New Convenient Synthesis of 8-C-Methylated Homoisoflavones and Analysis of Their Structure by NMR and Tandem Mass Spectrometry

Homoisoflavonoids are in the subclass of the larger family of flavonoids having one more alkyl carbon than flavonoids. Among them, 8-C-Methylated homoisoflavones have not been extensively studied for synthesis and biological evaluation. Author’s current objective is to synthesize 8-C-Methylated homoisoflavones by the reaction of 3-C-methylated dihydrochalcones with N,N’-dimethyl (chloromethylene) ammonium chloride generated in situ from DMF and PCl5 for one carbon extension at about room temperature. The 3-C-methylated dihydrochalcones were synthesized by the reduction of 3-Cmethylated chalcones, which were prepared from 3-C-methylated acetophenones and aromatic aldehydes in the presence of base. All the synthesized novel homoisoflavones’s structures were characterized by NMR and Tandem Mass Spectrometry.


Introduction
The homoisoflavonoids were classified into five groups based on their structures: sappanin-type (I), scillascillin-type (II), brazilin-type (III), caesalpin-type (IV), and protosappanin-type (V) [1], Figure 1 shows the structure of all the five types of homoisoflavonoids. 3-Benzylchromones, the sub-group of sappanin-type ho-moisoflavonoids, mainly found in the plants of Fabaceae (genus Cassia) and Asparagaceae families (genus Ophiopogon) [1] [2]. Almost all are hydroxy-substituted at C-5 in ring A, and usually hydroxyl-, methoxy-, and/or methylenedioxy-substituted at C-7 in ring A, as well as at C-2', C-3', and C-4' in ring B [3] [4] [5]. Additionally, methyl and/or formyl substituted can be found at C-6 and C-8 in ring A in compounds isolated from the genus Ophiopogon [5] [6] [7]. The methyl substituent at C-7 was reported from the genus Cassia [8]. Some naturally occurring 3-benzylchromene-4-ones are shown in Figure 2. Various 3-benzylchromene-4-ones have been reported with a broad range of bioactivities, including angioprotective, antiallergic and antihistaminic properties [9]. In view of the increasing interest on homoisoflavanoids, several organic chemists do the research on isolation, synthesis and applications of homoisoflavanoids and their derivatives since last two decades. In this context the author has synthesized several 8-C-methylated homoisoflavones which are not studied very well in the literature.
All the intermediates were confirmed by comparing the spectral data and melting points with the literature (Scheme 3).

H NMR and 13 C NMR of chalcones, dihydrochalcones and homoisoflavones
In the 1 H NMR spectra of the chalcones, the characteristic resonance signals for α and β protons appeared in the region δ 7.60 -7.90 and δ 7.70 -8.12 as doublets respectively. The H-5 aromatic protons were observed in the region δ 6.27 -6.29. The methoxyl and methyl groups on the aromatic rings displayed signals as singlets in the region δ 3.84 -3.98 and δ 1.93 to 2.04 respectively. The hydroxyl protons (OH at C-2) displayed signals in the region δ 13.89 -14.29. In the 13 C NMR Spectra of the chalcones, the resonance signals for the carbonyl carbons In the 1H NMR Spectra of the 8-C-methylated homoisoflavones, the characteristic resonance signals for the H-2 and H-9 were observed as singlets in the region δ 7.95 -8.14 and δ 3.45 -3.61 respectively. The aromatic protons of homoisoflavones were observed between δ 6.13 and δ 7.69 depending on the nature of the substituents on the aromatic rings. The methoxyl groups and methyl group on the aromatic rings displayed their proton signals in the region δ 3.84 -3.98 and δ 1.93 -2.04 as singlets respectively. In the 13 C NMR spectra of the homoisoflavones, the resonance signals for the carbonyl carbons (C=O) were located in the region δ 176.4 -180.8. The chemical shifts for the olefinic carbons, C-2 and C-3 were observed in the region δ 153.2 -154.8 and δ 120.8 -121.5 respectively. The carbon signal for C-9 was observed near δ 29.3 -30.4. The methoxyl and methyl group carbons attached to aromatic rings showed signals in the region δ 55.2 -55.9 and δ 7.2 -7.7 respectively. Figure 3 is the 1 H NMR Spectrum of 3-(3', 4', 5'-trimethoxybenzyl)-5, 7-dimethoxy-8-methyl-4H-chromen-4-one (9d), which is a typical spectrum of this group of compounds. Figure 4 is the 13 C NMR Spectrum of 3-(3', 4', 5'-trimethoxybenzyl)-5, 7-dimethoxy-8-methyl-4H-chromen-4-one (9d), which is a typical spectrum of this group of compounds.
Tandem Mass spectrometry of homoisoflavones For MS/MS analysis, a 4000 QTRAP mass spectrometer (AB SCIEX, Toronto, Canada) was used having Analyst 1.6.3 software. To tune the mass spectrometer, a 1000 ng/ml solution of pure compounds 9a-9i in acetonitrile (MeCN) were respectively injected into the source by continuous infusion. The mass spectrometer parameters were adjusted as source temperature 500 °C, Heater gas 60 (nitrogen) psi,  Nebulizer gas 40 (nitrogen) psi, Curtain gas 25 (nitrogen) psi, CAD gas 5 (nitrogen) psi, Ion Spray (IS) voltage 5500 volts, Source flow rate 20 µl/min without split.
APCI and ESI sources were tried for the ionization of homoisoflavonoids both in positive and negative ion modes. Base peak in positive mode gave the good intensity rather than in negative mode where base peak obtained with remarkably lower intensity. APCI and ESI produced very similar ions. Therefore, ESI in the positive ion mode was selected as the ion source for follow-up analyses. For full scan MS analysis, the spectra were recorded in the range of m/z 200 -500 Da, Figure 5 shows the parent ion (M+H) + of compound 9a at 341.2 . The isolation width of precursor ions was 3.0 mass units. During MS/MS product-ion analysis of compounds 9a to 9i, two common fragment ions at m/z 234.4 and 221.3 were observed which revealed that the major fragment ions occurred by the cleavage of C3-9 or C9-1' bonds to lose the B-ring (Scheme 3).   shows the daughter ions at 234.1 and 221.1 of compound 9a. Except 9H where first step was the conversion of methylenedioxy group at B-ring into a hydroxyl group, and then underwent the cleavage of C3-9 or C9-1' bonds to lose the B-ring. For compounds 9e, both (M + H) + and (M + 2H) + were obtained as prominent peak.

Conclusion
The author has developed a mild, efficient, and economical method for the synthesis of 8-C-methylated homoisoflavones using the PCl 5 /DMF complex. Operational simplicity, mild reaction conditions, short reaction times, and good yields are the notable advantages of this method. Study of biological activities and preclinical research of synthesized compounds are under progress.