PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Org Lett. Author manuscript; available in PMC 2014 April 8.
Published in final edited form as:
PMCID: PMC3979304
NIHMSID: NIHMS563421

An efficient and practical radiosynthesis of [11C]temozolomide

Abstract

An external file that holds a picture, illustration, etc.
Object name is nihms563421u1.jpg

Temozolomide (TMZ) is a prodrug for an alkylating agent used for the treatment of malignant brain tumors. A positron emitting version, [11C]TMZ, has been utilized to help elucidate the mechanism and biodistribution of TMZ. Challenges in [11C]TMZ synthesis and reformulation make it difficult for routine production. Herein we report a highly reproducible one-pot radiosynthesis of [11C]TMZ with a radiochemical yield of 17±5% and >97% radiochemical purity.

Chemotherapeutic treatment for advanced cancers persists as an area of intense research exploration. Existing treatment options, including surgery, radiation, and chemotherapy, seldom resulted in even one year of median improved survival for aggressive tumors such as malignant glioma.1,2 Temozolomide (TMZ) is the prodrug to an antineoplastic alkylating agent that has drawn interest among the research community due to growing evidence for its broad range of chemotherapeutic activity in both in vitro and preclinical in vivo studies. TMZ displays activity against tumors which have resistance to other antitumor agents, and shows potential to improve patient survival outcomes when used in conjunction with radiation and anti-angiogenic therapies.1,3 TMZ was approved for clinical use in the United States in 1999 and has since proven to be effective as an oral treatment for malignant glioma, malignant metastatic melanoma, and other adult central nervous system tumors.

TMZ is currently the standard of care for all newly diagnosed glioblastoma.4 Prior to the development of TMZ, de novo and acquired resistance to available chemotherapeutics, as well as complications from toxicity, prevented any single drug from attaining much more than a modest increase in overall survival rate.2

In comparison to other chemotherapies, TMZ is well-tolerated by patients and elicits less severe complications due to nonspecific toxicity. Its off-target toxicity is generally considered mild-to-moderate, and side effects are predictable and easily managed in both adult and pediatric patients, usually with the aid of anti-emetics.2 Patients who receive TMZ treatment also show improved Health-Related Quality-Of-Life (HR-QOL) scores—a common metric for cancer treatment efficacy assessed alongside side-effects—relative to similar antineoplastic alkylating agents.3

TMZ has physiochemical properties that account for its broad spectrum of efficacy and low toxicity. TMZ distributes widely throughout all tissues and penetrates the central nervous system, crossing the blood-brain barrier. Its pharmacokinetic profile is predictably described by a one-compartment open model, with dose-dependent linear increases in both Area Under the Curve (AUC) and peak plasma concentration (Cmax) values. TMZ is stabile in acidic conditions and its oral bioavailability is approximately 100%. Following absorption into the intestine, TMZ undergoes spontaneous non-enzymatic, pH-dependent hydrolysis to generate 5-(3-methyl-(triazen-1-yl)-imidazole)-4-carboxamide (MTIC), the pharmacologically-active alkylating agent. This reaction occurs through a base-catalyzed nucleophilic attack by water. MTIC then undergoes further hydrolysis in the presence of acid into 5-aminoimidazole-4-carboxamide (AIC) and a methyldiazonium cation 4 (Scheme 1). This cation irreversibly binds to DNA nucleotides via nucleophilic attack by guanine residues, resulting in DNA alkylation. Accumulation of methylated guanine residues leads to breaks in the daughter DNA strand, causing cell cycle arrest and cellular apoptosis.1,3

