Synthesis of [14C]-imexon

Article abstract of DOI:10.1002/jlcr.911

A four-step synthesis of [¹⁴C]-imexon is described, starting from [¹⁴C]-phosgene. The overall yield is 27% and the specific activity is 55 mCi/mmol. Copyright

Full text of DOI:10.1002/jlcr.911

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 165–170.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.911
Research Article
Synthesis of [14C]-imexon
1
2
2
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Bhumasamudram Jagadish , Bhashyam S. Iyengar , Aniko M. Solyom ,
William A. Remers2, Robert T. Dorr3, Jason Shin Yu4, Surendra Gupta4
and Eugene A. Mash1,*
1 Department of Chemistry, The University of Arizona, Tucson, AZ 85721-0041, USA
2 Department of Pharmacology and Toxicology, The University of Arizona, Tucson,
AZ 85721, USA
3 Department of Pharmacology, The University of Arizona, Tucson, AZ 85721, USA
4 American Radiolabeled Chemicals, Inc., St. Louis, MO 63146, USA
Summary
A four-step synthesis of [14C]-imexon is described, starting from [14C]-phosgene. The
overall yield is 27% and the specific activity is 55 mCi/mmol. Copyright # 2005 John
Wiley & Sons, Ltd.
Key Words: imexon; aziridine; sulfhydryl-binding; anticancer agent
Introduction
Imexon (4-imino-l,3-diazabicyclo[3.1.0]hexane-2-one, 1) is entering clinical trials as
a prospective anticancer agent. In a previous clinical trial based on its
immunomodulatory properties, imexon showed positive responses against a variety
of tumors.1 More recently, its activity in immunodeficient SCID mice and in cell
cultures suggests that immunomodulation is not responsible for cytotoxicity.2
*Correspondence to: Eugene A. Mash, Department of Chemistry, The University of Arizona, Tucson,
Arizona 85721-0041, USA. E-mail: emash@u.arizona.edu
Copyright # 2005 John Wiley & Sons, Ltd.
Received 5 November 2004
Revised 9 November 2004
Accepted 10 November 2004

