Synthesis and biological evaluation of santacruzamate A analogues for anti-proliferative and immunomodulatory activity

Abstract

Santacruzamate A (SCA) is a natural product isolated from a Panamanian marine cyanobacterium, previously reported to have potent and selective histone deacetylase (HDAC) activity. To optimize the enzymatic and cellular activity, 40 SCA analogues were synthesized in a systematic exploration of the zinc-binding group (ZBG), cap terminus, and linker region. Two cap group analogues inhibited proliferation of MCF-7 breast cancer cells, with analogous increased degranulation of cytotoxic T cells (CTLs), while one cap group analogue reduced CTL degranulation, indicative of suppression of the immune response. Additional testing of these analogues resulted in reevaluation of the previously reported SCA mechanism of action. These analogues and the resulting structure-activity relationships will be of interest for future studies on cell proliferation and immune modulation.

Keywords: Anti-proliferative activity; Enzyme assays; Immune modulation; Natural product analogues; Santacruzamate A.

Figure 1

Santacruzamate A (SCA, 1), SAHA, and the SCA-SAHA hybrid compound reported previously, and their overlaid 3D structures (SCA in yellow, SAHA in green, and SCA-SAHA hybrid in blue).

Figure 2

Effect of santacruzamate A and SAHA on the levels of acetylated histones H3K9, H3K23, and H4K12 in Molt-4 cells.

Figure 3

X-ray crystal structure of SAHA (green, left) bound to HDAC2 (gray, PDB: 4LXZ), with santacruzamate A (1) (yellow, right) overlaid over the binding conformation of SAHA with HDAC2. The zinc ion (dark gray) within the HDAC2 active site is shown with an idealized octahedral metal geometry (idealized metal geometries of the zinc ion are shown as yellow arrows and geometries not filled by HDAC2 are labeled G′, G″, G″′ and proceed clockwise starting from left) using the Chimera program. SAHA binds to the zinc ion via chelation with G′ and G″, while santacruzamate A likely binds only to G′. Hydrogen bonds are depicted as orange lines. The surface of the HDAC2 active site, within 4.1Å of the ligands, was generated via Chimera and colored according to the corresponding heteroatom on enzyme surface.

Scheme 1

Synthesis of santacruzamate A N-carbamate analogues. Reagent and conditions: (i) ClCO₂-R¹, K₂CO₃ or NaOH, THF, 0 °C to rt, 70–90%; (ii) Boc₂O, NaOH, THF, 97%; (iii) phenethylamine, EDC-HCl, TEA, cat. DMAP, CH₂Cl₂, 43–93%.

Scheme 2

Synthesis of santacruzamate A urea analogues. Reagents and conditions: (i) CS₂, NaOH, H₂O; (ii) 30% H₂O₂, KOH, H₂O; (iii) KNO₂, diluted H₂SO₄, 0 °C to rt; (iv) 68% ethylamine/H₂O, 100 °C; (v) phenethylamine, EDC-HCl, TEA, cat. DMAP, CH₂Cl₂, 0 °C to rt; (vi) 12a or 12b, cat. p-TsOH, NEAT 180 °C, 30 torr; (vii) 13a or 13b, phenethylamine, EDC-HCl, TEA, cat. DMAP, CH₂Cl₂, 0 °C to rt.

Scheme 3

Synthesis of santacruzamate A N-amide analogues. Reagents and conditions: (i) Ac₂O, AcOH, Et₂O; (ii) propionic anhydride or butyric anhydride, cat H₂SO₄, 100 °C; (iii) R²-COCl, NaOH, THF/H₂O, 0 °C to rt; (iv) phenethylamine, EDC-HCl, TEA, cat. DMAP, CH₂Cl₂.

Scheme 4

Synthesis of santacruzamate A thiol analogue. Reagents and conditions: (i) thioacetic acid, 0 °C to rt; (ii) thionyl chloride, DCM, refluxed for 2 h at 40 °C; (iii) 20a, H₂O, Na₂CO₃, 0 °C to rt; (iv) phenethylamine, EDC-HCl, TEA, cat. DMAP, CH₂Cl₂.

Scheme 5

Synthesis of santacruzamate A inverted ethyl carbamate analogue. Reagents and Conditions: (i) TEA, MeOH, reflux, 92%; (ii) methyl-4-hydroxybutanoate, N,N′-disuccinimdyl carbonate, TEA 79%; (iii) NHS-methyl-4-hydroxybutanoate, 68% ethylamine, TEA, THF; (iv) phenethylamine, EDC-HCl, TEA, DMAP, CH₂Cl₂ 56%.

Scheme 6

Synthesis of santacruzamate Acap group analogues. Reagent and conditions: (i) ethyl chloroformate, K₂CO₃, H₂O, 0 °C to rt; (ii) cap-group R¹, EDC-HCl, TEA, cat. DMAP, CH₂Cl₂; (iii) EDC-HCl, HOBt, TEA, phenethyl-OH, DCM, 0 °C to rt.

Scheme 7

Synthesis of santacruzamate A linker analogues. Reagents and conditions: (i) ethyl chloroformate, K₂CO₃, H₂O, 0 °C to rt; (ii) phenethylamine, aniline, benzenemethanamine, or benzenebutanamine, EDC-HCl, TEA, cat. DMAP, CH₂Cl₂, 0 °C to rt; (iii) ethylchloroformate, TEA, CH₂Cl₂.