Enantiospecific Synthesis of a Genetically Encodable Fluorescent Unnatural Amino Acid L-3-(6-Acetylnaphthalen-2-ylamino)-2-aminopropanoic Acid

doi:10.1021/jo2007626

J Org Chem. Author manuscript; available in PMC 2012 August 5.

Published in final edited form as:

J Org Chem. 2011 August 5; 76(15): 6367–6371.

Published online 2011 July 6. doi: 10.1021/jo2007626

PMCID: PMC3155268

NIHMSID: NIHMS309969

Enantiospecific Synthesis of a Genetically Encodable Fluorescent Unnatural Amino Acid L-3-(6-Acetylnaphthalen-2-ylamino)-2-aminopropanoic Acid

Zheng Xiang and Lei Wang^*

The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037

Email: lwang/at/salk.edu

The publisher's final edited version of this article is available at J Org Chem

Abstract

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Object name is nihms309969u1.jpg Object name is nihms309969u1.jpg

Fluorescent unnatural amino acids (Uaas), when genetically incorporated into proteins, can provide unique advantages for imaging biological processes in vivo. Synthesis of optically pure L-enantiomer of fluorescent Uaas is crucial for their effective application in live cells. An efficient six-step synthesis of L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap), a genetically encodable and polarity-sensitive fluorescent Uaa, has been developed. The synthesis takes advantage of a high-yield and enantiospecific Fukuyama-Mitsunobu reaction as the key transformation.

The past decade has witnessed the dramatic progress on site-specific incorporation of unnatural amino acids (Uaas) into proteins in live cells through the expansion of the genetic code.^1–4 Using engineered tRNA-synthetase pairs orthogonal to endogenous counterparts in the host cell, more than 70 Uaas have been incorporated into proteins in Escherichia coli, yeast, or mammalian cells, which enable new opportunities to study biological problems with expanding non-proteinogenic chemistries. Fluorescent Uaas (Figure 1), including L-dansylalanine (1),^5–7 L-(7-hydroxycoumrin-4-yl)ethylglycine (2)⁸ and L-Anap (3),⁹ are of particular interest for molecular and cellular biology, which may complement and enchance the widely used fluorescent proteins (FPs).¹⁰ These fluorescent Uaas can be incorporated at virtually any site of a protein, whereas the fusion of FPs is often limited to the N- or C-terminus or certain loop regions. In addition, fluorescent Uaas are much smaller than FPs, which helps to mitigate undesired perturbations to the structure and function of the target protein. Moreover, when developing fluorescent reporters, sensitivity to different environment cues such as pH and polarity can be chemically designed and synthesized¹¹ into the fluorescent Uaas, a flexibility not readily available for FPs.

FIGURE 1

Genetically Encoded Fluorescent UAAs

Despite the potential advantages, effective application of fluorescent Uaas in live cells faces multiple challenges. Efficient synthesis of optically pure Uaas is a foremost demand. When racemic Uaas are fed to cells, the D-enantiomer competes for cellular uptake yet cannot be incorporated into proteins by the ribosome.² This reduces the net intracellular concentration of the bioactive L-enantiomer, leading to lower incorporation efficiency. For fluorescent Uaas in particular, the intracellular D-enantiomer further increases background fluorescence, which is a critical issue for cell imaging with Uaas. Synthetic routes to enantiomerically pure 1 and 2 have been developed,^5,12 but only a racemic synthesis is available for Uaa 3.⁹ Anap is sensitive to polarity with changes in intensity and emission wavelength.⁹ Uaa 3, when selectively incorporated into proteins, has the potential to fluorescently report protein conformational changes, interactions, modifications, and activities in live cells. Herein, we report a concise and efficient synthetic approach of L-Anap, which offers significant improvements over the current synthesis.

L-Anap has been incorporated into proteins in Saccharomyces cerevisiae by Schultz and co-workers.⁹ They developed a synthetic approach of racemic anap, which gives 8.1% of total yield over 8 steps (Scheme 1A). The synthesis was based on the nucleophilic substitution of ethyl bromopyruvate oxime (6) developed by Gilchrist and co-workers.^13,14 Presumably due to the low nucleophilicity of the Burcherer reaction product of 4 and 5, compound 7 was synthesized in only 26% yield over two steps from compound 4.¹⁵ Another drawback of this synthesis resides in the nonstereoselective reduction of 7, which leads to the final racemic product.

