The body is made of many types of cells. In order to keep the body healthy and to function properly, cells grow, divide and produce more cells. In the adult male, the number of cells that are produced is usually balanced by the number of cells that die. In certain circumstances, the process of cell replacement may become error prone and cells become abnormal and divide in an uncontrolled way. These excess cells can therefore lead to the formation of a mass of tissue, called a tumour. Tumours can be benign or malignant; benign tumours do not spread from their original site, whereas malignant tumours (which are also known as cancers), spread (or metastasise) either by direct extension into surrounding tissues, or via the circulatory system i.e. the lymphatic or the blood systems. Malignant tumours have the ability to invade and damage surrounding tissues and organs (Page et al, 2002).
1.1 Prostate
The prostate is a male sex gland, which produces the bulk of the male ejaculate. It is approximately the size of a walnut and is located at the base of the bladder and in front of the rectum; it surrounds the upper part of the urethra (Figure 1). The prostate is a hormone-dependent organ, more specifically, it relies on androgens for its growth (Soronen et al, 2004).
Figure 1: Anatomy of the prostate (www.health.uab.edu)
1.2 Prostate cancer
Prostate cancer is one of the most common forms of cancer among men in the western world. According to the American Cancer Society, approximately 230,110 new cases of prostate cancer were diagnosed and 29,900 men died of the disease in 2004. Prostate cancer is the second leading cause of cancer deaths of men in the United States, after lung cancer, and the sixth leading cause of death of men (American Cancer Society, 2004).
1.2.1 Risk factors
The risk of carcinoma of the prostate has been linked with: age, ethnicity, family history, genetic susceptibility, hormonal and dietary factors.
1.2.1.1 Age
Prostate cancer has been shown to have a close link with age and the incidence rises with increased age. Only 1-2% cases of prostate cancers are diagnosed at less than 55 years of age, however men over the age of 50 are at high risk of prostate cancer. The evidence of prostate malignancy has been reported as 20% of men aged 50 to 60 years and 50% of those aged 70 to 80 years (Carter et al, 1990; Horwich, 1995).
1.2.1.2 Ethnicity
The mortality rates vary among different ethnic groups and countries. It has been reported that Black Americans are at the highest risk of prostate cancer in the world and Asians are at the lowest, however the morbidity and mortality rates increase for Asian immigrants to the western countries (American Cancer Society, 2004).
1.2.1.3 Family history and genetic susceptibility
A positive family history has been shown to be the strongest epidemiological risk factor for prostate cancer. The risk of prostate cancer in men with a father or brother affected by the malignancy is twice that of those who have no family history of prostate cancer; in addition, the risk of prostate cancer increases with increasing number of affected family members (Steinberg et al, 1990).
There have been seven candidate genes for prostate cancer that have been identified but their role in the development of prostate cancer is not fully understood (Simard et al, 2002). Among candidate genes, the activity of androgen receptor genes is governed by the length of CAG repeat units in the androgen receptor. The CAG sequence varies in length from 11 to 31 repeat in healthy men. Studies have suggested that an increased risk of prostate cancer is related to shorter CAG repeats (Crawford, 2003). SRD5A2 is another candidate gene for prostate cancer, which encodes the enzyme 5α-reductase (type 2) that catalyses the conversion of testosterone into the more potent androgen dihydrotestosterone. The catalytic activity of the enzyme is governed by the Ala49Thr variant and thus causes an increased risk of prostate cancer (Makridakis et al, 1999).
1.2.1.4 Diet
Diet is also an important risk factor in prostate cancer development, however, it should be noted that diet is not the only factor but is one of the important factors which play a role in the development of prostate cancer. Epidemiology studies have shown that diet with high fat content, in particular red meat and dairy product consumption can have a major impact on prevalence of prostate cancer (Whittemore et al, 1995). Beef and dairy products are a major source of dietary branched fatty acids; these fatty acids are oxidised by α-methyl-coenzyme-M-reductase, an enzyme that plays a key role in the peroxisomal oxidation of these fatty acids, and which has been found in prostate cancer patients only. It is hypothesised that hydrogen peroxide is generated by the oxidation, and which leads to the carcinogenic oxidative damage of the prostate (Gronberg, 2003).
The low incidence of prostate cancer in Japan compared with the U. S. A. may be due to difference in soybean products intake, which are a rich source of isoflavone. Isoflavone can inhibit protein tyrosine kinases that are important in cell proliferation and transformation and thus can limit the development and metastasis of prostate cancer (Tomoyuki, et al, 2002).
1.2.1.5 Hormonal and other factors
Luteinising hormone is produced by the stimulation of the pituitary gland by luteinising hormone releasing hormone, which is responsible for a cascade of events in the production of androgens which are derived from cholesterol (Figure 2). As mentioned earlier, the growth of the prostate is hormone-dependent or more specifically androgen-dependent. Androgens, for example testosterone and the more potent dihydrotestosterone, stimulate and control the development and maintenance of male characteristics as well as playing a crucial role in the development of prostate cancer (Hsing, 2001; Swerdloff et al, 2004).
Figure 2: Steroidal cascade for the biosynthesis of androgens.
1.3 Benign Prostatic hyperplasia
Hyperplasia is a general term referring to excess cell replication. Benign prostatic hyperplasia is the non-cancerous growth of the prostate; because the prostate gland surrounds the urethra, this abnormal growth most often results in lower urinary tract symptoms (Thrope et al, 2003). Benign prostatic hyperplasia is the most common non-cancerous growth of cells in men (Berry et al, 1984). Age and androgens for example testosterone and the more potent dihydrotestosterone, are major factors that cause benign prostatic hyperplasia (Oesterling, 1998; Montie et al, 1994).
