Design, synthesis and biological evaluation of AKT inhibitors bearing a piperidin-4-yl appendant

Compound 10h is a potent pan-AKT inhibitor showing cellular AKT inhibition and apoptosis induction in PC-3 prostate cancer cells.

Abstract

A series of AKT inhibitors possessing a piperidin-4-yl side chain was designed and synthesized. Some of them showed high AKT1 inhibitory activity and potent anti-proliferative effect on PC-3 prostate cancer cells in the preliminary screening. Further studies revealed the most potent compound, 10h, as a pan-AKT inhibitor. Compound 10h was able to inhibit the cellular phosphorylation of AKT effectively and induce apoptosis of PC-3 cells. It also showed high metabolic stability in human liver microsomes. Preclinical characterization of 10h, a promising lead AKT inhibitor, as a potential anti-prostate cancer therapeutic needs to be further investigated.

1. Introduction

Hyperactive aberration of PI3K-AKT signaling is observed in a wide variety of cancer types, which makes components of this signaling possible targets for cancer therapy.1 The serine/threonine kinase AKT, also known as protein kinase B (PKB), plays a critical role in this signaling and is hyper-activated in over 50% of human tumors.2 It regulates by phosphorylation over one hundred downstream substrates that are involved in the regulation of various cellular events, including cell survival, proliferation and metabolism. Glycogen synthase kinase 3 (GSK3) is the first reported substrate of AKT,3 and evaluation of the phosphorylation level of its β isoform is typically used as a tool to assess the intracellular inhibition level of AKT by small-molecule inhibitors.

Given that AKT is tightly linked to cell physiology and pathology, it has been catching the attention of the pharmaceutical world in the past decades. A number of AKT-targeted agents have entered clinical trials for an exploration of their therapeutic potential against various tumor types. For instance, AZD5363 (Fig. 1) was investigated for its safety and efficacy in prostate cancer in a phase 1 clinical trial (NCT01692262). In particular, since activation of the PI3K-AKT signaling through loss of the tumor suppressor PTEN was proven as a potential mechanism for the pathology of castration-resistant prostate cancer (CRPC),4 AKT inhibitors have been utilized in combination therapy for the treatment of CRPC, such as in a phase 2 trial of AZD5363 in combination with enzalutamide in metastatic CRPC (NCT02525068).

Fig. 1. ATP-competitive inhibitors of AKT in clinical trials.

Among all the AKT inhibitors reported, a specific type of ATP-competitive inhibitors, including GSK690693, AZD5363 and GDC0068, was well developed (Fig. 1).5 Analysis of the co-crystals of AZD5363 (ref. 6) or GDC0068 (ref. 7) in complex with AKT revealed that these agents bond to the hinge region of AKT. The modes of this ligand–protein interaction can be capitalized in the discovery of novel AKT inhibitors.

Our laboratory previously reported a series of AKT inhibitors with the general structure shown in Fig. 2.8 The representative compounds 5q and 5t in this series showed high AKT1 inhibition at a concentration of 50 nM with inhibitory rates of 69.7% and 65.5%, respectively. Both compounds also exhibited a moderate anti-proliferative effect on the prostate cancer cell line PC-3 with IC₅₀ values of 21.1 μM and 31.5 μM, respectively. In this work, structural modification was further performed. First, the hydrogen bond interactions, formed between the hydroxyethyl group of AZD5363 or the isopropylaminomethyl group of GDC0068 and the carboxyl residues of two glutamic acids (Glu 234 and Glu 278) near the hinge region, were critical for their ligand–protein binding affinity.6,7 Thus, one modification to our lead structure was to introduce an appendant to the bridging methylene between the carbonyl and the phenyl groups (Fig. 2). The criterion for this appendant was that it should provide a hydrogen bond donor to generate similar hydrogen bond interactions without introduction of chirality. After a literature survey, a piperidin-4-yl group which had been adopted by previously reported AKT inhibitors AI-1 (AKT IC₅₀ = 18 nM) and AI-2 (AKT IC₅₀ = 20 nM) was chosen (Fig. 2).9,10 This was a symmetric cyclic appendant with a terminal imine group as the hydrogen bond donor. The other modification focused on the hinge-binding pyrrolopyrimidine group; three nitrogen-containing bicyclic systems were attempted in this work. These modifications led to a series of N-heterocyclic based AKT inhibitors.

