Synthesis of Novel 4-Methylcoumarins and Comparative Specificities of Substituted Derivatives for Acetoxy Drug : Protein Transacetylase

Our laboratory has been credited for the discovery of a unique membrane bound enzyme termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic peracetates (PA) to certain functional proteins resulting in the modulation of their catalytic activity. In this report, we have synthesized eight novel 4-methylcoumarins and demonstrated the comparisons of acetoxy derivatives of 4-methylcoumarin with their propoxy and butoxy derivatives for the modulation of some receptor proteins such as cytochrome P-450 (Cyt.P450), NADPH cytochrome c reductase and cytosolic glutathione S-transferase (GST). The results clearly indicated that acetoxy derivatives have very high efficacy for the modulation of above mentioned functional proteins as compared to their other derivatives. We have also compared the acetoxy derivatives of 4-methylcoumarin with their acid substituted acetoxy derivatives and found that inclusion of carboxylic acid groups on the benzenoid rings of the coumarins system hardly affected TAase mediated catalytic activity. 396 Y. K. Tyagi et al.:


Introduction
In our earlier publications [1,2] evidences were given for the existence of a novel enzyme acetoxy drug: protein transacetylase (TAase) present in rat liver microsomes.The enzyme, transacetylse catalyzed the transfer of acetyl group from the various acetoxy drugs to specific enzyme proteins such as cytochrome P-450 (Cyt.P-450) [3], NADPH cytochrome c-reductase [4] and glutathione S-transferase [1,2,5].The simple assay procedures were developed by modifications in the literature procedures for the liver microsomal transacetylase assays [1].The TAase assay procedure was based on the irreversible inhibition of GST (protein substrate) with the use of various acetoxy derivatives of heterocyclic molecules (second substrates) [1,2].One of the products of the transacetylase reaction was identified as the hydroxyl derivatives of the respective acetoxy derivatives [1].The functional protein, NADPH cytochrome c-reductase assay was based on the activation of an enzyme participating in hepatic microsomal electron transport and Cyt.P-450 assay was based on the inhibition of liver microsomal Cyt.P-450, linked mixed function oxidases (MFO) by acetoxy derivatives of heterocyclic molecules catalyzing the epoxidation of aflatoxin B 1 (AFB 1 ) [3].Apart from these activities we have also used the polyphenolic peracetates (PA) to modify another protein called Nitric Oxide Synthase (NOS).As enhanced intracellular levels of Nitric Oxide (NO) helps vasorelaxation in asthmatic patients.The intracellular level of NO alteration is controlled by the mechanism involved in the activation of NOS.Our studies, for the first time highlighted the role of PA as the novel potent agent for enhancing the intracellular levels of NO [6].We had also demonstrated TAase catalyzed modification of Protein Kinase C (PKC) activity in lymphocytes of asthmatic patients by PA.The PKC activity of lymphocytes of asthmatic patients was noticed to proportionally increase with severity of the disease [7].Acetoxy derivatives were found to exert inhibitory action on PKC while hydroxyl derivatives of the respective compounds did not show any activity.We had also highlighted the activation of human platelets NOS by PA catalyzed by Transacetylase (TAase) [6].Purified human placental TAase mediated acetylation of nNOS by DAMC was demonstrated [8].It was thought interesting to examine more and more acetoxy drugs to find out a better substrate for the transacetylase mediated biological actions.In this connection, eight novel 4-methylcoumarins were synthesized and characterized by spectral data (UV, IR, 1 H NMR and 13 C NMR) to examine structure activity relationship (SAR) of acetoxy, propoxy and butoxy derivatives of 4methylcoumarin on liver microsomal Cyt.P-450 linked MFO, NADPH cytochrome creductase and cytosolic glutathione S-transferase (GST).
Structure of the compounds was confirmed by the 1 H NMR, 13 C NMR, UV, IR and data for these compounds are given in the chemistry section.The IR spectrum of compound 3 showed two characteristic absorptions at 3751 and 3304 cm -1 for hydroxyl groups and two other absorptions appeared at 1769 and 1699 cm -1 for carbonyl groups.The 1 H NMR spectrum showed characteristic peak of 4-methylcoumarin at δ 6.91 for C-3 proton.Finally the structure was supported by its 13 C NMR spectrum, C-2 appeared at δ 158.28 and other carbonyl carbon appeared at δ 167.80 for COOH.Compound 4 had two characteristic absorptions at 3368 and 1701 cm -1 for hydroxyl and carbonyl groups respectively in its IR spectrum.The 1 H NMR spectrum showed characteristic singlet of 4methylcoumarin at δ 6.21 for C-3 proton.Finally the structure was confirmed by its for carbonyl carbons.In its 1 H NMR spectrum, a characteristic peak of 4methylcoumarin appeared at δ 6.32 for C-3 proton.Finally the structure was confirmed by its 13 C NMR spectrum, C-2 appeared at δ 158 and carbonyl for COOH appeared at δ 200.26.The IR spectrum of compound 9 showed three characteristic absorptions at 3675, 3447 and 1702 cm -1 , two for hydroxyl group and one for carbonyl group respectively.In its 1 H NMR spectrum, a characteristic peak of 4methylcoumarin was appeared at δ 6.20 for C-3 proton.Finally the structure was confirmed by its 13 C NMR spectrum, C-2 appeared at δ 