flask and refluxed under nitrogen overnight. The reaction mixture was filtered to remove formed inorganic salt and the solvent was evaporated. The obtained brown yellow liquid was dissolved in 40 ml of CH2Cl2 and washed three times with water to remove all water soluble material. Solution was dried with anhydrous sodium sulfate, filtered and CH2Cl2 was removed by rotary evaporation. The obtained yellow brown liquid was chromatographed on silica gel (eluant CH2Cl2:MeOH 9:1). A pale yellow liquid was obtained. Yield: 2,8g (69%) 1H NMR (CDCl3): δ 1,81 (m, J=7.1Hz, 4H, -CH2-CH2-CH2-); δ 3,25 (t, J=7.5, 4H, -S-CH2-); δ 3,45 (t, J=7.3, 4H, -CH2-CO-) 13C NMR (CDCl3): δ 31,7 (-S-CH2-); δ 32,3 (-CH2-CH2-CH2-); δ 58,9 (-CH2-OH); δ 112,3 (C=C); δ 121,7 (-CN) IR (KBr): 3405 (-OH); 2945; 2876; 2221 (-CN); 1654; 1045; 976; 884; 563 MS (EI 70 eV): [M+]258

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11.26 Preparation of [2,3,7,8,12,13,17,18-octakis[(2-hydroxyethyl)thio]-5,10,15,20- porphyrazinato]magnesium.

NC S

SNC N

N

N

N

N

N

N

N

R

R

R

RR

R

R

R

Mg

R=

OH

OH

SOH

PrOMgPrOH

Procedure: 0,41g (17 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 25 ml of dry propanol were placed in flask and refluxed overnight under nitrogen. The reaction mixture was cooled down and 1g (4,3 mmol) of 1,2-dicyano-1,2bis(2-hydroxyethylthio)ethene dissolved in 20 ml of dry propanol was added to reaction mixture and the mixture was refluxed under nitrogen for 8 hours. The reaction mixture was filtered hot and the residues were washed with fresh propanol. The solvents from the collected extracts were evaporated and the residue was chromatographed on silica gel (eluant CH2Cl2:MeOH 4:1). A blue liquid was obtained. Yield: 0,66g (65%) 1H NMR (CD3OD): δ 3,85 (t, J=5,86Hz ,16 H, -S-CH2-); δ 4,06 (t, J=5,86Hz, 16H, -CH2-OH) 13C NMR (CD3OD): δ 39,4 (-CH2-); δ 61,7 (-CH2-OH); δ 79,2 (ring); δ 140.,1 (ring) IR (KBr): IR: 3642 (O-H); 2532(C-H); 1386; 1323; 1267; 1174; 945; 921; 887; 675; 543; 432 MS(ESI): [M+Cl]- =980, [M+H2PO4]-=1041

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11.27 Preparation of [2,3,7,8,12,13,17,18-octakis[(2-hydroxypropyl)thio]-5,10,15,20- porphyrazinato]magnesium.

NC S

SNC N

N

N

N

N

N

N

N

R

R

R

RR

R

R

R

Mg

R= S

OH

OH

OH

PrOMgPrOH

Procedure: 0,37g (15 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 25 ml of dry propanol was placed in flask and refluxed overnight under nitrogen. The reaction mixture was cooled down and 1g (3,8 mmol) of 1,2-dicyano-1,2-bis(3-hydroxypropylthio)ethene dissolved in 20 ml of dry propanol was added and mixture was refluxed for 8 hours. The reaction mixture was filtered hot and the residues were washed with propanol and solvents from collected extracts was evaporated residue was chromatographed on silica gel (eluant CH2Cl2:MeOH 9:1). A blue liquid was obtained. Yield: 0,68g (67%) 1H NMR (CD3OD): δ1,80 (m, J=7,1Hz, 4H, , -CH2-CH2-CH2-); δ 3,34 (t, J=7,5Hz,4H, -S-CH2-); δ 3,5 (t, J=7.5Hz, 4H, -CH2-CO-) 13C NMR (CD3OD): δ 31,1(-S-CH2-); δ 33,4 (-CH2-CH2-CH2-); δ 60,0 (-CH2-OH); δ 156,6 (ring); δ 140,3 (ring) IR(KBr): 3331 (-OH); 2920; 2876; 1052; 1025; 987; 745; 567 MS(ESI): [M+Cl]-= 1091

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11.28 Preparation of [2,3,7,8,12,13-hexa[(2-hydroxyethyl)thio]-17,18di(4-methoxy-4-oxo-1-butyl)thio]-5,10,15,20- porphyrazinato]magnesium.