Fig. 2. Design of novel ATK inhibitors based on our and others' previous studies.

2. Results and discussion

2.1. Chemistry

Compounds 10a–10l were prepared following the route shown in Scheme 1. Halogen groups like chloride or bromide were first introduced at the C-5 position of a commercially available 4-chloropyrazolopyrimidine (1) via free radical halogenation. Aromatic nucleophilic substitution of the chloride from the C-4 position of intermediate 2 by tert-butyl piperazine-1-carboxylate under microwave irradiation afforded intermediate 3, which was de-protected with trifluoroacetic acid (TFA) to give intermediate 4. Finally, condensation with the appropriate carboxylic acid (5) followed by de-protection of the Boc protective group afforded compounds 10a–10l.

Scheme 1. Reagents and conditions: (a) NCS or NBS, DMF, r.t., 12–18 h; (b) tert-butyl piperazine-1-carboxylate, DMF, microwave, 150 °C, 20 min; (c) TFA, DCM, r.t., 4 h; (d) HBTU, DIPEA, DMF, r.t., 6 h; (e) TFA, DCM, r.t., 4 h.

This synthetic route was successfully applied to the preparation of compounds 14a–14d and 18a–18d using 6-chloro-9H-purine (11) and 4-chloroquinazoline (15) as starting materials, respectively (Scheme 1).

4-Chlorobenzylcyanide (6) was used as a starting material for the preparation of building block 5 (Scheme 2). The two hydrogen atoms of the methylene group of 6 were first abstracted by sodium hydride, followed by replacing the two chlorine atoms from Boc-protected bis(2-chloroethyl)amine (8) to afford the substituted 4-phenylpiperidine-4-carbonitrile as intermediate 9. Subsequent hydrolysis generated carboxylic acid as intermediate 5.

Scheme 2. Reagents and conditions: (a) aq. NaOH, (Boc)₂O, DCM, r.t., 12 h; (b) NaH, DMF, 0–5 °C; 80 °C, 12–18 h; (c) NaOH, ethanol, 60 °C, 48 h.

2.2. AKT inhibitory activity and anti-proliferative effect on PC-3 cells

Preliminary screenings were carried out for the newly synthesized compounds, including the evaluation of AKT inhibitory activity at a compound concentration of 1 μM and its anti-proliferative effect on a prostate cancer cell line of PC-3 (Table 1).

Table 1. Preliminary screenings for the new compounds: AKT1 inhibition and anti-proliferation in PC-3 cells.

Code	Structure		AKT1 inhibition			Anti-proliferative effect on PC-3 cells b (IC50/μM)
	R1	R2	%, @1 a μM	%, @0.1 a μM	IC50 b /nM
10a	H	4-F	7.4	—	—	>40
10b	H	4-Cl	38.7	—	—	>40
10c	H	4-Br	11.6	—	—	>40
10d	H	3,4-diCl	52.1	—	—	>40
10e	Cl	4-F	86.7	60.9	—	37.4 ± 2.2
10f	Cl	4-Cl	91.9	83.4	66.6 ± 2.1	19.4 ± 1.6
10g	Cl	4-Br	95.4	84.5	32.9 ± 1.9	32.4 ± 2.1
10h	Cl	3,4-diCl	94.3	83.7	24.3 ± 1.6	3.7 ± 0.9
10i	Br	4-F	93.2	53.3	—	36.6 ± 2.4
10j	Br	4-Cl	96.7	86.7	26.9 ± 1.7	9.8 ± 1.2
10k	Br	4-Br	96.5	90.6	51.9 ± 2.1	15.0 ± 1.3
10l	Br	3,4-diCl	93.8	90.9	30.5 ± 1.8	10.3 ± 1.1
14a	—	4-F	94.2	—	—	>40
14b	—	4-Cl	45.6	—	—	>40
14c	—	4-Br	68.0	—	—	>40
14d	—	3,4-diCl	51.4	—	—	33.9 ± 2.2
18a	—	4-F	23.0	—	—	13.7 ± 1.3
18b	—	4-Cl	34.2	—	—	>40
18c	—	4-Br	6.1	—	—	22.8 ± 1.6
18d	—	3,4-diCl	21.9	—	—	14.0 ± 1.8
GSK690693			99.5	98.5	21.3 ± 2.1	14.1 ± 1.6

^a n = 3.