1.4 5α-Reductase
The enzyme 5α-reductase is a membrane bound nicotinamide adenine dinucleotide phosphate (NADPH)-dependent protein which catalyses the conversion of testosterone into the more potent androgen dihydrotestosterone (Figure 3). Dihydrotestosterone is the androgen with the highest affinity for the androgen receptors within androgen-dependent cells and is primarily responsible for the effect of androgens in androgen-dependent organs.
Two isozymes of 5α-reductase have been cloned, expressed and characterised; the two isozymes differ in pH optima, sensitivity to inhibitors and tissue distribution (Jenkins et al, 1992, Ernesto et al, 2004). The type-2 isozyme is found predominantly in genital skin, male accessory sex glands, and in the prostate, including benign prostatic hyperplasia and prostate adenocarcinoma tissues; whilst the type 1 isozyme is found in non-genital skin, in the liver and in the brain (Melcangi et. al, 1998; Thigpen et al, 1993).
The normal activity of 5α-reductase routinely maintains testosterone mediated biological functions, such as anabolic actions and spermatogenesis of humans as well as dihydrotestosterone mediated effects such as increased facial and body hair, acne and prostate enlargement (Li et. al, 1995).
Testosterone Dihydrotestosterone
Figure 3. Conversion of Testosterone to Dihydrotestosterone
.
1.4.1 Mechanism of 5α-reductase
The catalytic conversion of testosterone to dihydrotestosterone involves direct donation of hydride from NADPH to the C5 position of testosterone, leading to formation of an enolate that would be stabilised by some electrophilic residue. Then enzyme mediated tautomerism leads to the product dihydrotestosterone with release of NADP+. The 5α-hydrogen of dihydrotestosterone was originated from the 4-pro-S proton of the nicotinamide ring (Figure 4) (Li et. al, 1995; Abul-Hajj, 1972).
Figure 4: Where E is enzyme, NADPH is adenine dinucleotide phosphate and DHT is dihydrotestosterone.
1.5 5α-reductase inhibitors
As mentioned earlier, prostate is an androgen-dependent organ, and testosterone and dihydrotestosterone are the two major androgens which play a crucial role in the development of prostate cancer. Abnormally high 5α-reductase activity in humans results in excessive dihydrotestosterone levels and can play a role in development of prostate caner. The use of 5α-reductase inhibitors can be a logical treatment and preventive action for prostate cancer and benign prostatic hyperplasia. The study of inhibition of 5α-reductase has lasted more than two decades; consequently, numerous non-steroidal and steroidal compounds have been designed and synthesised as inhibitors of this enzyme.
According to the kinetic mechanism of testosterone reduction catalysed by the enzyme 5α-reductase, three different types of inhibitors could be conceived: inhibitors competitive with NADPH and testosterone (type A) should interact with the free enzyme; inhibitors competitive with testosterone (type B) should fit the enzyme-NADPH complex, and inhibitors fitting the enzyme-NADP+ (type C) should be uncompetitive versus testosterone.
1.5.1 Steroidal inhibitors of 5α-reductase
Steroidal inhibitors are an important class of compounds which show inhibitory activity against 5α-reductase. Several different types of compound have been reported, including natural, as well as synthetic, steroidal compounds (Voigt et al, 1973; Frye, 1996).
i. Natural steroidal inhibitors
Among the natural steroidal inhibitors of 5α-reductase, progesterone (Figure 5) was reported as the most potent inhibitor of 5α-reductase (type B), which effectively inhibited the enzyme by 93% with a Ki approximately equal to 0.7 μM (Frye, 1996). Several other steroids from natural sources including deoxycorticosterone, estradiol and hydrocortisone were assayed for enzyme inhibitory activity and it was found that estradiol (Figure 6) and other steroids resembling estradiol had no inhibitory effect (Voigt et al, 1970). It was hypothesised that the necessary structural features for inhibition of the enzyme are 3-keto-Δ4 structure and a 17β-side chain containing one or more oxygen functional groups (Voigt et al, 1973).
Figure 5: Progestrone
Figure 6: Estradiol
4-Androsten-3-one-17β-carboxylic acid (Figure 7), a metabolite of deoxycorticosterone, has been reported as a potent natural steroidal competitive inhibitor (type B) of 5α-reductase with 88% inhibitory activity and no known hormonal activity (Voigt et al, 1973; Li et al, 1995).
Figure 7: -Androsten-3-one-17β-carboxylic acid
ii. 4-Azasteroid inhibitors
Azasteroids are another important class of steroidal inhibitors of 5α-reductase. Among the 4-azasteroids, 17β-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (DMAA) (Figure 8), is a potent irreversible inhibitor of 5α-reductase. The DMAA is competitive with testosterone with Ki equal to 5nM for rat prostatic 5α-reductase. DMAA has moderate affinity for the rat prostate androgen receptor with IC50 equal to 3μM. DMAA inhibits both membrane bound as well as solubilised 5α-reductase (Liang et al, 1981). Because of the reported hepatotoxicity DMAA was not clinically investigated for the inhibition of 5α-reductase (Kedderis et al, 1990).
Figure 8: DMAA
A series of 4-azasteroids has been prepared and tested for the inhibition of human and rat prostatic 5α-reductase. High inhibitory activity has been reported with a group of 17β-substituted 4-methyl-4-aza-5α-androstan-3-ones (Figure 9) (Rasussan et al, 1984). The potency of the 17β-substituted 4-methyl-4-aza-5α-androstan-3-ones is based upon the ability of the A-ring amide to interact with 5α-reductase and thought to mimic the enolate like transition state. Enhanced inhibition of 5α-reductase has been reported with 4-N containing alkyl substitutions and on incorporation of lipophilic substituted semipolar groups at C17, on 4-azasteroids (Rasussan et al, 1986; Liang et al, 1984).
