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Tetrahedron Lett. Author manuscript; available in PMC 2013 June 20.
Published in final edited form as:
Tetrahedron Lett. 2012 June 20; 53(25): 3144–3146.
doi:  10.1016/j.tetlet.2012.04.044
PMCID: PMC3398704
NIHMSID: NIHMS374795

Facile Synthesis of Mutagen X (MX): 3-Chloro-4-(dichloromethyl)-5-hydroxy-5H-furan-2-one

Abstract

3-Chloro-4-(dichloromethyl)-5-hydroxy-5H-furan-2-one (Mutagen X, MX) was synthesized in six steps from commercially-available and inexpensive starting materials (27% overall yield). This synthesis enables the preparation of MX analogs and does not require the use of chlorine gas, as do previously reported methods.

Keywords: Mutagen X, Furanone, Chlorine, Mucochloric Acid, Mutagenicity

Introduction

Halogenated organic substance containing vinyl chloride and chlorinated hydroxyfuranone functionalities such as mucochloric acid (1) and Mutagen X (2, MX) are contaminants in chlorinated water and industrial chemical waste (Figure 1).1 Compounds 1 and 2 were discovered in the late 1970s, and shown to be mutagenic in the Ames assay.2 MX was isolated initially from chlorine-bleached pulp mill effluents in 1979.3 One fraction among the different concentrates showed consistent mutagenicity in Salmonella typhimurium strain TA100. Later the mutagenic compound was identified as MX.4 These halogenated compounds were also isolated from chlorine-disinfected or treated drinking water.5 They are formed by the reaction of Cl2 with humic acids derived from microorganisms present in soil and water.6 MX was shown to be present in detectable limits in these drinking water sources and at levels as high as 310 ng/L.4 Though the concentration of MX in drinking water is typically 100- to 1000-fold lower than other common chlorinated by-products of concern such as trihalomethanes, it is believed that MX is more mutagenic. Smeds et al. analyzed drinking water samples from 35 locations and reported that MX accounted for up to 67% of the overall mutagenicity (S. typhimurium TA100).7 Similar results were also obtained by Wright et al. among 88 samples taken from 36 towns in Massachusetts (USA).8

Figure 1
Mucochloric acid (1) and Mutagen X (2).

MX, in some model systems, was particularly potent relative to other halogenated compounds in inducing DNA damage and altering pathways involved in cell growth.4 MX was also found to be mutagenic in mammalian cell assay in vitro and in vivo.9 In studies performed by Komulainen et al. MX was found to be a potent carcinogen in rodents.10 There has been speculation that MX reacts directly with the aminopurine functionality of adenosines.5 Because we observed that MX reacted covalently with the active site lysine of an enzyme in heme biosynthesis that we are investigating, we sought to prepare larger quantities of MX to explore its chemical reactivity and stability.

Two distinct methods have been reported to synthesize MX. The first, by Padmapriya et al. in 1985, involved five steps starting from tetrachloroacetone (3, Scheme 1).11 In 1995, Franzén et al.12a modified Padmapriya’s synthesis by addition of H2SO4 to the metal catalyst in the olefin chlorination of 4 to give 5, Jinqu et al.12b later used UV-light instead of a metal catalyst in the chlorination procedure. We found that the methods for the chlorination of olefin 4 were not generally reproducible, even repeating exactly the methods and stoichiometry of reagents used. The second general procedure for the preparation of MX was reported by Lalonde et al., in 1990.13 Key intermediate 4-(hydroxymethyl)-2(5H)-furanone (6), made in 2 steps, was utilized to assemble MX in 8 steps going through olefin 7 and vinylchloride 8 with an overall yield of 4%. In our work, we sought to improve the overall yield and reduce the number of chemical steps, while removing the use of chlorine gas altogether.

Scheme 1
(a) Ph3P=CHCO2Me, THF, 84%. (b) (i) Cl2 gas, FeCl3. (ii) Et3N, 80%. (c) (i) LiOH, quantitative. (ii) KHCO3, 65%. (d)(i) PCC. (ii) PCl5, 60%. (e) (i) Cl2 gas, FeCl3. (ii) Et3N, 48%. (f) (i) NBS, light. (ii) Hg(OAc)2, H2O, acetone, 20%.

Our initial concept was to utilize triethyl-2-chloro-2-phosphonoacetate (9) in a Horner-Wadsworth-Emmons (HWE) reaction14 to install the chlorine in the desired the α-position. Compound 3 was reacted with 9 under basic conditions but reaction was not observed to give 10 (Scheme 2), which we attributed to competition between deprotonation of the acidic hydrogens and quenching of the reactivity of 3 with deprotonation of phosphonoacetate 9.