NC S

SNC

OH

OH

NC

NC

S(CH2)3

O

O

S(CH2)3

O

O

N

N

N

N

N

N

N

NMg

R

R

R'

R'

R

RR

R

R

R'

PrOMgPrOH

Procedure: The experiment was made in three different molar ratios of reactants:

- experiment 1: 0,1g (4,38 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 25 ml of dry propanol, 0,5g (1,46 mmol) 1,2-dicyano-1,2-bis(4-methoxy-4-oxo-1-butyl)thio ethylene, 0,33g (1,46 mmol) of 1,2-dicyano-1,2bis(2-hydroxyethyl)thioethene

- experiment 2: 0,09g (4,11 mmol) of magnesium turnings, , 0,01g (0,07 mmol) of iodide and 25 ml of dry propanol, 0,75g (2,14 mmol) of (2Z)- 1,2-dicyano-1,2-bis(4-methoxy-4-oxo-1-butyl)thio ethylene, 0,12g (0,54 mmol) of 1,2-dicyano-1,2bis(2-hydroxyethyl)thioethene

- experiment 3: 0,24g (10,2 mmol) of magnesium turnings, , 0,01g (0,07 mmol) of iodide and 25 ml of dry propanol, 0,75g (2,19mmol) of (2Z)- 1,2-dicyano-1,2-bis(4-methoxy-4-oxo-1-butyl)thio ethylene, 0,08g (0,36 mmol) 1,2-dicyano-1,2bis(2-hydroxyethyl)thioethene

The magnesium turnings were refluxed overnight under nitrogen, the reaction mixture was cooled down and a propanol solution of dinitriles was added. The reaction mixture was refluxed under nitrogen for 10 hours. Then the mixture was filtered on hot and the residue was washed few times with fresh propanol. Filtrates were evaporated and residues were chromatographed on silica gel (eluant CH2Cl2:MeOH 9:1) Yield: reaction failed

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11.29 Preparation of [2,3,7,8,12,13-hexa[(2-hydroxypropyl)thio]-17,18di[(4-methoxy-4-oxo-1-butyl)thio]-5,10,15,20- porphyrazinato]magnesium.

NC S

SNC

NC

NC

S(CH2)3

O

O

S(CH2)3

O

O

N

N

N

N

N

N

N

NMg

R

R

R'

R'

R

RR

R

OH

OH

R

R'

PrOMgPrOH

Procedure: 0,21g (8,7 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 25 ml of dry propanol was placed in a round bottomed flask and refluxed under nitrogen overnight. The reaction mixture was cooled down and the mixture of dinitriles dissolved in 10 ml of dry propanol was added and the mixture was refluxed for 8 hours. The reaction mixture was filtered on hot and the residues were washed with fresh propanol. The collected filtrates was rotary evaporated and blue residue was chromatographed on silica gel (eluant CH2Cl2:MeOH 4:1). Yield: Another product.

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11.30 Preparation of 1,2-dicyano-1,2-bis(2,2-diethoxyethyl)thio ethylene.

S-NC

NC S- Na+

Na+

Cl

O

O

SNC

NC S

O

O

O

O

Et

Et

Et

Et

NaIacetone

Procedure: 2g (10,7 mmol) of Na NMT, 0,1g (0,66 mmol) of sodium iodide, 4,63g (23,6 mmol) of 2-chloro-1,1-diethoxyethane and 30 ml of dry acetone were placed in the round bottomed flask and refluxed overnight under nitrogen. The reaction mixture was cooled down and filtered to remove formed inorganic salt. The filtrate was rotary evaporated and the residue was dissolved in 40 ml of CH2Cl2 and washed three times with 40 ml of water. The organic solution was dried with anhydrous sodium sulfate, filtered and rotary evaporated: The residue was chromatographed on silica gel (eluant CH2Cl2:MeOH 9:1). A yellow liquid was obtained Yield: 2,8g (71%) 1H NMR (CDCl3): δ 1,17 (t, J=6,5Hz, 12H, -CH3); δ 3,32 (d, J=4,7Hz, 4H, -S-CH2-); δ 3,57 (q, J=6.5Hz, 8H, -O-CH2-); δ 4,62 (t, J=4.7Hz, 2H, -CH-(OET)2) 13C NMR (CDCl3): δ 15,3 (-CH3); δ 31,7 (-S-CH2-); δ 62,2 (-O-CH2-CH3); δ 100,4 (-CH-(OET)2); δ 112,3 (C=C); δ 121,8 (-CN) IR (KBr): 2964 (C-H); 2931 (C-H); 2815 (-O-CH-O-); 2229 (-CN); 1506; 1371; 1302; 1246;1182; 770; 600; 569;454 MS (EI 70eV): [M+] 374

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11.31 Preparation of 1,2-dicyano-1,2-bis(2-oxoethyl)thio ethylene.