Figure 9: 4-Azasteroids inhibitors of 5α-reductase.
Finasteride [17β-(N-tert-butylcarbamoyl)-4-aza-5α-androstan-1-en-3-one] (Figure 10) has been used for the treatment of benign prostatic hyperplasia. Finasteride was the first 5α-reductase inhibitor approved for the treatment of benign prostatic hyperplasia (Tian et al, 1994). It has been reported that finasteride significantly decreases the levels of serum as well as in-prostatic dihydrotestosterone. In addition it decreases the size of the prostate (Kaplan, 2001). Finaseteride is a selective inhibitor of human 5α-reductase type 2 with a reported IC50 equal to 0.18nM, Ki equal to 69nM and is a weak inhibitor of human 5α-reductase type 1 with a reported Ki equal to 300nM (Frye, 1996; Ernesto et al, 2004).
Figure 10: Finasteride
Dutasteride [17β- N-(2,5-bis(trifluoromethyl)phenyl)-4-aza-5α-androstan-1-en-3-one] (Figure 11) is another potent competitive 4-azasteroidal inhibitor of 5α-reductase. Dutasteride is the first compound with inhibitory activity for both isozymes of 5α-reductase (Roehrborn et al, 2002). The Ki values for human 5α-reductase type 1 and 2 are 2.4nM and 0.5nM respectively. In comparison to finasteride, dutasteride is 60 fold more potent in its initial Ki versus 5α-reductase type1 (Frye, 1996).
Figure 11: Dutasteride
iii. 6-Azasteroids inhibitors:
6-Azasteroids (Figure 12) are competitive inhibitors of 5α-reductase. 6-Azasteroids represent a more substrate like mimic of the transition state catalysed by 5α-reductase than the 4-azasteroids since they retain the Δ4 unsaturation (Frye, 1996).
Figure 12: 6-Azasteroidal inhibitors
iv. 3-Carboxysteroid inhibitors
3-Carboxysteroidal inhibitors are an important class of steroidal inhibitors of 5α-reductase. These inhibitors mimic the enolate-like transition state of conversion of testosterone to dihydrotestosterone catalysed by 5α-reductase, by incorporating a sp2-hybridised centre at C-3 and C-4 and most critically, an anionic carboxylic acid at C-3 as a charge replacement for the enolate oxyanion, showing uncompetitive kinetics (Li et al, 1995). A number of 3-carboxysteroidal inhibitors (Figure 13) of 5α-reductase has been synthesised and showed potency for inhibition of 5α-reductase (Frye, 1996; Holt et al, 1990).
Figure 13: 3-Carboxysteroids
1.5.2 Non-steroidal inhibitors of 5α-reductase
Due to potential undesired hormonal action exhibited by steroidal compounds, researchers focused their research in synthesising non-steroidal inhibitors of 5α-reductase. A number of non-steroidal inhibitors of 5α-reductase have been reported (Halgunset et al, 1987). Non-steroidal inhibitors of 5α-reductase are classified according to their structure.
Those which are structural analogues of azasteroids, act as competitive inhibitors vs. testosterone include benzo[f]quinolinones, pyridones, benzo[c]quinolinones and benzo[c]quinolizinones.
Those which are structural analogues of ONO-3805 (Figure 14) act as non-competitive inhibitors vs. testosterone.
Figure 14: ONO-3805
Benzo[f]quinolinone
Benzo[f]quinolinone have been formally derived by removing the D ring from
4-azasteroids and substitution of the C ring with an aromatic one (Jones et al, 1993). Almost all of these compounds are 5α-reductase type-1 selective. LY191704 (Figure 15) has been reported as a potent inhibitor of 5α-reductase type-1. The reported IC50 for human 5α-reductase type-1 and type-2 are respectively 8.6nM and 1750nM (Jones et al, 1993; Frye, 1996).
Figure 15: LY191704
Pyrodines
Pyrodines (Figure 16) have been derived by formal replacement of the B and C ring of 4-azasteroids with an acyclic linker. Pyrodines have been reported to show a weak inhibitory activity against both 5α-reductase type-1 and type-2 (Hartmann et al, 1994).
Figure 16: Pyrodines
Benzo[c]quinolinones
Benzo[c]quinolinones (Figure 17) are formally derived from 6-azasteroids by removing ring D of the steroidal system. These compounds are weak and less selective inhibitors of 5α-reductase type-1 with a reported Ki equal to 920nM (Frye, 1996; Ernesto et al, 2004).
Figure 17: Benzo[c]quinolinone
Non-steroidal aryl acids
The tricyclic non-steroidal aryl acids are formally derived by removal of the D ring from androstenecaroboxylic acid inhibitors. These compounds are selective inhibitors of 5α-reductase type-1. Bromo-9,10-dihydrophenanthrene-2-carboxylic acid (Figure 18) has been reported as a potent non-steroidal inhibitor of this class with a reported Ki equal to 26nM (Abell et al, 1996). It has been reported that introduction of double to the B ring of compound LY1109 (Figure 19) results in loss of selectivity towards 5α-reductase type-1 and favours an increased potency towards 5α-reductase type-2 with a reported Ki equal to 260nM for human 5α-reductase type-2 (Ernesto et al 2004; Abell et al, 1996).
