Facile synthesis of mutagen X (MX): 3-chloro-4-(dichloromethyl)-5-hydroxy- 5H-furan-2-one

Article abstract of DOI:10.1016/j.tetlet.2012.04.044

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.

Full text of DOI:10.1016/j.tetlet.2012.04.044

Tetrahedron Letters 53 (2012) 3144–3146
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Facile synthesis of mutagen X (MX):
3-chloro-4-(dichloromethyl)-5-hydroxy-5H-furan-2-one
Venkata Velvadapu a, Mark E. McDonnell a, Eileen K. Jaffe b, Allen B. Reitz a,
⇑
a Fox Chase Chemical Diversity Center, Inc., 3805 Old Easton Road, Doylestown, PA 18902, USA
b Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
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.
Received 13 March 2012
Revised 5 April 2012
Accepted 9 April 2012
Available online 14 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Mutagen X
Furanone
Chlorine
Mucochloric acid
Mutagenicity
Introduction
MX, in some model systems, was particularly potent relative to
other halogenated compounds in inducing DNA damage and alter-
Halogenated organic substances 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 (Fig. 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 dif-
ferent concentrates showed consistent mutagenicity in Salmonella
typhimurium strain TA100. Later the mutagenic compound was
identified as MX.4 These halogenated compounds were also iso-
lated 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 trihalome-
thanes, 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 Massachu-
setts (USA).8
ing pathways involved in cell growth.4 MX was also found to be
mutagenic in mammalian cell assays in vitro and in vivo.9 In stud-
ies 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 Be-
cause we observed that MX reacted covalently with the active site
lysine of an enzyme in heme biosynthesis that we are investigat-
ing, 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. modi-
fied Padmapriya’s synthesis by the addition of H2SO4 to the metal
catalyst in the olefin chlorination of 4 to give 5, and Jinqu et al. la-
ter used UV-light instead of a metal catalyst in the chlorination
procedure.12 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
Cl
Cl
Cl
Cl
Cl
O
O
O
O
HO
HO
MX(2)
Mucochloric acid (1)
Figure 1. Mucochloric acid (1) and Mutagen X (2).
⇑
Corresponding author. Tel.: +1 215 589 6435; fax: +1 215 489 4920.
E-mail addresses: areitz@fc-cdci.com, reitz12000@yahoo.com (A.B. Reitz).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.04.044

V. Velvadapu et al. / Tetrahedron Letters 53 (2012) 3144–3146
3145
O
O
ref. 11, 12
Cl
O
OMe
Cl
OMe
Cl
a
b
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
c
3
4
5
2
Cl
Cl
f
ref. 13
HO
Cl
e
Cl
d
Cl
O
O
O
O
O
O
7
8
6
Scheme 1. Reagents and conditions: (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%.
O
Cl
Cl
Cl
Cl
Cl
O
O
P
O
OEt
Cl
Cl
CO2H
Cl
EtO
EtO
a
Cl
Cl
+
Cl
O
OEt
CHO
O
HO
Cl
9
Cl
Cl
Cl
Cl
10
B
A
3
Figure 2. MX in ring-closed (A) and open chain (B) forms.
Scheme 2. Reagents and conditions: (a) NaH, THF, 0 °C; or n-BuLi, À78 °C.
(AIBN). The crude reaction mixture was then treated with HCl/
dioxane in water under reflux conditions to hydrolyze the anomer-
ic 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.
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 two steps, was utilized to assemble
MX in eight steps going through olefin 7 and vinylchloride 8 with
an overall yield of 4%. In our work, we sought to improve the over-
all yield and reduce the number of chemical steps, while removing
the use of chlorine gas altogether.
Evaluation of the 1H 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 (Fig. 2). MX ex-
ists 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
ambient temperature. In contrast to this previous report,21 we
observed that the only form of MX at pH of 7.4 in D2O was the
closed ring lactone A. In fact, in all tested acidic and neutral
solutions of D2O, MX existed as the closed ring lactone.
Our initial concept was to utilize triethyl-2-chloro-2-phospho-
noacetate (9) in a Horner–Wadsworth–Emmons (HWE) reaction14
to install the chlorine in the desired a
-position. Compound 315 was
reacted with 9 under basic conditions but reaction was not ob-
served 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.
Installation of the
a-chlorine on the olefin was achieved first by
the HWE reaction of 9 with 1,3-diacetoxyacetone (11) to yield
a
-
chloroester 12 in 80% yield (Scheme 3),16 followed by treatment
of 12 with catalytic PTSA under reflux conditions17 in EtOH to fur-
nish 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 cen-
ter was achieved with refluxing in CCl4, with 2 equiv of N-bromo-
succinimide (NBS) and a catalytic amount of azobisisobutyronitrile
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 Forma-
tion of these lactams was similar to the reductive amination of
mucochloric acid as previously reported.24 These results suggested
Cl
Cl
O
a
OEt
b
HO
AcO
AcO
AcO
AcO
O
O
O
12
11
13
Cl
Cl
Cl
Cl
Cl
c
Cl
d
O
O
O
HO
O
MX (2)
8
Scheme 3. Reagents and conditions: (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.

