Research paper
Synthesis, bioconversion, pharmacokinetic and pharmacodynamic evaluation of N-isopropyl-oxy-carbonyloxymethyl prodrugs of CZh-226, a potent and selective PAK4 inhibitor

https://doi.org/10.1016/j.ejmech.2019.111878Get rights and content

Highlights

  • In an attempt to improve bioavailability of compound 3, we synthesised a series of prodrugs of compound 3 by masking its terminal nitrogen of the piperazine moiety.

  • Most synthesised prodrugs of 3 have low or no inhibition of PAK4 activity.

  • The stability of synthetic prodrugs was evaluated in PBS, SGF, SIF, rat plasma and liver S9 fraction.

  • Prodrug 19 could be quickly converted to parent drug 3 in rats, providing higher exposure of 3 compared to its direct administration.

  • When given via oral route at daily doses of 25 and 50 mg/kg, the prodrug 19 was effective and well tolerated in mouse model of HCT-116 and B16F10.

Abstract

We have previously disclosed compound 3 (CZh-226), a potent and selective PAK4 inhibitor, but its development was delayed due to poor oral pharmacokinetics. In an attempt to improve this issue, we synthesised a series of prodrugs by masking its terminal nitrogen of the piperazine moiety. Most synthesised prodrugs of 3 have low or no inhibition of PAK4 activity. The stability of synthetic prodrugs was evaluated in PBS, SGF, SIF, rat plasma and liver S9 fraction. Of these, prodrug 19 was not only stable under both acidic and neutral conditions but also could be quickly converted to parent drug 3 in rat plasma and liver S9 fraction. Such effective conversion into parent drug 3 was observed in rats, providing higher exposure of 3 compared to its direct administration. When given via oral route at daily doses of 25 and 50 mg/kg, the prodrug 19 was effective and well tolerated in mouse model of HCT-116 and B16F10.

Introduction

p21-Activated kinase (PAK) was initially identified as an effector of Rho GTPases that are involved in a variety of cellular functions, including cellular morphology, adhesion, migration, proliferation and survival. The PAK family members are classified into group I (PAK1-3) and group II (PAK4-6) based on their structures, substrate specificity and sequence homology [1,2]. Among all of the PAKs, PAK4 has a place at critical nodal points in multiple signaling pathways, including the Ras-ERK, Wnt/β-catenin, and androgen receptor/estrogen receptor (AR/ER)-dependent pathway [[3], [4], [5], [6], [7], [8]]. Hence, PAK4 has emerged as an attractive target in the field of cancer therapy [9,10]. Moreover, early studies on PAK1/2 revealed that PAK2 inhibition correlates with increased acute cardiovascular toxicity [11]. Consequently, there has been great interests in the development of selective PAK4 inhibitors.

Few inhibitors of PAK4 have been reported, and their use has been limited by their weak potency and/or lack of PAK1 selectivity, sometimes combined with unsuitable pharmacokinetic properties [12]. Among those, 1 (PF-3758309, Fig. 1) from Pfizer [13] and the structurally distinct quinazoline 2 (GNE-2861, Fig. 1) from Genentech [14] are probably the most studied. Although designed to be a PAK4 inhibitor, compound 1 is, in fact, a multiple-target kinase inhibitor, which potently inhibits all group II PAK members, including PAK1, AMPK, CHK2, RSK, PKC, and SRC [13]. Researchers from Genentech succeeded in identifying a type I1/2 PAK4 inhibitors, such as GNE-2861, with the common structural feature of a tertiary propargyl alcohol. GNE-2861 is a potent and reasonably selective PAK4 inhibitor, but suffers poor permeability and moderate micromolar cell activity, and therefore could not be used as an in vivo tool orally [14]. There remains a clear need for selective oral PAK4 inhibitors to advance in vivo studies.

Our early study discovered, along with optimization, compound 3 (CZh-226, Fig. 1) as a selective PAK4 inhibitor, guided by X-ray crystallography and a structure-based drug design (SBDD) approach [15]. While showing remarkable PAK4 selectivity (346-fold vs PAK1), favorable kinase selectivity profile, and good cell metastatic activity in vitro, compound 3 was suboptimal due to low metabolic stability in human/rat hepatocytes and/or poor permeability, leading to poor rat pharmacokinetics (PK). This fact necessitates administration at very high systemic intraperitoneal or intravenous doses to observe efficacy, severely limiting its potential use as an in vivo probe.