Figure 18: Bromo-9,10-dihydrophenanthrene-2-carboxylic acid
Figure 19: Compound LY1109
1.6 Aim of Project:
Coumarins are an important class of organic compounds. Coumarin contains a benzene ring fused with a α-pyrone ring (Gilchrist, 1984). A large number of coumarins occur as natural products and have also been synthesised as potential drug substances (Khan et al, 2005; Hoult et al, 1996). Most of the coumarins are intensely fluorescent and possess photophysical activity. Coumarins have a wide range of uses; they are used as anticoagulant, additives in food and cosmetics; in the preparation of insecticides; optical brighteners and dispersed fluorescent and laser dyes (Azuma et al, 2003; Estevez-Braun et al, 1997).
The aim of this project is to synthesise the coumarin derivatives (Figure 20) using modified Pechmann reaction followed by the reduction of the C=C bond using suitable reducing conditions (Scheme 1). The synthesised products will be purified and characterised by using NMR, GC-MS, IR and UV-visible.
Figure 20: Coumarin backbone
Scheme 1: Synthesis of coumarin derivatives (where R=alkyl or aryl group; a= β- keto ester/ H+; b= H2/ Pd)
2.0 Experimental
2.1 Discussion
Coumarins can be synthesised by several methods such as the Pechmann reaction, Perkin reaction or by Knoevenagel condensation (Joule et al, 1995; Woods et al, 1962; Potdar et al, 2001; Murray et al, 1983; Brufola et al, 1996).
i. Perkin reaction
Perkin condensation is the simplest way for synthesis of coumarins (Joule et al, 1995). An aliphatic acid anhydride and an aromatic aldehyde condense in the presence of the salt of the acid to yield coumarins (Scheme 2). Due to weak basicity of the carboxylate anion, elevated temperatures are generally required.
Scheme 2: Perkin condensation for synthesis of coumarin
ii. Knoevenagel condensation
This reaction involves condensation between an aldehyde and a malonic acid derivative or related compound (Scheme 3). The base catalyst used is generally an amine or its salt. Refluxing of the reactants is required for most preparations using this reaction (Joule et al, 1995; McCrae 1973).
The amine abstracts a proton from active methylene to yield a carbanion, which with some steps analogous to Aldol condensation gives coumarin derivatives.
Scheme 3: Knoevenagel condensation
iii- Pechmann reaction
The Pechmann reaction is the most widely applied method for the synthesis of coumarins. Harsh reaction conditions, difficulty in purification and problems of low yields have been reported while synthesising the coumarins using other methods, and is not the case in the Pechmann reaction.
Pechmann reaction (Scheme 4) involves condensation of phenols with β-ketoesters under acid catalysed conditions to give coumarins (Joule et al, 1995). Experimental data from syntheses of large number of coumarins shows certain limitations of this reaction. It is quite apparent from experimental results that a compound must have a nucleophilic substituent groups distributed around the periphery of the ring in such a manner that the β-ketoester may displace a ring proton activated by the combined ortho effect of one of the nucleophilic substituents and the para effect of the other nucleophilic substituent. One of these groups must be phenolic. Resorcinol works best to give better yields (Woods et al, 1962).
The first step of the synthesis is believed to be the electrophilic attack on the benzene ring ortho to the phenolic oxygen by the protonated ketone carbonyl, and then it undergoes ring closure to give coumarins (Scheme 2).
Scheme 4: Pechmann synthesis of 7-hydroxy-4-methyl coumarin
A modified Pechmann reaction was used in this project. Trifluoroacetic acid was used along with concentrated sulfuric acid to synthesise 7-hydroxy 4-methyl coumarin, 3-hydroxy-8,9,10,11-tetrahydro-7H-cyclohepta[c]chromen-6-one and 7-hydroxy-4-propyl-coumarin.
Hydrogenation of 7-hydroxy-4-methyl coumarin and 3-hydroxy-8,9,10,11-tetrahydro-7H-cyclohepta[c]chromen-6-one was done using activated palladium catalyst.
The characterisation of all synthesised products was done by using 1H NMR, 13C NMR, FTIR, UV spectroscopy, and GC-MS.
i. Synthesis of 7-hydroxy-4-methyl-2H-chromen-2-one
The synthesis of 7-hydroxy-4-methyl 2H-chromen-2-one was carried out using modified Pechmann reaction (Scheme 5) using trifluoroacetic acid along with concentrated sulfuric acid. It was observed that the solvent system ethyl acetate and hexane (7:4) for recrystallisation of the crude product did not work properly; a yellow coloured solid was obtained after recrystallisation. On doing thin layer chromatography (TLC) it was observed that some of the starting material resorcinol was also present in the product. The product was insoluble in almost all the solvents but was soluble in acetone and in methanol only when a very little quantity was added.
In the second batch of 7-hydroxy-4-methyl-2H-chromen-2-one it was observed that no precipitates were obtained after quenching the reaction mixture but an oily liquid was obtained. On addition of water it gave precipitates.
The problems that occurred during the synthesis were probably due to improper stirring during the course of the reaction, as the reaction was carried out at very low temperature; difficulty in stirring the reaction mixture was also observed due to the use of a large amount of concentrated acid. In order to overcome these problems smaller quantities of the reagents were used and fine powdered resorcinol was used. To overcome the problem of excess acid, water was added after quenching the reaction mixture.
These modifications worked and gave a product with improved purity and the product was soluble in acetone and methanol even when large quantity of product was added.
Scheme 5: Synthesis of 7-hydroxy-4-methyl-2H-chromen-2-one using modified
Pechmann condensation.
ii. Synthesis of 7-hydroxy-4-methyl-chromen-2-one
In order to reduce the 7-hydroxy-4-methyl-2H-chromen-2-one activated palladium on charcoal was used as a catalyst (Scheme 6). The hydrogenation was continued over the period of a week. It was observed by using TLC and from GC-MS that even after one week there was some of the unreduced starting material. The hydrogenation was continued for one more week, and the balloon was refilled with hydrogen gas on a daily basis.