SCHEME 1

Synthetic Approach for D,L-Anap (A) and Synthetic Strategy for LAnap (B)

To synthesize sufficient amounts of L-anap, we decided to develop an efficient and enantiospecific synthetic pathway toward L-anap (Scheme 1B). Our synthetic strategy involves Fukuyama-Mitsunobu reaction^16–18 as the key step, which couples compound 10 with N-trityl-L-serine methyl ester 11¹⁹ to give intermediate 12. This approach has two features: First, both the nucleophile and the electrophile are channeled to facilitate the intermolecular coupling by tuning the electronic effect of the protecting groups. o-Nitrobenzenesulfonyl group makes 10 a good nucleophile under Mitsunobu condition and can be removed easily under mild conditions. Trityl group of compound 11 can conduct the reactive intermediate through the intermolecular coupling and circumvent β-elimination as the side reaction. Second, the Mitsunobu reactions of 11 with different nucleophiles have been proved to be free of racemization,¹⁹ therefore intermediate 12 are expected to be synthesized enantiospecifically.

We commenced with the synthesis of 6-acyl-2-naphthylamine (13) via two pathways (Scheme 2). In approach A, the selective Heck reaction^20–22 and Cu(I)-catalyzed C-N^23,24 bond formation were employed to introduce the acetyl and amino groups. 6- Bromo-2-naphthol (14) was transformed to triflate 15, which was coupled with butyl vinyl ether through Cabri’s procedure,^20–22 followed by treatment with acid, to give intermediate 16. Under Buchwald’s condition,²⁵ compound 16 underwent the C-N bond formation to give intermediate 17 in 75% yield. After deprotection, compound 13 was obtained in 54% yield over 4 steps. Although this approach provided gram-scale synthesis of 13 with satisfactory yield, approach B, which took three steps less than A, was explored in parallel. Approach B features the direct conversion of hydroxyl group of compound 4 to amino group. Although this transformation via Burcherer reaction²⁶ has been reported by Cho and his coworkers,²⁷ the procedure involves stirring the reaction mixture at 140 °C for 96 hours in a steel-bomb reactor, which is not viable in many biology laboratories. Inspired by recent advances in one-pot alkylation-Smiles rearrangment-hydrolysis²⁸ sequence, we applied Mizuno and Yamano’s procedure to the transformation of 4 to 13. Monitoring the reaction with TLC showed the complete conversion of the alkylation and Smiles rearrangement steps. However, the in situ hydrolysis of 19 under basic condition was incomplete and the final product 13 was difficult to separate from intermediate 19. Therefore, the crude product of 19 was separated and hydrolyzed under acidic condition, which provided compound 13 in 73% yield.

SCHEME 2

Synthesis of Compound 13

With compound 13 in hand, we set out to explore the Fukuyama-Mitsunobu reaction (Scheme 3). Compound 13 was treated with o-nitrobenzenesulfonyl chloride and pyridine to give compound 10. In the presence of diisopropyl azodicarboxylate and triphenylphosphine, compounds 10 and 11 were coupled to give intermediate 12 in 88% yield over two steps. Sequential deprotection of trityl group and o-nitrobenzensulfonyl group furnished L-anap methyl ester (20). Deprotection of 20 under acidic condition^29,30 furnished L-anap as its hydrochloride salt. The enantiomeric purity of 3 was examined with the Mosher method,³¹ and the dr value was above 40:1, which indicates that less than 3% racemization had occurred during deprotection (see Supporting Information).

SCHEME 3

Synthesis of L-Anap

In conclusion, we developed an efficient synthetic route to enantiomerically pure L-anap, which gives 51% of total yield. Compared to the previous method, our synthetic approach not only afforded a bioactive L-enantiomer but also enabled a 12-fold increase in yield with fewer steps. All the reactions of our approach proceeded under mild conditions as well as in gram-scale. The general approach demonstrated here can also be applied to the synthesis of other 3-N-aryl-2,3-diaminoproprionic acids, which serve as important building blocks for medicinal small molecules.^32–34 The application of L-anap to image protein modifications and activities in mammalian cells is undergoing and will be reported in due course.

