The nuclear export inhibitor aminoratjadone is a potent effector in extracellular-targeted drug conjugates

Abstract

The concept of targeted drug conjugates has been successfully translated to clinical practice in oncology. Whereas the majority of cytotoxic effectors in drug conjugates are directed against either DNA or tubulin, our study aimed to validate nuclear export inhibition as a novel effector principle in drug conjugates. For this purpose, a semisynthetic route starting from the natural product ratjadone A, a potent nuclear export inhibitor, has been developed. The biological evaluation of ratjadones functionalized at the 16-position revealed that oxo- and amino-analogues had very high potencies against cancer cell lines (e.g. 16R-aminoratjadone 16 with IC₅₀ = 260 pM against MCF-7 cells, or 19-oxoratjadone 14 with IC₅₀ = 100 pM against A-549 cells). Mechanistically, the conjugates retained a nuclear export inhibitory activity through binding CRM1. To demonstrate a proof-of-principle for cellular targeting, folate- and luteinizing hormone releasing hormone (LHRH)-based carrier molecules were synthesized and coupled to aminoratjadones as well as fluorescein for cellular efficacy and imaging studies, respectively. The Trojan-Horse conjugates selectively addressed receptor-positive cell lines and were highly potent inhibitors of their proliferation. For example, the folate conjugate FA-7-Val-Cit-pABA-16R-aminoratjadone had an IC₅₀ of 34.3 nM, and the LHRH conjugate d-Orn-Gose-Val-Cit-pABA-16R-aminoratjadone had an IC₅₀ of 12.8 nM. The results demonstrate that nuclear export inhibition is a promising mode-of-action for extracellular-targeted drug conjugate payloads.

Fig. 1. Structures and SAR of ratjadones A–D and related CRM1 inhibitors.

Fig. 2. Inhibition of nuclear export by ratjadone A derivatives in recombinant HeLa cells. (A) Functional domains in fluorescent translocation biosensor system. (B) Cellular distribution of biosensor in untreated (left) and ratjadone A treated HeLa cells (right). (C) IC₅₀ values of nuclear export inhibitory activity. (D) Fluorescence microscopy pictures of biosensor expressing HeLa cells treated with 1, 16, and 17.

Scheme 1. Unsuccessful strategies for the selective derivatization of ratjadone A. Reagents and conditions: (a) TBSOTf (2.3 equiv.), 2,6-lutidine (2.6 equiv.), (CH₂Cl₂), –100 °C, 3 h, 62% brsm; (b) MsCI (3.3 equiv.), Py (4.0 equiv.), (CH₂Cl₂), 23 °C, 3.5 h, 98%; (c) NaN₃ (3.0 equiv.), (DMF); (d) DIAD (1.1 equiv.), PPh₃ (1.15 equiv.), DPPA (1.0 equiv.), (THF); (e) mCPBA (1.0 equiv., NaHCO₃ (1.05 equiv.), (CH₂Cl₂ : H₂O/3 : 1), 0 °C or –10 °C; (f) DMDO (1.1 equiv.), (CH₂Cl₂), –30 °C; (g) O₃, (CH₂C1₂ : MeOH/9 : 1), –78 °C, PPh₃ (xs); (h) OsO₄ (0.01 equiv.), NMO (1.0 equiv.) or PNO (1.0 equiv.), (CH₂Cl₂), 0 °C or –10 °C.

Scheme 2. Semi-synthesis of C16-aminoratjadones from (+)-ratjadone A. Reagents and conditions: (a) IBX (1.1 equiv.), (DMSO, 0.075 M), 23 °C. Addition of 16 h + 23 °C. 24 h, 75% brms 13, 8% brms 14, 15% brms 15. (b) (NH₄)OAc (10 equiv.), NaCNBH₃ (2.0 equiv.), (MeOH), 23 °C, 13 h + 40 °C, 2 h, 34% brsm 16, 21% brms 17, 8% brsm 18, 2% brsm 19.

Scheme 3. Synthesis of C16-aminoratjadones bearing short terminal alkyne moieties and enzymatically cleavable Val-Cit-pABA or disulphide linkers. Reagents and conditions: (a) 22 (1.1 equiv.), NMM (3.0 equiv.), (CH₂C1₂). 23 °C, 1.5 h, 65% for 20; (b) 24 (1.1 equiv.), NMM (3.0 equiv.), (CH₂C1₂), 23 °C, 20 h; 90% for 21 and 69% for 22; (c) 27 or 28 (1.1 equiv.), NMM (6.0 equiv.), (DMF), 23 °C. 26 h, 76% for 25 and 66% for 26; (d) 30 (1.0 equiv.), TSTU (1.0 equiv.), NMM (5.0 equiv.), (DMF), 23 °C, 15 h, 65% for 29; (e) 32 (1.0 equiv.), (MeCN : PBS (pH = 7.45)/1 : 2), 0 °C to 23 °C, 2.5 h, 70% for 31.