Even at the end of the second week of hydrogenation some of the starting material was present in the product. The compound was then columned to obtain a pure hydrogenated product. Silica was used as a stationary phase and a mixture of ethyl acetate and hexane (50:50) was used as mobile phase for separation. This gave a pure product with only one spot on the TLC plate and gave a cleaner GC-MS with only one peak.
Scheme 6: Hydrogenation of 7-hydroxy-4-methyl-2H-chromen-2-one.
iii. Synthesis of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one
The synthesis of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one was carried out using modified version of Pechmann condensation (Scheme 7). The product was synthesised twice. In order to avoid problems faced during the synthesis of 7-hydroxy-4-methyl-2H-chromen-2-one, the same modifications were applied for the synthesis of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one. These modifications resulted in a much cleaner and pure product with only one spot on the TLC plate and one peak in the GC-MS. No problems were observed after using the above mentioned modifications. The solvent system for recrystallisation worked very well, and pure product with comparable good % yield (66%) was obtained.
Scheme 7: Synthesis of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-
one using modified Pechmann condensation.
iv. Synthesis of 3-hydroxy-7,8,9,10,11,11a-hexahydrocyclohepta[c]chromen-6(6aH)-one
The hydrogenation of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one was carried out using palladium as the catalyst (Scheme 8). The hydrogenation was continued over the period of one week with continuous stirring. After one week the product was analysed by GC-MS and it was found that only a small fraction of the reactant was hydrogenated. The hydrogenation was continued for another week with continual refilling of hydrogen gas on daily basis. But this did not work and even at the end of second week of hydrogenation only a small fraction of the reactant was hydrogenated. In order to separate hydrogenated product from starting material by column chromatography, TLC was carried out using different solvent systems. But only one spot showing the starting material was observed. It was concluded that the hydrogenated product can not be separated from starting material and the mixture of both was used for the characterisation.
Scheme 8: Hydrogenation of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-
6(7H)-one
v. Synthesis of 7-hydroxy-4-propyl-2H-chromen-2-one
7-Hydroxy-4-propyl-2H-chromen-2-one was synthesised using modified Pechmann condensation (Scheme 9). The same modifications were applied to the synthetic procedure as mentioned in the synthesis of 7-Hydroxy-4-methyl-2H-chromen-2-one. No problems were observed in the synthesis of the product. Pure product, with comparable good % yield, was obtained giving only one spot on the TLC plate and one peak in the GC-MS.
Scheme 9: Synthesis of 7-hydroxy-4-propyl-2H-chromen-2-one by using modified
Pechmann condensation
2.2 Materials and methods
Chemicals: All chemicals were purchased from Sigma-Aldrich. TLC: precoated silica gel plates were used. GC-MS: Hawlett Packard 5971 series mass selective detector and Hawlett Packard 5890 series II gas chromatograph were used. IR: Infrared spectra were recorded Perkin Elmer Fourier Transform Infrared spectrophotometer on NaCl plates. UV: UV spectra were recorded on Varian Cary 100 Scan series. HPLC: Perkin Elmer 250 LC pump was used, Spherisorb 250X 4.60 mm column was used and UV (226nm) detector was used. NMR: 1H NMR and 13C NMR were recorded on 400Mhz Jeol NMR spectrometer, the chemical shifts were expressed in pp and the coupling constant J is expressed in Hz. M.P: Melting point were recorded on electrothermal digital melting point apparatus.
2.2.1 Synthesis of 7-hydroxy-4-methyl-2H-chromen-2-one
Resorcinol (4.11g, 37.0mmol) was dissolved in hot ethylacetoacetate (5.63g, 40.0mmol) and the solution was cooled to 0 oC. While stirring, a mixture of trifluoroacetic acid (7.2ml, 90.0 mmol) and concentrated sulfuric acid (12.4ml, 124.0 mmol) was added in a dropwise manner in such a way that the temperature did not exceed 10oC. The reaction mixture was then allowed to warm to room temperature and stirring was continued overnight. The reaction mixture was quenched cautiously with ice and water was added to the reaction flask. After quenching, the reaction mixture was allowed to stir at room temperature for a further 2h. The precipitate formed was collected by suction filtration and recrystallised from a solution of ethyl acetate and hexane mixture (7:4) to give a yellow coloured solid (4.65g, 71 %; m.p. 201-204 oC; lit. m.p. 185-190 0C(Aldrich)).
υ(max) (film) cm-1: 3416.2 (OH), 2923.45 (CH aliphatic), 1679.2 (C=O ester), 1599.6 ( C=C aromatic), 1132.5 (C-O); δH (CD3OD): 7.5 (1H, d, J= 8.6 C8-H), 6.7 (1H, dd, J= 2.5, J=6.2 C6-H), 6.6 (1H, d, J=2.4 C5-H), 5.9 (1H, d, J=1.28 C3-H), 1.9 (1H, m, J=2.2, C11-H); δC (CD3OD): 161.12 (C=O), 160.31 (C=C), 155.64, 153.13, 126.59, 112.95, 112.76, 111.11 (aryl C), 102.59 (C-O), 17.82 (CH3); λmax (nm) : 322; GCMS: tR 17.583 min., m/z 176 (M+) 176(Base Peak); Rf : 0.47 (EA/Hex= 50:50)
2.2.2 Synthesis of 7-hydroxy-4-methyl-chromen-2-one
7-Hydroxy-4-methyl-2H-chromen-2-one (1.00g, 5.68 mmol) was dissolved in 40ml of methanol in a round bottom flask. Activated 5% palladium on charcoal (0.1239g) was transferred to round bottom flask. The flask was then sealed with septum, Air was removed from the flask and flask was flushed with hydrogen gas three times. A balloon filled with hydrogen gas was set with the reaction flask to supply hydrogen gas to the reaction mixture. The reaction mixture was left stirring for one week. The hydrogenated product was then filtered and dried to give a white coloured solid (0.39g, 39%; m.p. 110-116 oC).