Experimental Section

1-(6-Bromonaphthalen-2-yl)ethanone 16

To a solution of 6-bromo-2-naphthol 14 (2.231 g, 10 mmol) in CH₂Cl₂ (50 mL) was added DIPEA (1.92 mL, 11 mmol) and PhNTf₂ (3.930 g, 11 mmol) at room temperature. The reaction mixture was stirred under nitrogen for 12 hours and concentrated under vacuum. The residue was filtered through a short silica gel column and eluted with EtOAc/hexanes (1/19). The solution was concentrated under vacuum to give the crude product of 15 as yellow oil. 15 was dissolved in DMF (20 mL). Et₃N (2.79 mL, 20 mmol), butyl vinyl ether (6.47 mL, 50 mmol), dppp (113.4 mg, 0.275 mmol) and Pd(OAc)₂ (56.1 mg, 0.25 mmol) were added to the solution sequentially. The mixture was stirred under nitrogen at 80 °C for 2 hours and then cooled to 0 °C. 2 M HCl solution (20 mL) was added to the reaction mixture slowly and the mixture was stirred for half an hour. Water (120 mL) and EtOAc (50 mL) were added to the mixture. The two phases were separated and the aqueous phase was washed twice with EtOAc (50 mL). The organic phases were combined and washed with brine (30 mL), dried over anhydrous Na₂SO₄, concentrated and purified with flash chromatography (EtOAc/hexanes = 1/7) to give compound 16 (1.798 g, 72%) as white solid. R_f = 0.23 (EtOAc/hexances = 1/6). ¹H NMR (500 MHz, CDCl₃): δ = 8.41 (s, 1 H), 8.05 (d, J = 1.5 Hz, 1H), 8.04 (s, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.62 (dd, J = 9.0, 2.0 Hz, 1H), 2.71 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ = 197.8, 136.6, 134.9, 131.2, 131.1, 130.4, 130.1, 127.6, 125.2, 123.0, 26.8. HRMS (EI): calcd for C₁₂H₉BrO: 247.9831, found 247.9832.

tert-Butyl 6-acetylnaphthalen-2-ylcarbamate 17

To the mixture of compound 16 (1.993 g, 8 mmol), tert-butyl carbamate (1.125 g, 9.6 mmol), CuI (76.2 mg, 0.40 mmol) and K₂CO₃ (2.211 g, 16 mmol) was added N,N′-dimethylethylenediamine (86.1 μL, 0.80 mmol) and toluene (16 mL). The mixture was stirred under nitrogen at 110 °C for 24 hours. After cooling to room temperature, the reaction mixture was filtered and washed with EtOAc (20 mL). The filtrate was concentrated and purified by flash chromatography (EtOAc/hexanes = 1/3) to give compound 17 (1.719 g, 75%) as white solid. R_f = 0.29 (EtOAc/hexanes = 3/7). ¹H NMR (500 MHz, CDCl₃): δ = 8.37 (s, 1H), 8.07 (s, 1H), 7.99 (dd, J = 9.0, 1.5 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.42 (dd, J = 8.5, 2.0 Hz, 1H), 6.85 (s, 1H), 2.70 (s, 3H), 1.56 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ = 198.1, 152.7, 138.5, 136.7, 133.3, 130.7, 130.0, 128.9, 127.9, 124.8, 119.9, 114.0, 81.2, 28.5, 26.7. HRMS (ESI-FT): [M+H]⁺ calcd for C₁₇H₂₀NO₃, 286.1438; found 286.1441.