3146
V. Velvadapu et al. / Tetrahedron Letters 53 (2012) 3144–3146
15. (a) Aranda, G.; Bertranne-Delahaye, M.; Azerad, R.; Maurs, M.; Cortés, M.;
Cl
Cl
Ramirez, M.; Vernal, G.; Prangé, T. Synth. 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 h at 0 °C and 0.5 h 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 h. The
reaction was quenched with sat NH4Cl (50 mL) and the aqueous layer was
separated and worked up in EtOAc (2 Â 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
Cl
O
Cl
Cl
N
Ph(CH2)3NH2
Cl
O
Na(OAc)3BH, CHCl3
O
HO
Ph
2, MX
14
Figure 3. Reductive amination of MX with phenylpropylamine.
80% yield. IR (film) 1730, 1640, 739 cmÀ1 1H NMR (300 MHz) d 5.01 (s, 2H),
;
that in biological systems, 1 or 2 could react with nucleophiles
(DNA or aminoacids) to form a Schiff base as the first step followed
by subsequent modifications (Fig. 3).25
In conclusion a facile synthesis of MX has been developed with
an overall yield of 27% in six steps, starting with 9. A favorable as-
pect of the synthetic route presented is that a variety of MX ana-
logs can be prepared, without the use of chlorine gas.
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 d 170.0, 169.9 162.0, 138.3, 126.3, 62.5, 61.6, 60.4, 20.3
(2C), 13.7. HRMS (FAB) calcd for C11H15ClO6+H+ = 279.0629, found 279.0637.
17. Balasubramaniam, R. P.; Moss, D. P.; Wyatt, J. K.; Spence, J. D.; Gee, A.; Nantz,
M. H. 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 h. 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 cmÀ1
;
Acknowledgments
1H NMR (300 MHz) d 4.99 (s, 2H), 4.73 (d, J = 5.1 Hz, 2H), 3.46(br s, 1H); 13C
NMR d 169.4, 160.3, 117.2, 70.8, 57.5. HRMS (FAB) calcd for C5H5ClO3+H+
=
The authors would like to thank Dr. Jeff Pelletier for helpful dis-
cussions. We also acknowledge the support of the National Insti-
tutes of Health (1 R43 AI084224-01).
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 mortar and pestle and then suspended into DCM
a
(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 eight matched those reported in Ref. 13.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
04.044.
References and notes
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 24 h 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 (3 Â 5 mL). The combined organic layers were washed with water
(5 mL), brine (5 mL), and dried over Na2SO4. The solvent was evaporated under
reduced pressure and purified via flash column chromatography (hexanes/
EtOAc 1:4, four 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 Ref. 13.
1. (a) Singer, S. J.; Spengler, F.; Chavez, J. T.; Kusmierek, T. Carcinogenesis 1987, 8,
745–747; (b) Cheng, K. C.; Preston, D. S.; Cahill, M. K.; Dosanjh, B.; Singer, L. A.
Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9974–9978; (c) Fedtke, N.; Boucheron, J. A.;
Turner, M. J., Jr.; Swenberg, J. A. Carcinogenesis 1990, 11, 1279–1285; (d)