Scheme 1
Hydrolysis of TMZ to MTIC

The mechanism of TMZ is well understood, making [11C]TMZ a useful radiopharmaceutical to observe tumor cells via transfer of the [11C]methyl group to tumor cell DNA in vivo. Also, the tissue distribution and broad-spectrum anti-tumor properties of TMZ provide utility as a diagnostic and prognostic agent.5 PET imaging has been used with [11C]TMZ to confirm the drug’s therapeutic action and evaluate its metabolic activity, pharmacokinetics, and biodistribution.6 However, challenges with reproducibly synthesizing [11C]TMZ, as well as with formulating the final product in an injectable solution that maintains its stability, have limited its accessibility. The first radiochemical route to [11C]TMZ 10, published by Brown et al.7 (Scheme 2), utilized conventional synthetic chemistry methods initially described by Wang et al.8 Alternative approaches to a cycloaddition with MTIC 2 have been attempted using 1,1′-carbonyldiimidazole, 4-nitrophenyl chloroformate, and chloroformic acid trichloromethyl ester without success.10 Although the Brown group successfully radiolabeled [3-N-11C-methyl]TMZ7 10 using diazoimidazole 9 and [11C]MIC 8,9 a simpler synthetic method would benefit other researchers hoping to access [11C]TMZ.

Scheme 2
Synthesis of [11C]TMZ via cycloaddition of 8 and 9

[11C]MIC is not routinely produced at most PET radiotracter facilities. In conventional chemistry, the cycloaddition of diazoimidazole precursor and methyl isocyanate is slow, with the fastest reported syntheses taking as long as one day using > 150-fold excess of MIC.11 The long reaction time and high molar ratio of MIC are incompatible with the short half-life of carbon-11 (t1/2 = 20.4 min) and the substoichiometric amounts of [11C]MIC utilized in radiochemistry. Illustrating this point, the Brown group reported a 70% drop in chemical yield when optimizing synthesis parameters for radiochemistry by raising the reaction temperature and shortening the reaction time.

In addition, [11C]MIC is synthesized from [11C]CH3I 7 using a heated silver cyanate column; this added step decreases the final radiochemical yield due to losses incurred during chemical transformation and radioactive decay. In order to simplify the synthetic parameters and reduce the total time required to synthesize [11C]TMZ, we sought a new approach to circumvent the use of MIC.

Given the option to pursue either [3-N-11C-methyl]TMZ or [4-11C-carbonyl]TMZ as our target tracer, we chose the former for two reasons. First, the 3-N-methyl carbon is incorporated into guanine residues during DNA methylation as demonstrated by Saleem et al.12 Radiolabeling at the 3-N position is critical to [3-N-11C-methyl]TMZ function as a tumor imaging agent; synthesizing [4-11C-carbonyl]TMZ results in the loss of the carbon-11 radionuclide as expelled [11C]CO2 6.12 Second, 3-N-methylation expands the synthetic possibilities to include SN2 methylation of the TMZ desmethyl analogue, nortemozolomide (norTMZ) 14, with [11C]CH3I.

In order to access the 3-N-methylation route to [3-N-11C-methyl]TMZ, we first synthesized desmethylTMZ according to a published patent13i (Scheme 3). Diazotization of aminoimidazole 3 afforded diazoimidazole 9.14 In parallel, N-(tert-butoxycarbonyl) glycine 11 was treated with triethylamine and ethylchloroformate to give 12. Compound 12 was immediately treated upon isolation with 9 to furnish compound 13 at a total yield of 44% starting from compound 9. Deprotection of 13 with 3 N HCl produced 14 as a pink solid that is stable for at least six months when stored at 2–4 °C.

Scheme 3
Synthesis of nortemozolomide and [11C]temozolomide

We anticipated two methylation sites on the norTMZ 14 molecule, at the 3-N heterocyclic position and the 8-N primary carboxamide position. We expected 3-N- methylation to proceed through an anion that is more stable than the primary amide anion produced by 8-N amide deprotonation. Synthesis conditions for 3-N- methylation of norTMZ were screened using substoichiometric [13C]CH3I in order to preoptimize for radiochemistry with [11C]CH3I. We tested several bases including cesium carbonate, potassium tert-butoxide, and NaH for their ability to selectively deprotonate either the 3-N or 8-N positions of norTMZ. We found that both cesium carbonate and potassium tert-butoxide preferentially deprotonated the 8-N amide position, resulting in undesirable 8-N-methylation product 15. Although deprotonation can also occur at the 8-N amide position with NaH, we found NaH most effectively deprotonated the 3-N carboxamide. The amount of NaH used is critical in favoring methylation at the 3-N position over methylation at the 8-N position. An excess of base is often used in radiochemistry and under these conditions we observed only deprotonation at the 8-N carboxamide position of 14. Use of 1 equivalent of NaH at the same temperature resulted in the formation of the 8-N[13C]methylated product 15 and 3-N-methyl 16 in a 1:1 ratio. Selective 3-N-methylation occurred using < 1 equivalent of NaH in the presence of [13C]CH3I at 45 °C in DMF to furnish [3-N-13C-methyl]TMZ 16 as the major product (Figure 1.) The respective positions of 13C-labeling were confirmed by 13C-NMR (Figure 1).