6-Acyl-2-naphthylamine 13

To a solution of compound 17 (1.427 g, 5 mmol) in CH₂Cl₂ (15 mL) at 0 °C was added TFA (5 mL) dropwise. The reaction mixture was stirred at room temperature for 3 hours and concentrated under vacuum. The residue was dissolved in dichloromethane (100 mL) and washed with saturated Na₂CO₃ aqueous solution and brine. The organic phase was dried over Na₂SO₄ and concentrated to give compound 13 (0.919 g, 99%) as yellow solid. ¹H NMR (500 MHz, CDCl₃): δ = 8.31 (d, J = 1.5 Hz, 1H), 7.92 (dd, J = 8.5, 1.5 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 6.97 (dd, J = 8.5, 2.5 Hz, 1H), 6.95 (d, J = 2.0 Hz, 1H), 4.08 (br s, 2 H), 2.66 (s, 3 H); ¹³C NMR (125 MHz, CDCl₃): δ = 198.0, 146.9, 137.8, 131.5, 131.3, 130.6, 126.6, 126.1, 124.8, 118.9, 108.0, 26.6.

6-Acyl-2-naphthylamine 13

To a solution of compound 4 (558.6 mg, 3 mmol) in DMA (6 mL) was added NaOH (360 mg, 9 mmol). The resulting mixture was stirred at room temperature for one hour. 2-Bromo-2-methylpropanamide (1.494 g, 9 mmol) was added to the mixture. After stirring at room temperature overnight, TLC showed complete conversion of compound 4. NaOH (1.080 g, 27 mmol) was added and the resulting mixture was stirred at 50 °C for 5 hours. Water (60 mL) was added to the mixture and was extracted with CH₂Cl₂ (25 mL) for three times. The combined organic phase was concentrated and redissolved in ethanol (15 mL) and 6 M HCl solution (15 mL). The mixture was refluxed until TLC showed complete conversion of compound 19. After the mixture was cooled to room temperature, most of EtOH was removed under reduced pressure. The residue was diluted with EtOAc and water. The aqueous solution was neutralized with 1 M NaOH solution. The mixture was extracted with EtOAc for three times. The combined organic layers were dried over anhydrous Na₂SO₄, concentrated and purified by flash chromatography (EtOAc/hexanes = 1/1) to give 13 (406.8 mg, 73%) as yellow solid.

(S)-Methyl 3-(N-(6-acetylnaphthalen-2-yl)-2-nitrophenylsulfonamido)-2- (tritylamino)propanoate 12

To a solution of 13 (555.7 mg, 3 mmol) in CH₂Cl₂ (30 mL) was added pyridine (0.27 mL, 3.3 mmol) and o-NsCl (698.1 mg, 3.15 mmol) sequentially at 0 °C. The reaction was allowed to stir at room temperature overnight. The reaction mixture was washed with 1 M HCl solution (20 mL), water and brine, and dried over Na₂SO₄. The solution was concentrated and the resulting red solid was dissolved in toluene (30 mL) and stirred at 0 °C. To this was added 11 (2.169 g, 6 mmol) and PPh₃ (1.574 g, 6 mmol). DIAD (1.18 mL, 6 mmol) was added dropwise to the solution. The reaction mixture was stirred overnight at room temperature. The solution was concentrated and purified by flash chromatography (EtOAc/hexanes = 2/3) to give compound 12 (1.892 g, 88 %) as red solid. R_f = 0.28 (EtOAc/hexanes = 1/1). ¹H NMR (500 MHz, CDCl₃): δ = 8.43 (s, 1H), 8.06 (dd, J = 8.5, 1.5 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.88 (s, 1H), 7.60 (dd, J = 8.0, 1.5 Hz, 1H), 7.56 (dt, J = 8.0, 1.0 Hz, 1H), 7.45 (dd, J = 8.5, 2.0 Hz, 1H), 7.42 (dd, J = 8.0, 1.0 Hz, 1H), 7.33-7.29 (m, 1H), 7.28-7.26 (m, 6H), 7.11-7.05 (m, 9H), 4.36 (dd, J = 15.0, 4.5 Hz, 1H), 4.29 (dd, J = 14.5, 7.0 Hz, 1H), 3.53 (br s, 1H), 3.16 (s, 3 H), 2.72 (s, 3H), 2.66 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃): δ = 197.8, 173.0, 145.5, 138.6, 135.7, 135.4, 133.9, 132.3, 131.8, 131.7, 131.2, 131.0, 129.7, 128.8, 128.69, 128.66, 128.0, 127.9, 127.0, 126.5, 125.0, 124., 71.4, 56.7, 56.6, 52.0, 26.9. HRMS (ESI-FT): [M+Na]⁺ calcd for C₄₁H₃₅N₃O₇SNa, 736.2089; found 736.2086. [α]_D²³ + 69.6 (c 1.00, CHCl₃).

