An Historical Study Of Organometallic Compounds Biology Essay

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1.0 History of metallic compounds as medicinal agents: In the discovery of metal based drugs during the last century medicinal chemists took challenges to develop the safer and effective drugs. During this drug discovery process scientist has developed a highly sophisticated technology in order to predict the outcome of their research. A brief historic is presented hereafter:

1910 a landmark year:

Paul Ehrlich invented an organoarsenic compound called Salvarsan1, 4 (Arsphennamine) and used as an anti-syphilis medication2 (Syphilis is a disease caused by parasite sphirochete Treponema palladium).

Figure 1.1 First organoarsenic drug

1912:

Paul Ehrlich invented an another organoarsenic compound called Neosalvarsan4 which superseded the more toxic and less water soluble Salvarsan. Both of these drugs were easily oxidised in air and had to be sealed under inert atmosphere3.

Figure 1.2 Anti-syphilis drug

1915:

New organoarsenic drug called Mapharsen4 was invented. This is much more stable in air and widely used in the treatment of syphilis. By the discovery of Penicillin by Alexander Fleming in 1928, the use of organoarsenic drugs came into an end.

Figure 1.3 Predominantly used drug for syphilis

1965:

Barnett Rosenberg discovered the effective medicinal use of Cisplatin5, Cisplatinum, or cis-diamminedichloroplatinum(II) (CDDP) in the treatment of cancer (antitumoral properties).

Figure 1.4 Platinum-based chemotherapy drug 

Present studies:

Now a days the studies on Dichlotitanocene [Titanocene(IV)dichloride] shows that the molecule is a potent anticancer drug6. At present the studies on the mechanism of drug action is under clinical trials. It also shows antiviral and insecticidal attributes.

Figure 1.5 Titanium-based chemotherapy drug

1.1 Revolution in organometallics:

Ferrocene is an organometallic chemical compound in which two planar cyclopentadienyl rings sandwiches the central metal (Iron, Fe+2) so they are also known as sandwich compounds or metallocenes. Ferrocene was discovered and characterized in 1951. After this discovery, the rapid acceleration was found in the study of organometallic compounds.

Two scientist Ernst Otto Fisher and Geoffrey Wilkinson awarded Noble prize in chemistry 1973 for their studies in organometallics so called sandwich compounds.

1.1.1 Structure and bonding in Ferrocene:

The Iron atom in Ferrocene has +2 oxidation state. The each cyclopentadienyl (Cp) rings has a negative charge by which each cyclopentadienyl acts as a monoanionic ligand, thus the number electrons on each cyclopentadienyl ring is six. The twelve electrons, six from each cyclopentadienyl ring bonds with six d-electrons of Fe+2, making a complex having 18 electrons, noble gas electronic configuration.

The Molecular Orbital diagram best describes the bonding in Ferrocene.

Figure 1.6 Molecular Orbital diagram of Ferrocene (FeCp)

1.1.2 Reactivity and Aromaticity of Ferrocene:

Reactivity:

The chemistry of Ferrocene resembles that of Benzene. It undergoes Friedel-Crafts acylation7 and alkylation8, formylation9, sulfonation10, metalated with n-butyllithium11, 12, phenylsodium13 and mercuric acetate11.

The anionic character of the five membered rings (Cp) makes Ferrocene more

nucleophilic, so Ferrocene undergoes electrophilic substitution more readily than

Benzene. The reactivity of Ferrocene is 60 times faster than Benzene14.

Aromaticity:

The aromaticity of Ferrocene is mainly due to the effects of their C-C σ bonds and Fe lone pairs. The cyclopentadienyl rings of Ferrocene has six π electrons which are electronically analogous to benzene ring.

Thus Ferrocene can act as an effective phenyl bioisostere15.

1.1.3 Spectroscopic features:

In the 1H NMR spectrum, cyclopentadienyl (Cp) appears as a 'singlet' within a range of 4.0-4.5 ppm as the molecule shows symmetry (having equivalent chemical environiment). The 1H NMR spectrum of benzene is within a range of 7.2-7.4 ppm, appears as a singlet. Even though the 1H's of Ferrocene has 3 ppm upfield then Benzene, this difference is not the result in any decrease in the aromaticity of Ferrocene which can be explained by the fact that high electron density around the cyclopentadienyl rings results in greater shielding of ring hydrogen's.

Figure 1.7 Ferrocene Spectral features

1.1.4 Applications of Ferrocene and its derivatives:

As a new medicine:

These are used in the preparation of new drugs such as Ferrocenyl-penicillin16, Ferrocenyl-cephalosporin16, 17, Ferrocenyl hybrid of penicillin and cephalosporin16, 17, 18 and Ferroquine19.

Figure 1.7 Ferrocene an effective phenyl bioisostere in synthesizing new drugs

Organic Synthesis:

In organic synthesis Ferrocene and their derivatives are used Chiral catalyst19, 20.

Material science:

In material science these are used as a Fuel additives and as Conducting materials21, 22.

Bio-analysis:

Ferrocene derivatives are used in DNA'e-sensor23' and Glucose sensor21, 22.