SNC

NC S

O

O

O

O

Et

Et

Et

Et

SNC

NC SO

OHClH2O/MeOH

Procedure: 2g (5,3 mmol) of 1,2-dicyano-1,2-bis(2,2-diethoxyethyl)thio ethylene was dissolved in 30 ml of methanol. Then to the methanolic solution was added 20 ml of 10% HCl and the reaction mixture was stirred for three hours. 10% of aqueous solution of NaOH was added to the reaction mixture and pH of the solution was increased to value 6,5 and stirred for 20 min. Methanol was evaporated to precipitate the product. The product was filtered and crystallized from CH2Cl2 n-hexane system to obtain white crystals, which were filtered and washed with hexane and dried on air. Yield: 0,98g (82%) 1H NMR (CDCl3): δ 3,49 (d, J=2,6Hz, 4H, -CH2-); δ 10,10 (t, J=2,6Hz, 2H, -CHO) 13C NMR (CDCl3): δ 46,7 (-CH2-); δ 112,3 (C=C); δ 122,5 (-CN); δ 198,3 (-CHO) IR (KBr): 2996; 2931; 2215 (-CN); 1752 (-CO-); 1523; 1323; 1256; 1156; 945; 881; 840; 667;526 MS (EI 70eV): [M+] 226 Melting point: 154°C

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11.32 Preparation of 1,2-dicyano-1,2-bis(2-iminododecahydro-closo-dodecaborateethyl)thio ethylene.

SNC

NC SO

O BNH3MeOH

SNC

NC SNH

NH

-4

Procedure: 1,76 (4,4 mmol) of ammoniaundecahydro-closo-dodecaborate TBA salt and 0,35 g (4,4 mmol) of pyridine were dissolved in 30 ml of methanol. 0,5g (2,2 mmol) of 1,2-dicyano-1,2-bis(2-oxoethyl)thio ethylene dissolved in 10 ml of methanol was added drop wise to the stirred solution over 1 hour. The reaction mixture was stirred for 3 hours. Diethyl ether was added to the reaction mixture to precipitate the product, which was filtered and crystallized from CH2Cl2 n-hexane system. The unreacted BNH3 was obtained. Yield: reaction failed

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11.33 Preparation of 6-hydroxy-6,7-dihydro-5H-1,4-dithiepine-2,3-dicarbonitrile.

S

SNC

NC

OH

S-NC

NC S- Na+

Na+

O

Cl

ethanol

Procedure: A 500 ml three necked round bottom flask equipped with a reflux condenser, two hoses connected to a peristaltic pump and a fast external stirrer was filled with 150 ml of dry ethanol and 5g of NaOH was dissolved in it. The solvent was heated up to 50°C and vigorously stirred. Separately solutions 1g (5,3 mmol) of Na MNT in 100 ml of dry ethanol and 0,49g (5,3 mmol) of epichlorohydrine in 100 ml of dry ethanol were pumped at this step from the peristaltic pump. The solutions were pumped over 4 hours, then the reaction mixture was stirred and refluxed for 10 hours. Then the reaction mixture was evaporated. Only a mixture of polymeric material was obtained. Yield: reaction failed.

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11.34 Preparation of 1,3-ditosylacetone.

O

OHHO

S OO

Cl

CH3

O

OOTsTs

Procedure: Experiment 1: A round bottom flask was filled with 40 ml of dry pyridine. Then the reaction flask was cooled down below 0°C in a ice salt bath. 16,9g (88 mmol) of tosyl chloride was added in portion to the cooled pyridine and the reaction was stirred for half an hour. Then 2g (22 mmol) of dihydroxyacetone was added to the reaction mixture in portions. The reaction mixture was stirred for 2 hours, then the solution was poured over water with ice and vigorously stirred. At this step solution turned dark brown. Yield: reaction failed. Experiment 2: 10,6 g (55 mmol) of tosyl chloride and 6,6g (83,5 mmol) of dry pyridine were dissolved in 50ml of dry chloroform. The reaction mixture was cooled down below 0°C in a salt-ice bath and stirred vigorously. 2g (22,2 mmol) of dihydroxyacetone was added to the stirred solution and stirred for 2 hours. The chloroform solution was washed three times with 40 ml of water. The organic solution was dried with anhydrous sodium sulfate, filtered and rotary evaporated. The obtained solid was dissolved in hot acetone, cooled down and some water was added to crystallize product. Yield: reaction failed. The obtained product was tosyl acid.

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11.35 Preparation of pyridinum 5,6-dicyanopyrazine-2-olate.