Scheme 4. Semi-synthesis of alkyne-linked C16-aminoratjadones. Reagents and conditions: (a) NH₄OAc (2.0 equiv.), NaBH₃CN (2.0 equiv.), (MeOH), 23 °C, 4h, 68% of 33 as 2 : 1-mixture of diastereomers; (b) 24 (1.1 equiv.), NMM (6.0 equiv.), (CH₂Cl₂), 23 °C, 20 h, 62% of 34 as a 2 : 1 mixture of diastereomers; (c) propargylamine (2.0 equiv.), NaBH₃CN (2.0 equiv.), (MeOH), 23 °C, 4 h, 99% of 35 as a 2 : 1-mixture of diastereomers.

Fig. 3. Structure of biotin-PEG₃-16S-aminoratjadone conjugate 36.

Scheme 5. Synthesis of azido-folate carrier molecules in solution. Reagents and conditions: (a) (1) Pip (xs), (CH₂Cl₂), 23 °C, 3 h, (2) Fmoc-(Asp(OtBu))₃-OH (1.2 equiv.), EDCl HCl (1.1 equiv.), HOAt (1.1 equiv.), NMM (6.0 equiv.), (THF), 0 °C + 23 °C, 24 h, 58% of 38; (b) (1) TFA (30 equiv.), (CH₂C1₂), 23 °C, 22 h, (2) p-azidobenzoic acid (1.1 equiv.), HATU (1.1 equiv.), HOAt (1.1 equiv.), NMM (10.0 equiv.), (DMF), 23 °C, 24 h, 88% of 39 and 85% of 40; (c) (1) Et₂NH (xs), (DMF), 23 °C, 20 h, (2) FA-γ-OSu (6.0 equiv.), NMM (6.0 equiv.), (DMSO), 23 °C, 14 h, 48% of FA-N₃-1; (d) (1) Et₂NH (xs), (CH₂Cl₂), 23 °C, 9 h, (2) Fmoc-(Asp(OtBu))₃-OH (1.2 equiv.). EDCI (1.2 equiv.), HOAt (1.2 equiv.), NMM (6.0 equiv.), (THF), 30 min preactivation at 23 °C, then 23 °C, 5 h, 75% of 41; (e) (1) TFA (50 equiv.), (CH₂Cl₂), 23 °C, 20 h, (2) Et₂NH (xs), (DMF), 23 °C, 20 h, (3) FA-γ-OSu (6.0 equiv.), NMM (6.0 equiv.), (DMSO), 23 °C, 16 h, 32% of FA-N₃-2.

Fig. 4. Folate-based carrier molecules and folate–fluorescein conjugates synthesized in this study. Reagents and conditions: (a) FA-N₃ (1.1 equiv.), CuSO₄ (0.05 equiv.), TBTA (0.1 equiv.), NaAsc (0.5 equiv.), DiPEA (6.0 equiv.), (DMSO : H₂O :  ^tBuOH/2 : 1 : 1), 23 °C, 2–24 h; (b) FA-N₃ (1.1 equiv.), (DMSO), 23 °C, 4–20 h.

Fig. 5. Confocal fluorescence microscopy. (A) Imaging of KB 3.1 and A-549 cells with folate–fluorescein conjugate FA-1-FITC (8.6 μM, 5 min, 37 °C). KB 3.1 cells were imaged in the presence (left) of absence (middle) of an excess of folic acid. (B) Imaging of KB 3.1 cells treated with 1 μM of six folate–fluorescein conjugates at two different time points (30 min and 72 h). All experiments were conducted in folate-free RPMI medium with 10% folate-containing FCS. Merge F + BF: merge fluorescence signal + bright field.

Fig. 6. Fluorescence intensity per cell determined by flow cytometry of KB 3.1 cells that were labelled with six different folate–fluorescein conjugates.

Fig. 7. Novel folate–aminoratjadone conjugates with non-cleavable and enzymatically cleavable linkers synthesized in this study.

Fig. 8. Novel LHRH derivatives and LHRH-aminoratjadone conjugates synthesized in this study.

Fig. 9. Human plasma stability of selected folate–aminoratjadone conjugates and reference compounds (10 μg mL^–1 in human plasma at 37 °C and pH = 7.4).