The medicinal chemistry of imidazotetrazine prodrugs

Abstract

Temozolomide (TMZ) is the standard first line treatment for malignant glioma, reaching "blockbuster" status in 2010, yet it remains the only drug in its class. The main constraints on the clinical effectiveness of TMZ therapy are its requirement for active DNA mismatch repair (MMR) proteins for activity, and inherent resistance through O6-methyl guanine-DNA methyl transferase (MGMT) activity. Moreover, acquired resistance, due to MMR mutation, results in aggressive TMZ-resistant tumour regrowth following good initial responses. Much of the attraction in TMZ as a drug lies in its PK/PD properties: it is acid stable and has 100% oral bioavailability; it also has excellent distribution properties, crosses the blood-brain barrier, and there is direct evidence of tumour localisation. This review seeks to unravel some of the mysteries of the imidazotetrazine class of compounds to which TMZ belongs. In addition to an overview of different synthetic strategies, we explore the somewhat unusual chemical reactivity of the imidazotetrazines, probing their mechanisms of reaction, examining which attributes are required for an active drug molecule and reviewing the use of this combined knowledge towards the development of new and improved anti-cancer agents.

Scheme 1

Prodrug activation of TMZ.

Figure 1

Reaction of TMZ, MTIC and CH₂N₂ in D₂O systems. (A) Products of reaction of TMZ with phosphate buffer pD = 7.8 showing deuterium incorporation into methanol and methyl phosphate; (B) MTIC in Na₂CO₃ (10% in D₂O) showing the intact isotopic configuration of the MTIC methyl and deuterium incorporation into the methanol product of hydrolysis; (C) Products of condensing diazomethane into Na₂CO₃ (10% in D₂O) [15,16,17].

Figure 2

Reaction of MTZ with phosphate buffer pD = 7.8, 37 °C. (A) ¹H-NMR spectrum after 14 days; (B) The same sample spiked with authentic chloroethanol (7).

Scheme 2

The aqueous chemistry of MTZ.

Figure 3

Comparison of reaction of ETZ and TMZ with DNA, showing the unreactivity of ETZ up to 5 mM, sites of GN7 alkylation produced in the 276-bpBamHI-Sal1 fragment of pBR322, by the piperidine cleavage method. Adapted from reference [24].

Scheme 3

The aqueous chemistry of ETZ.

Figure 4

¹H-NMR investigation of the reaction of ETZ with phosphate buffer (pH 7.8) in a two phase system (D₂O/CDCl₃). Spectra A–C CDCl₃ layer, spectra D–G, D₂O layer. (A) At the end of the imidazotetrazine hydrolysis reaction identifying the putative ethene peak; (B) after addition of Br₂, the arrow indicates the position of a new peak; (C) sample of spectrum B after adding authentic dibromoethane; (D) at the end of the imidazotetrazine hydrolysis reaction; (E–G) sample of spectrum D after sequentially spiking with authentic AIC, ethylphosphate and ethanol (note the shift in the AIC signal after adding the acidic ethyl phosphate).

Scheme 4

The aqueous fate of the 3-trifluoroethylanalogue 1n.

Figure 5

pH Dependence of the pseudo-first order rate constants for hydrolysis of TMZ and MTIC. Adapted from reference [16].

Figure 6

¹⁵N-NMR spectra of TMZ. (A) ¹⁵N{¹H} in DMSO-d₆; (B) with full ¹H nOe in TFA. Both experiments were acquired in the presence of Cr(acac)₃ and referenced to external CH₃NO₂ [26].

Figure 7

Dimers seen in Lowe’s crystal structure of TMZ [33]. Strong H-bonds are shown in green.

Figure 8

Temozolomide–salicylic acid co-crystal [35]. Strong H-bonds are shown in green.

Figure 9

A tetrameric unit of TMZ in the crystal structure of TMZ·HCl·2H₂O [27]. Strong H-bonds are shown in green.

Figure 10

Cucurbit[n]uril (14) and uptake of TMZ into GBM cells [38].

Scheme 5

Initial synthesis of TMZ from AIC∙HCl and routes to AHX [20,25,26,40,41,42].

Scheme 6

Masked methyl isocyanate approach to TMZ [46].

Scheme 7

TMS deprotection to give TMZ [48].

Scheme 8

Routes to TMZ via urea 21 [47,49,50].

Scheme 9

Synthesis of imidazotriazinones [25].

Scheme 10

Synthesis of TMZ∙HCl from diazo-ICN (31) [47].

Scheme 11

Summary of routes to amide alternatives [47].

Scheme 12

Wanner and Koomen’s route to TMZ.

Scheme 13

Schering-Plough industrial route to TMZ [54].

Scheme 14

Schering-Plough alternative route to TMZ [55,56].

Scheme 15

Common routes to isocyanates.

Scheme 16

Urea pyrolysis to give labelled methylisocyanates [26].

Scheme 17

Thermolysis of 4,4'-methylenebis(phenylisocyanate) (50) [59].

Scheme 18

Alternative routes to isocyanates used in labelling studies [8].

Figure 11

Summary of sites that have been isotopically labelled in TMZ [7,8,25,26,60,61,62,63].

Figure 12

Pyrazolo analogues of MTZ and TMZ [64].

Scheme 19

Heteroanalogues of imidazotetrazines [65,66].

Scheme 20

Synthesis of pyridopyrrolotetrazinones [72,73].

Figure 13

Unstable tetrazepines [74], and pyrazolotetrazinone herbicidal agents [76].

Scheme 21

Synthesis of 6- and 8-analogues via substituted imidazoles [19].

Scheme 22

General synthesis of esters and amides via MTZ acid chloride [78].

Scheme 23

Synthesis of amides from MTZ carboxylic acid [45].

Figure 14

TMZ hexyl ester [80], and β-lactam antibiotics [81].

Scheme 24

Glycol linker dimer [82].

Scheme 25

Formation of thiazoles and imidazoles [83].

Scheme 26

Construction of oxazoles and oxadiazoles [83].

Scheme 27

Example of steric effects on the diazo-IC–isocyanate coupling reaction [43,84,85].

Scheme 28

Synthesis of nortemozolomide [86,87].

Scheme 29

Methylation of nortemozolomide [63,87].

Scheme 30

Alkylation of nortemozolomide [84,87].

Figure 15

Compounds resistant to MGMT effects [85,88,89].

Scheme 31

Summary of alkyldiazonium ions derived from early imidazotetrazines.

Figure 16

(Left) Comparison of reaction of TMZ, MTZ and a disulphide-linked dimer 105 with DNA showing the enhanced reactivity of the episulphonium prodrug, sites of DNA alkylation detected by the Taq DNA polymerase stop method (adapted from reference [90]); (Right) Reaction of Aziridinium Prodrugs (DP86 and DP68, 107a and 108a) showing the increased reactivity of the bisimidazotetrazine. Sites of GN7 alkylation on the upper strand of pBR322 DNA detected by the piperidine cleavage method. Arrows indicate the position and sequence context of the alkylated guanines. Adapted from reference [93].

Scheme 32

Generation of episulfonium ions from a sulfur-linker dimer [90].

Figure 17

Nitrogen mustards, resulting aziridinium ions and novel imidazotetrazine dimers.

Scheme 33

Synthesis of N-linked dimers and momomers from diisocyanates and isocyanates [93,94,95].

Scheme 34

Cyclisation of β-aminoisocyanates.

Scheme 35

Aziridinium release [93].

Figure 18

Effect of electron-withdrawing capabilities of substituent X on the activity of aniline dimers 109 [95]. The differences between sets of lines indicate the limited MMR/MGMT effects.