^bIC₅₀ data are the means of three independent experiments.

Compounds (14a–14d) containing a purine ring exhibited higher AKT1 inhibitory activity than their pyrazolopyrimidine analogues (10a–10d) and quinazoline analogues (18a–18d) but lacked anti-proliferative activity. Compounds (18a–18d) bearing a quinazoline structure showed low AKT1 inhibition but high anti-proliferation, while pyrazolopyrimidine-derived compounds (10a–10d) lacked both AKT1 inhibitory activity and cytotoxicity. However, introduction of a halogen substituent to the C-3 position of the pyrazolopyrimidine ring significantly elevated inhibitory activity against both AKT1 and PC-3 cells. Compounds (10e–10l) with a halogen substitution (Cl or Br) at the C-3 position of the pyrazolopyrimidine ring exhibited both high AKT inhibition and high growth inhibition in PC-3 cells than did their hydrogen substituted counterparts (10a–10d).

For compounds (10a–10l) based on a pyrazolopyrimidine ring with or without a halogen substituent at the C-3 position, substitutions on the terminal phenyl ring had different effects. All of the 4-F substituted compounds (10a, 10e and 10i) showed weaker AKT1 inhibition at 1 μM than their chlorine- or bromine-substituted analogues. This tendency was confirmed at a diluted concentration of 0.1 μM as compounds 10e and 10l showed inferior AKT1 inhibition than their chlorine- or bromine-substituted analogues 10f–10h and 10j–10l. A similar trend was also observed on the potency against PC-3 cells, as compounds (10f–10h and 10j–10l) with chlorine or bromine substitution all showed higher anti-proliferative activity than their 4-F substituted analogues (10e and 10i).

Based on the preliminary data on both AKT1 inhibition and PC-3 cell growth inhibition, compounds (10f–10h and 10j–10l) were selected for the determination of AKT1 inhibitory IC₅₀ values (Table 1). In comparison with the positive drug GSK690693, these compounds all showed slightly lower AKT1 inhibitory activity. Among them, compound 10h showed the highest AKT1 inhibitory activity with an IC₅₀ value of 24.3 nM and was most potent against PC-3 cells with an IC₅₀ value of 3.7 μM. This cellular potency was nearly 4-fold more effective than that of GSK690693 (IC₅₀ = 14.1 μM).

2.3. Kinase selectivity study of selected compound 10h

Compound 10h was further evaluated for its selectivity against a panel of 16 kinases (Fig. 3) at the concentration of 1 μM. This compound showed high AKT1 selectivity over 11 kinases (Abl, B-Raf, CDK1, CHK1, GSK3β, JAK1, PDK1, PKCα, RAF-1 and ROCK1). However, compound 10h showed moderate inhibitory activity against MNK1 (47%) and Aurora A (64%), and high inhibitory activity against RSK1 (83%), PKA (99%) and p70S6K (99%). The lack of selectivity against the latter three kinases was mainly due to the high overall ATP binding site homology (>70%) of these kinases with AKT1.11

Fig. 3. Selectivity assay for 10h in 16 human kinases and three AKT kinase isoforms (n = 3).

At the concentration of 10 nM, compound 10h showed pan-inhibitory activity to all three AKT isoforms with moderate to high inhibition of AKT2, AKT1 and AKT3 by 38%, 83% and 93%, respectively (Fig. 3). A more than 2-fold selectivity of AKT1 and AKT3 compared to AKT2 was also observed.