By the use of sensors numerous numbers of individual tests can be done simultaneously within no time.

Figure 1.8 Schematic representation of the e-Sensor DNA Detection System.

Homogeneous Catalysis:

Ferrocene and its derivatives are used in Olefin polymerization24 and Hdroformylation20- 22.

Figure 1.9 The use of Vinyl ferrocene in the synthesis of Dendrimers.

Bioconjugates:

Ferrocene and their derivatives can conjugate with aminoacids, carbohydrates, proteins, DNA and RNA. As the Ferrocene derivatives conjugates with the DNA and RNA they can be utilized for the treatment of cancer20-22.

Figure 1.10 Ferrocene derivative as an aminoacid bioconjugator.

Chapter 2: Roles of HDACs and HAT in gene regulation

2.0 Introduction to HDACs:

The proteins which are closely colligated with DNA molecules are 'Histones'. These histones are accountable for the structure for the structure of chromatin and play measurable roles in

Gene expression

Cell cycle progression

DNA repair

Differentiation

All these cellular process are largely interceded by the enzymes such as Histone acetylase and Histone deacetylases (HDACs) 27-29.

The acetylation of histones by histone acetylase is a major modification which impacts the gene transcription. This gene transcription is controlled by the reverse action of Histone acetyltransferase (HATs). Histone deacetylase (HDACs) removes the acetyl group from hyperacetylated histones. By this deacetylation the histones return to its basal state, accompanying the suppression of gene transcription36.

Figure 2.1 Acetylation and Deacetylation of Lysine residue

2.1 Mechanism of chromatin dynamics:

In the resting cell the DNA is tightly wounded to the core histone proteins by which the RNA polymerase cannot bind with the DNA in order to perform transcription. This conformation of chromatic structure is described as closed or suppression of gene expression. To initiate the transcription, a proinflammatory transcription factors NF-kB are activated. These factors bind to the specific recognition sequence in DNA and large co-activator molecules (CREB-binding protein or CPB) which switches on gene transcription by activating histone acetyltransferase (HATs) 30-33.

Histone actyltransferase acetylates the Lysine residues present on the histones which are bounded to DNA. The acetyl moiety donor, Acetyl coenzyme A is the high energy donor for histone acetyltransferase which transfers the acetyl moiety to Ɛ-NH3+ group of the lysine residues of histone N-terminal domains. This acetyl group which is introduced to lysine neutralizes the positive charge and increases the hydrophobicity. By this consequence, the condensed chromatin (heterochromatin) is transformed into a more relaxed structure called euchromatin which is associated with greater levels of gene transcription40.

The case is reversed in the presence of histone deacetylase (HDACs) which removes the acetyl group and re-establishes the positive charge in histones39.

Figure 2.2 Conversion of heterochromatin to euchromatin39.

CREB-binding protein (CBP) interacts with several transcription factors which include cyclic AMP response element binding protein (CREB), nuclear factor (NF)-kB, activator protein (AP)-1 and signal transduction activated transcription factors (STATs). CPB have intrinsic histone acetyltransferase (HAT) activity which results in opening up of chromatin structure32, 36, and 39.

Figure 2.3 Gene repression and Gene transcription39.

2.2 HDACs Biology:

HDACs are the family of enzymes that are not only found in animals but also in plants, fungi and bacteria. The HDACs are mostly localized in the nucleus and most likely acetylates nucleosomal histones leading to transcriptional activation. Some of the HDACs are localized in the cytoplasm and their main function is to acetylated newly synthesized histones before chromatin assembly during DNA replication38.

Several transcriptional regulators have been found to possess intrinsic HAT activity.

Gcn5p: Gcn5p acts as a transcription co-activator which is required for expression of several genes. Gcn5p is a multisubunit protein complex that contains Ada1p, Ada2p, Ada3p/Ngg1p, Ada5p/spt20p. This protein complex acts as a bridge between transactivators and general transcription.

PCAF: PCAF (p300/CBP Associating Factor) is required for the transcriptional activation of many genes. PCAF assist the Gcn5p-related HAT domain to recognize nucleosomal substrate.

p300/CBP: p300 and CREB-binding protein (CBP) interacts with phosphorylated cAMP and many other transcriptional factors . It plays an important role in suppressing tumorgenesis.

SRC-1 and ACTR: SRC-1 (Steroid Receptor Coactivator-1) facilitates ligand-inducibletranscription. ACTR is essential for transcription of downstream genes.

2.3 HDACs Classification: The classification of HDACs are mainly established upon

Homology to Yeast HDACs

Cellular localization

Number of catalytic active sites

Size

Based upon the above criteria HDACs are classified into four classes in which each differ structurally, functionally and phylogenetically30, 37.

2.4 HDACs and HDACi Pharmacology:

The main application of HDACs inhibitors in pharmacology is in the treatment of cancer, and the main aim is to relate a specific tumor type with a specific gene expression profile, by which we can define the alteration responsible for each cancer.

HDAC inhibitors have a standard, modular construction with substrate acetyl-lysine and show structural similarity with that of HDAC acetyl-lysine substrate25.

HDACs inhibitor consists of a metal binding moiety, linker and capping group34, 35.