υ(max) (film) cm-1: 3394.2 (OH), 2923.69 (CH aliphatic), 1718.48 (C=O ester), 1600.7 ( C=C aromatic), 1157.6 (C-O); δH (d-acetone): 8.5 (1H, s, O-H), 7.0 (1H, d, J= 8.2 C5-H), 6.7 (1H, dd, J= 2.5, J=7.2 C6-H), 6.4 (1H, d, J=2.4 C8-H), 3.1 (1H, dd, J= 6.5 C4-H), 2.7 (3H, m, J= 7.1, J= 8.6, J=5.5, J= 10.2 C11-H), 2.5(1H, dd, J=7.14, J= 8.6, C3-H); δC (d-acetone): 167.67 (C=O), 157.42, 152.25, 127.44, 119.19., 111.47, (aryl C), 103.54 (C-O), 36.72 (CH2), 19.72 (CH3); λmax (nm) : 328; GCMS: tR 15.13 min., m/z 178 (M+) 163 (Base Peak); Rf : 0.65 (EA/Hex= 50:50)
2.2.3 Synthesis of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one
Resorcinol (0.77, 6.95mmol) was dissolved in hot methyl-2-oxo-cycloheptane carboxylate (1.30g, 7.65mmol) and the solution was cooled to 0 oC. While stirring, a mixture of trifluoroacetic acid (1.4ml, 17.5mmol) and concentrated sulfuric acid (2.4ml, 24mmol) was added in a dropwise manner in such a way that the temperature did not exceed 10oC. The reaction mixture was then allowed to warm to room temperature and stirring was continued overnight. The reaction mixture was quenched cautiously with ice and water was added to the reaction flask. After quenching, the reaction mixture was allowed to stir at room temperature for a further 2h. The precipitates formed was collected by suction filtration and recrystallised from solution of ethyl acetate and hexane mixture (7:4) to give a blenched almond coloured solid (1.12g, 66.2%; m.p. 180-186 0C; lit. m.p. 189-190 oC
υ(max) (film) cm-1: 3213.61 (OH), 2923.18 (CH aliphatic), 1679.32 (C=O ester), 1613.47 ( C=C aromatic), 1095.97 (C-O); δH (CD3OD): 7.6 (1H, d, J= 8.8 C4-H), 6.7 (1H, dd, J= 2.4, J=6.4 C1-H), 6.6 (1H, d, J=2.4 C2-H), 2.8 (6H, m, J=5.4C9-11-H), 1.5 (4H, m, J=5.5, C8,12-H); δC (CD3OD): 161.44 (C=O), 160.16 (C=C), 154.51, 153.97, 125.83, 124.45, 112.57 (aryl C), 102.51 (C-O), 31.96, 27.69, 26.29, 25.89, 25.19 (CH2); λmax (nm) : 328; GCMS: tR 21.099 min., m/z 230 (M+), 230 (Base Peak); Rf : 0.68 (EA/Hex= 50:50)
2.2.4 Synthesis of 3-hydroxy-7,8,9,10,11,11a-hexahydrocyclohepta[c]chromen-6(6aH)-one
3-Hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one (0.2g, 0.87 mmol) was dissolved in 40ml of methanol in a round bottom flask. Activated 5% palladium on charcoal (0.0213g) was transferred to round bottom flask. The flask was then sealed with septum, Air was removed from the flask and flask was flushed with hydrogen gas three times. A balloon filled with hydrogen gas was set with the reaction flask to supply hydrogen gas to the reaction mixture. The reaction mixture was left stirring for one week. The mixture of hydrogenated product and starting material was then filtered and dried to give a blenched almond coloured solid (0.14g; m.p. 160-166 oC).
2.2.5 Synthesis of 7-hydroxy-4-propyl-2H-chromen-2-one
Resorcinol (0.82g, 7.38mmol) was dissolved in hot ethyl butryl acetate (1.27g, 8.02mmol) and the solution was cooled to 0 oC. While stirring, a mixture of trifluoroacetic acid (2.0ml, 25.0 mmol) and concentrated sulfuric acid (2.5ml, 25.0 mmol) was added in a dropwise manner in such a way that the temperature did not exceed 10oC. The reaction mixture was then allowed to warm to room temperature and stirring was continued overnight. The reaction mixture was quenched cautiously with ice and water was added to the reaction flask. After quenching, the reaction mixture was allowed to stir at room temperature for a further 2h. The precipitates formed was collected by suction filtration and recrystallised from solution of ethyl acetate and hexane mixture (7:4) to give a yellow coloured solid (0.98g, 64 %; m.p. 142-148 oC)
υ(max) (film) cm-1: 3416.2 (OH), 2923.27 (CH aliphatic), 1699.84 (C=O ester), 1617.79 (C=C aromatic), 1140.06 (C-O); δH (d-acetone): 7.5 (1H, d, J= 8.7 C8-H), 6.7 (1H, dd, J= 2.4, J=6.4 C5-H), 6.6 (1H, d, J=2.4 C6-H), 5.9 (1H, s, C3-H), 2.7 (2H, t, J=7.6, C11-H), 1.9 (2H, m, J= 2.2, C12-H), 1.6 (3H, m, J=7.6, C13-H); δC (d-acetone): 160.99 (C=O), 160.49 (C=C), 156.67, 155.97, 126.42, 112.8, 112.19, 110.19 (aryl C), 102.83 (C-O), 33.51, 21.83, 13.43 (CH2); λmax (nm) : 327; GCMS: tR 23.579 min., m/z 204 (M+) 204(Base Peak); Rf : 0.54 (EA/Hex= 50:50)
2.3 UV studies of 7-hydroxy-4-methyl-2H-chromen-2-one
All the coumarins, synthesised by modified Pechmann condensation since resorcinol has been used as a starting material, contain acidic proton. When the acidic proton of phenolic ring is withdrawn by base, it gives phenoxide containing C=O in conjugation with the π electrons of the aromatic ring. The absorption of the π electrons gives maxima in the UV range of electromagnetic radiation. To study the effect of addition of base to coumarins; different concentrations of sodium hydroxide was used.
