Synthesis and biological evaluation of p38α kinase-targeting dialkynylimidazoles

Abstract

Based on the mild, thermal rearrangement of 1,2-dialkynylimidazoles to reactive carbene or diradical intermediates, a series of 1,2-dialkynylimidazoles were designed as potential irreversible p38 MAP kinase α-isoform (p38α) inhibitors. The synthesis of these dialkynylimidazoles and their kinase inhibition activity is reported. The 1-ethynyl-substituted dialkynylimidazole 14 is a potent (IC₅₀ = 200 nM) and selective inhibitor of p38α. Moreover, compound 14 covalently modifies p38α as determined by ESI-MS after 12 h incubation at 37 °C. The unique kinase inhibition, covalent kinase adduct formation, and minimal CYP450 2D6 inhibition by compound 14 demonstrate that dialkynylimidazoles are a new, promising class of p38α inhibitors.

p38 MAP kinase (p38α) belongs to a family of serine/theronine kinases that serve as important mediators of inflammatory cytokines including tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β).^1,2 Elevated levels of the pro-inflammatory cytokines are associated with a number of diseases, such as toxic shock syndrome, rheumatoid arthritis, osteoarthritis, diabetes and inflammatory bowel disease.³ Therefore, inhibition of p38α is considered to be a potential therapeutic strategy.⁴ A number of p38α inhibitors have been synthesized and characterized.⁵ Although these compounds show good inhibition of p38α, many also inhibit other protein kinases with similar or greater potency.⁶

There has been a growing interest in irreversible inhibitors of protein kinases,⁷ and a number of these drugs are in clinical trials.⁸ Advantages of irreversible kinase inhibition include increased selectivity,⁹ duration,¹⁰ and therapeutic utility, especially against kinases that are resistant to competitive, ATP-binding pocket-targeting drugs.¹¹ Additionally, irreversible inhibitors and related selective, covalent kinase modifying small molecules are of interest as probes for chemical genetics studies.¹² While certain natural products and ATP analogs irreversibly inhibit kinases,¹³ none are selective towards p38α. Thus, there is a need to develop selective and irreversible inhibitors that target p38α. We have discovered a novel thermal cyclization and rearrangement of 1,2-dialkynylimidazoles (DAIms) (Scheme 1). Mild thermolysis of DAIms in the presence of chlorinated solvents or HCl leads to the isolation of imidazo[1,2-a]pyridine (ImPy) products, which may result from trapping of an initially-formed diradical intermediate via aza-Bergman cyclization.¹³

Scheme 1.

Thermal cyclization and rearrangement of 1,2-dialkynylimidazoles

Thermolysis under neutral conditions in non-halogenated solvents affords products derived from trapping cyclopentapyrazine (CyPP) carbene intermediates by H-atom abstraction, C–H bond insertion, and alkene addition reactions.^14–16 The CyPP carbene is proposed to be derived from an intermediate cyclic cumulene that results from collapse of the diradical.¹⁴ Non-covalent association between DAIms and a kinase may facilitate the rate-determining aza-Bergman cyclization.

The formation of reactive diradical and carbene intermediates under mild conditions from DAIms has led us to propose that DAIms can be designed to undergo kinase binding-induced cyclization and covalent inactivation of specific kinase targets. Specifically, the structural similarity between DAIms and the known p38α inhibitors such as SB-203580¹⁷ and RWJ-67657¹⁸ (Figure 1) has inspired the design and inhibition studies of p38α-targeting DAIms described here.

Figure 1.

Examples of 4,5-diarylimidazole p38a inhibitors.

An initial route to kinase-targeting dialkynylimidazoles is shown in Scheme 2. The known 4(5)-(4-fluorophenyl)-5(4)-(4-pyridyl)imidazole 1¹⁹ was protected with trityl group. Interestingly, this reaction only afforded one regioisomer, which was assigned as the 5-(4-fluorophenyl)-4-(4-pyridyl)imidazole 2 based on COSY and NOESY NMR. Compound 2 was deprotonated with n-BuLi at 0 °C, and quenched with I₂ to give the 2-iodo-imidazole 3, which was deprotected in aqueous TFA to afford 4. Coupling of the lithium anion of imidazole 4, formed by deprotonation with LHMDS, with phenyl(phenylethynyl)-iodonium tosylate²⁰ afforded a 15 % yield of a 1:1 mixture of the regioisomeric N-alkynyl-2-iodoimidazoles 5 and 6. The separated regioisomers were subjected to Sonogashra coupling with various terminal acetylene partners to provide the regioisomeric dialkynylimidazoles 7 and 8. The regiochemical assignments within this series were made based on the X-ray crystal structure of 7b shown in Figure 2.²¹

Scheme 2.

Reagents and conditions: (a) Et₃N, Ph₃CCl, CH₂Cl₂ (58 %); (b) i) n-BuLi, ii) I₂, THF, 0 °C (60 %); (c) TFA, H₂O (83 %); (d) LHMDS, PhI⁺CCPhTsO⁻; (e) RCCH, Pd(PPh₃)₄, CuI, Et₃N.

Figure 2.

X-ray crystal structure of dialkynylimidazole 7b.

Although providing access to select kinase-targeting dialkynylimidazoles, the synthetic route shown in Scheme 2 suffers from a number of limitations associated with the alkynyliodonium coupling reaction. Only the phenylethynyl and TMS-ethynyl iodonium reagents could be employed in this coupling,²² and even in these cases, the yields are poor and mixtures of regioisomers are produced.

