INTRODUCTION:

Mitragynine was mainly eliminated via O-demethylation to a more polar 9-hydroxycorynantheidine by CYP2D6 in human liver microsome^1-2. It was previously found that the 9-hydroxycorynanthedine was a partial agonist with relative potency twice of the mitragynine³. 9-hydroxycorynantheidine was previously reported could be prepared by EtSH and AlCl₃ in CH₂Cl₂ with high yield at reasonable cost⁴. However, the excess of EtSH have disagreeable odor even in low concentrations. This had resulted in additional costs to provide environmentally acceptable operation to prevent air pollution⁵. The penetrating EtSH had been replaced by the odorless C₁₂H₂₅-SH (lauryl mercaptan) in demethylation of simple aryl methyl ether compound in the recent publication^5-6_. This publication mentioned only the yield, which is excellent, but no other details are given; the information which is lack off and this knowledge gap need to be filled.

The C₁₂H₂₅-SH and 1-dodecane methyl thiol-ether formed in the reaction was soluble in organic solvents and they are contained in the same phase as the product^5-6. Thus, an additional separation step is necessary to remove it. On the other hand, the C₁₂H₂₅-SH is a surfactant, which may cause difficulties in the phase separation step⁵.

Demethylation by BBr₃ could be performed at or below room temperature^7-8. It can be used for the selective demethylation of aryl methyl ethers effectively in the presence of many functional groups including methylenedioxy groups and biphenyl ethers without affecting these group or causing any decomposition under mild condition^7-8. BBr₃ is highly sensitive to moisture and reacts rapidly with water to give B(OH)₃ and HBr^7-9. Cleavage of ether requires one mole of BBr₃ per mole of ether^7-9. It may also be preferably to use additional BBr₃ for substrates containing potentially coordinating functional groups (COOH, NHR, etc.) to improve the deprotection^7-9. With aryl alkyl ethers, BBr₃ in CH₂Cl₂ selectively affords the alkyl bromide and aromatic alcohol^7-10. The first step (Scheme 1) in this ether cleavage reaction is an acid-base reaction between BBr₃ (a Lewis acid) and the ether (a Lewis base) followed by the loss of bromide ion. Bromide ion then participates in an S_N2 reaction, attacking the methyl carbon and displacing oxygen to give an alkyl borate ester. Hydrolysis of this ester (Scheme 1) gives the aromatic alcohol^7-9.

ArOCH₃ + BBr₃---à ArOBBr₂ + CH₃Br ------------- (1)

ArOBBr₂ + 3H₂O --à ArOH + B(OH)₃ (Boric Acid) ----(2)

Scheme 1. Demethylation using boron tribromide

We intend to investigate various method of demethylation to prepare 9-hydroxycorynangtheidine including the BBr₃methodwhich previously not been reported in the reaction with mitragynine. Our semisynthesis aim is to provide a new odorless dealkylation process with a cheaper reagent and can be performed at milder conditions with higher yields, conversion rate and selectivity. Our interest in future is to prepare various derivatives based on 9-hydroxycorynantheidine including the ester derivative of 9-hydroxycorynantheidine which could be the safer prodrugs and could be hydrolyzed in vivo by hydrolase to 9-hydroxycorynanthedine.

Approximately 5-10 equivalent of BBr₃ was used by this designated method for the selective cleavage of 9-methoxy group of mitragynine picrate to prepare the desmethyl 9-hydroxycorynantheidine picrate (2). We performed the experiments under various reactions conditions: temperature (0, 10, 25^oC) and time (60 and 180 minutes) (Tab. 1).We manage to obtain the desmethyl product in good yields (> 80 %) and high percentage of conversion (> 90 %) with negligible percentage of impurities even after only 60 minutes at 0^oC (Tab. 1). The progress of reaction was monitored by TLC and GCMS. After 3 h, the spot corresponding to the starting material, mitragynine, was disappeared and TLC showed only one intense spot at hR_f = 56 (1:1 EtOAc-hexanes, UV radiation at 254 nm). Confirmation by GCMS showed the similar trend after 3 h. The spectral data of the compound obtained (3)¹¹ agreed with those in the literature for 9-hydroxycorynantheidine⁴. This had indicate that 3 h is the required reaction time for full conversion of mitragynine to 9-hydroxycorynantheidine.