Scheme 2
(a) NaH, THF, 0° C; or n-BuLi, −78° C.

Installation of the α-chlorine on the olefin was achieved first by the HWE reaction of 9 with 1,3-diacetoxyacetone (11) to yield α-chloroester 12 in 80% yield (Scheme 3),16 followed by treatment of 12 with catalytic PTSA under reflux conditions17 in EtOH to furnish lactone 13 in 85% yield.18 Primary alcohol 13 was oxidized using PCC and then treated with PCl5 to afford dichloromethyl compound 8 in 80% yield.19 Bromination of 13 at the anomeric center was achieved with refluxing in CCl4, with two equivalents of N-bromosuccinimide (NBS) and a catalytic amount of azobisisobutyronitrile (AIBN). The crude reaction mixture was then treated with HCl/dioxane in water under reflux conditions to hydrolyze the anomeric bromide to the corresponding alcohol, thus affording 2 in 50% yield over the two steps.20 The use of HCl/dioxane did not result in the formation of appreciable side products, and purification was relatively straight-forward.

Scheme 3
(a) (EtO)2P(O)CH(Cl)CO2Et (10), NaH, THF, 80%. (b) PTSA, EtOH, 85%. (c) (i) PCC. (ii) PCl5, 80% over two steps (d) (i) NBS, AIBN (cat), CCl4. (ii) HCl, dioxane, 50% over two steps.

Evaluation of the H-1 NMR (300-MHz) of MX in CDCl3, D2O, and DMSO-d6 confirmed the dependency of solvent on the equilibrium of the open-chain and the ring-closed forms of MX (Figure 2). MX exists as a 1:1 ratio of the ring-closed (A) and the open-chain forms (B) in DMSO-d6 at ambient temperature, and in CDCl3 and D2O the predominant form observed was the ring closed form at room temperature.21 In contrast to this previous report, we observed that the only form of MX at pH of 7.4 was the closed ring lactone A. In fact, in all tested acidic and neutral solutions of D2O, MX existed as the closed ring lactone.

Figure 2
MX in ring-closed (A) and open chain (B) forms.

There are three sites in MX that can react with nucleophiles such as amines, namely the lactone carbonyl, hemiacetal carbon, and the dichloromethyl substituent. When MX was treated with 3-phenylpropylamine under conditions of reductive amination,22 five membered ring lactam 14 was isolated in 50% yield.23 Formation of these lactams was similar to the reductive amination of mucochloric acid as previously reported.24 These results suggested that in biological systems, 1 or 2 would react with nucleophilic bases pair (DNA or aminoacids) to form a Schiff base as the first step and then propagate subsequent modifications (Figure 3).25

Figure 3
Reductive amination of MX with phenylpropylamine.

In conclusion a facile synthesis of MX has been developed with an overall yield of 27% in six steps, starting with 9. A favorable aspect of the synthetic route presented is that a variety of MX analogs can be prepared, without the use of chlorine gas.

Supplementary Material

01

Acknowledgments

The authors would like to thank Dr. Jeff Pelletier for helpful discussions. We also acknowledge the support of the National Institutes of Health (1 R43 AI084224-01).