2.4. Influence of 10h on cellular AKT and GSK3β phosphorylation

Compound 10h was further evaluated for its impact on cellular AKT signaling in PC-3 cells via an immunoblotting assay (Fig. 4A). In contrast to GSK690693 which enhanced AKT activation in this assay, a high inhibition of AKT phosphorylation by 10h was observed after 24 hours of treatment at all three concentrations. A similar phenomenon also occurred on reported AKT inhibitors based on a diphenylmethylamine scaffold12 and indicated an involvement of an additional functional mechanism for compound 10h to overcome the positive feedback loop which was usually evoked to fight back the AKT inhibition within the cells. In the meantime, 10h showed weaker inhibition on the phosphorylation of GSK3β, a downstream effector of AKT, compared with efficient inhibition of GSK3β phosphorylation by GSK690693. This was consistent with the distinct function of the two compounds in AKT phosphorylation as GSK3β was directly downstream and suppressed by activated AKT within cells.

Fig. 4. (A) Effects of 10h on AKT signaling within PC-3 cells; (B and C) apoptosis induction by 10h in PC-3 cells.

2.5. Apoptotic induction of 10h in PC-3 cells

Compound 10h was further evaluated for its apoptosis inductive effect on PC-3 cells via an annexin V-FITC/propidium iodide (PI) binding assay (Fig. 4B and C). After 48 hours of treatment, a mild degree of apoptosis was observed in a dose-dependent manner. The percentage of apoptotic cells increased by 1.32–27.83% when compared to the DMSO control.

2.6. Stability in human liver microsomes

Liver microsomes are subcellular fractions derived from the endoplasmic reticulum of the liver. They contain a variety of metabolic enzymes (cytochrome P450) and are used as an in vitro model for the prediction of in vivo metabolic stability in liver. Compound 10h showed high metabolic stability with more than 80% total remaining after a one-hour treatment in human liver microsomes. The calculated half-life (T_1/2) and clearance rate (CL_int(mic)) were more than 145 min and less than 9.6 μL min^–1 mg^–1, respectively (Table 2).

Table 2. Metabolic stability of 10h in human liver microsomes.

Code	T 1/2 (min)	CLint(mic) (μL min–1 mg–1)	CLint(liver) (mL min–1 kg–1)	Remaining (T = 60 min)
10h	>145	<9.6	<8.6	81.8%
Testosterone	16.9	82.2	74.0	8.1%
Diclofenac	10.2	136.2	122.6	1.5%
Propafenone	7.7	180.9	162.8	0.5%

3. Conclusion

In this work, the discovery of a potent AKT targeted inhibitor 10h showing efficient anti-prostate cancer potency in PC-3 cells is described. This compound belongs to a series of N-heterocyclic based AKT1 inhibitors with the structural feature of a piperidin-4-yl side chain. Compounds based on a 3-halogenic pyrazolopyrimidine ring in this series showed high AKT1 inhibition and a potent anti-proliferative effect on PC-3 cells, of which 10h showed the highest potency in both AKT1 inhibition (IC₅₀ = 24.3 nM) and PC-3 cell proliferation (IC₅₀ = 3.7 μM). In a further characterization of the kinase selectivity profile, compound 10h showed potent pan-AKT inhibitory activity as well as inhibitory activity against kinases of the AGC family. 10h could moderately inhibit cellular AKT phosphorylation and induce apoptosis of PC-3 cells. It also showed high metabolic stability in human liver microsomes. Therefore, further evaluation of 10h is warranted for its potential application as an anti-prostate cancer therapeutic.

4. Materials and methods

4.1. Chemistry

All reagents and solvents were purchased from commercial suppliers and used without further purification. All reactions were monitored by thin layer chromatography (TLC). Silica gel GF254 plates were used and visualized with UV light. Column chromatography was performed with silica gel using the solvent systems as indicated in the synthetic procedures. NMR spectra were recorded on a Bruker AVANCE DRX400 or DRX600 spectrometer using tetramethylsilane (TMS) as an internal standard in DMSO-d₆. Chemical shifts are reported in parts per million (ppm). Coupling constants (J) are given in Hz. The mass spectra (MS) were measured with an API 4000 mass spectrometer. All of the melting points were determined using a Büchi capillary melting point apparatus and were uncorrected.