DOE optimization of organic synthesis condition shown that time was a more significant factor than temperature for overall performance of this demethylation approach of BBr₃in terms of yields, selectivity and conversion rate (table-2) (Fig 1-3). The contour plot of Fig. 1 showed that to achieve higher yields, combination of both time and temperature is crucial and significant. On the contrary, the contour plot of Fig. 2 indicated that to achieve higher conversion rate, only the time factor is important whereas the contour plot of Fig. 3 showed that selectivity is higher at higher temperature.

By employing the best synthetic condition (entry 2) from Tab. 1, we investigate other potential demethylation reagents (Tab. 2). We have demonstrated that the foul-smelling EtSH in published method can be replaced by C₁₂H₂₅-SH /NaOMe or BBr₃ which proved to be highly selective, almost shared the same percentage in selectivity and yields compared to the well-known regioselective biological enzyme, CYP2D6 in demethylation of mitragynine picrate (Tab. 2). We believe that eliminating the stench of thiols is an important part of sulphur chemistry and that the use of these odourless thiols will greatly improve the physical environment and can provide a friendly green environment for researcher to work with. In this study, one main improvement is in the purification of product. The mitragynine picrate crystal was used as the precursor and after conversion to 9-hydroxycorynanthedine picrate, the synthetic product can be purified via crystallization under methanol without the necessity for column chromatography which is laborious. This help to fill the current knowledge gap of difficult purification when using C₁₂H₂₅-SH as synthetic reagents. Further application of this demethylation approach to the synthesis of a variety of ester derivatives based on position carbon 9 is in progress.

In summary, an efficient approach using BBr₃and NaOMe/ C₁₂H₂₅-SH to prepare the 9-hydroxycorynantheidine at higher yield, conversion rate and selectivity (> 90 %) within shorter reaction times (180 min) under relatively mild reaction conditions (0-25⁰C) had been developed.

Table 1: Effect of temperature and time towards yields, selectivity and conversion using BBr₃ approach

Entry	Temperature ^oC	Time, min	Equivalent mole of reactant, mmol	Equivalent mole of reactant consumed, mmol	Equivalent mole of product obtained, mmol	Yields, %	Conversion, %	Selectivity, %
1	0	60	0.05	0.045	0.040	80	90	89
2	0-25	180	0.05	0.05	0.047	94	100	94
3	0	180	0.05	0.05	0.043	86	100	86
4	10	180	0.05	0.05	0.046	92	100	92
5	0-25	60	0.05	0.046	0.044	88	92	96

Table 2: Comparison of the various demethylation approach towards yields, selectivity and conversion using the condition from entry 2, Tab. 1

Entry	Temperature, ^oC	Time, min	Equivalent mole of reactant, mmol	Equivalent mole of reactant consumed, mmol	Equivalent mole of product obtained, mmol	Yields, %	Conversion, %	Selectivity, %
1 BBr₃	0-25	180	0.05	0.05	0.046	94	100	94
2 C₁₂H₂₅-SH + NaOMe	0-25	180	0.05	0.05	0.047	94	100	94
3 C₁₂H₂₅-SH + AlCl₃	0-25	180	0.05	0.043	0.042	84	86	98
4 TBAF	0-25	180	0.05	0.010	0.009	18	20	90
5 CYP2D6	0-25	180	0.05	0.05	0.049	98	100	98

EXPERIMENTAL:

General Experimental Procedures

Boron Tribromide were purchased from commercial sources and used as received. Melting points are uncorrected IR spectra were recorded as KBr pellets on on a Thermal Scientific Nicolet 6700 spectrophotometer with Omnic software. Preparative TLC was performed on a 0.50 mm thick Si gel 60 F254 layer with a fluorescent indicator coated on 20 cm x 20 cm glass sheets. Visualization was accomplished with UV light (254 nm). UV spectra were measured on a Shimadzu UV 160-A Double Beam Spectrophotometer. CHN were obtained using Perkin Elmer CHN Analyzer Model 2400-2. GC-ECD spectra were taken using HP 6890A GC system, 7673B Auto sampler with HP-5MS Polyimide coated Capillary column (30 m x 0.25 mm i.d. x 0.1 μm), heated from 100^oC to 280^oC at 10^oC / min, 10 Kpa helium at 1.00 mL/min flow rate and injection volume I μL with split ratio 5 : 1. ¹H NMR spectra and ¹³C NMR spectra were recorded on a 400 MHz and 100 MHz nmr spectrometer respectively using CDCl₃ as solvent.