N

NNC

NC O

NC NH2

NH2NC

O

OHH

O

pyridinemethanol NH

Procedure: 2g (18,5 mmol) of diaminomaleonitrile, 2,9g (37 mmol) of pyridine and 40 ml of methanol were placed in a round bottomed flask. 3,4g (37 mmol) of glyoxalic acid two hydrate dissolved in 20 ml of methanol was added dropwise from dropping funnel over one hour. The reaction mixture was stirred overnight. The reaction mixture was filtered and the filtrate was rotary evaporated to the dryness. The residue was dissolved in hot acetone, cooled down and stirred with active carbon for half an hour. The mixture was filtered and the solvent was rotary evaporated to the dryness. The residue was crystallized from 10 ml of methanol. Yield: 3,7g (89%) 1H NMR (acetone d6): δ 7,75 (t, J=7,08Hz, 2H, ar); δ 8,19 (t, J=7,82Hz,1H, ar), δ 8,29 (s, 1H, ar –N=CH-COH-); δ 8,81 (d, J=4,4Hz, 2H, ar); δ 9,17 (s, 1H, H+-) 13C NMR (acetone d6): δ 114,7 (C=C); δ 115,6 (-CN); δ 116,3 (ar), δ 127,4 (ar); δ 133,8 (C=C); δ 143,3 (ar); δ 144,6 (-N=CH-COH-); δ 145,6 (ar); δ 165,6 (ar C-OH) IR (KBr): 3441 (N-H); 3101 (C-H); 2220 (-CN); 1565; 1397; 1223; 1165; 974; 761; 686; 639; 596; 534; 496; 464; 419 MS (ESI): [A]-=145, [K]+= 80 Melting point: 168°C

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11.36 Preparation of acetamidoacetimidosuccinonitrile (Hinkel 1937).

NC

NH2

NH2

NC CH3

O

O

CH3

OCN

NH HN

NC

CH3

O

H3C

Oacetic

anhydride

Procedure: 2g (18,5 mmol) of diaminomaleonitrile and 10 ml of acetic anhydride were placed in a round bottom flask and heated at 100°C over half an hour. The rest of acetic anhydride from the produced solid mass was evaporated in high vacuum. The rest was dissolved in acetone and decolorized by stirring with active carbon for half an hour. The mixture was filtered, the solvent was rotary evaporated and the residue was crystallized from hot methanol, filtered washed with fresh methanol and dried on air. Yield: 2,6g (73%); (lit 96%) 1H NMR (acetone d6): δ 2,28 (s, 6H, -CH3): 5,75 (br, 2H, NH) 13C NMR (acetone d6): δ 22,6 (-CH3); δ 113,0 (C=C); δ 123,9 (-CN); δ 168,4 (-CO-) IR (KBr): 3250 (N-H); 3000 (C-H); 2224 (-CN); 1740 (-CO-); 1611; 1524; 1370; 1215;994;753; 689; 600 MS (EI 70 eV): [M+] 192 Melting point: 225°C decomposition; (lit: 224°C decomposition)

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11.37 Preparation of acetamidoiminosucinonitrile (Hinkel 1937).

NC

NH2

NH2

NC CH3

O

O

CH3

O

NC

NH2

NH

NCO

CH3

aceticanhydride

Procedure: 2g (18,5 mmol) of diaminomaleonitrile and 10 ml of acetic anhydride was heated up to 50°C for half an hour. The rest of the acetic anhydride was evaporated in high vacuum. The residues were dissolved in 20 ml of hot acetone, cooled down and stirred with active carbon for half an hour. The mixture was filtered, the solvent was rotary evaporated and the solid residue was crystallized from hot ethanol, filtered and dried on air. Yield: 2,05g (76%) (lit: 98%) 1H NMR (acetone d6): δ 2,85 (s, 3H, -CH3); δ 5,89 (br, 2H, -NH2); δ 8,45 (br, 1H, -NH-) 13C NMR (acetone d6): δ 24,4 (-CH3); δ 88,2 (C=C); δ 113,0 (-CN); δ 116,5 (-CN); δ 128,2 (C=C); δ 167,3 (-CO) IR (KBr): 3434; 3339; 3244; 2206 (-CN); 1680 (-CO-); 1628 (C=C); 1507; 1372; 1302; 1246; 1182; 1044; 770; 600; 570 MS (EI 70 eV): [M+] 150 MS (ESI): [M-H]-=149, [M+Na]+=173 Melting point: 172°C decomposition; lit: 164°C decomposition

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11.38 Preparation of1,2-dicyano-1-(acetylamino)-2-[(2-hydroxy-2-oxo-ethen)amino]ethylene.