Guengrich, F. P. Chem. Res. Toxicol. 1992, 5, 852–855.
2. Loper, J. C. Mutat. Res. 1980, 76, 241–268.
3. (a) Holmbom, B. R.; Voss, R. H.; Mortimer, R. D.; Wong, A. Tappi J. 1981, 64, 172–
174; (b) Holmbom, B. IARC Sci. Publ. 1990, 104, 333–340.
4. Mcdonald, T. A.; Komulainen, H. J. Environ. Sci. Health 2005, 23(2), 163–214.
5. (a) Hemming, J.; Holmbom, B.; Reunanen, M.; Kronberg, L. Chemosphere 1986,
15, 549–556; (b) Meier, J. R.; Knohl, R. B.; Coleman, W. E.; Ringhand, H. P.;
Munch, J. W.; Kaylor, W. H.; Streicher, R. P.; Kopfler, F. C. Mutat. Res. 1986, 363–
373.
21. LaLonde, R. T.; Cook, G. P.; Carlton, H. W.; Babish, J. G. Environ. Mol. Mutagensis.
1991, 17, 40–48.
22. Baxter, E. W.; Reitz, A. B. Reductive aminations of carbonyl compounds with
borohydride and borane reducing agents In Organic Reactions; Wiley: New
York, 2002; Vol. 59, pp 1–714.
6. (a) Kringstad, K. P.; Ljungquist, P. O.; de Sousa, F.; Strömberg, L. Environ. Sci.
Technol. 1983, 17, 553–555; (b) Fielding, M.; Horth, H. Wat. Supply 1986, 4103–
4126.
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 24 h. 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
7. Smeds, A.; Vartiainen, T.; Maki-Paakkanen, J.; Kronberg, L. Sci. Technol. 1997, 31,
1033–1039.
8. Wright, J. M.; Schwartz, J.; Vartiainen, T.; Maki-Paakkanen, J.; Altshul, L.;
Harrington, J. J. Environ. Health Perspect. 2002, 110, 157–164.
9. (a) Chang, L. W.; Daniel, F. B.; De Angelo, A. B. Teratogen. Carcinogen. Mutagen.
1981, 11, 103–114; (b) Jansson, K.; Hyttinen, J. M. T. Mutat. Res. 1994, 322, 129–
132; (c) Harrington, B. K.; Doerr, C. L.; Moore, M. M. Mutat. Res. 1995, 348, 105–
110; (d) Furihata, C.; Yamashita, M.; Kinae, N.; Matsushima, T. Water Sci.
Technol. 1992, 25, 341–345; (e) Jansson, K. Mutat. Res. 1995, 299, 25–28.
10. Komulainen, H.; Kosma, V.; Vaittinen, L.; Vartiainen, T.; Tuomisto, J. J. Natl.
Cancer Inst. 1999, 89, 848–856.
11. Padmapriya, A. A.; Just, G.; Lewis, N. G. Can. J. Chem. 1985, 63(4), 828–832.
12. (a) Franzén, R.; Kronberg, L. Tetrahedron. Lett. 1995, 36(22), 3905–3908; (b)
Jinqu, Z.; Zhen, Z.; Huixian, Z.; Minmin, Y. Synth. Commun. 1995, 25, 3401–
3405. Note: We couldn’t validate this experimental due to the lack of UV
reactor setup..
(film) 3068, 3033, 1684, 1496,1446 cmÀ1 1H NMR (300 MHz) d 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 d 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) calcd for
C
14H14Cl3NO+H+ = 318.0219 found 318.0216.
24. Zhang, J.; Blazeka, P. G.; Davidson, J. G. Org. Lett. 2003, 5(4), 553–556.
25. (a) Munter, T.; Curieux, F. L.; Sjoholm, R.; Kronberg, L. Chem. Res. Toxicol. 1998,
11, 226–233; (b) Franzen, R.; Tanabe, K.; Morita, M. Chemosphere 1998, 36(13),
2803–2808.
13. LaLonde, R. T.; Perakyla, H.; Hayes, M. P. J. Org. Chem. 1990, 55(9), 2847–2855.
14. Maryanoff, B. E.; Reitz, A. B. Chem. Rev. 1989, 89, 863–927.