The metal binding moiety coordinates with catalytic metal atom within the HDAC active site.

Linker is structurally related to carbon chain present in acetyl-lysine substrate.

Capping group interacts with active site of the enzyme

Figure 2.4 SAHA an example of HDAC inhibitor showing capping group,

linker and metal binding moiety.

Suberoyl Anilide Hydroxamic Acid (SAHA or Vorinostat) contains

Hydroxamic acid group which coordinates with Zinc atom at the base of active

site.

Linker lies in a confined hydrophobic channel

Capping group interacts with amino acids.

Figure 2.5 Molecular mechanisms of HDAC inhibitors in anticancer effects.

Transcriptional activation in chromatin (HAT) can lead to cell growth arrest, differentiation and/or apoptosis and finally results in the inhibition of tumor growth. Transcriptional repression in chromatin (HDAC) can lead to cell growth and tumor growth. These HDAC inhibitor can directly inhibit HDAC and/or indirectly activate HAT.

2.5 Class I and II histone deacetylase inhibitors (HDACi):

Hydroxamic acid derived compounds: All these compounds have a hydroxamic- acid group in common and differ mainly in the linker and the capping groups.

Examples:

Suberoylanilide hydroxamic acid (SAHA), Trichostatin (TSA),

M-carboxycinnamic acid bis-hydroxamide(CBHA), Azelaic bis-hydroxamic acid

(ABHA), NVP-LAQ824, LBH589, Oxamflatin, PXD101, Scriptaid and

Pyroxamide.

Cyclic tetrapeptides: All these type of compounds are currently in phase II clinical trials. These are used as monotherapy and in combination therapy to treat hematological and solid malignancies.

Examples:

Depsipeptide (FK228, FR901228), Apicidine, Trapoxin, HC-toxin, Chlamydocin,

Depudesin and CHAPS.

Short-chain fattyacids: These drugs are weaker then hydroxamic acid derivatives

and cyclic tetrapeptides. These have therapeutic effects as monotherapy in myelodyspastic syndrome.

Examples:

Valproic acid (VA), Phenyl butyrate (PB), Phenyl acetate (PA), Sodium butyrate

(SB) and AN-9 (Pivanex).

Synthetic pyridyl carbamate derivative: Promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF1 1.

Example: MS-275.

Synthetic benzamide derivatives: Mediates G1 cell cycle arrest, inhibits proliferation and induces apoptosis in vitro and in vivo.

Example: CI-994 (N-acetyldinaline).

Ketones: These are non-peptide macrocyclic histone deacetylase (HDAC)

Inhibitor.

Example: α-ketomides and Trifluoromethyl ketone.

A relative wide range of structures have been identified that are able to inhibit the activity of class I, II and IV HDACs. They are derived both from natural sources and from synthetic routs. Some examples are listed below27;

Synthetic HDAC inhibitors:

Figure 2.6 Synthetic HDAC inhibitors

HDAC inhibitors from natural sources:

Figure 2.7 HDAC inhibitors from natural sources

2.6 Interactions of HDAC8 (Class I) with Inhibitor and Substrate26:

Figure 2.8 Representing TSA and Hydroxamic acid derived compounds interactions

with HDAC-8 (Class I).

Chapter-3 Microwaves in Medicinal Chemistry

3.1 Introduction to Microwave Chemistry

The field of chemical sciences where microwave irradiations are used in a chemical reaction is known as 'Microwave Chemistry'. These microwaves generate heat by rapid collisions in polar molecules in a solvent or conducting ions in a solid by acting as high frequency electrical fields. Such type of heating is called a dielectric heating where the reaction mixtures are homogenously heated without contact to a wall of the vessel.

Microwave chemistry and microwave assisted synthesis is an effective tools in medicinal chemistry41.

3.2 Microwave-assisted reactions and processes: background

The foundations of microwave technology started before World War II. Since 1970's the use of microwaves in the food industry drastically increased. During the 1980's, application of microwaves in the laboratory and industrial processes emerged. First chemical reactions were reported in the year 1986. Performing chemical reactions with a kitchen microwave created problems such as reproducibility and safety42. Limitations with a kitchen microwave was that

No temperature control

No stirring

Limitations to the power applied and

Placement of the reaction vessel.

However the use of kitchen microwave is not suitable for the conduction of chemical reaction according to GLP (good laboratory practice) requirements.

3.2.1 Conventional heating Vs microwave heating:

In a conventional heating energy is transferred via thermal conduction. The temperature on the outside surface is greater than the internal temperature (temperature in the reaction).

In a microwave heating the energy is transferred kinetically as the vessel is transparent to microwave. As the reactant mixture absorbs microwave energy, superheating occurs in the system. Microwave radiation results excitation in the molecules aligning their dipoles within the external field. Strong agitation causes an intense internal heating. The microwave heating shows a non-thermal microwave effect (microwave energy directly couples to energy modes within the molecule).