(S)-Methyl 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoate 20

To a solution of 12 (1.428 g, 2 mmol) in CH₂Cl₂ (16 mL) was added TFA (2.0 mL) and water (2.0 mL) at 0 °C. The reaction mixture was stirred at room temperature for 3 hours and concentrated under vacuum. The residue was dissolved in DMF (10 mL). Thiophenol (440.4 mg, 4.0 mmol) and K₂CO₃ (2.76 g, 20 mmol) were added to the solution sequentially. The reaction mixture was stirred at room temperature for two hours. Water (80 mL) was added to the mixture and the solution was extracted with EtOAc (40 mL) for three times. The combined organic phase was washed with brine (40 mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated and purified by flash chromatography (EtOAc/hexanes = 1/1, then MeOH/MeOH = 1/20) to give compound 20 (459.6 mg, 80%) as yellow solid. R_f = 0.15 (MeOH/CH₂Cl₂ = 1/20). ¹H NMR (500 MHz, CDCl₃): δ = 8.29 (s, 1 H), 7.92 (dd, J = 9.0, 2.0 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 6.95 (dd, J = 8.5, 2.0 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 4.75 (br s, 1H), 3.82 (br s, 1H), 3.78 (s, 3H), 3.65-3.63 (m, 1H), 3.35 (dd, J = 12.0, 7.5 Hz, 1H), 2.66 (s, 3H), 1.70 (br s, 2H); ¹³C NMR (125 MHz, CDCl₃): δ = 197.9, 147.9, 138.0, 131.2, 131.0, 130.4, 126.3, 126.2, 124.9, 118.9, 104.4, 53.6, 52.5, 46.9, 26.5. HRMS (ESI-FT): [M+H]⁺ calcd for C₁₆H₁₉N₂O₃, 287.1390; found 287.1395. [α]_D²⁴ +74.0 (c 1.00, CHCl₃).

L-Anap 3

A solution of compound 16 (286.3 mg, 1 mmol) in 2 M HCl solution (10 mL) was stirred at 60 °C for 8 hours. The reaction mixture was lyophilized to give yellow solid. After washing with diethyl ether, the solid was dried under vacuum to give L-anap (306.8 mg, 99%) as yellow solid. ¹H NMR (500 MHz, D₂O): δ = 7.82 (s, 1H), 7.45 (dd, J = 8.5, 2.0 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 6.87 (dd, J = 8.5, 2.0 Hz, 1H), 6.71 (s, 1H), 3.53-3.51 (m, 1H), 3.47 (dd, J = 13.0, 4.5 Hz, 1H), 3.23 (dd, J = 13.0, 7.0 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d6): δ = 197.1, 169.5, 148.0, 137.6, 130.5, 130.4, 130.2, 125.7, 125.5, 124.1, 119.0, 102.9, 51.5, 42.9, 26.4. HRMS (ESI-FT): [M+H]⁺ calcd for C₁₅H₁₇N₂O₃, 273.1234; found 273.1239. [α]_D²⁵ + 74.2 (c 1.07, DMSO).

Supplementary Material

1_si_001

Click here to view.^{(3.9M, pdf)}

Acknowledgments

We thank Dr. Michael Burkart, Mr. Christopher Vickery and Dr. Jing Xu for help with the HRMS and optical rotation. This work was supported by the March of Dimes Foundation (5-FY08-110), California Institute for Regenerative Medicine (RN1-00577-1), and National Institutes of Health (1DP2OD004744-01).