4.1.1. General procedure for the synthesis of intermediate 2

To a solution of 4-chloro-1H-pyrazolo[3,4-d]pyrimidine (1, 3.1 g, 20.0 mmol) in N,N-dimethylformamide (DMF, 15 ml), N-chlorosuccinimide (NCS, 2.8 g, 21.0 mmol) or N-bromosuccinimide (NBS, 3.7 g, 21.0 mmol) was added. This mixture was stirred at room temperature for 12–18 hours. Ice-cold water (150 ml) was poured into the reaction mixture, and the precipitate was filtered, washed with water (2 × 20 ml), and dried to give intermediate 2.

3,4-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (2a)

Light yellow solid, yield 74%. ¹H NMR (400 MHz, DMSO-d₆) δ 14.77 (s, 1H), 8.88 (s, 1H).

3-Bromo-4-chloro-1H-pyrazolo[3,4-d]pyrimidine (2b)

Off-white solid, yield 94%. ¹H NMR (400 MHz, DMSO-d₆) δ 14.13 (s, 1H), 8.06 (s, 1H).

4.1.2. General procedure for the synthesis of intermediates 3, 12 and 16

A solution of intermediate 2 (15.2 mmol), N,N-diisopropylethylamine (DIPEA, 4.0 g, 30.4 mmol) and tert-butyl piperazine-1-carboxylate (3.1 g, 16.7 mmol) in DMF (15 ml) was heated at 150 °C under microwave irradiation for 20 min. This reaction mixture was cooled and poured into ice-cold water (150 ml) followed by extraction of ethyl acetate (EA, 3 × 30 ml). The organic extracts were combined, washed with saturated aqueous NH₄Cl (2 × 30 ml) and brine (2 × 30 ml), dried over anhydrous Na₂SO₄, filtered, condensed under reduced pressure, and purified by column chromatography (petroleum ether (PE)/EA = 9/1) to obtain intermediate 3.

Intermediates 12 and 16 were prepared in a similar procedure as described above using commercially available 6-chloro-9H-purine (11) and 4-chloroquinazoline (15) as starting materials, respectively.

tert-Butyl 4-(9H-purin-6-yl)piperazine-1-carboxylate (12)

Off-white solid, yield 91%. ¹H NMR (400 MHz, DMSO-d₆) δ 13.09 (s, 1H), 8.23 (s, 1H), 8.15 (s, 1H), 4.21 (br s, 4H), 3.44 (br s, 4H), 1.43 (s, 9H).

tert-Butyl 4-(quinazolin-4-yl)piperazine-1-carboxylate (16)

Off-white solid, yield 88%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.09–7.93 (m, 2H), 7.72 (t, J = 7.7 Hz, 1H), 7.49–7.15 (m, 1H), 4.26–4.14 (m, 4H), 3.62 (br s, 4H), 1.44 (s, 9H).

4.1.3. General procedure for the synthesis of intermediates 4, 13 and 17

To a solution of intermediate 3 (1.0 mmol) in dichloromethane (DCM, 6 ml), trifluoroacetic acid (TFA, 2 ml) was added. This mixture was stirred at room temperature for 4 hours, and the precipitate formed was collected by filtration and dried to give intermediate 4. Intermediates 13 and 17 were prepared in a similar procedure as described above.

3-Chloro-4-(piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine (4a)

Off-white solid, yield 68%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.36 (d, J = 5.7 Hz, 1H), 3.93–3.79 (m, 4H), 3.12–3.00 (m, 4H).

3-Bromo-4-(piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine (4b)

Off-white solid, yield 88%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 1H), 3.96–3.74 (m, 4H), 3.20–3.07 (m, 4H).

4-(Piperazin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine (4c)

Off-white solid, yield 84%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.99 (s, 2H), 8.97 (s, 1H), 8.63 (s, 1H), 4.32 (s, 4H), 3.35 (s, 4H).

6-(Piperazin-1-yl)-9H-purine dihydrochloride (13)

Off-white solid, yield 88%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.71 (br s, 2H), 8.45 (s, 1H), 8.39 (s, 1H), 4.52 (s, 4H), 3.28 (s, 4H).