Figure 1
13C-NMR spectra for methylation of norTMZ with [13C]CH3I

The optimized reaction conditions were subsequently applied to carbon-11 radiochemistry. [11C]CO2 was produced through the 14N(p,α)11C nuclear reaction in an Eclipse 11-MeV cyclotron (Siemens) and was reduced to [11C]CH3I using a TRACERlab FX-MeI unit (GE Healthcare). [11C]CH3I was trapped in a TRACERlab FX-M reactor (GE Healthcare) preloaded with a solution containing excess norTMZ 14 and 0.7 eq. NaH, (60% w/w dispersion in mineral oil) in dry DMF that had stirred at −5 °C for 1 min prior to trapping. The solution was heated to 45 °C for 5 min, then cooled to room temperature and quenched with 0.5% aq. AcOH. The reaction mixture was purified by reverse-phase semi-preparative HPLC and eluted with a mobile phase of 0.5% AcOH in H2O: EtOH (95:5). The desired fraction was collected and aliquots were used to establish the chemical and radiochemical purity by analytical HPLC. The identity of the product was confirmed by analytical HPLC with additional co-injection of TMZ reference standard (Figure 2A–C) and with radio-thin-layer chromatography (Figure 2D). The average radiochemical yield was 17 ± 5% (decay-corrected to trapped [11C]CH3I; n = 6). Chemical and radiochemical purities were ≥ 97% in all instances. Specific activity measurements by HPLC reached the limit of detection, but were at least 3 Ci/μmol at the end of synthesis. The average time required for the synthesis from end of cyclotron bombardment to end of synthesis and purification was 30 min—an improvement over the previously reported synthesis time of 47 minutes using [11C]MIC.7

Figure 2
[11C]Temozolomide chromatography and radio TLC

We observed chemical hydrolysis of [3-N-11C-methyl]TMZ in the final formulation according to analytical HPLC, corroborating previous reports that the compound decomposes at physiological pH.15 Our purification and preparative method was designed to mitigate this issue; we monitored the stability of the radiopharmaceutical in an undiluted semi-preparative HPLC fraction and found it to be stable for at least 75 minutes in the acidic HPLC mobile phase. The mobile phase is composed of reagents that fall within USP safety limits for human injection; as such, buffering and salinating the mobile phase in accordance with these standards would afford a solution of [11C]TMZ 10 that could immediately be filtered for injection without reformulation.

TMZ is a front-line treatment for various types of cancer, and its application as a radiopharmaceutical makes it a novel ligand for imaging tumor cells in vivo. In this paper, we report a direct methylation of its 3-N-desmethyl analogue, norTMZ 14, using [11C]CH3I 7 to produce [3-N-11C-methyl]TMZ 10 in high chemical and radiochemical purity. Our synthesis is practical, efficient, reproducible, and can be applied for oncological PET imaging in humans.

Supplementary Material

Supplementary Information

Footnotes

Supporting Information Available. The experimental procedure and compound characterization data are available free of charge via the Internet at http://pubs.acs.org.