CN

N NH

NC

O

CH3

O

HO

CN

H2N NH

NC

O

CH3

O O

HO H

H2SO4methanol

Procedure: 1g (6,6 mmol) of mono acetylated DAMN and 0,61g (6,6 mmol) of glyoxalic acid mono hydrate were dissolved in methanol. A drop of concentrated sulfuric acid was added to the stirred solution. The reaction mixture was heated at 60°C in the next 24 hours. The reaction mixture was cooled down. The methanol was evaporated and the residue was dissolved in 40 ml of CH2Cl2 and washed three times with 40 ml of water. The organic layer was dried with anhydrous sodium sulfate, filtered and rotary evaporated. The solid residue was crystallized from hot carbon tetrachloride, filtered and dried on air. The structure of the product was not determined, spectral characteristic: 1H NMR (CDCl3): δ 1,61 (br, 2H); δ 2,51 (s, 2H); δ 4,07 (s, 2H), δ 4,45 (br, 1H) 13C NMR (CDCl3): δ 25,2; δ 54,9; δ 115,3; δ 118,1; δ 132,4; δ 156,4; δ 156,8; δ 158,7 IR (KBr): 3460; 3346; 2960; 2224; 1626; 1559; 1500; 1429; 1399; 1279; 1186; 1010; 778; 673; 624 MS (EI 70 eV): [M+] 207 Melting point: 134°C

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11.39 Preparation [octakis acetyloaminoporphyrazinato] magnesium.

CN

NH HN

NC

CH3

O

H3C

O

N

N

N

N

N

N

N

NMg

R

R

R

R

R

RR

R

R= NH

O

CH3

PrOMgPrOH

Procedure: 0,5 g (20 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 40ml of dry propanol were placed in a round bottom flask and refluxed under nitrogen overnight. The reaction mixture was cooled down and 1g (5,2 mmol) of di acetylated DAMN dissolved in 10 ml of dry propanol was added to the reaction mixture and the mixture was refluxed under nitrogen for 24 hours. The reaction solvent was evaporated and the residue was analyzed. No porphyrazine was found Yield: Reaction failed.

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11.40 Preparation [tetrakis-β-amino-tetrakis-β’-acetamido porphyrazinato] magnesium.

CN

H2N HN

NC

CH3

O

N

N

N

N

N

N

N

NMg

R

R

R

R

R

RR

R

R= NH

O

CH3R= NH2

PrOMgPrOH

Procedure: 0,64 g (26 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 40ml of dry propanol were placed in a round bottom flask and refluxed under nitrogen overnight. The reaction mixture was cooled down and 1g (0,64 mol) of acetamidoiminosucinonitrile dissolved in 10 ml of dry propanol was added and refluxed for 24 next hours. After this time the reaction solvent was rotary evaporated and the residue was analyzed. No porphyrazine was found. Yield: reaction failed

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11.41 Preparation of 1,2-dicyano-1-amino-2[(4-formylphenyl)methylen amino]ethylene.

NC NH2

NH2NC

H

O H

O NC N

NH2NC

H

H

O

H2SO4methanol

Procedure: 3g (27 mmol) of DAMN, 3,4g (27 mmol) of terephthalaldehyde and 50 ml of methanol was placed in a round bottom flask. A drop of concentrated sulfuric acid was added to the stirred solution and the reaction mixture was stirred over 10 hours. The product precipitated as yellow crystals, the reaction mixture was filtered, the product was washed with fresh methanol and dried on air. Yield: 4,7 g (77%) 1H NMR (CDCl3): δ 6,32 (br, 2H, -NH2); δ 8,01 (d, J=8,3Hz, 2H, ar); δ 8,25 (d, J=8,3Hz, 2H, ar); δ 8,45 (s, 1H, -N=CH-); δ 10,11 (s, 1H, -CHO) 13CNMR (CDCl3): δ 107,2 (C=C); δ 113,9 (C=C); δ 118,2 (-CN); δ 121,5 (-CN); δ 132,3 (-CH- ar); δ 132,8 (-CH-ar); δ 133,7 (C ar); δ 138,6 (-C-CO ar); δ 142,1 (-N=C-); δ 191,2 (-CHO) IR (KBr): 3422 (-NH2); 3305 (-NH2); 3202 (C-H); 2238 (-CN); 1688 (-CO-); 1621 (C=N); 1605 (ar C-C); 1395; 1308; 1206; 946; 816; 739; 558; 499 MS (ESI): [M-H]-= 223 MS (EI 70 eV): [M+] 224 Melting point: 209°C decomposition

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11.42 Preparation of 1,2-dicyano-1-amino-2{[4-(undecahydro-closo-dodecaborate)imino]methyl]phenylmethylen amino}ethylene.