157.99 and other carbonyl carbons appeared at δ 164.68.Compound 10 had two characteristic absorption at 3476 and 1699 cm -1 for hydroxyl and carbonyl groups respectively in its IR spectrum.The 1 H NMR spectrum showed characteristic peak of 4-methylcoumarin at δ 6.20 for C-3 proton.Finally the structure was confirmed by its 13 C NMR spectrum, C-2 appeared at δ 158.86 and two other carbonyl carbons appeared at δ 162.59 and 166.64.Compound 12 had three characteristic absorptions at 1792, 1761, 1699 cm -1 for acetoxy, carboxylic and C-2 carbonyl groups respectively in its IR spectrum.The 1 H NMR spectrum showed characteristic singlet of 4methylcoumarin, appeared at δ 6.31 for C-3 proton.Finally the structure was confirmed by its 13 C NMR spectrum, C-2 appeared at δ 158.45 and other carbonyls appeared at δ 161.16 and 166.16.The IR spectrum of compound 11 showed two characteristic absorptions which appeared at 3394 and 1700 cm -1 for hydroxyl and carbonyl groups respectively.The 1 H NMR spectrum showed characteristic singlet of 4-methylcoumarin at δ 6.31 for C-3 proton.Finally the structure was supported by its 13 C NMR spectrum, CH 2 carbon appeared at δ 62.24 and C-2 appeared at δ 158.80.The other carbonyl groups appeared at δ 163.68 and 172.21.
For the structure activity relationship, number of acetoxy, propoxy and butoxy derivatives of 4-methylcoumarin were synthesized and examined with the liver microsomal Transacetylase (TAase) for comparative specificities of acyl groups of coumarin derivatives.The catalytic activity of TAase was governed by carbonyl group in pyran ring of coumarin nucleus [2].For the TAase catalytic activity, the inhibition of GST under the conditions of assay was considered to be proportional to the TAase activity as demonstrated in our earlier publications [1,2,5].The results in table 1 show the TAase catalyzed inhibition of cytosolic GST by polyphenolic acetates, 7,8-diacetoxy-4-methylcoumarin-5-carboxylic acid (4) and 7,8-diacetoxy-4-methyl-coumarin-6-carboxylic acid (6) causes significant inhibition of TAase which is quite identical to that of the model acetoxy drug 2. The extent of inhibition of GST by 7-acetoxy-4-methylcoumarin-6-carboxylic acid ( 10) is similar to that of the 7-acetoxy-4-methylcoumarin (8).These results clearly indicate that the addition of carboxylic group on the benzenoid ring hardly affect on TAase mediated catalytic activity.The transfer of acetyl group from polyphenolic acetates to receptor proteins catalyzed by the novel enzyme acetoxy drug: protein TAase has also been established by our earlier studies [10,11].DAMC, 2 a model acetoxy drug was found to modulate the activities of enzyme proteins, such as cytochrome P-450 linked MFO, NADPH cytochrome c reductase and cytosolic GST catalyzed by TAase [3,4].Our previous studies have highlighted the structural features of polyphenolic acetates determining the specificity for liver microsomal TAase [12,13].
The acetoxy coumarins were also examined for the ability to irreversibly activate liver microsomal NADPH cytochrome c reductase, which is taken as another yardstick to assess the activity and specificity of TAase (Table 2).It is interesting to note that various concentrations of acetoxy coumarins cause the activation of the reductase (catalyzed by TAase) in tune with their specificities of TAase measured in terms of inhibition of cytosolic GST.
These coumarin derivatives were also used as substrates for the inhibition of liver microsomal catalyzed AFB 1 binding to DNA in vitro.The pattern of inhibition of AFB 1 -DNA binding by acetoxy derivatives of 4-methylcoumarin and their acid containing acetoxy derivative followed similar trend as observed earlier.
Accordingly, compound 2, 4 and 6 inhibited liver microsomal cytochrome P-450 catalyzed AFB 1 epoxidation (measured as binding to DNA) to a similar extent.
Similar trend also persisted for compounds 8 and 10 (Table 3).We have also examined propoxy and butoxy derivative of 4-methylcoumarin for the first time for the modulation of above described functional proteins.The results presented in Table 1- Further, the obtained results supported by PM3 optimized structure, suggested that the electron charge distribution and bond order about the active O-Ac bond nearly remain same in all the systems considered here.The longest axis measurement (6.86 cm) for the PM3 optimized geometry of DAMC ( 2) is almost equal to 7,8-diacetoxy coumarin-6-carboxylic acid (6.952 cm) and 7,8-diacetoxy coumarin-5-carboxylic acid (6.950 cm) is found to be much smaller than that of 7,8dipropoxy 4-methylcoumarin (9.4 cm) and 7,8-dibutoxy-4-methylcoumarin (10.966 cm).This is in agreement with an observed result that compounds 2, 4 and 6 shows higher TAase catalytic activity as compared to compounds 11, 12 and 13.This is because of the possible hindrance to the propoxy and butoxy groups from the accessing the active site of TAase resulting in the decreased rate of transfer of the propyl and butyl groups to the enzyme.Further the PM3 optimized geometries have revealed that the acetoxy groups in compound 2, 4 and 6 are bent towards the oxygen heteroatom of the pyran ring.We have further observed that in compound 2, 4 and 6, the C-7 acetoxy group helps the C-8 acetoxy group to orient itself most favorably towards the oxygen heteroatom of the coumarins moiety and this phenomenon, presumably controls the TAase mediated transfer of acetyl group to the functional proteins.