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B. JAGADISH ET AL.
Although imexon has an aziridine ring, it does not alkylate DNA nor does it
react with lysine, a model compound for the free amino groups of proteins.3 It
does react readily with cellular thiols, including glutathione and cysteine,3 and
it depletes them in a dose- and time-dependent manner.4 This depletion results
in an increase in the level of reactive oxygen species in cells, which damage
mitochondrial but not nuclear DNA.4 The mitochondrial membrane potential
of imexon-sensitive cells undergoes a significant decrease, and cytochrome C is
released from the mitochondria. This effect induces apoptotic cell death.5
The mode of action described above accounts for many of the properties of
imexon, but it leaves unanswered a number of important questions, including
(1) What is the distribution of imexon among important tissues such as liver
and kidney? (2) What is the metabolic fate of imexon? (3) How strongly does
imexon bind to serum proteins? (4) Is the depletion of glutathione and other
thiols the result of direct interaction, or is it controlled by reaction of imexon
with enzymes such as glutathione S-transferase 2 and glutathione peroxidase 1
that are essential to the maintenance of glutathione levels? Many such
questions are best addressed by experiments based on the use of radiolabeled
imexon.
Imexon 1 was first prepared by Bicker et al.6 by cyclization of the
carboxamide 2 with KOH in anhydrous methanol. Two methods have been
described for the synthesis of 2. In the first method 2-cyanoaziridine 3 is reacted
with isocyanic acid.7 In the second method aziridine 3 is reacted with phenyl
chloroformate and the resulting carbamate 4 is treated with ammonia to give 2.8
Recently, Remers et al.9 reported a new synthesis of imexon. Trichlor-
oacetylisocyanate was reacted with 3 to give 5. Compound 5 was converted to
2 by treatment with ammonia in ethanol. Cyclization was achieved by
treatment of 2 with benzyltrimethylammoniurn hydroxide in absolute ethanol.
Results and discussion
Keeping in mind the availability of 2-cyanoaziridine 3 and the relative
efficiencies of the reaction pathways described above, we planned to
incorporate the 14C label at the carbonyl carbon atom. Radiolabeled isocyanic
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SYNTHESIS OF [14C]–IMEXON
167
acid and trichloroacetyl isocyanate are not commercially available. Isocyanic
acid is unstable above 08C, and preparation of radiolabeled trichloroacetyl
isocyanate presented an undesirable synthetic challenge. On the other hand, the
14C-labeled carbamate 4 might be prepared by treating 3 with 14C-labeled phenyl
chloroformate, which in turn could be obtained from phenol and [14C]-phosgene.
We first optimized the synthesis of non-radioactive imexon 1 on a
micromolar scale. Phenyl chloroformate was prepared in situ from phenol
and phosgene (20% in toluene) according to an analogous procedure for
making phenyl chlorothionoformate.10 Thus, reaction of 3 with phenyl
chloroformate gave the carbamate 4 in 67% yield. Carboxamide 2 was
obtained in 80% yield by treatment of an ether solution of 4 with liquid
ammonia. Cyclization of 2 with benzyltrimethylammonium hydroxide (40% in
methanol, Triton B) in absolute ethanol produced imexon 1 in 71% yield.
[14C]-Imexon was prepared in a similar manner from [14C]-phosgene as
described in the experimental section and depicted in Scheme 1. The yield of
[14C]-imexon was 7 mg (63 mmol, 3.5 mCi, 27% over four chemical steps
starting from [l4C]-phosgene). The product [14C]-imexon was shown to be
chemically and radiochemically pure (Figure 1).
Experimental
Materials
2-Cyanoaziridine (2) was prepared by a literature procedure.11 [14C]-Phosgene,
which is commercially available, was prepared from [14C]-carbon monoxide
and chlorine.12, y
Methods
1H-NMR and 13C-NMR spectra were obtained at 300 and 75 MHz,
respectively, using a Varian Unity-300 spectrometer. Proton NMR spectra
were referenced to tetramethylsilane (0 ppm) or to the residual HOD in D2O
solvent (4.63 ppm). Carbon-13 NMR spectra were referenced to the CDCl3
signal (77.0 ppm) or to the DMSO-d6 signal (39.5 ppm). Mass spectra were
obtained from the Mass Spectrometry Lab in the Department of Chemistry at
The University of Arizona, Tucson, Arizona.
Phenyl 2-cyano-1-aziridine-carboxylate ð½14Cꢀ-4Þ. [14C]-Phosgene (see Scheme
1, 22 mCi, specific activity 55 mCi/mmol, 0.40 mmol) was condensed into
350 ml of toluene using vacuum line techniques and the solution kept under
y [l4C]-Calcium carbonate was prepared by absorption of [14C]-carbon dioxide into aqueous calcium chloride
solution. Pyrolysis of a mixture of [14C]-calcium carbonate and zinc powder in a ratio of 1:2 at 7008C for
30 min gave [14C]-carbon monoxide. [14C]-Phosgene was synthesized by the photolysis of [14C]-carbon