Figure 3.1 Microwave heating Vs Conventional heating

3.3 Comparison between bond energies of selected covalent bonds and the energy content of microwave photons of different frequencies:

Energy [eV]

C-C bond

3.61

C=C bond

6.35

C-O bond

3.74

C=O bond

7.71

C-H bond

4.28

O-H bond

4.80

Hydrogen bond

0.04 - 0.44

Microwaves f = 300 MHz

1.2 - 10-6

Microwaves f = 2.45 GHZ

1.0 - 10-5

Microwaves f = 300 GHz

1.2 - 10-3

Chapter 4

EXPERIMENTAL PROCEDURE, RESULTS AND DISCUSSION:

4.1 Synthesis of 6-Methylsulfanyl-1-ferrocenyl-hexan-1-one:

a.Safety:

The chemicals and solvents used in this part of the experiment are at best harmful and at worst highly toxic. It is important to work at all times in the fume cupboard and to wear rubber gloves and a lab-coat when handling the solvents and chemicals.

b.Reaction conditions:

c. General experimental procedure: 6-bromo-1-oxohexyl ferrocene (0.184g, 0.51mmol), sodiumthiomethoxide (0.04g,0.57mmol) and tetrahydronfuran (3 mL) was subjected to microwave irradiation by ramping to 150oC and held at that temp. for 20 min at 150 W,

1 atm. The crude of the reaction mixture was extracted with ethyl acetate (30 mL) and washes with deionised water (20 mL) and brine (3x20 mL), the organic layer is dried using magnesium sulphate and concentrated under reduced pressure, brown oil (0.125g, 87%).

d.Results and Discussion:

δ1H NMR (δ, 270.0MHz, CDCl3); 1.48-1.80(6H,m,3xCH2), 2.11(3H,s,SCH3), 2.53(2H,t,J=8.10Hz,CH2), 2.71(2H,t,J=8.10Hz,CH2),4.20(5H,s,Fc), 4.49(2H,t,J2.70Hz,Fc),4.79(2H,t,J2.70Hz,Fc); δC13NMR (δ,67MHz,CDCl3); 24.12, 28.75, 29.10, 30.67, 39.53, 69.31, 69.74, 72.11, 77.01, 116.27, 157.17. HRMS (m/z, HNESP); C17H22FeOS [M+H] + = Calc. 330.27 observed. 331.0816.

Sodiumthiomethoxide generates the Na+ and MeS-. This MeS- acts on the CH2Br group present on (1) and liberates Br- giving raise to 6-Methylsulfanyl-1-ferrocenyl-hexan-1-one. The Br- and Na+ forms a salt NaBr which can be removed during the aqueous washes. The percentage purity of the compound is 92% by HPLC.

4.2 Synthesis of 1-Ferrocenyl-6-(pyrimidin-2-ylsulfanyl)-hexan-1-one.

a.Safety:

The chemicals and solvents used in this part of the experiment are at best harmful and at worst highly toxic. It is important to work at all times in the fume cupboard and to wear rubber gloves and a lab-coat when handling the solvents and chemicals.

b.Reaction conditions:

c.General experimental procedure:

In a dried microwave tube 2-mercaptopyrimidine (61mg,0.55mmol), sodiumhydride (24mg, 0.6mmol) and tetrahydrofuran (2mL) are added and maintained for 5hrs at room temperature. To this reaction mixture 6-bromo-1-oxohexylferrocene (180mg, 0.49mmol) and DMF (1mL) are added and subjected to microwave irradiation by ramping to 180oC and held at that temp. for 60 mins at 150W, 1 atm. pressure. The process of this reaction was monitored by TLC. After total consumption of (1), the crude of reaction mixture was extracted with ethyl acetate (50 mL) and washed with deionised water (15mL) and brine (2x15mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Dark brown crude (134mg, 80%).

d.Results and Discussion:

δ H 1HNMR (δ,270 MHz, CDCl3); 0.81(2H,m,CH2), 1.20(2H,m,CH2), 1.74(2H,m,CH2), 2.65(2H,t,CH2), 3.14(2H,t,CH2), 4.10(5H,s,Cp), 4.43(2H,t,J2Hz,Cp), 4.72(2H,t,J2Hz,Cp), 6.8(1H,t,Mc), 8.44(2H,s,Mc);

δC 13C NMR(δ,68MHz, CDCl3); 23.12, 27.75,28.10, 29.67, 38.54, 68.32, 68.74, 71.11, 100.00, 155.27, 156.17, 210.12;

HRMS (m/ z, HNESP) [M+H]+ for C20H22N2OSFe= Calc. 394.30 observed. 395.0877.

Sodiumhydride reacts with 2-mercaptopyrimidine, liberates hydrogen gas and forms a sodium salt of marcaptopyrimidine, this reacts with (1) under microwave conditions givs raise to 1-Ferrocenyl-6-(pyrimidin-2-ylsulfanyl)-hexan-1-one.The percentage purity of the compound is 90% by HPLC.