Footnotes

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References and Notes

1. (a) Singer SJ, Spengler F, Chavez JT, Kusmierek T. Carcinogenesis. 1987;8:745–747. [PubMed](b) Cheng KC, Preston DS, Cahill MK, Dosanjh B, Singer LA. Proc Natl Acad Sci USA. 1991;88:9974–9978. [PubMed](c) Fedtke N, Boucheron JA, Turner MJ, Jr, Swenberg JA. Carcinogenesis. 1990;11:1279–1285. [PubMed](d) Guengrich FP. Chem Res Toxicol. 1992;5:852–855. [PubMed]
2. Loper JC. Mutat Res. 1980;76:241–268. [PubMed]
3. Holmbom BR, Voss RH, Mortimer RD, Wong A. Tappi. 1981;64(3):172–174.(b) Holmbom B. IARC Sci Publ. 1990;104:333–340. [PubMed]
4. Mcdonald TA, Komulainen H. J Envi Sci & Health. 2005;23(2):163–214. [PubMed]
5. Hemming J, Holmbom B, Reunanen M, Kronberg L. Chemosphere. 1986;15:549–556.(b) Meier JR, Knohl RB, Coleman WE, Ringhand HP, Munch JW, Kaylor WH, Streicher RP, Kopfler FC. Mutat Res. 1987:363–373. [PubMed]
6. Kringstad KP, Ljungquist PO, de Sousa F, Strömberg L. Environ Sci Technol. 1983;17:553–555. [PubMed](b) Fielding M, Horth H. Wat Supply. 1986;4:103–126.
7. Smeds A, Vartiainen T, Maki-Paakkanen J, Kronberg L. Sci Technol. 1997;31(4):1033–1039.
8. Wright JM, Schwartz J, Vartiainen T, Maki-Paakkanen J, Altshul L, Harrington JJ. Environ Health Perspect. 2002;110(2):157–164. [PMC free article] [PubMed]
9. Chang LW, Daniel FB, De Angelo AB. Teratogen Carcinogen Mutagen. 1981;11:103–114. [PubMed](b) Jansson K, Hyttinen JMT. Mutat Res. 1994;322:129–132. [PubMed](c) Harrington BK, Doerr CL, Moore MM. Mutat Res. 1995;348:105–110. [PubMed](d) Furihata C, Yamashita M, Kinae N, Matsushima T. Wat Sci Tech. 1992;25:341–345.(d) Jansson K. Mutat Res. 1995;299:25–28. [PubMed]
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11. Padmapriya AA, Just G, Lewis NG. Can J Chem. 1985;63(4):828–832.
12. Franzén R, Kronberg L Tetrahedron Lett. 1995;36(22):3905–3908.
Jinqu Z, Zhen Z, Huixian Z, Minmin Y Synth Commun. 1995;25(21):3401–3405.
. Note: We couldn’t validate this experimental due to the lack of UV reactor setup.
13. LaLonde RT, Perakyla H, Hayes MP. J Org Chem. 1990;55(9):2847–2855.
14. Maryanoff BE, Reitz AB. Chem Rev. 1989;89:863–927.
15. Aranda G, Bertranne-Delahaye M, Azerad R, Maurs M, Cortés M, Ramirez M, Vernal G, Prangé T Syn Comm. 1997;27(1):45–60.
. (b) Trichloro- and pentachloroacetone were formed and identified as impurities.
16. Procedure and spectral data for 12: To a suspension of sodium hydride (60% dispersion in oil, 1.0 g, 25.73 mmol) in THF (150 mL) at 0° C was added triethyl-2-chloro-2-phosphonoacetate (6.35 g, 24.58 mmol) dropwise. The reaction solution was allowed to stir for 1 hr at 0° C and 0.5 hr at room temperature. The resulting yellow solution was cooled to 0° C and 1,3-diacetoxyacetone (4.21 g, 24.58 mmol) was added. The resulting solution was slowly allowed to warm to ambient temperature and stirred for 20 hr. The reaction was quenched with sat NH4Cl (50 mL) and the aqueous layer was separated and worked up in EtOAc (2 × 100 mL). The combined organic layers were washed with water (50 mL), brine (50 mL) and dried over Na2SO4. The solvent was reduced under reduced pressure and purified via flash column chromatography (hexanes: EtOAc 5:1) to obtain 5.4 g of 12 as a yellow oil in 80% yield. IR (film) 1730, 1640, 739 cm−1; 1H NMR (300-MHz) δ 5.01 (s, 2H), 4.92 (s, 2H), 4.31 (q, J = 7.1 Hz, 2H), 2.11 (d, J = 13.1 Hz, 3H), 2.05 (s, 3H), 1.45 – 1.22 (m, 3H); 13C NMR δ 170.0, 169.9 162.0, 138.3, 126.3, 62.5, 61.6, 60.4, 20.3 (2C), 13.7. HRMS (FAB) calc’d for C11H15ClO6+H+ = 279.0629, found 279.0637.
17. Balasubramaniam RP, Moss DP, Wyatt JK, Spence JD, Gee A, Nantz MH. Tetrahedron. 1997;53:7429–7444.