References

1. Johansson F, Ekman S, Blomquist E, Henriksson R, Bergström S, Bergqvist M. A Review of Dose-dense Temozolomide Alone and in Combination with Bevacizumab in Patients with First Relapse of Glioblastoma. Anticancer Res. 2012;32(9):4001–4006. [PubMed]
2. Friedman HS, Kerby T, Calvert H. Temozolomide and treatment of malignant glioma. Clin Cancer Res. 2000;6(7):2585–2597. [PubMed]
3. Darkes MJM, Plosker GL, Jarvis B. Temozolomide: a review of its use in the treatment of malignant gliomas, malignant melanoma and other advanced cancers. Amer J Cancer. 2002;1(1):55–80.
4. Surawicz TS, Davis F, Freels S, Laws ER, Menck HR. Brain tumor survival: results from the National Cancer Data Base. J Neurooncol. 1998;40(2):151–160. [PubMed]
5. Neidle S, Thurston DE. Chemical approaches to the discovery and development of cancer therapies. Nat Rev Cancer. 2005;5(4):285–296. [PubMed]
6. Brock C, Matthews J, Brown G, Luthra S, Brady F, Newlands E, Price P. The kinetic behavior of temozolomide in man. 1996:475.
7. Brown GD, Luthra SK, Brock CS, Stevens MFG, Price PM, Brady F. Antitumor Imidazotetrazines. 40. 1 Radiosyntheses of [4-11C-Carbonyl] and [3-N-11C-Methyl]-8-carbamoyl-3-methylimidazo [5,1-d]-1, 2, 3, 5-tetrazin-4(3H)-one (Temozolomide) for Positron Emission Tomography (PET) Studies. J Med Chem. 2002;45(25):5448–5457. [PubMed]
8. Wang Y, Stevens MFG. Synthetic studies of 8-carbamoylimidazo-[5, 1-d]-1, 2, 3, 5-tetrazin-4(3H)-one: A key derivative of antitumour drug temozolomide. Bioorg Med Chem Lett. 1996;6(2):185–188.
9. Brown GD, Henderson D, Steel C, Luthra S, Price PM, Brady F. Two routes to [11C-carbonyl] organo-isocyanates utilizing [11C] phosgene ([11C] organo-isocyanates from [11C] phosgene) Nucl Med Biol. 2001;28(8):991–998. [PubMed]
10. Wang Y, Stevens MFG, Chan T, DiBenedetto D, Ding Z, Gala D, Hou D, Kugelman M, Leong W, Kuo S. Antitumor imidazotetrazines. 35. New synthetic routes to the antitumor drug temozolomide. J Org Chem. 1997;62(21):7288–7294. [PubMed]
11. Stevens MFG, Hickman JA, Stone R, Gibson NW, Baig GU, Lunt E, Newton CG. Antitumour imidazotetrazines. 1. Synthesis and chemistry of 8-carbamoyl-3-(2-chloroethyl) imidazo [5, 1-d]-1, 2, 3, 5-tetrazin-4(3H)-one, a novel broad-spectrum antitumor agent. J Med Chem. 1984;27(2):196–201. [PubMed]
12. Saleem A, Brown GD, Brady F, Aboagye EO, Osman S, Luthra SK, Ranicar ASO, Brock CS, Stevens MFG, Newlands E. Metabolic activation of temozolomide measured in vivo using positron emission tomography. Cancer Res. 2003;63(10):2409. [PubMed]
13. Methods and intermediates for the synthesis of 4-oxo-3,4-dihydro-imidazo [5,1-d] [1,2,3,5] tetrazines. WO/2011/107. WO Patent. 2011;726Methods and intermediates for the synthesis of 4-oxo-3,4-dihydro-imidazo [5,1-d] [1,2,3,5] tetrazines. WO/2011/107. WO Patent. 2011:726.The authors of the patent state that the compound reported by Wang, et al. Bioorg Med Chem Lett. 1996;6(2):185–188.) was not properly characterized and was thus misinterpreted to be nortemozolomide.
14. Shealy YF, Struck RF, Holum LEEB, Montgomery JA. Synthesis of Potential Anticancer Agents. XXIX. 5-Diazoimidazole-4-carboxamide and 5-Diazo-v-triazole-4-carboxamide1, 2. J Org Chem. 1961;26(7):2396–2401.
15. Denny BJ, Wheelhouse RT, Stevens MFG, Tsang LLH, Slack JA. NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry. 1994;33(31):9045–9051. [PubMed]