Design, Synthesis and Biological Evaluation of Prodrugs of 666-15 as Inhibitors of CREB-Mediated Gene Transcription

Abstract

cAMP-response element binding protein (CREB) is a transcription factor involved in multiple cancers. Chemical inhibitors of CREB represent potential anticancer agents. We previously identified 666-15 as a potent CREB inhibitor. While 666-15 showed efficacious anticancer activity in vivo through intraperitoneal (IP) injection, its oral bioavailability is limited. To increase its oral bioavailability, we describe synthesis and evaluation of prodrugs based on 666-15. The amino acid esters were attempted, but they were not stable for detailed characterization. The corresponding sulfate and phosphates were prepared. The sulfate of 666-15 was too stable to release 666-15 while the phosphates were converted into 666-15 with half-lives of ∼2 h. Phosphate 3 was also a potent CREB inhibitor with anti-breast cancer activity. Furthermore, compound 3 showed much improved oral bioavailability at 38%. These studies support that 3 can be used as an oral CREB inhibitor while IP administration of 666-15 is preferred for in vivo applications.

Figure 1

In vivo pharmacokinetic properties of 666-15. (A) Chemical structure of 666-15. (B) Plasma concentration of 666-15 in mice treated with a single dose of 666-15 by PO or IV (n = 3). The mice were treated with a single dose of 666-15 and the blood was collected at indicated time points post drug administration. The plasma was prepared to determine the concentration of 666-15 by LC-MS/MS.

Figure 2

Chemical structures of designed 666-15 prodrugs 1–4.

Scheme 1. Synthesis of Compound 1

Scheme 2. Synthesis of Compounds 2–4

Figure 3

Hydrolysis of compounds 2–4 in tissue culture media. The indicated compounds (100 μM for 2 and 3, 25 μM for 4) were incubated with DMEM supplemented with 10% FBS at 37 °C along with an internal standard naphthol AS-E (100 μM). At different time points, an aliquot was taken and mixed with acetonitrile to precipitate proteins. The supernatant was analyzed by RP-HPLC. The t_1/2 was calculated using one-phase exponential decay protocol.

Figure 4

Single-dose pharmacokinetics of compound 3 in mice. A single dose of 3 at the indicated dose level was administered to mice by IP, IV, or PO (n = 3). At different time points post drug administration, the blood was collected and the plasma levels of 3 (A) and 666-15 (B) were determined by LC-MS/MS. The plasma concentrations of 3 in the PO and IV groups were not detectable beyond 2 and 4 h post dosing, respectively. The remaining 3 detected at ≥8 h post dosing in the IP group was approaching the lower limit of detection.