4-(Piperazin-1-yl)quinazoline dihydrochloride (17)

Off-white solid, yield 84%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.02 (s, 2H), 8.95 (s, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.10–8.02 (m, 2H), 7.74 (ddd, J = 8.4, 5.6, 2.7 Hz, 1H), 4.37 (s, 4H), 3.34 (s, 4H).

4.1.4. General procedure for the synthesis of intermediate 8

To a solution of bis(2-chloroethyl)amine hydrochloride (7, 8.0 g, 44.8 mmol) in DCM (20 ml) at room temperature, aqueous NaOH (6 M, 20 ml) was added followed by addition of Boc₂O (9.8 g, 44.8 mmol). Then, the reaction mixture was stirred for 12 hours and extracted with DCM (2 × 30 ml). The organic extracts were combined, washed with brine (30 ml), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give intermediate 8 as a colorless oil which was used directly for the next step without further purification.

4.1.5. General procedure for the synthesis of intermediate 9

To a solution of substituted phenylacetonitrile 6 (14.8 mmol) and intermediate 8 (16.3 mmol) in DMF (25 mL) on an ice bath, sodium hydride (1.1 g, 44.4 mmol) was added in portions. Then, the reaction mixture was heated at 80 °C for 12–18 hours. This mixture was then cooled and quenched by dropwise addition of H₂O (20 ml). After extraction with EA (3 × 30 ml), the organic extracts were combined, washed with brine (2 × 30 ml), dried over Na₂SO₄, filtered, concentrated under reduced pressure, and purified using column chromatography (PE/EA = 20/1) to afford intermediate 9.

tert-Butyl 4-cyano-4-(4-fluorophenyl)piperidine-1-carboxylate (9a)

White solid, yield 43%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.43 (s, 4H), 3.76 (d, J = 13.6 Hz, 2H), 2.98 (br s, 2H), 2.33 (d, J = 13.6 Hz, 2H), 1.76–1.68 (m, 2H), 1.39 (s, 9H).

tert-Butyl 4-(4-chlorophenyl)-4-cyanopiperidine-1-carboxylate (9b)

White solid, yield 52%. ¹H NMR (600 MHz, DMSO-d₆) δ 7.57 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 4.13 (br s, 2H), 3.01 (br s, 2H), 2.11 (d, J = 13.2 Hz, 2H), 1.90 (td, J = 12.9, 4.2 Hz, 2H), 1.41(s, 9H).

tert-Butyl 4-(4-bromophenyl)-4-cyanopiperidine-1-carboxylate (9c)

White solid, yield 40%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.64 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 4.12 (d, J = 12.8 Hz, 2H), 3.01 (br s, 2H), 2.12 (d, J = 12.8 Hz, 2H), 1.90 (td, J = 13.1, 4.2 Hz, 2H), 1.41 (s, 9H).

4.1.6. General procedure for the synthesis of intermediate 5

To a solution of intermediate 9 (6.6 mmol) in ethanol (10 ml), aqueous NaOH (10 M, 10 ml) was added. The reaction mixture was stirred at 60 °C for 48 hours, cooled and concentrated under reduced pressure. The residue was adjusted to pH 5–6 with aqueous HCl (1 M), and the white solid precipitate was collected by filtration, washed with H₂O (2 × 30 ml), and dried to afford the intermediate 5.

1-(tert-Butoxycarbonyl)-4-(4-fluorophenyl)piperidine-4-carboxylic acid (5a)

White solid, yield 86%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (s, 1H), 7.43 (s, 4H), 3.77 (d, J = 13.6 Hz, 2H), 2.97 (br s, 2H), 2.34 (d, J = 13.6 Hz, 2H), 1.77–1.69 (m, 2H), 1.39 (s, 9H).

1-(tert-Butoxycarbonyl)-4-(4-chlorophenyl)piperidine-4-carboxylic acid (5b)

White solid, yield 75%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.81 (s, 1H), 7.42 (s, 4H), 3.76 (d, J = 13.6 Hz, 2H), 2.98 (br s, 2H), 2.33 (d, J = 13.6 Hz, 2H), 1.74–1.68 (m, 2H), 1.40 (s, 9H).