An improved synthetic route to these dialkynylimidazoles employing the recently reported copper-catalyzed N-alkynylation of imidazoles with bromoalkynes was devised (Scheme 3). Treating 4-fluorophenylimidazole 9²³ with TIPS-protected bromo-acetylene in the presence of catalytic CuI and 2-acetyl-cyclohexanone as ligand affords a 9:1 mixture of regioisomeric alkynylimidazoles 10b and 10a, respectively, in 79 % yield.²² Iodination of the 2-position of 10b affords the 2-iodoimidazole 11, which undergoes Sonogashira coupling with O-TIPS-protected homopropargyl alcohol to give the dialkynylimidazole 12 in 73 % yield.²⁴ Deprotonation of 12 with n-BuLi followed by iodine quench affords the 5-iodoimidazole 13 in 74 % yield. A final Suzuki-Miyaura coupling of the 5-iodo imidazole 13 with pyridine-4-boronic acid followed by TBAF deprotection gives **the dialkynyl-imidazole 14.²⁵ Mild thermolysis of 14 at 80° C under acidic conditions in the presence of chloride afforded 15, the product of HCl addition to the diradical, in 50 % yield.

Scheme 3.

Reagents and conditions: (a) BrCCTIPS, CuI, AcC, Cs₂CO₃, dioxane, 50 °C overnight followed by reflux for 4 h (79 %, 1:9 10a/10b); (b) i) n-BuLi, ii) I₂, THF, −78 °C (91 %); (c) TIPSOCH₂CH₂CCH, Pd(PPh₃)₄, CuI, Et₃N (73 %); (d) i) n-BuLi, ii) I₂, THF, −78 °C (74 %); (e) pyridine 4-boronic acid, Pd(PPh₃)₄, K₂CO₃ (41 %); (f) TBAF, THF, −78 °C (89 %); (g) Me₄NCl, TfOH, DMF, 80 °C, 5 days (50 %).

All of these 1,2-dialkynylimidazoles were assayed against p38α MAPK at a fixed time-point of 60 min (Table 1).²⁶ Compounds 7a–c and 8a–c display modest inhibition at 10 μM concentration. In this series there is little difference in activity between the 1-alkynyl-5-fluorophenyl regioisomers 7a–c and the 1-alkynyl-5-pyridylisomers 8a–c, in contrast to reported 1-substituted pyridylimidazole p38α inhibitors.²⁸ Interestingly, the 1-ethynyl-substituted analog 14 is a potent inhibitor of p38α. Compound 14 completely inhibits p38α at 10 μM (Table 1), and has an IC₅₀ for p38α of 200 nM.²⁷ In comparison, the IC₅₀ of 14 against p38 (5.4 μM) is >25-fold higher. Dialkyny-limidazole 14 was also assayed at concentration of 20 μM against a panel of 53 additional human kinases. Only one kinase, (MAPK4/HGK) was strongly inhibited (>90% inhibition at 20 μM, IC₅₀ = 4.2 μM), while six additional kinases were moderately inhibited (between 50–90% inhibition, see Supporting Information). The cyclized 15 also inhibited p38α (IC₅₀ = 370 nM).

Table 1.

In vitro activity of 1,2-dialkynylimidazoles against p38α

Compound	p38α % inhibition (@ 10 μM)a
7a	19
8a	28
7b	63
8b	83
7c	53
8c	75
14	100

^aTests were carried out in duplicate.

Dialkynylimidazole 14 (100 μM) was incubated with non-phosphoryated p38α (5 μM) at 37 °C in 50 mM HEPES, 10 mM MgCl₂, 2 mM DTT, 1 mM EGTA, pH 7.5 for 12 h, followed by extensive dialysis, and the sample was analyzed by ESI- MS. A new peak in the mass spectrum at m/z = 41896, which corresponds to addition of a single molecule of 14 (MW = 331) to p38α, was observed (~25 % adduct) (Figure 3). Under identical conditions but with 1 mM DTT present, the adduct was the predominant species observed (Supporting Information).

Figure 3.

(a) ESI-MS spectrum of unphosphorylated p38α incubated for 12 h at 37 °C; (b) ESI-MS spectrum of unphosphorylated p38α incubated with dialkynylimidazole 14 for 12 h at 37 °C, followed by extensive dialysis.

A common concern for pyridinylimidazole MAPK inhibitors such as RWJ 67657 and SB-203580 is their inhibition of cytochrome P₄₅₀ (CYP450) enzymes, which may be linked to hepatotoxicity.²⁹ Interestingly, the dialkynylimidazole 14 displays a much lower level of inhibition of CYP450 2D6 (4% inhibition at 10 μM) compared to SB-203580 (78% inhibition at 10 μM).

In summary, novel p38α-targeting dialkynylimidazoles were designed, synthesized and evaluated. Although 1-phenethynyl-substituted dialkynylimidazoles 7a–c and 8a–c are only modest inhibitors of p38α, the 1-ethynyl-substituted dialkynylimidazole 14 is a potent and selective inhibitor. Commensurate with the increased facility of rearrangement of 1-ethynyl-substituted dialkynylimidazols relative to 1-phenethynyl analogues,¹⁶ compound 14 forms a covalent adduct with p38α. However, the conditions for p38α adduct formation (12 h at 37 °C) are much milder than those required for cyclization/trapping of 14 to afford 15 (5 days at 80 °C), indicating that the kinase may facilitate the cyclization of 14. Further studies on the site and mechanism of this covalent modification of p38α by 1-ethynyl-substituted dialkynylimidazoles are on-going. The unique kinase inhibition, covalent kinase adduct formation, and minimal CYP450 2D6 inhibition by compound 14 demonstrate that dialkynylimidazoles are a new, promising class of p38α inhibitors.