Prodrug approach is a well-established strategy to alter the pharmacokinetic and tissue distribution of drug molecules [16,17]. For all the reasons stated above, an orally bioavailable prodrug of compound 3 would pave the way for the development of this promising PAK4 inhibitor. As predicted by XenoSite (XenoSite is an in silico method that predicts the sites of cytochrome P450-mediated metabolism of druglike molecules.) [18], the terminal nitrogen of the piperazine of compound 3 was identified as a labile metabolic hotspot (Fig. S1, supporting information). Furthermore, it is a relatively strong base (pKa = 7.93), which implies that more than 80% of compound 3 is ionized at physiological pH. This resulting temporary positive charge contributes significantly to low membrane permeability. Therefore, the rational design of the prodrug approach is based on the masking of the positive charge at the terminal nitrogen of the piperazine with a variety of capping groups.

As depicted in Fig. 2, we utilized three types of amine promoieties including simple alkyl carbamates (1118), isopropyl-oxy-carbonyloxymethyl (POC) (1920), and pivaloyl-oxy-methyl (POM) (2122). The carbamate functionality has been utilized in many prodrug strategies designed for secondary amine [19,20]. While simple esterification fails in the case of compound 3, as the rate of hydrolysis of simple alkyl carbamate-based prodrugs is very slow. A common strategy for increasing the rate of hydrolysis of carbamate-based prodrugs is to use a spacer group to connect the parent drug with a lipophilic carboxylate ester. Carboxylesterase may mediate the hydrolysis of the carboxylate esters then trigger a cascade reaction, providing an effective way to release the desired parent drug, with concomitant release of carbon dioxide and nontoxic byproducts (Fig. 2) [19]. Two examples of such prodrug moieties are isopropyl-oxy-carbonyloxymethyl (POC) and pivaloyl-oxy-methyl (POM).

In the present work, we report the synthesis and comprehensive characterization of prodrugs of derived from 3. The lead prodrug, N-isopropyl-oxy-carbonyloxymethyl-3 (prodrug 19), was determined to be chemically stable, yet exhibited excellent release of 3 in rat plasma and rat liver S9 fraction, which was further evaluated in rat and showed increased plasma exposure following oral administration. When compared to equimolar parent drug 3, prodrug 19 showed 4.2-fold improvement in oral bioavailability in the rat. Finally, the prodrug 19 was evaluated in the mouse model of HCT-116 and B16F10 cancer cells following oral administration to assess its in vivo efficacy.

Section snippets

Chemistry

In all of the prodrugs sythesized herein, a promoiety was incorporated into the terminal nitrogen of the piperazine of compound 3. Compound 3 was prepared as previously described in detail [15]. As shown in Scheme 1, the synthesis of intermediates 7a-b, 9a-b was achieved by a three-step procedure [21]. It started from the reactions of appropriate 1-chloroalkyl carbonochloridates (4a-b) with ethanethiol in the presence of triethylamine to yield the corresponding O-(1-chloroalkyl)-S-ethyl

PAK4 inhibitory activity of prodrugs

The inhibitory potency against PAK4 of compound 3 and related prodrugs 11–22 were evaluated as described in our early study [15]. As expected, 3 exhibited potent PAK4 inhibition with an IC50 value of 18 nM. Not surprisingly, most prodrugs failed to reveal such inhibitory potency (Table 1). According to the co-crystal structure of compound 3/PAK4 (PDB 5XVG), the lack of inhibitory potency of the prodrugs is likely due to the inability of capping NH on the piperazine ring to form a

Conclusions

We succeeded in rational design, synthesis, and pharmacokinetic and pharmacodynamic evaluation of a series of carbamates as prodrugs of compound 3, a potent and selective PAK4 inhibitor but with poor pharmacokinetic property. As the best case, prodrug 19 was found to be chemically stable, along with efficient release of 3 in rat plasma and liver S9. Prodrug 19 showed 3.7-fold enhanced Cmax, 4-fold enhanced AUC0-t and 5.2-fold longer half-life, compared with that of parent drug 3, respectively.

Chemistry

All reagents and solvents were obtained from commercial suppliers and used without further purification unless otherwise indicated. Anhydrous solvents were dried and purified by conventional methods prior to use. Column chromatography was carried out on silica gel (200–300 mesh). All reactions were monitored by thin layer chromatography (TLC) on silica gel plates with fluorescence F-254 and visualized with UV light. 1H NMR and 13C NMR spectral data were recorded in DMSO‑d6 on a Bruker ARX-600

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Grant 81872729, 81230077) and Program for Innovative Research Team of the Ministry of Education, and Program for Liaoning Innovative Research Team in University.

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