4.3 Synthesis of 2-(6-Ferrocenyl-hexylsulfanyl)-pyrimidine:

a.Safety:

The chemicals and solvents used in this part of the experiment are at best harmful and at worst highly toxic. It is important to work at all times in the fume cupboard and to wear rubber gloves and a lab-coat when handling the solvents and chemicals.

b.Reaction conditions:

c.General experimental procedure:

In a dried microwave tube 2-mercaptopyrimidine (61mg, 0.55mmol), sodiumhydride (24mg,0.6mmol) and tetrahydrofuran (2mL) are added and maintained for 5hrs at room temperature. To this reaction mixture 6-bromohexylferrocene(150mg,0.43mmol) and DMF(1mL) are added and subjected to microwave irradiation by ramping to 180oC and held at that temp. for 60 mins at 150W, 1 atm. pressure. The process of this reaction was monitored by TLC. After total consumption of (8), the crude of reaction mixture was extracted with ethyl acetate (50 mL) and washed with demonized water (15mL) and brine (2x15mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Dark brown crude (129mg, 80%).

d.Results and Discussion:

δH 1HNMR (δ,270 MHz, CDCl3); 0.88(2H,m,CH2), 1.46(2H,m,CH2), 1.51(2H,m,CH2), 1.74(2H,m,CH2), 2.32(2H,t,CH2), 3.17(2H,t,CH2), 4.03(2H,t,J2Hz,Cp), 4.04(2H,t,J2Hz,Cp), 4.08(5H,s,Cp), 6.95(1H,t,Mc), 8.50(2H,s,Mc); δC 13C NMR(δ,76MHz, CDCl3); 28.74, 29.07, 29.13, 29.05, 30.84, 30.97, 67.00, 68.04, 68.44, 89.35, 116.22, 157.14.

HRMS (m/ z, HNESP) [M+H]+ for C20H24N2SFe= Calc. 380.33 observed. 381.1082.

Sodiumhydride reacts with 2-mercaptopyrimidine, liberates hydrogen gas and forms a sodium salt of marcaptopyrimidine, this reacts with (8) under microwave conditions givs raise to 2-(6-Ferrocenyl-hexylsulfanyl)-pyrimidine. The percentage purity of the compound is 88% by HPLC.

4.4 Synthesis of

1-(6-Oxo-6-Ferrocenyl-hexyl)-1-H-[1,2,3]triazole-4-carboxylicacid hydroxamide.

a.Safety:

The chemicals and solvents used in this part of the experiment are at best harmful and at worst highly toxic. It is important to work at all times in the fume cupboard and to wear rubber gloves and a lab-coat when handling the solvents and chemicals.

b.Reaction conditions:

c.General experimental procedure:

(i).Synthesis of (10).

In a dried RBF 6-bromo-1-oxohexylferrocene (363mg,1mmol), sodium azide (130mg, 2mmol) and DMF (2.5mL) are taken and the reaction mixture should be warmed to 90oC for approximately 90mins. The process of this reaction was monitored by TLC. After total consumption of (1), the crude of reaction mixture was extracted with ethyl acetate (75 mL) and washed with deionised water (25mL) and brine (4x25mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Orange oil (296mg, 91%).

Results and Discussion:

δH 1HNMR (δ,400 MHz, CDCl3); 0.89(2H,m,CH2), 1.30(2H,m,CH2), 1.72(2H,m,CH2), 2.70(2H,J7.1Hz,t,CH2), 3.30(2H,J7.3Hz,t,CH2), 4.15(5H,s,Cp), 4.50(2H,t,J4Hz,Cp), 4.77(2H,t,J4Hz,Cp);

δC 13C NMR(δ,400MHz, CDCl3); 23.85, 26.55, 28.76, 39.30, 51.23, 69.22, 69.67, 72.11, 80.00, 204.03.

Sodiumazide produces N3- anion which replaces the Br - from (1) forming (10). The Br- and Na+ forms a salt NaBr which can be removed during the aqueous washes. The percentage purity of the compound is 98% by HPLC and gave excellent yield.

(ii).Synthesis of (11).

To this crude methylpropiolate (77mg,0.92mmol), 1:1 t-BuOH/H2O(1:1mL), N-N-diisopropylethylamine(0.18mL,1.01mmol) and copper iodide (80mg,0.4mmol) were added and the reaction mixture should be warmed to 60oC for approximately 60mins. The process of this reaction was monitored by TLC. After total consumption of (10), the crude of reaction mixture was quenched with saturated ammonium hydroxide, extracted with ethyl acetate (75 mL) and washed with deionised water (25mL) and brine (3x25mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Yellowish solid (300mg, 77%).

Results and Discussion:

δH 1HNMR (δ,300 MHz, MeOH-d); 1.40(2H,m, CH2), 1.71(2H,m, CH2), 1.98(2H,m, CH2), 2.76(2H,J7.2Hz,t, CH2), 3.89(3H,S,OCH3), 4.19(5H,s,Cp), 4.56(2H,J7.5Hz,t,CH2), 4.79(2H,t,J2.1Hz,Cp), 4.85(2H,t,J2.1Hz,Cp), 8.67(1H,s,CH);

δC 13C NMR (δ,300MHz,MeOH-d); 24.81, 27.15, 30.99, 40.14, 51.49, 52.00, 70.54, 70.96, 73.78, 79.89, 129.63, 140.38, 162.37, 207.35

HRMS (m/z, HNESP) [M+H]+ for C20H23N3O3Fe= Calc. 409.26, observed. 410.1162.

