Rational Design of Azastatin as a Potential ADC Payload with Reduced Bystander Killing

Abstract

Auristatins are a class of ultrapotent microtubule inhibitors, whose growing clinical popularity in oncology is based upon their use as payloads in antibody-drug conjugates (ADCs). The most widely utilized auristatin, MMAE, has however been shown to cause apoptosis in non-pathological cells proximal to the tumour ("bystander killing"). Herein, we introduce azastatins, a new class of auristatin derivatives encompassing a side chain amine for antibody conjugation. The synthesis of Cbz-azastatin methyl ester, which included the C2-elongation and diastereoselective reduction of two proteinogenic amino acids as key transformations, was accomplished in 22 steps and 0.76 % overall yield. While Cbz-protected azastatin methyl ester (0.13-3.0 nM) inhibited proliferation more potently than MMAE (0.47-6.5 nM), removal of the Cbz-group yielded dramatically increased IC₅₀ -values (9.8-170 nM). We attribute the reduced apparent cytotoxicity of the deprotected azastatin methyl esters to a lack of membrane permeability. These results clearly establish the azastatins as a novel class of cytotoxic payloads ideally suited for use in next-generation ADC development.

Keywords: Antibodies; Cytotoxicity; Diastereoselectivity; Medicinal chemistry; Total synthesis.

Figure 1

The structural evolution of auristatins. While dolastatin 10, their natural predecessor, has failed to gain clinical relevance, three antibody–drug conjugates armed with a second generation auristatin have received marketing authorization and are used in cancer treatment.

Figure 2

Azastatin‐OMe (7), the target molecule of the work outlined herein. The Val‐Dil core common to dolastatin 10 and its derivatives is highlighted in blue, two key structural elements at the N‐ and C‐termini are marked green. The additional amino moiety intended for antibody conjugation is drawn in purple.

Figure 3

Retrosynthetic analysis of the target molecule. Both of the key γ‐amino acids dolaisoleucine and 4‐aminodolaproine were to be obtained from their respective α‐amino acid homologues (stage 1) in an analogous fashion: C2‐elongation was to yield β‐keto esters (stage 2), which were to be reduced diastereoselectively to furnish highly chiral β‐methoxy esters (stage 3).

Scheme 1

Synthesis of Dov‐Val‐Dil‐OtBu, 15. Reagents and conditions: a) tBuOAc, HNiPr₂, nBuLi, THF, 84 %. b) NaBH₄, SiO₂, DCM/iPrOH 15 : 1, 83 % (5 : 1 mixture of diastereoisomers). c) MeOTf, LiHMDS, DMEU, THF, 60 %. d) 1. Cyclohexene, Pd/C, MeOH. 2. HCl/Dioxane, 90 %. e) Cbz‐Val‐OH, BEP, NEtiPr₂, DCM, 85 %. f) H₂, Pd/C, MeOH. g) 17, DECP, NEt₃, DCM, 54 % over two steps. h) CH₂O, H₂, Pd/C, H₂O/MeOH, 99 %. i) H₂, Pd/C, EtOAc/MeOH 3 : 1, 53 %.

Scheme 2

A) Synthesis of key intermediate 22. B) Rationalization of the syn‐selectivity of the reduction of 9 b by means of the Cram chelate model. Reagents and conditions: a) Boc₂O, NaOH, H₂O/THF. b) Ac₂O, pyridine, DCM. c) C₆F₅OH, DCC, EtOAc, 60 % over three steps. d) EtO₂C‐CH(Me)‐CO₂H, iPrMgBr, THF, 72 % (15 % of unreacted 20). e) NaBH₄, MnCl₂, MeOH, 95 %. 35 % of (R,R)‐22 upon purification by flash chromatography on silica.

Scheme 3

Stereochemical characterization of both major diastereomers of 22 by means of ¹H‐NMR and ¹H‐¹H nuclear Overhauser effect (NOE) spectroscopy. Selected spacial interactions are symbolized by red arrows. Reagents and conditions: a) TFA/DCM 1 : 1. b) Cs₂CO₃, MeOH.

Scheme 4

Synthesis of C‐terminal dipeptide 26. Reagents and conditions: a) NaH, Me₂SO₄, THF/DMF 3 : 1, 80 %. b) K₂CO₃, MeOH/H₂O 40 : 1, 97 %. c) DIAD, DPPA, PPh₃, THF, 85 %. d) H₂, Pd/C, EtOAc. e) CbzCl, NEt₃, DCM. f) LiOH, MeOH/H₂O, 1 : 1. g) H₂N‐Phe‐OMe x HCl, HATU, NEtiPr₂, DCM, 58 % over four steps.

Scheme 5

Ligation of the N‐terminal tripeptide 15 and C‐terminal dipeptide 26 to yield Cbz‐Azastatin‐OMe, 27. The final deprotection yielded both Azastatin‐OMe, 7 and its N‐ethyl analogue, 28. Reactions and conditions: a) DCM/TFA 1 : 1. b) DCM/TFA 8 : 3. c) DECP, NEt₃, DME, 75 % over three steps. d) H₂, Pd/C, EtOH, 98 % (mixture of 7 and 28).