Footnotes

Supporting Information Available: general experimental information and spectra for all new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

References

1. Wang L, Brock A, Herberich B, Schultz PG. Science. 2001;292:498. [PubMed]

2. Wang L, Schultz PG. Angew Chem Int Ed Engl. 2005;44:34. [PubMed]

3. Wang Q, Parrish AR, Wang L. Chem Biol. 2009;16:323. [PMC free article] [PubMed]

4. Liu CC, Schultz PG. Annu Rev Biochem. 2010;79:413. [PubMed]

5. Summerer D, Chen S, Wu N, Deiters A, Chin JW, Schultz PG. Proc Natl Acad Sci U S A. 2006;103:9785. [PubMed]

6. Wang Q, Wang L. J Am Chem Soc. 2008;130:6066. [PubMed]

7. Wang W, Takimoto JK, Louie GV, Baiga TJ, Noel JP, Lee KF, Slesinger PA, Wang L. Nat Neurosci. 2007;10:1063. [PMC free article] [PubMed]

8. Wang J, Xie J, Schultz PG. J Am Chem Soc. 2006;128:8738. [PubMed]

9. Lee HS, Guo J, Lemke EA, Dimla RD, Schultz PG. J Am Chem Soc. 2009;131:12921. [PMC free article] [PubMed]

10. Tsien RY. Angew Chem Int Ed Engl. 2009;48:5612. [PubMed]

11. Lee JS, Kim YK, Vendrell M, Chang YT. Mol Biosyst. 2009;5:411. [PubMed]

12. Brun MP, Bischoff L, Garbay C. Angew Chem Int Ed Engl. 2004;43:3432. [PubMed]

13. Gilchrist TL, Roberts TG. J Chem Soc, Perkin Trans 1. 1983:1283.

14. Gilchrist TL, Lemos A. Tetrahedron. 1992;48:7655.

15. Li JR, Zhang G. J Chem Crystallogr. 2005;35:789.

16. Fukuyama T, Jow CK, Cheung M. Tetrahedron Lett. 1995;36:6373.

17. Swamy KC, Kumar NN, Balaraman E, Kumar KV. Chem Rev. 2009;109:2551. [PubMed]

18. Kan T, Fukuyama T. Chem Commun (Camb) 2004:353. [PubMed]

19. Cherney RJ, Wang L. J Org Chem. 1996;61:2544.

20. Cabri W, Candiani I, Bedeschi A, Santi R. J Org Chem. 1990;55:3654.

21. Cabri W, Candiani I, Bedeschi A, Penco S, Santi R. J Org Chem. 1992;57:1481.

22. Cabri W, Candiani I. Acc Chem Res. 1995;28:2.

23. Surry DS, Buchwald SL. Chemical Science. 2010;1:13. [PMC free article] [PubMed]

24. Evano G, Blanchard N, Toumi M. Chem Rev. 2008;108:3054. [PubMed]

25. Klapars A, Huang X, Buchwald SL. J Am Chem Soc. 2002;124:7421. [PubMed]

26. Seeboth H. Angew Chem Int Ed. 1967;6:307.

27. Kim HM, Kim BR, An MJ, Hong JH, Lee KJ, Cho BR. Chemistry. 2008;14:2075. [PubMed]

28. Mizuno M, Yamano M. Org Lett. 2005;7:3629. [PubMed]

29. Burkart DJ, McKenzie AR, Nelson JK, Myers KI, Zhao X, Magnusson KR, Natale NR. Org Lett. 2004;6:1285. [PubMed]

30. Sakai R, Koike T, Sasaki M, Shimamoto K, Oiwa C, Yano A, Suzuki K, Tachibana K, Kamiya H. Org Lett. 2001;3:1479. [PubMed]

31. Dale JA, Dull DL, Mosher HS. J Org Chem. 1969;34:2543.

32. Lauffer DJ, Mullican MD. Bioorg Med Chem Lett. 2002;12:1225. [PubMed]

33. Wang JY, Guo XF, Wang DX, Huang ZT, Wang MX. J Org Chem. 2008;73:1979. [PubMed]

34. Yang SM, Scannevin RH, Wang B, Burke SL, Huang Z, Karnachi P, Wilson LJ, Rhodes KJ, Lagu B, Murray WV. Bioorg Med Chem Lett. 2008;18:1140. [PubMed]