Typical procedure for demethylation

1. BBr₃: To a pre-cooled stirred solution of 1 (99 %, 31.40 mg, 0.05 mmol) in CH₂Cl₂ (10 mL) was added a solution of BBr₃ (125mg, 0.50 mmol) in 4Å molecular sieve dried CH₂Cl₂(20 mL) under a gentle flow of nitrogen. After the reaction was stirred at 0^oC for 60 min. The ice bath was removed and the reaction is allowed to progress for another two hr. The reaction was gthen poured into a mixture of 80 g of ice and 5 ml of concentrated NH₄OH (28-30%, pH 10). The whole mixture was extracted successively with CH₂Cl₂ (3x20 mL) followed by EtOEt (3x 20 ml) in 100 ml separating funnel. The combined organic layer was washed with brine and dried over anhydrous Na₂SO₄ and evaporated to afford 33 mg of crude product. Purification of the crude desmethyl mitragynine was performed by recrystallized 3 times the crude product from methanol to give (2) (28.26 mg, 90 %) as an amorphous yellowish off-white powder. The 9-hydroxycorynantheidine picrate (2) obtained was converted to 9-hydroxycorynantheidine (3) by adding a hot saturated acetone solution of (2) to an excess of dilute aqueous ammonia and the liberated base was extracted with ether. The washed and dried ether extract was observed as yellow amorphous powder (3).

2. C₁₂H₂₅-SH + NaOMe: To a pre-cooled stirred solution of 1 (99 %, 31.40 mg, 0.05 mmol) in C₁₂H₂₅-SH (10 mL) was added NaOMe(5.5 mL, 0.10 mmol) under a gentle flow of nitrogen. After the reaction was stirred at 0^oC for 60 min. The ice bath was removed and the reaction is allowed to progress for another two hr. at 25^oC. The reaction mixture was then extracted successively with CH₂Cl₂ (3x20 mL). The combined organic layer was washed with brine , dried over anhydrous Na₂SO₄ and evaporated to obtain crude product. Purification of the crude desmethyl mitragynine was performed by recrystallized 3 times the crude product from methanol to give (2) (27.63 mg, 88%) as an amorphous yellowish off-white powder. The 9-hydroxycorynantheidine picrate (2) obtained was converted to 9-hydroxycorynantheidine (3) by adding a hot saturated acetone solution of (2) to an excess of dilute aqueous ammonia and the liberated base was extracted with ether. The washed and dried ether extract was observed as yellow amorphous powder (3).

3. C₁₂H₂₅-SH + AlCl₃: To a pre-cooled stirred solution of 1 (99 %, 31.40 mg, 0.05 mmol) in C₁₂H₂₅-SH (10 mL) was added AlCl₃(13.34 mg, 0.10 mmol) under a gentle flow of nitrogen. After the reaction was stirred at 0^oC for 60 min. The ice bath was removed and the reaction is allowed to progress for another two hr. at 25^oC. The reaction mixture was then extracted successively with CH₂Cl₂ (3x20 mL). The combined organic layer was washed with brine and dried over anhydrous Na₂SO₄ and evaporated to afford 33 mg of crude product. Purification of the crude desmethyl mitragynine was performed by recrystallized 3 times the crude product from methanol to give (2) (22.61 mg, 72%) as an amorphous yellowish off-white powder. The 9-hydroxycorynantheidine picrate (2) obtained was converted to 9-hydroxycorynantheidine (3) by adding a hot saturated acetone solution of (2) to an excess of dilute aqueous ammonia and the liberated base was extracted with ether. The washed and dried ether extract was observed as yellow amorphous powder (3).