NC N

NH2NC

H

O

H

NC N

NH2NC

H

NH

H

BNH3methanol

-2

Procedure: Experiment1: 0,5g (2,2 mmol) of 1,2-dicyano-1-amino-2[(4-formylphenyl)methylen amino]ethylene was dissolved in 35 ml of methanol, 0,9g (2,2 mmol) of ammoniaundecahydro-closo-dodecaborate TBA salt and few drops of the aqueous solution of NaOH 5% were added to the solution. The mixture was stirred for 4 hours. The solvent was evaporated and the residue was analyzed. Only unreacted material was found in the reaction mixture. Yield: reaction failed Experiment2: 0,9 g (2,2 mmol) of ammoniaundecahydro-closo-dodecaborate TBA salt was dissolved in dry THF and cooled down in a ice salt bath. 0,05g (2,2 mmol) of sodium hydride was added to solution and stirred for half an hour. 0,5g (2,2 mmol) of (2Z)-2-amino-3-{[(1E)-(4-formylphenyl)methylene]amino} but-2-enedinitrile was added to the solution and the reaction mixture was stirred over 4 hours. The solvent was evaporated and the residue was analyzed. No product was found. Only unreacted material was found in the reaction mixture. Yield: reaction failed.

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11.43 Preparation of [tetrakis(β-p-formyl phenyl methin)octakis amino porphyrazinato] magnesium

N

N

N

N

N

N

N

NMg

R

R

R

R

R

RR

R

R= R= NH2

NC N

NH2NC

H

H

O

H

H

O

N

PrOMgPrOH

Procedure: 0,42 g (17,5 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 50 ml of dry propanol were placed in a round bottom flask and refluxed under nitrogen overnight. The reaction mixture was cooled down and 1g (4,4 mmol) of (2Z)-2-amino-3-{[(1E)-(4-formylphenyl)methylene] amino}but-2-enedinitrile dissolved in 20 ml of dry propanol was added, and the reaction mixture was refluxed for 24 hours. The reaction mixture was cooled down, the solvent was rotary evaporated and the residue was analyzed. No porphyrazine was found. Yield: reaction failed.

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11.44 Preparation of 1,2-dicyano-1-amino-2-[(4-carboxyphenyl)methylene amino] ethylene.

NC N

NH2NC

H

HO

O

H

HO

O

ONC NH2

NH2NC

H2SO4methanol

Procedure: 3g (27 mmol) of DAMN and 3,7g (27 mmol) of 4-formylbenzoic acid was dissolved in 50 ml of methanol and to the stirred solution a drop of concentrated sulfuric acid was added. The mixture was stirred for 2 hours. The product precipitated as yellow crystals, which was filtered, washed with fresh methanol and dried on the air. Yield: 4,5 g (69%) 1H NMR (DMSO-d6): δ 6,08 (br, 2H, -NH2) δ 7,96 (d, J=8,55, 2H, ar); δ 8,12 (d, J=8,55, 2H, ar); δ 8,29 (s, 1H, -N=CH-) 13C NMR (DMSO-d6): δ 106,2 (C=C); 114,0 (C=C); δ 118,2 (-CN); δ 121,5 (-CN); δ 129,9 (-C-COOH ar); δ 131,4 (C ar); δ 131,9 (C ar); δ 137,2 (C ar); δ 142,3 (-N=CH-); δ 170,8 (-COOH) IR (KBr): 3426 (N-H); 3326 (N-H); 2845 (C-H); 2209 (-CN); 1725 (-CO-); 1622 (-N=CH-); 1303; 1265; 1186; 988; 754; 895 MS (ESI): [M+Na]+ = 263; [M-H]- = 239 MS (EI 70 eV): [M+] 240 Melting point: 189°C decomposition

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11.45 Preparation of [tetrakis(β-p-carboxy phenyl methin)octakis amino porphyrazinato] magnesium

N

N

N

N

N

N

N

NMg

R

R

R

R

R

RR

R

R= R= NH2

H

HO

O

N

NC N

NH2NC

H

HO

O

PrOMgPrOH

Procedure: 0,4g (16,6 mmol) of magnesium turnings, 0,01g (0,07 mmol) of iodide and 50 ml of dry propanol were placed in a round bottomed flask and refluxed under nitrogen over night. The reaction mixture was cooled down and 1 g (4,1 mmol) of 1,2-dicyano-1-amino-2-[(4-carboxyphenyl)methylene amino] ethylene dissolved in 10 ml of dry propanol was added to the reaction mixture and the mixture was refluxed for 24 hours. The reaction mixture was cooled down, the solvent was rotary evaporated and the residue was analyzed. No porphyrazine was found. Yield: reaction failed.

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11.46 Preparation of S-4-((E)-{[(Z)-2-amino-1,2-dicyanovinyl]imino}methyl) benzoyl mercaptoundecahydro-closo-dodecaborate.