General
Melting points were determined in sulphuric acid bath and were uncorrected.
The UV and IR Spectra were recorded on Beckman DU-64 Spectrophotometer and Perkin-Elmer model 1720 FTIR spectrophotometer respectively.The 1 H NMR spectra were recorded either at Shimartzu or Bruker AC-250 instrument at 60 and 300 MHz respectively.The 13 C NMR spectra were recorded on same instrument as 1 H NMR spectral recordings at 62.8 and 75 MHz respectively.TMS has been used as an internal standard for both 1 H and 13 C NMR spectral recordings.The chemical shift values are on δ scale.The mass spectra were recorded on a Varian Mat 311, a mass spectrometer at 70 eV.The physical and spectral data of DHMC and DAMC have been published previously [14].
To an ice cold H 2 SO 4 (5.0 ml), solution of 2,3,4-trihydroxy benzoic acid (1.0 gm., 0.0058 mM) in ethyl acetoacetate (2.8 ml, 0.0174) was added slowly with constant stirring.After completion of the addition, reaction mixture was stirred for 6 hrs at room temperature and the progress of the reaction was monitored on TLC.
Aqueous work up of the reaction mixture yielded white colored product of m.p. 253

Animals
Male albino rats of wistar strain weighing around 180-200 g. fed on rat chow supplied by Hindustan Lever Ltd., Mumbai (India) were used.

Preparation of microsomes and cytosol
Rats were killed by decapitation, liver removed and 30% homogenate (w/v) was prepared in 10 mM phosphate buffer containing 0.25 M sucrose and 1.4 mM βmercaptoehtanol, pH adjusted to 7.0.The homogenate was centrifuged at 10,000g for 30 min.and the obtained supernatant was spun at 100,000 g for 1 h in the Beckman ultracentrifuge Model L-7.The cytosolic fraction was set-aside at -20 °C.
requirement, were preincubated at 37 °C in a shaker water bath.The aliquots (0.5) mL portion) were removed periodically into spectrophotometer cuvette (1 cm light path) containing 0.1 mM EDTA, 36 mM cytochrome C and 1mM NADPH in a total volume of 1 mL.The progress of the reaction was followed by monitoring absorption at 550 nm.In the control samples, acetoxy drugs were replaced by DMSO.The increment in reductase activity due to acetoxy drug over the control was expressed as percent activation.

