Expanding the scope of native chemical ligation - templated small molecule drug synthesis via benzanilide formation

Abstract

We describe a reaction system that enables the synthesis of Bcr-Abl tyrosine kinase inhibitors (TKI) via benzanilide formation in water. The reaction is based on native chemical ligation (NCL). In contrast to previous applications, we used the NCL chemistry to establish aromatic rather than aliphatic amide bonds in coupling reactions between benzoyl and o-mercaptoaniline fragments. The method was applied for the synthesis of thiolated ponatinib and GZD824 derivatives. Acid treatment provided benzothiazole structures, which opens opportunities for diversification. Thiolation affected the affinity for Abl1 kinase only moderately. Of note, a ponatinib-derived benzothiazole also showed nanomolar affinity. NCL-enabled benzanilide formation may prove useful for fragment-based drug discovery. To show that benzanilide synthesis can be put under the control of a template, we connected the benzoyl and o-mercaptoaniline fragments to DNA and peptide nucleic acid (PNA) oligomers. Complementary RNA templates enabled adjacent binding of reactive conjugates triggering a rapid benzoyl transfer from a thioester-linked DNA conjugate to an o-mercaptoaniline-DNA or -PNA conjugate. We evaluated the influence of linker length and unpaired spacer nucleotides within the RNA template on the product yield. The data suggest that nucleic acid-templated benzanilide formation could find application in the establishment of DNA-encoded combinatorial libraries (DEL).

Fig. 1. (A) Mechanism of native chemical ligation (NCL). (B) Small molecule drugs containing benzanilide units. (C) NCL-based small molecule drug synthesis via benzanilide formation.

Fig. 2. Chemical structures of nilotinib (1), ponatinib (2), and GZD824 (3), as well as their thiolated derivatives 1a–3a.

Scheme 1. Formation of the core benzanilide structure of nilotinib-SH (1a). Reagents and conditions: KOtBu, THF, rt, 3 h, Ar, 97%.

Scheme 2. Synthesis of ponatinib-SH (2a) and GZD824-SH (3a) via NCL-type reaction. Reagents and conditions: (a) NBS, AIBN, AcOH, 80 °C, 26 h, 72%; (b) 1-methylpiperazine, DIPEA, DMF, rt, 1 h, 91%; (c) tBuSH, Cs₂CO₃, DMF, rt, 1 h, 83%; (d) (i) Na₂S₂O₄, MeOH, H₂O, rt, 1 h; (ii) conc. HCl, rt, 3 h, 86%; (e) TFMSA, TFA, thioanisole, rt, 30 min, 100%; (f) (i) ethynyltrimethylsilane, Pd(PPh₃)₂Cl₂, CuI, NEt₃, THF, rt, 17 h, Ar; (ii) K₂CO₃, MeOH, rt, 30 min, 88%; (g) 3-bromoimidazo[1,2-b]pyridazine or 5-bromo-1H-pyrazolo[3,4-b]pyridine, Pd(PPh₃)₂Cl₂, CuI, DIPEA, DMF, 80 °C, 5 h, Ar, 99% 15/75% 16; (h) NaOH, MeOH, H₂O, 60 °C, 3 h, 67% 17/90% 18; (i) thiophenol, HATU, DIPEA, DMF, 45 °C, 3 h, 100% 19/56% 20; (j) transfer buffer (4 M GnHCl, 100 mM NaH₂PO₄/Na₂HPO₄, 12.5 mM TCEP, pH 7.2), MeCN, 40 °C, 24 h, Ar.

Scheme 3. Schematic representation of the RNA-templated benzanilide formation via NCL-type reaction.

Scheme 4. Synthesis of PNA-linked ponatinib fragments. (A) Acceptor conjugate. (B) Donor conjugates. Reagents and conditions: (a) HATU, NMM, DMF, rt, 30 min; (b) TFMSA, TFA, TIS, rt, 3 h; (c) HATU, NMM, NMP, rt, 30 min; (d) TFA, TIS, EDT, H₂O, rt, 3 h. Apostrophe = Bhoc protecting group.

Fig. 3. UPLC analysis of crude and HPLC-purified PNA acceptor conjugate 23a.

Scheme 5. Synthesis of DNA-linked ponatinib fragments. (A) Acceptor conjugates. (B) Donor conjugates. Reagents and conditions: (a) (i) CuSO₄, THPTA, sodium ascorbate, phosphate buffer (100 mM NaH₂PO₄/Na₂HPO₄, pH 7.4), H₂O, DMSO, 30 °C, 3 h, Ar; (ii) TCEP, rt, 30 min, 28%; (b) (i) H₂O, DMSO, rt, 22 h; (ii) TCEP, rt, 30 min, 32%; (c) CuSO₄, THPTA, sodium ascorbate, phosphate buffer (100 mM NaH₂PO₄/Na₂HPO₄, pH 7.4), H₂O, DMSO, 60 °C, 6 h, Ar, 37%; (d) HATU, DIPEA, H₂O, DMF, rt, 4 h, 15%. DNA1 = 5′-TTGAACTCTGCTTAAATCCAG-3′. DNA2 = 5′-CCGCTGAAGGGCTT-3′.

Scheme 6. RNA-templated benzanilide formation via NCL-type reaction between the various acceptor and donor conjugates.

Fig. 4. (A) Product yields after 180 min for background reactions of DNA donor conjugates 30a or 30b with acceptor conjugates 23a –23c. (B–E) Fold change of product formation upon introduction of unpaired template nucleotides (underlined in B) relative to seamless annealing (0 nt) for transfer reactions of DNA donor conjugates 30a or 30b with DNA acceptor conjugate 23b (C), DNA acceptor conjugate 23c (D), or PNA acceptor conjugate 23a (E). Error bars show the standard deviation of triplicate measurements. For details, see Tables S3–S6. Mean yields given in numbers over the bars. (F) Product yields after 720 min for the indicated transfer reactions in presence and absence of RNA template. Conditions: 10 mM MOPS, 200 mM NaCl, 2 mM TCEP, pH 7.2, [A] = 1 μM, [D] = 2 μM, [RNA] = 0/1 μM, 37 °C. BG = background; nt = nucleotides.