monoxide with a slight excess of chlorine gas under incandescent light over 8 h. The overall yield of [14C]-
phosgene from [14C]-carbon dioxide is about 85%.
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B. JAGADISH ET AL.
Scheme 1.
Figure 1. HPLC UV trace (lower panel) and radiochromatogram (upper panel)
of a mixture of pharmaceutical grade imexon and synthetic [14C]-imexon
(concentrations 20.0 and 22.6 ppm, respectively). Area under the curve for the
UV peak 6055.7 mAU (100%). Area under the curve for the 14C peak
138 082 cpm (97.2%)
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SYNTHESIS OF [14C]–IMEXON
169
nitrogen. This solution was added slowly via syringe to a solution of phenol
(37.6 mg, 0.40 mmol) in 380 ml of 1N NaOH at ꢁ208C. The mixture was
allowed to attain room temperature and stirring was continued for 2 h. The
intermediate phenyl chloroformate, [14C]-7, was not isolated. A solution of 2-
cyanoaziridine (3, 27 mg, 0.40 mmol) in 450 ml of 2N Na2CO3 and 800 ml of
ether were added to the solution of [14C]-7 and the resulting mixture was
stirred for 2 h. The reaction mixture was then diluted with ether (30 ml) and
water (15 ml), the phases were separated, and the aqueous phase extracted with
ether (2 ꢂ 15 ml). The organic phases were combined, washed with brine
(20 ml), and dried over MgSO4. The mixture was filtered and volatiles removed
by evaporation. The light yellow residual oil was fractionated by gravity
column chromatography on silica gel. Elution with 10% ether/pentane gave
1.8 mCi (8%) of the by-product [14C]-diphenyl carbonate as a white solid.
Further elution with 40% ether/pentane gave 17 mCi (77%) of [14C]-4 as a
viscous oil which solidified upon storage at ꢁ208C. The thin layer
chromatographic mobility of this material was identical to that of an authentic
standard (Rf=0.49 on 0.25 mm silica gel plates eluted with 1:2 ether:pentane).
Spectral data for non-radioactive 4: mp 56–588C; IR cmꢁ1 3108, 3060, 2371,
2255, 1742, 1591, 1489, 1308, 1201; lH NMR (CDC13) d 2.78 (1, d, J=5.7 Hz),
2.82 (1, d, J=3 Hz), 3.21 (1, dd, J=6, 3.3 Hz), 7.16 (2, d, J=7.8 Hz), 7.27 (1, t,
J=7.2 Hz), 7.40 (2, t, J=7.8 Hz); 13C NMR (CDCl3) d 22.4, 31.8, 115.7, 120.9,
126.4, 129.5, 150.4, 158.5; HRMS (FAB+) calculated for C10H9N2O2
189.0664, found 189.0663.
2-Cyano-1-aziridine-carboxamide-ð½14Cꢀ-2Þ. To A solution of phenyl carba-
mate [14C]-4 (34.2 mg, 0.18 mmol, 10 mCi) in diethyl ether (5 ml) was added
liquid ammonia (0.5 ml) and the reaction mixture stirred for 2 h at the boiling
temperature of ammonia and then allowed to come to ambient temperature
overnight. The solvent was removed at reduced pressure and the residue
washed with 40% diethyl ether/pentane. The carboxamide [14C]-2 was
obtained as a white solid (18 mg, 0.162 mmol, 8.9 mCi) in 89% yield.
Spectral data for non-radioactive 2: mp 70–728C; IR cmꢁ1 3388, 3203, 2362,
2259, 1690, 1625, 1399, 1004; 1H NMR (CDCl3) d 2.55 (1, d, J=3.9 Hz), 2.58
(1, d, J=6.3 Hz), 3.06 (1, dd, J=6.6, 3.6 Hz), 5.31 (2, br s); 13C NMR (DMSO-
d6) d 21.4, 30.8, 118.3, 162.0; HRMS (FAB+) calculated for C4H6N3O
112.0511, found 112.05114.
4-Imino-1,3-diazabicyclo[3.1.0]hexan-2-one ð½14Cꢀ-imexon; ½14Cꢀ-1Þ. To a solu-
tion of carboxamide [14C]-2 (18 mg, 162 mmol, 8.9 mCi) in absolute ethanol
(200 ml) in a 2 ml vial fitted with a Teflon lined screw cap was added
benzyltrimethylammonium hydroxide (40% solution in methanol, Triton B,
7.36 ml, 16 mmol). The reaction mixture was agitated using a vortex mixer at
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B. JAGADISH ET AL.
30 min intervals for 3 h and then left overnight. Ethanol (200 ml) was then
added, the reaction mixture was agitated, centrifuged, and the solvent
decanted from an off-white precipitate. This process was repeated three times
and the remaining solid dried under vacuum. Analysis of this solid by HPLC-
MS confirmed its chemical identity as imexon. The yield of [14C]-1 was 7 mg
(63 mmol, 3.5 mCi, 39%). The chemical and radiochemical purity of this
product were >97%.
Spectral data for non-radioactive 1: mp >2508C; IR cmꢁ1 3300-2500 (b),
1
1800-1400 (b), 1296, 12343, 1181, 1013, 820; H NMR (D2O) d 2.39 (1, d,
J=3 Hz), 2.55 (1, d, J=5.4 Hz), 3.59 (1, dd, J=5.4, 3.3 Hz); HRMS (FAB+)
calculated for C4H6N3O 112.0511, found 112.0511.
Conclusion
In summary, we have synthesized [14C]-imexon starting from [14C]-phosgene
and 2-cyanoaziridine in an overall yield of 27% with a specific activity of
55 mCi/mmol. The experimental procedures are simple and efficient. To the
best of our knowledge this is the first reported synthesis of [14C]-imexon.
Acknowledgements
This work was supported by grant CA-17094 (to RTD) and by Core Support
Grant CA-23074 (to the Arizona Cancer Center) from the National Cancer
Institute, NIH, Bethesda, MD, USA.
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