NC N

NH2NC

H

HO

O

NC N

NH2NC

H

S

O

1.(COCl)22.BSH

Procedure: 0,5 g (2,0 mmol) of 1,2-dicyano-1-amino-2-[(4-carboxyphenyl)methylene amino] ethylene was placed in a round bottomed flask filled with 20 ml of oxalyl chloride and refluxed for 3 hours. After this time the excess of the oxalyl chloride was evaporated in high vacuum. Separately in another flask 1g (3 mmol) of mercaptoundecahydro-closo-dodecaborate TMA salt was dissolved in 30 ml of dry acetonitrile together with 0,24 g (3 mmol) of pyridine and stirred for half an hour. The acetonitrile solution was added to the former prepared acid chloride and stirred for 4 hours. The acetonitrile was evaporated, the residue was dissolved in fresh 10 ml of acetonitrile and 30 ml of diethyl ether was added to precipitate the product, which was filtered and crystallized from hot water. The obtained containing boron material was unreacted BSH. Yield: reaction failed.

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11.47 Preparation of N-(-1,2-dicyano-2-{[(4-formylphenyl)methylene]amino} vinyl)acetamide.

CN

HNH2N

NC

O

CN

HNN

NC

OH

O

H

methanol

Procedure: 0,5 g (3,3 mmol) of N-[(Z)-2-amino-1,2-dicyanovinyl]acetamide and 0,45 g (3,3 mmol) terephthalaldehyde was dissolved in 50 ml and stirred for 5 hours. The product precipitated and then the product was filtered and crystallized from hot acetonitrile, filtered, washed with fresh cold acetonitrile and dried on air. Yield: 0,92 g (93%) 1H NMR (DMSO-d6): δ 2,97 (s, 3H, -CH3); δ 8,06(d, J=3,9, Hz, 2H, ar); δ 8,36 (d, J=3,9Hz, 2H, ar); δ 8,66 (s, 1H, -N=CH-); δ 10,08 (s, 1H, -CHO); δ 10,88 (br, 1H, -NH-) 13C NMR (DMSO-d6): δ 23,7 (-CH3); δ 103,1 (-C=C-); δ 114,4 (-CN); δ 115,0 (-CN); δ 128,3 (ar); δ 129,0 (-C=C-); δ 130,2 (ar); δ 130,5 (ar), δ 138,2 (ar); δ 138,7 (-N=CH-); δ 154,1 (-CO-); δ 193 (-CHO) IR (KBr): 3415 (N-H); 3305 (C-H); 2239 (-CN); 1688 (-CO-); 1622 (-CO-); 1573; 1553; 1397; 1308; 946; 815; 739; 557; 497 MS (EI 70 eV): [M+]266 Melting point: 209°C decomposition

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11.48 Preparation of N-{(2Z)-1,2-dicyano-2-[({4-[ammoniaundecahydro-closo-dodecaborateimino)methyl]phenyl}methylene)amino]vinyl}acetamide.

CN

HNN

NC

OH

O

H

CN

HNN

NC

OH

N

H

NaOHmethanol

-2

Procedure: Experiment 1: 0,45 g (1,1 mmol) of ammoniaundecahydro-closo-dodecaborate TBA salt was dissolved in methanol together with 0,3 (1,1 mmol) of N-(-1,2-dicyano-2-{[(4formylphenyl)methylene] amino} vinyl)acetamide and a few drops of a 5% Na OH aqueous solution was added to initiate the reaction. The mixture was stirred for 4 hours. The solvent was evaporated and the residue was analyzed. Only starting material was found. Yield: reaction failed. Experiment 2: 0,45 g (1,1 mmol) of ammoniaundecahydro-closo-dodecaborate TBA salt was dissolved in dry DMF and 0,043 g (60 % suspension in oil) of sodium hydride was added and stirred for 1 hour. A solution of N-((Z)-1,2-dicyano-2-{[(1E)-(4-formylphenyl)methylene]amino}vinyl) acetamide in 10 ml of dry DMF was added drop wise to the reaction mixture and the reaction mixture was stirred in next 4 hours. 40 ml of diethyl ether was added to the solution to precipitate the product. The precipitate was filtered and analyzed. Only starting materials was found. Yield: reaction failed.

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11.49 Preparation of 4-[(undecahydro-closo-dodecaborateimino)methyl] benzaldehyde.

O

O NH

O

BNH3MeOH

-2

Procedure: Experiment 1: 0,75 (1,8 mmol) of ammoniaundecahydro-closo-dodecaborate TBA salt was dissolved in 25 ml of methanol together with 0,25 g (1,8 mmol) of terephthalaldehyde. Few drops of 5% NaOH aqueous solution were added to the stirred solution to initiate the reaction. The reaction mixture was stirred in the next 4 hours, then the solvent was evaporated and the residue was analyzed. No product was found. Yield: reaction failed. Experiment 2: 0,75 g (1,8 mmol) of ammoniaundecahydro-closo-dodecaborate TBA salt was dissolved in dry THF and 0,073 g (60 % suspension in oil) of sodium hydride was added to the solution and stirred for 1 hour. Next 0,25 g (1,8 mmol) of terephthalaldehyde was added and the reaction was stirred for 4 hours. The solvent was rotary evaporated and the residue was analyzed. No product was found. Yield: reaction failed.

