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Complete structural determination of 1,2-Benzo-8-(Alanyl)-3-Phenoxazone by NMR techniques (HSQC & HMBC).

Abstract. -- The structure of 1,2-Benzo-8-(Alanyl)-3-Phenoxazone was determined by use of heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC). Both HSQC and HMBC experiments optimize direct and long range [H.sup.1]-[C.sup.13] connections respectively rather than [C.sup.13]-[H.sup.1] connections. In this study HMBC experiments allows the assignment of quaternary carbons C-3 and C-17 with protons that are two and three bonds away ([.sup.2,3][J.sub.C-H]). The HSQC experiments show the direct [H.sup.1]-[C.sup.13] connections for C-15, C-16, C-4, C-6, C-11, C-14 and C-9 by using [C.sup.13] satellites in proton spectrum to acquire correlation. This study confirms the attachment of alanyl group at C-8 and the position of C-3 in the form of C = O of BLP.


It has been shown that 1-nitroso-2-naphthol qualitatively and quantitatively reacts with various phenolic compounds in the presence of HN[O.sub.3] (Bhansali 1971). In order to study the mechanism of reaction, nitroso naphthol was reacted with tyramine and 1,2-Benzo-8-(2-aminoethyl)-3-phenoxazone (BAP) was produced. This compound was characterized by one and two-dimensional NMR spectroscopy and mass spectrometry (Donaldson & Lenon 1979). BAP is an analog of actinomycin D and is a very potent antitumor agent which is also very toxic for human use. Moreover, BAP was selected by the National Cancer Institute (NCI) of Bethesda, Maryland, for screening against HIV activity during its drug development program. Similarly, the reaction of nitroso naphthol with tyrosine produced 1,2-Benzo-8-(alanyl)-3-phenoxazone, (BLP) and was characterized by one and two-dimensional NMR spectroscopy and mass spectrometry (Bhansali & Kook 1993). This compound was screened against various cell lines of prostrate cancer and HIV activity by the National Cancer Institute in its drug development laboratories and has shown very promising results. Further, this compound received a patent by the U.S. Patent Office in 1994.

Moreover, heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC) experiments are quite useful in sequencing carbon atoms through proton determination especially for the end parts of the molecule. Because of the usefulness of this compound, it is considered of importance to confirm its absolute configuration by HSQC and HMBC experiments.




BLP was run in DMSO-[d.sub.6] on the BrukerAMX500 HMBC and HSQC experiments were used to determine the complete structure of BLP. The former is the long-range [.sup.1]H-[.sup.13]C experiment which allows the assignment of quaternary carbons with protons which are two or three bonds away ([.sup.2,3]J C-H), while by HSQC experiment the direct [.sup.1]H-[.sup.13]C connections were obtained by using [.sup.13]C satellites in proton spectrum to acquire correlation.


All carbons and protons can be assigned by HSQC (Figure 1, Table 1). The HMBC experiment demonstrates that the H-14 (Figure 2, Table 2) has long range coupling with C-3 carbonyl group (182.9 ppm) and C-12 (132.2 ppm), on the other hand, H-4 could be related via long-range correlation to C-2 (132.1 ppm), this confirms the position of C-3 carbonyl. For confirmation of the position of the substituent at C-8, the HMBC experiment also shows clearly the long-range correlations of the beta proton H-15 (3.2 ppm) of the amino acid part of the molecule with the carboxyl group of the amino acid C-17 (170.5 ppm), and the C-16 (52 ppm), and C-8 (135 ppm). This does not make the assignment of the peak at 135 ppm to C-8, because it still could be the C-7 of the other isomer. But protons H-7 (7.48 ppm) and H-9 (7.76 ppm) shows ([.sup.3]JC-H) correlation to C-6a (143.0 ppm) which pins down the assignment making it the 8-isomer. This in conjunction with the correlation of C-8 with H-15 (2.96 ppm) completes the match.

The present data generated by HMQC and HMBC experiments support the previous data on characterization of 1,2-Benzo-8-alanyl-3-phenoxazone by [.sup.1]H-NMR, [.sup.1]H-COSY, J resolved 2D and 13[C.sup.-1]H heteronuclear correlation experiments. Further, this study concludes the complete structural determination of the compound.
Table 1. [.sup.1]H-[.sup.13]C--NMR (HSQC) Data of BLP

Position [.sup.1]H([.sup.3,4]J Hz) [.sup.13]C

 1 130.8
 1a 147.1
 2 132.1
 3 182.9
 4 6.43 106.6
 4a 151.3
 6 7.45 116.2
 6a 143.0
 7 7.48 133.3
 8 135.0
 9 7.78 130.5
 9a 131.6
11 8.59 (1.3, 1.7, 7.9) 124.3
12 7.88 (1.3, 7.9, 9.1) 132.2
13 7.81 (7.2, 9.1) 132.5
14 8.15 (1.3, 2.3, 7.2) 125.4
15 3.16, 3.22 (4.5, 6.32, 8.0) 35.1
16 4.33 (4.5, 6.2) 53.1
17 170.5

Table 2. NMR (HMBC) Data of BLP


14 C-3, C-12
15 C-17, C-16, C-8
 6 C-9a
 7 C-6a, C-15
 9 C-6a, C-15
11 C-1a, C-13
 4 C-2
13 C-11


This study was supported by a grant from Research Centers in Minority Institutions (RCMI) Grant No. 2G12RR03045-06.


Bhansali, K. G. 1971. Use of 1-Nitroso-2-Naphthol in quantitative determination of medicinal phenolic compounds. J. Pharm. Sci., 61:146.

Bhansali, K. G. & A. M. Kook. 1987. Characterization of 1,2-Benzo-8-(2-aminoethyl)-3-phenoxazone. J. Pharm. Sci., 76:654-657.

Bhansali, K. G. & A. M. Kook. 1993. Synthesis and characterization of a series of 5H-Benzo[a]-phenoxazin-5-one derivatives as potential antiviral/antitumor agents. Heterocycles, 1239-1251.

Donaldson, S. S. & R. A. Lenon. 1979. Alteration of nutritional status. Impact of chemotherapy and radiation therapy. Cancer, 2036-2052.

K. G. Bhansali, S. G. Milton and F. Matloubimoghaddam*

College of Pharmacy and Health Sciences, Texas Southern University

Houston, Texas 77004 and *Department of Chemistry, Sharif University of Technology, Tehran, Iran
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Author:Bhansali, K.G.; Milton, S.G.; Matloubimoghaddam, F.
Publication:The Texas Journal of Science
Date:Nov 1, 1997
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