Statistical analysis
The final values are the mean of three observations and standard deviation was calculated with the help of following website: A semi empirical procedure was used to study the enzyme mediated transacetylation reactions of various acetoxycoumarins.The molecular spatial structure and bond length analysis of these systems was theoretically estimated using HyperChem Release 5.1 Pro Quantum Chemistry Package [17].The preliminary PM3 optimized structures were subjected to a systematic conformational search and the lowest energy conformation obtained was then fully optimized without geometric constraints using RHF/PM3 semi empirical methods [18].

Conclusion
Eight novel 4-methylcoumarins were synthesized and their structures were confirmed by the spectral data.The results presented in this communication revealed that the ortho diacetoxy system is better than those of ortho dipropoxy and dibutoxy system with respect to catalytic activity of glutathione S-transferase, NADPH cytochrome c-reductase, AFB 1 -DNA binding and cytochrome P-450 linked MFO.Introduction of carboxylic group at any position in the benzenoid ring of the coumarin moiety is ineffective in the modulation of aforesaid functional proteins.

5 (
compounds 7,8-dipropoxy-4-methylcoumarin-5carboxylic acid, 11 7,8-dipropoxy-4-methylcoumarin-6-carboxylic acid, 12 and 7,8dibutoxy-4-methylcoumarin, 13) clearly indicate that propoxy compounds showed less degree of modulation and butoxy derivative exhibited much lesser degree of modulation as compared to acetoxy derivative in the modulation of aforesaid functional proteins.These observations clearly indicated that the compound containing acetoxy groups could actively facilitate the TAase catalyzed transfer of acetyl groups to the proteins while the propoxy and butoxy groups at the same positions (C-7 and C-8) on the benzenoid ring contribute less to the TAase catalytic activity because of transfer of propoxy and butoxy group from propoxy and butoxy derivatives to proteins with slower rate as compared to acetoxy derivatives which in turn showed less activity.Here it can be explained that as the numbers of carbon atoms in the acyl group at 7 th and 8 th position of benzenoid ring increases, the steric hindrance at carbonyl group of acyl group increases.As a result the tendency of attacking the carbonyl group by nucleophilic amino group of lysine residues of our enzyme decreases.Thus the acetoxy derivatives are better substrates for TAase than propoxy and butoxy derivatives.
[ 3 H] AFB 1 -(G) was obtained from Moravek Biochemicals, Brea, CA (USA).Ethoxyresorufin and pentoxyresorufin were purchased from Fluka Chemicals.All other chemicals were of high grade and were procured from local suppliers.

Test compound Concentration (µM) Activation of NADPH cytochrome C-reductase Standard deviation
Inhibition of liver microsomes catalyzed AFB 1 -DNA binding in vitro by substituted 4-methylcoumarins # Comparison of relative specificity to acetoxy/propoxy/butoxy derivatives of 4-methylcoumarins to liver microsomes to acetoxy drug: Protein transacetylase.#Units of TAase activity expressed in terms of % inhibition of GST under condition of the assay.#Valuesaremean of three observation with variation <5% # Number in bold letters denotes the test compound and preincubated for 10 min.Tab.2.Activation of liver microsomal NADPH cytochrome c reductase by various derivatives of 4-methylcoumarin catalyzed by microsomal acetoxy drug: protein transacetylase # Values are mean of three observations with variation less than <5%.#Number in bold letters denotes the test compound and preincubated for 10 min.Tab.3.#Comparison of TAase catalyzed modulation of rat liver microsomes mediated aflatoxin B 1 binding to DNA in vitro by acetoxy/propoxy derivatives of 4-methylcoumarin.Inhibition of AF B 1 binding to DNA (Pmoles/mg/30 min).Concentration of test compound was 100 µM.#Values are mean of three observations with variation <5%.#Number in bold letters denotes the test compound number.The TAase-mediated inhibition of liver microsomal cytochrome P-450 linked mixed function oxidase (MFO) by acetoxy derivative of 4-methylcoumarin is shown in Table3, 4 and 5.For this purpose, test compounds were preincubated with liver microsomes, followed by the addition of ethyoxyresorufin/pentoxyresorufin and NADPH in order to assay the activity of ethoxyresorufin deethylase (EROD) and pentoxyresorufin (PROD).The results are shown in Table4and 5.These results conclusively ratify the inferences drawn earlier on the specificity of these compounds to TAase.