The alkynyl methyl ester reacts with azide forming a 1,2,3-triazole with a methyl ester. The proton on the triazole appears at a downfield as a singlet. The percentage purity of the compound is 93% by HPLC.

(iii)Synthesis of (12).

In a oven dried RBF hydroxylamine hydrochloride (14g, 200mmol), potassium hydroxide (12g, 200mmol) should be added slowly to methanol(150mL) and the reaction mixture should be warmed to 40oC for approximately 20 mins. After this time has elapsed the reaction mixture should then be cooled to 0oC. The solid residue should then be filtered using a Buchner funnel. The actual filtrate should then be added to methyl ester precursor (300mg, 0.91mmol), potassium hydroxide (1.2g) and stirred at room temperature for 24 hours. The reaction mixture is washed with saturated ammonium chloride and extracted with ethyl acetate(75mL) and washed with deionised water (25mL) and brine (3x25mL), the organic layer is dried with magnesium sulphate. Crashed out using ethyl acetate/hexane 1:25 (160mg, 53%).

Results and Discussion:

δH 1HNMR (δ,400 MHz, DMSO-d6); 1.33(2H,m,CH2), 1.56(2H,m,CH2), 1.89(2H,m,CH2), 3.30(2H,J7.4Hz,t,CH2), 4.13(5H,s,Cp), 4.29(2H,t,J4Hz,Cp), 4.40(2H,t,6.96Hz,,CH2), 4.49(2H,t,J4Hz,Cp), 8.53(1H,s,OH), 9.01(1H,s,CH), 11.23(1H,s,NH);

δC 13C NMR(δ,500MHz,MeOH-d); 27.58, 28.16, 30.68, 30.93, 67.57, 70.16, 70.51, 82.38, 127.12, 142.15, 160.36, 160.67, 210.10.

Anal.calc: C,55.63; H,5.4; N,13.66. Found C,54.45; H,5.74; N,14.44.

HRMS (m/ z, HNESP) [M+H]+ for C19H22N4O3Fe= Calc. 410.25, observed= 411.1727.

The methyl ester reacts with hydroxylamine hydrochloride forming hydroxamic acid. The yield was low and the purity of the compound was good, 89% by HPLC.

4.5 Synthesis of 1-(6-Ferrocenyl-hexyl)-1H-[1,2,3]triazole-4-carboxylicacid

hydroxamide:

a.Safety:

The chemicals and solvents used in this part of the experiment are at best harmful and at worst highly toxic. It is important to work at all times in the fume cupboard and to wear rubber gloves and a lab-coat when handling the solvents and chemicals.

b.Reaction conditions:

c.General experimental procedure:

(i) Synthesis of (13).

In a dried RBF 6-bromohexylferrocene (350mg,1mmol), sodium azide (130mg,2mmol) and DMF (2.5mL) are taken and the reaction mixture should be warmed to 90oC for approximately 90mins. The process of this reaction was monitored by TLC.

After total consumption of (8), the crude of reaction mixture was extracted with ethyl acetate (75 mL) and washed with deionised water (25mL) and brine (4x25mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Orange oil (283mg, 91%).

Results and Discussion:

δH 1HNMR (δ,400 MHz, CDCl3); 1.35(2H,m,CH2), 1.58(2H,m,CH2), 1.60(2H,m,CH2), 2.16(2H,m,CH2), 2.32(2H,J7.1Hz,t,CH2), 3.26(2H,J7.4Hz,t,CH2), 4.02(2H,t,J4Hz,Cp), 4.04(2H,t,J4Hz,Cp), 4.08(5H,s,Cp);

δC 13C NMR(δ,400MHz, CDCl3); 26.40, 28.61, 28.87, 29.28, 30.80, 51.20, 66.86, 67.85, 68.27, 88.94;

Sodiumazide produces N3- anion which replaces the Br - from (8) forming (13). The Br - and Na+ forms a salt NaBr which can be removed during the aqueous washes. The percentage purity of the compound is 98% by HPLC and gave excellent yield.

(ii) Synthesis of (14).

To this crude methylpropiolate (77mg,0.92mmol), 1:1 t-BuOH/H2O (1:1mL),

N-N-diisopropylethylamine (0.18mL, 1mmol) and copper iodide (80mg,0.4mmol) were added and the reaction mixture should be warmed to 60oC for approximately 60mins. The process of this reaction was monitored by TLC. After total consumption of (13), the crude of reaction mixture was quenched with saturated ammonium hydroxide, extracted with ethyl acetate (75mL) and washed with deionised water (25mL) and brine (3x25mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo.

Yellowish solid (288mg, 80%).

Results and Discussion:

δH 1HNMR (δ,400 MHz, CDCl3); 1.20(2H,m,CH2), 1.33(2H,m,CH2), 1.47(2H,m,CH2), 1.92(2H,m,CH2), 2.30(2H,J7.3Hz,t,CH2), 3.13(2H,J7.4Hz,t,CH2), 3.86(3H,s,OCH3), 3.95(2H,t,J4Hz,Cp), 4.02(2H,t,J4Hz,Cp), 4.07(5H,s,Cp), 8.05(1H,s,CH); HRMS (m/ z, HNESP) [M+H]+ for C20H25N3O2Fe= Calc. 395.25 observed. 396.1371.

