Design and synthesis of triarylacrylonitrile analogues of tamoxifen with improved binding selectivity to protein kinase C

Abstract

The clinical selective estrogen receptor modulator tamoxifen is also a modest inhibitor of protein kinase C, a target implicated in several untreatable brain diseases such as amphetamine abuse. This inhibition and tamoxifen's ability to cross the blood brain barrier make it an attractive scaffold to conduct further SAR studies toward uncovering effective therapies for such diseases. Utilizing the known compound 6a as a starting template and guided by computational tools to derive physicochemical properties known to be important for CNS permeable drugs, the design and synthesis of a small series of novel triarylacrylonitrile analogues have been carried out providing compounds with enhanced potency and selectivity for PKC over the estrogen receptor relative to tamoxifen. Shortened synthetic routes compared to classical procedures have been developed for analogues incorporating a β-phenyl ring, which involve installing dialkylaminoalkoxy side chains first off the α and/or α' rings of a precursor benzophenone and then condensing the resultant ketones with phenylacetonitrile anion. A second novel, efficient and versatile route utilizing Suzuki chemistry has also been developed, which will allow for the introduction of a wide range of β-aryl or β-heteroaryl moieties and side-chain substituents onto the acrylonitrile core. For analogues possessing a single side chain off the α- or α'-ring, novel 2D NMR experiments have been carried out that allow for unambiguous assignment of E- and Z-stereochemistry. From the SAR analysis, one compound, 6c, shows markedly increased potency and selectivity for inhibiting PKC with an IC₅₀ of 80nM for inhibition of PKC protein substrate and >10μM for binding to the estrogen receptor α (tamoxifen IC₅₀=20μM and 222nM, respectively). The data on 6c provide support for further exploration of PKC as a druggable target for the treatment of amphetamine abuse.

Keywords: Amphetamine; Estrogen receptor α (ERα); Protein kinase C (PKC); SAR; Tamoxifen; Triarylacrylonitrile derivatives.

Figure 1

Clinical triphenylethylene SERMS

Figure 2

Assignment of aromatic regions of NMR spectra for compounds 3a and 4a hydrochloride salts. A. Structures of 3a and 4a with labeled aromatic protons; B. Assigned ¹H aromatic regions for 3a and 4a; C. 2D ¹H-¹H TOCSY spectra for 3a (left) and 4a (right); D. 2D ¹H-¹³C HSQC spectra for 3a (left) and 4a (right).

Figure 3

Compound 6c dose dependently inhibits PMA-stimulated MARCKS phosphorylation. A. Representative Western blot of pMARCKS (top row) with GAPDH loading control (bottom row). Concentrations in μM are given above the lanes. V1 and V2 are vehicle; P1and P2 are PMA control. The molecular weight markers of 95 and 34 kDa are shown. B. Dose response curve calculated from pMARCKS Western blot analysis. PMA control is calculated as PMA values minus vehicle control and set at 100 %. n=4–6.

Scheme 1

Reagents and conditions. (i) excess BrCH₂CH₂Br, Cs₂CO₃, ACN, reflux, 1–2 d (63–70% yield); (ii) for 2a: 1a, 4-(2-chloroethyl)morpholine·HCl, Cs₂CO₃, ACN, reflux, 21 h (85% yield). For 2b: 1b, NHMe₂·HCl, K₂CO₃, acetone, reflux, 16 h (76% yield); (iii) 20 equiv. PhCHLiCN, THF, −78 ºC to rt, 20 – 48 h (86 – 100% yield); (iv) for 5a: 1c, Et₂NCH₂CH₂Cl·HCl, Cs₂CO₃, ACN, reflux, 18 h (92% yield). For 5b: 1c, 4-(2-chloroethyl)morpholine·HCl, Cs₂CO₃, ACN, reflux, 18 h (90% yield). For 5c: 1d, N-methylpiperazine, ACN, reflux, 2 h (86% yield).

Scheme 2

Reagents and conditions. (i) NaH, (EtO)₂P(O)CH₂CN, THF, reflux, 20 h (89% yield); (ii) Br₂, 1,2-DCE, −25 ºC – rt, 4 h (59% yield); (iii) thiophen-2-ylboronic acid, Pd(PPh₃)₄, K₂CO₃, 1:1 toluene/2-propanol, reflux, 2.5 d (87%); (iv) BBr₃, DCM, rt, 16 h (98% yield); (v) Me₂NCH₂CH₂Br·HBr, Cs₂CO₃, ACN, reflux, 18 h (42% yield).