Since coumarins absorb UV radiations, UV-Visible spectroscopy can be used for the quantification of coumarins. In order to use UV spectroscopy for quantitative measurements of coumarins, it is very much important to calculate the limit of detection for all derivatives of coumarins using UV-Vis. spectrophotometer.
2.3.1 Effect of addition of base
7-Hydroxy-4-methyl-2H-chromen-2-one contains a hydroxyl substituent; addition of sodium hydroxide will displace the proton from the ring resulting in a shift in UV maximum.
Different concentrations of sodium hydroxide were added to the solution of 7-hydroxy-4-methyl-2H-chromen-2-one and the UV absorption was recorded (Table 1).
Table 1
No.
Sample
Wavelength (nm)
Absorbance
1
7-OH-Coumarin
322
0.3184
2
7-OHCoumarin + 1drop 1M NaOH
368
0.2741
3
7-OH-Coumarin + 1drop 2M NaOH
364
0.4161
4
7-OH-Coumarin + 1drop 3M NaOH
365
0.4844
5
7-OH-Coumarin + 1drop 4M NaOH
365
0.506
6
7-OH-Coumarin + 1drop 5M NaOH
365
0.5043
From the results in table 1 it is clear that addition of NaOH to 7-Hydroxy-4-methyl-2H-chromen-2-one results in displacement of UV maximum at higher wavelength.
2.3.2 Limit of detection
Limit of detection (LOD) is the concentration of analyte that can be detected with reasonable certainty for a given analytical procedure (Miller et al, 1986). Mathematically it can be represented as,
y = yb + 3sb ------------------- (1)
Where y, yb and sb are signal of sample at LOD, blank signal and standard deviation of the blank respectively.
For determination of limit of detection of 7-hydroxy-4-methyl-2H-chromen-2-one by using UV spectrophotometer, a series samples with different concentrations of 7-hydroxy-4-methyl-2H-chromen-2-one, were scanned at 322nm (Table 2). On the basis of results statistical calculations were done to calculate the limit of detection of 7-hydroxy-4-methyl-2H-chromen-2-one.
Table 2
Concentration x (μg/ml)
Absorbance (y)
0.010
0.0121
0.100
0.0491
0.125
0.0389
0.250
0.0434
0.500
0.0687
1.000
0.1112
2.000
0.1855
4.000
0.4835
6.000
0.5926
8.000
0.6523
10.000
0.7944
12.500
1.0273
15.000
1.3876
17.500
1.4302
20.000
1.6093
According to the equation of line,
y= mx + b -------------- (2)
Where y is the absorbance, m is the slope of the line, x is the concentration of sample and b is the y-intercept of the line. In order to calculate the limit of detection we need to calculate, all the associated values i.e., the values of y, m, b, r (correlation coefficient) and sb (sy/x).
Where,
r = Σi {(xi-xavg)(yi-yavg)}/ {[ Σi(xi-xavg)2][ Σi(yi-yavg)2]}1/2 ---- (3)
m = Σi {(xi-xavg)(yi-yavg)}/ Σi(xi-xavg)2 -------- (4)
sy/x = { Σi(yi- Å·)2/ n-2)}1/2 --------------- (5)
The calculations for the above mentioned values are,
xavg= 6.47 μg/ml
yavg= 0.57
x²
x-xavg
y-yavg
(x-xavg)²
(y-yavg)²
(x-xavg)(y-yavg)
0.00
-6.46
-0.554
41.68
0.31
3.57
0.01
-6.37
-0.517
40.52
0.27
3.29
0.02
-6.34
-0.527
40.20
0.28
3.34
0.06
-6.22
-0.522
38.63
0.27
3.25
0.25
-5.97
-0.497
35.59
0.25
2.97
1.00
-5.47
-0.455
29.87
0.21
2.48
4.00
-4.47
-0.380
19.94
0.14
1.70
16.00
-2.47
-0.082
6.08
0.01
0.20
36.00
-0.47
0.027
0.22
0.00
-0.01
64.00
1.53
0.087
2.35
0.01
0.13
100.00
3.53
0.229
12.49
0.05
0.81
156.25
6.03
0.462
36.41
0.21
2.79
225.00
8.53
0.822
72.83
0.68
7.01
306.25
11.03
0.864
121.76
0.75
9.54
400.00
13.53
1.044
183.18
1.09
14.12
Σ=1308.84
0.00
0.000
681.77
4.51
55.19
By putting the calculated values in the equations we get,
r = 0.9949
m = 0.081
b = yb= 0.04
In order to calculate sb i.e. sy/x, first the value of Å· will be determined.
x
Å·
y-Å·
(y-Å·)²
0.01
0.043
-0.03
0.00
0.10
0.050
0.00
0.00
0.13
0.052
-0.01
0.00
0.25
0.063
-0.02
0.00
0.50
0.083
-0.01
0.00
1.00
0.123
-0.01
0.00
2.00
0.204
-0.02
0.00
4.00
0.366
0.12
0.01
6.00
0.528
0.06
0.00
8.00
0.690
-0.04
0.00
10.00
0.852
-0.06
0.00
12.50
1.054
-0.03
0.00
15.00
1.257
0.13
0.02
17.50
1.459
-0.03
0.00
20.00
1.661
-0.05
0.00
Σ
8.486
0.00
0.05
s y/x = sb = 0.06.