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12 Summary. The goal of this work was to synthesize water soluble boronated porphyrazines. However the goal of the work was not achieve. Two boron clusters, mercaptoundecahydro-closo-dodecaborate and ammoniaundecahydro-closo-dodecaborate, were chosen for attachment to the porphyrazine. Their sodium salts are very well soluble in water. Both of the clusters can be reacted with activated carboxylic acids. Additionally the ammoniaundecahydro-closo-dodecaborate can react with aldehydes and form Schiff base. These reactions give opportunity to attach the boron cluster the porphyrazine systems. First the synthetic route was investigated, which starts with Na MNT. NaMNT was reacted with three different methyl chloro or bromo acid esters and then cyclized. Only the butyl ester derivative was able to cyclization. The probable reason is sterical. The obtained porphyrazine ester derivative was first hydrolyzed, activated by transferring it into acid derivative and then reacted with BSH. The obtained mixture product was separated by HPLC method. After separation the obtained two main fraction was not found expected. The probable reason was that the obtained product was a mixture of not fully substituted porphyrazines which creates problems during the purification process. It was decided to prepare first a boronated building block in the second synthetic attempt, because of the troubles with purification of the substituted porphyrazine. This attempt was started first by reacting BSH with chloracetyl chloride and the obtained product was reacted with Na NMT. This prepared building block was reacted to the porphyrazine system. However this reaction failed also probably for the same reasons, sterical reasons like in the first synthetic attempt. For this reason the next synthetic attempt was made by reacting Na MNT with methyl 4-chlorobutanoate, then hydrolyzing to its acid derivative, activating and reacting with BSH. This building block was also cyclized but also in this case the boronated porphyrazine system was not obtained. In this case the reason of unsuccessful experiment can be that the basic condition of the cyclization reaction can causes the decomposition of building block or also in this case the reason can be sterical. It was decided to prepare porphyrazines with different side chains to avoid the sterical difficulties. Few different dinitriles were chosen for cross cyclization together with (2Z)-2,3-bis(4-(methyl butanoate)thio) but-2-enedinitrile, which should be the linker to the boron cluster. Cross cyclization was made in different conditions and in different dinitriles combinations. However porphyrazines with different side chains were not obtained. Even if two different dinitriles were

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cyclized only a porphyrazine with one type of side chain was obtained. Here the problem could be that chosen dinitriles had completely different activity in a cyclization reaction. The BNH3 boron cluster compounds was also used in the synthesis of boronated porphyrazines. Aliphatic aldehyde was chosen as linker between the porphyrazine system and the boron cluster which can form in reaction with amino group the Shiff base. First the protected aldehyde group was attached to the dinitrile then de-protected and finally reacted with BNH3. However this reaction also failed. The probable reason is that BNH3 is a strong nucleophile and can react with α proton of the aldehyde instead of carbonyl group. The DAMN derivatives were used as a macrocyclization precursors instead of the Na MNT. The amino group of DAMN were reacted with different aldehydes. However none of them were successfully reacted with boron clusters BSH and BNH3. None of the DAMN derivatives was successfully transferred to the porphyrazine system. The reason can be that during the cyclization both amino groups should be protected by methylation.

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13 Abbreviations. BNCT Boron Neutron Capture Therapy BSH mercaptoundecahydro-closo-dodecaborate PDT Photo Dynamic Therapy BPA p-borono phenylalanine amino acid HpD hematoporphyrin derivatives BNH3 ammoniaundecahydro-closo-dodecaborate. DAMN diaminomaleonitrile B12H12 dodecahydrododecaborate Na MNT disodium salt of dithiolodimaleodinitrile IR Infra Red Spectroscopy 1H NMR Proton-1 Nuclear Magnetic Resonance Spectroscopy 13C NMR Carbon-13 Nuclear Magnetic Resonance Spectroscopy 11B NMR Boron Nuclear Magnetic Resonance Spectroscopy HPLC High Pressure Liquid Chromatography TLC Thin Layer Chromatography MS Mass Spectroscopy THF Tetrahydrofurane DMF Dimethylformamide DMSO Dimethyl Sulfoxide TMA Tetramethylammonium cation TBA Tetrabutylammonium cation

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UV-Vis Ultra Violet and Visible Light Spectroscopy rt Retention Time MPA Porphyrazine with the metal cation H2Pz Porphyrazine without the metal cation H2PA Substituted porphyrazine without metal cation Ts Tosyl group

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