The alkynyl methyl ester reacts with azide forming a 1,2,3-triazole with a methyl ester. The proton on the triazole appears at a downfield as a singlet. The percentage purity of the compound is 90% by HPLC.

(iii) Synthesis of (15).

In a oven dried RBF hydroxylamine hydrochloride (14g, 200mmol), potassium hydroxide (12g, 200mmol) should be added slowly to methanol(150mL) and the reaction mixture should be warmed to 40oC for approximately 20 mins. After this time has elapsed the reaction mixture should then be cooled to 0oC. The solid residue should then be filtered using a Buchner funnel. The actual filtrate should then be added to methyl ester precursor (300mg, 0.91mmol), potassium hydroxide (1.2g) and stirred at room temperature for 24 hours. The reaction mixture is washed with saturated ammonium chloride and extracted with ethyl acetate (75mL) and washed with deionised water (25mL) and brine (3x25mL), the organic layer is dried with magnesium sulphate. Crashed out using ethyl acetate/hexane 1:25(152mg, 53%).

Results and Discussion:

δH 1HNMR (δ,400 MHz, DMSO-d6); 1.11(2H,m,CH2), 1.22(2H,m,CH2), 1.43(2H,m,CH2), 1.82(2H,m,CH2), 2.25(2H,J7.4Hz,t,CH2), 4.01(2H,t,J4Hz,Cp), 4.05(2H,t,J4Hz,Cp), 4.07(5H,s,Cp), 4.38(2H,J7.8Hz,t,CH2), 8.53(1H,s,CH), 9.01(1H,s,OH), 11.21(1H,s,NH);

δC 13C NMR(δ,500MHz,MeOH-d); 27.25, 29.84, 30.10, 31.17, 32.01 49.43, 68.06, 69.08, 69.40, 127.07, 210.01;

Anal.calc: C,57.59; H,6.10; N,14.14. Found C,58.24; H,6.63; N,13.63.

HRMS (m/ z, HNESP) [M+H]+ for C19H24N4O2Fe= Calc. 396.26 observed. 397.1325.

The methyl ester reacts with hydroxylamine hydrochloride forming hydroxamic acid. The yield was low and the purity of the compound was good, 92% by HPLC.

4.6 Synthesis of Click compounds by using 6-Bromo-1-oxohexylferrocene as SM:

a.Safety:

The chemicals and solvents used in this part of the experiment are at best harmful and at worst highly toxic. It is important to work at all times in the fume cupboard and to wear rubber gloves and a lab-coat when handling the solvents and chemicals.

b.Reaction conditions:

c.General experimental procedure:

(i).Synthesis of (10).

In a dried RBF 6-bromo-1-oxohexylferrocene (363mg, 1mmol), sodium azide (130mg, 2mmol) and DMF (2.5mL) are taken and the reaction mixture should be warmed to 90oC for approximately 90mins. The process of this reaction was monitored by TLC. After total consumption of (1), the crude of reaction mixture was extracted with ethyl acetate (75 mL) and washed with deionised water (25mL) and brine (4x25mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Orange oil (296mg, 91%).

Results and Discussion:

δH 1HNMR (δ,400 MHz, CDCl3); 0.89(2H,m,CH2), 1.30(2H,m,CH2), 1.72(2H,m,CH2), 2.70(2H,J7.1Hz,t,CH2), 3.30(2H,J7.3Hz,t,CH2), 4.15(5H,s,Cp), 4.50(2H,t,J4Hz,Cp), 4.77(2H,t,J4Hz,Cp);

δC 13C NMR(δ,400MHz, CDCl3); 23.85, 26.55, 28.76, 39.30, 51.23, 69.22, 69.67, 72.11, 80.00, 204.03.

Sodiumazide produces N3- anion which replaces the Br - from (1) forming (10). The Br- and Na+ forms a salt NaBr which can be removed during the aqueous washes. The percentage purity of the compound is 98% by HPLC and gave excellent yield.

(ii)Synthesis of (18):

To (10) propargrylbenzoate (76mg,0.47mmol), 1:1 tBuOH/H2O (0.5:0.5 mL), N-N-diisopropylethylamine (0.08mL,0.46mmol) and copper iodide (40mg,0.2mmol) were added and the reaction mixture should be warmed to 60oC for approximately 60 mins. The process of this reaction was monitored by TLC. After total consumption of (10), the crude of reaction mixture was quenched with saturated ammonium hydroxide, extracted with ethyl acetate (50 mL) and washed with deionised water (15mL) and brine (3x15mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Yellowish solid (198mg, 89%).

Results and Discussion:

δH 1HNMR (δ,300 MHz, CDCl3); 1.4(2H,m,CH2), 1.73(2H,m,CH2), 2.0(2H,m,CH2), 2.26(2H,t,CH2), 4.14(5H,s,Cp), 4.26(2H,J2.1Hz,t, CH2), 4.46(2H,J2.1Hz,t, CH2), 4.73(2H,t,CH2), 5.45(2H,s,CH2), 6.7(1H,s,CH), 7.53(5H,s,Ph).