Now the absorbance at LOD will be,
y = yb + 3 sb
y = 0.22
Now the limit of detection is,
LOD = y- b/ m
LOD = 2.22 μg/ml.
Hence the limit of detection for 7-hydroxy-4-methyl-2H-chromen-2-one is 2.22 μg/ml while using UV spectroscopy for the measurement.
2.4 UV studies of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-
one
2.4.1 Addition of base
3-Hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one contains hydroxyl group, addition of sodium hydroxide displace a proton from the ring and results a shift in the UV maximum at a higher wavelength.
Different concentrations of sodium hydroxide were added to the solution of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one and the UV absorbance was recorded (Table 3).
Table 3
No.
Sample
Wavelength (nm)
Absorbance
1
3-OH-Coumarin
328
0.2604
2
3-OH-Coumarin + 1drop 2M NaOH
368
0.2637
3
3-OH-Coumarin + 1drop 3M NaOH
368
0.2684
4
3-OH-Coumarin + 1drop 4M NaOH
368
0.2784
5
3-OH-Coumarin + 1drop 5M NaOH
368
0.3294
From the results in table 2 it is clear that addition of NaOH to 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one, results in displacing the UV maximum at a higher wavelength.
2.4.2 Limit of detection
For determination of limit of detection of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one by using UV spectrophotometer, a series samples with different concentrations of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one, were scanned at 328nm (Table 4). On the basis of results statistical calculations was done to calculate the limit of detection of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one.
Table 4
Concentration x (μg/ml)
Absorbance y
0.10
0.02
0.13
0.03
0.25
0.04
0.50
0.04
1.00
0.05
2.00
0.83
4.00
0.12
6.00
0.42
8.00
0.67
10.00
0.80
12.00
0.73
14.00
0.98
16.00
1.22
18.00
1.36
20.00
1.55
Same calculations have been applied as in case of 7-hydroxy-4-methyl-2H-chromen-2-one.
xavg= 7.47 μg/ml
yavg= 0.59
x²
x-xavg
y-yavg
(x-xavg)²
(y-yavg)²
(x-xavg)(y-yavg)
0.01
-7.37
-0.571
54.24
0.33
4.20
0.02
-7.34
-0.561
53.88
0.31
4.12
0.06
-7.22
-0.552
52.06
0.30
3.98
0.25
-6.97
-0.547
48.51
0.30
3.81
1.00
-6.47
-0.541
41.80
0.29
3.50
4.00
-5.47
0.239
29.87
0.06
-1.31
16.00
-3.47
-0.471
12.01
0.22
1.63
36.00
-1.47
-0.171
2.15
0.03
0.25
64.00
0.54
0.079
0.29
0.01
0.04
100.00
2.54
0.209
6.43
0.04
0.53
144.00
4.54
0.139
20.57
0.02
0.63
196.00
6.54
0.389
42.71
0.15
2.54
256.00
8.54
0.629
72.85
0.40
5.37
324.00
10.54
0.769
110.99
0.59
8.10
400.00
12.54
0.959
157.13
0.92
12.02
Σ=1541.34
0.00
0.000
705.44
3.97
49.43
By putting the calculated values in the equations we get,
r = 0.9843
m = 0.07
b = yb= 0.07
In order to calculate sb i.e. sy/x, first the value of Å· will be determined.
x
Å·
y-Å·
(y-Å·)²
0.01
0.068
-0.05
0.00
0.10
0.075
-0.04
0.00
0.13
0.077
-0.04
0.00
0.25
0.085
-0.04
0.00
0.50
0.103
-0.05
0.00
1.00
0.138
0.69
0.48
2.00
0.208
-0.09
0.01
4.00
0.348
0.07
0.01
6.00
0.488
0.18
0.03
8.00
0.628
0.17
0.03
10.00
0.768
-0.04
0.00
12.50
0.944
0.04
0.00
15.00
1.119
0.10
0.01
17.50
1.294
0.07
0.00
20.00
1.469
0.08
0.01
Σ
7.812
1.05
0.59
s y/x = sb = 0.213.
Now the absorbance at LOD will be,
y = yb + 3 sb
y = 0.71
Now the limit of detection is,
LOD = y- b/ m
LOD = 9.13 μg/ml.
Hence the limit of detection for 3-Hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one is 9.13 μg/ml while using UV spectroscopy for the measurement.
2.5 Conclusion and future work
During the course of study derivatives of coumarins were synthesised using modified Pechmann synthesis and hydrogenation of those products was performed. All the products were characterised using spectroscopic techniques.
Effect of addition of was base to coumarins was studied and the limit of detection using UV spectroscopy was worked out for 7-Hydroxy-4-methyl-2H-chromen-2-one and 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one for the quantitative analysis of coumarins.
Further work can be done on hydrogenation of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-6(7H)-one (Scheme 10) using different catalysts as well as enzyme inhibition assays can be done for testing the inhibitory activity of synthesised products against 5α-reductase. A series of coumarin derivatives can be synthesised to study the trend.
Scheme 10: Hydrogenation of 3-hydroxy-8,9,10,11-tetrahydrocyclohepta[c]chromen-
6(7H)-one using different catalysts.