4. TBAF: To a pre-cooled stirred solution of 1 (99 %, 31.40 mg, 0.05 mmol) in CH₂Cl₂ (10 mL) was added TBAF(130.73 mg, 0.50 mmol) under a gentle flow of nitrogen. After the reaction was stirred at 0^oC for 60 min. The ice bath was removed and the reaction is allowed to progress for another two hr. at 25^oC. The reaction mixture was then extracted successively with CH₂Cl₂ (3x20 mL). The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄ and evaporated off to obtain crude product. Purification of the crude desmethyl mitragynine was performed by recrystallized 3 times the crude product from methanol to give (2) (5.60 mg, 18%) as an amorphous yellowish off-white powder. The 9-hydroxycorynantheidine picrate (2) obtained was converted to 9-hydroxycorynantheidine (3) by adding a hot saturated acetone solution of (2) to an excess of dilute aqueous ammonia and the liberated base was extracted with ether. The washed and dried ether extract was observed as yellow amorphous powder (3).

5. CYP2D6: 0.25 mg/ml of human liver microsomes, phenotyped for CYP2D6 activity (Gentest Corp., Woburn, MA, 7 pmol), were placed in a shaking incubator at 37 ± 1^oC with sodium phosphate buffer (75 mM, pH 7.4), MgCl₂ (3 mM), EDTA (1 mM), and mitragynine picrate (99%, 31.40mg) in a total volume of 200 μl, was preincubated for 2–3 min in a water bath at 37 °C and the reactions were initiated by the addition of 50 μl of 5 mM NADPH. In addition, control reactions were carried out without enzyme or NADPH. Reactions were stopped after 25 min or 45 min by the addition of equal volume of cold methanol (1/1) and centrifuged at 9000×g for 20 min, 5 °C. Incubations were performed in duplicate. The pure Desmethyl mitragynine picrate (2) obtained was (30.77 mg, 98%). The 9-hydroxycorynantheidine picrate (2) obtained was converted to 9-hydroxycorynantheidine (3) by adding a hot saturated acetone solution of (2) to an excess of dilute aqueous ammonia and the liberated base was extracted with ether. The washed and dried ether extract was observed as yellow amorphous powder (3).

Spectral data of principle compound:

9-hydroxycorynanthedine (3). Yellow amorphous powder. UV (MeOH, λm(nm)): 294, 226; IR (KBR, cm^-1): 3213 (C-OH), 1636(C-NH), 1695 (C=O); GCMS m/z (%) = 384(M⁺) (100), 369(35), 255(30), 200(76); Anal. Calcd for C₂₂H₂₈N₂O₄: C, 68.75; H, 7.29; N, 7.30. Found: C, 68.54; H, 7.32; N, 7.24; 1H NMR (400 MHz, CDCl₃): δ 7.68 (1H, br.s, N-H), 7.43 (1H, s, H-17), 6.89 (1H, dd, J= 7.6, 7.6 Hz, H-11), 6.84 (1H, d, J= 7.2 Hz, H-12), 6.47 (1H, d, J= 7.1 Hz, H-10), 3.71 (6H, s, 17-OCH₃ and 22-OCH₃), 3.19 (1H, m, H-6), 3.13 (1H, br.d, J=11.3 Hz, H-3), 3.04 (2H, m, H-15 and H-21), 2.94 (2H, m, H-5 and H-6), 2.56 (1H, m, H-5), 2.08 (1H, m, H-14), 2.44 (1H, m, H-21), 1.76 (2H, m, H-14 and H-19), 1.69 (1H, m, H-20), 1.22 (1H, m, H-19), 0.86 (3H, dd, J=7.4, 7.4 Hz, H-18); ¹³C NMR (100 MHz, CDCl₃): δ 169.4(C-22), 160.7(C-17), 154.5(C-9), 137.3(C-13), 133.7(C-2), 121.9(C-11), 117.7(C-7), 111.6(C-16), 107.9(C-8), 104.3(C-10), 99.8(C-12), 61.7(17-OCH₃), 61.3(C-3), 57.8(C-21), 22.8(C-5), 51.5(22-OCH₃), 39.9(C-20), 32.0(C-15), 29.8(C-14), 22.8(C-6), 14.2(C-19), 12.9(C-18).

ACKNOWLEDGMENTS:

This work was supported by USM Research University Grant (RUT), Universiti Sains Malaysia, Penang, Malaysia.

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Received on 08.02.2013 Modified on 06.04.2013

Asian J. Research Chem. 6(9): September 2013; Page 863-867