MS (ESI +ve) m/z Calc= 485.35, observed= 486.3 [M+H]+

The alkynyl group of propargrylbenzoate reacts with azide forming a 1,2,3-triazle. The proton on the triazole appears at a downfield as a singlet. The percentage purity of the compound is 94% by HPLC.

(iii)Synthesis of (19):

To this crude Propargrylamine (26mg,0.47mmol),1:1 tBuOH/H2O (0.5:0.5 mL), N-N-diisopropylethylamine (0.09mL,0.5mmol) and copper iodide (40mg,0.2mmol) were added and the reaction mixture should be warmed to 60oC for approximately 60 mins. The process of this reaction was monitored by TLC. After total consumption of (10), the crude of reaction mixture was quenched with saturated ammonium hydroxide, extracted with ethyl acetate (50 mL) and washed with deionised water (15mL) and brine (3x15mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Brown crude (149mg, 88%).

Results and Discussion:

δH 1HNMR (δ,300 MHz, CDCl3); 0.82(2H,m,CH2), 1.21(2H,m,CH2), 1.32(2H,m,CH2), 1.65(2H,t,CH2), 1.9(2H,t,CH2), 2.3(2H,t,NH2), 2.64(2H,J2.1,t,Cp), 4.12(5H,s,Cp), 4.43(2H,J2.1,t,Cp), 4.70(2H,s,CH2), 6.1(1H,s,CH).

MS (ESI +ve) m/z Calc= 380.26, observed= 381.2 [M+H]+

The alkynyl group of propargrylamine reacts with azide forming a 1,2,3-triazle. The proton on the triazole appears at a downfield as a singlet. The percentage purity of the compound is 84% by HPLC.

4.7 Synthesis of Click compound by using 6-Bromohexylferrocene as SM:

a.Safety:

The chemicals and solvents used in this part of the experiment are at best harmful and at worst highly toxic. It is important to work at all times in the fume cupboard and to wear rubber gloves and a lab-coat when handling the solvents and chemicals.

b.Reaction conditions:

c.General experimental procedure:

(i).Synthesis of (13).

In a dried RBF 6-bromohexylferrocene (210mg,0.6mmol), sodium azide (78mg,1.2mmol) and DMF (1.5 mL) are taken and the reaction mixture should be warmed to 90oC for approximately 90 mins. The process of this reaction was monitored by TLC. After total consumption of (8), the crude of reaction mixture was extracted with ethyl acetate (50 mL) and washed with deionised water (15mL) and brine (4x15mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Orange oil (148mg, 89%).

Results and Discussion:

δH 1HNMR (δ,400 MHz, CDCl3); 1.35(2H,m,CH2), 1.58(2H,m,CH2), 1.60(2H,m,CH2), 2.16(2H,m,CH2), 2.32(2H,J7.1Hz,t,CH2), 3.26(2H,J7.4Hz,t,CH2), 4.02(2H,t,J4Hz,Cp), 4.04(2H,t,J4Hz,Cp), 4.08(5H,s,Cp);

δC 13C NMR(δ,400MHz, CDCl3); 26.40, 28.61, 28.87, 29.28, 30.80, 51.20, 66.86, 67.85, 68.27, 88.94;

Sodiumazide produces N3- anion which replaces the Br - from (8) forming (13). The Br - and Na+ forms a salt NaBr which can be removed during the aqueous washes. The percentage purity of the compound is 98% by HPLC and gave excellent yield.

(ii).Synthesis of (20):

To (13) Propargrylamine (26mg,0.47mmol),1:1 tBuOH/H2O (0.5:0.5 mL), N-N-diisopropylethylamine (0.09mL,0.5mmol) and copper iodide (40mg,0.2mmol) were added and the reaction mixture should be warmed to 60oC for approximately 60 mins. The process of this reaction was monitored by TLC. After total consumption of (13), the crude of reaction mixture was quenched with saturated ammonium hydroxide, extracted with ethyl acetate (50 mL) and washed with deionised water (15mL) and brine (3x15mL), the organic layer is dried with magnesium sulphate and concentrated in vacuo. Brown crude (149mg, 86%).

Results and Discussion:

δH 1HNMR (δ,400 MHz, CDCl3); 1.20(2H,m,CH2), 1.33(2H,m,CH2), 1.47(2H,m,CH2), 1.92(2H,m,CH2), 2.43(2H,t,NH2), 2.73(2H,t,CH2), 2.99(2H,t,CH2), 3.76(2H,t,CH2), 4.0(2H,J7.4Hz,t,CH2), 4.13(2H,J7.4Hz,t,CH2), 4.37(5H,s,Cp) 5.7(1H,s,CH).

MS (ESI +ve) m/z Calc= 366.28, observed= 367.2 [M+H]+.

The alkynyl group of propargrylamine reacts with azide forming a 1,2,3-triazle. The proton on the triazole appears at a downfield as a singlet. The percentage purity of the compound is 88% by HPLC.

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