18. Procedure and spectral data for 13: To a solution of 12 (5.5 g, 19.73 mmol) in 90% EtOH (100 mL) was added PTSA (0.38 g, 2 mmol). The resulting solution was heated to reflux for 72 hr. The solution was concentrated and dissolved in EtOAc (100 mL) and washed with sat. NaHCO3 (30 mL), water (20 mL), brine (20 mL) and dried over Na2SO4. The solvent was reduced under reduced pressure and purified via flash column chromatography (hexanes:EtOAc 1:4) to obtain 2.5 g of 13 as a yellow oil in 85% yield. IR (film) 3340, 1722, 744 cm−1; 1H NMR (300MHz) δ 4.99 (s, 2H), 4.73 (d, J = 5.1 Hz, 2H), 3.46(bs, 1H); 13C NMR δ 169.4, 160.3, 117.2, 70.8, 57.5. HRMS (FAB) calc’d for C5H5ClO3+H+ = 148.9999, found 149.0000.
19. Procedure for 8: PCC (3.2 g, 14.7 mmol) was dispersed on solid NaCl (15 g) by grinding together in a mortar and pestle and then suspended into DCM (50 mL). A solution of 13 (1 g, 6.71 mmol) in DCM (15 mL) was then added to the above suspension. After 3 h the DCM layer was filtered through a short plug of silica and washed with DCM (25 mL). To this solution PCl5 (6 g, 28.85 mmol) was added and stirred for 20 min at rt. Solid NaHCO3 (20 g) was then added followed by water (200 mL) slowly. This biphasic solution was stirred approximately 4 h until all the CO2 evolution ceased. The DCM layer was separated and the aqueous layer was extracted with DCM (30 mL). The combined organic layers were washed with water (30 mL), brine (30 mL) and dried over Na2SO4. The solvent was concentrated under reduced pressure and filtered through a plug of silica using Ether (50 mL). After concentration of the organic layer 1 g of 8 was obtained as brown color oil in 80% yield. Spectral data of 8 matched those reported in reference 13.
20. Procedure for 2: To a solution of 8 (0.5 g, 2.48 mmol) in CCl4 (25 mL) was added NBS (0.88 g, 4.96 mmol), cat AIBN (10–20 mg) and refluxed for 24h under N2. The resulting solution was cooled filtered through a plug of silica using DCM (10 mL). This solution was concentrated and redissolved in 80% Dioxane (15 mL) and 5% HCl (5 mL) solution. This solution was refluxed for 2 h after which the solution was concentrated and the aqueous layer was extracted with EtOAc (3X5 mL). The combined organic layers were washed with water (5 mL), brine (5 mL) and dried over Na2SO4. The solvent was reduced under reduced pressure and purified via flash column chromatography (hexanes:EtOAc 1:4, 4 drops of HCl) to obtain 0.26 g of 2 as a yellow oil in 50% yield. Spectral data of 2 matched those reported in reference 13.
21. LaLonde RT, Cook GP, Carlton HW, Babish JG. Env Mol Mutagensis. 1991;17:40–48. [PubMed]
22. Baxter EW, Reitz AB. Organic Reactions. Vol. 59. Wiley; New York: 2002. Reductive Aminations of Carbonyl Compounds with Borohydride and Borane Reducing Agents; pp. 1–714.
23. Procedure and spectral data for 14: Sodium triacetoxyborohydride (25 mg, 0.16 mmol) was slowly added to a mixture of MX (25 mg, 0.11 mmol) and 3-phenylpropylamine (16 mg, 0.12 mmol) in chloroform (1 mL) acetic acid (0.01 mL) and 22 mg of molecular sieves. The reaction mixture was stirred at rt for 24h. The reaction mixture was partitioned between water (1 mL) dichloromethane (2 mL), the phases were separated and the organic phase was washed once with water (1 mL). The organic phase was concentrated under reduced pressure and purified via flash column chromatography (Hexanes: EtOAc 4:1) to obtain 16 mg of 14 as a yellow oil in 50% yield. IR (film) 3068, 3033, 1684, 1496,1446 cm−1; 1H NMR (300MHz) δ 7.32-7.17 (m, 5H), 6.74 (s, 1H), 4.19 (s, 1H), 3.57 (t, J = 7.5 Hz, 2H), 2.70–2.71 (m, 2H), 2.15-1.94 (m, 2H); 13C NMR δ 164.1, 144.3, 140.8, 128.5 (2C), 128.3 (2C), 126.2, 126.1, 62.8, 48.2, 43.1, 33.0, 29.7. HRMS (FAB) calc’d for C14H14Cl3NO+H+= 318.0219 found 318.0216.
24. Zhang J, Blazeka PG, Davidson JG. Org Lett. 2003;5(4):553–556. [PubMed]
25. (a) Munter T, Curieux FL, Sjoholm R, Kronberg L. Chem Res Toxicol. 1998;11:226–233. [PubMed](b) Franzen R, Tanabe K, Morita M. Chemosphere. 1998;36(13):2803–2808. [PubMed]