4-(4-Bromophenyl)-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (5c)

White solid, yield 87%. ¹H NMR (600 MHz, DMSO-d₆) δ 12.80 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 3.76 (d, J = 13.8 Hz, 2H), 2.97 (br s, 2H), 2.34 (d, J = 13.8 Hz, 2H), 1.72–1.66 (m, 2H), 1.41 (s, 9H).

4.1.7. General procedure for the synthesis of compounds 10, 14 and 18

To a solution of intermediate 5 (1.0 mmol) in DCM (6 ml), HBTU (417 mg, 1.1 mmol) and DIPEA (388 mg, 3.0 mmol) were added. After stirring at room temperature for 15 min, intermediate 4 (1.0 mmol) was added. The reaction mixture was stirred for 6 hours and washed with saturated NH₄Cl (3 × 20 ml) and brine (30 ml). The organic layer was concentrated under reduced pressure to give an oily residue. This residue was dissolved in DCM (4 ml), and TFA (2 ml) was added. After stirring for 4 hours at room temperature, the mixture was concentrated under reduced pressure, adjusted with saturated Na₂CO₃ to pH 9, and extracted with DCM (3 × 20 ml). The organic extracts were combined, washed with brine (2 × 20 ml), and concentrated under reduced pressure to give a solid residue which was purified by column chromatography (DCM/MeOH = 50/1 ramping to 20/1) to give compounds 10a–10l. Compounds 14a–14d and 18a–18d were prepared in a similar procedure as mentioned above.

(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-fluorophenyl)piperidin-4-yl)methanone (10a)

White solid, yield 64%. Mp: 112–115 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.20 (s, 1H), 8.11 (s, 1H), 7.36 (dd, J = 8.5, 5.2 Hz, 2H), 7.14 (t, J = 8.6 Hz, 2H), 3.69 (br s, 8H), 2.38 (d, J = 13.3 Hz, 2H), 2.05–1.84 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 173.46, 157.08, 155.04, 154.69, 140.40, 133.41, 126.93, 115.80, 115.58, 99.78, 49.11, 43.92, 42.77, 35.57. HRMS (ESI) m/z calculated for C₂₁H₂₅FN₇O [M + H]⁺: 410.2099, found 410.2098.

(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-chlorophenyl)piperidin-4-yl)methanone (10b)

White solid, yield 67%. Mp: 120–123 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.21 (s, 1H), 8.12 (s, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 4.05–3.45 (m, 8H), 3.20–3.04 (m, 4H), 2.42 (d, J = 13.5 Hz, 2H), 2.01 (br s, 2H). ¹³C NMR (100 MHz, MeOD) δ 172.82, 157.09, 155.04, 154.70, 142.69, 133.40, 132.97, 129.19, 126.69, 99.79, 48.88, 43.84, 42.43, 34.52. HRMS (ESI) m/z calculated for C₂₁H₂₅ClN₇O [M + H]⁺: 426.1804, found 426.1799.

(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-bromophenyl)piperidin-4-yl)methanone (10c)

White solid, yield 62%. Mp: 149–152 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.21 (s, 1H), 8.12 (s, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 3.67 (br s, 8H), 3.04 (br s, 4H), 2.37 (d, J = 12.9 Hz, 2H), 1.95 (t, J = 14.7 Hz, 2H). ¹³C NMR (100 MHz, MeOD) δ 173.17, 157.09, 155.04, 154.69, 143.66, 133.42, 132.12, 127.06, 120.63, 99.79, 49.31, 43.33, 41.81, 35.35. HRMS (ESI) m/z calculated for C₂₁H₂₅BrN₇O [M + H]⁺: 472.1278, found 472.1282.

(4-(1H-Pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(3,4-dichlorophenyl)piperidin-4-yl)methanone (10d)

White solid, yield 52%. Mp: 174–177 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.20 (d, J = 10.6 Hz, 1H), 8.13 (s, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.51 (d, J = 1.9 Hz, 1H), 7.26 (dd, J = 8.4, 1.9 Hz, 1H), 3.80 (br s, 4H), 3.55 (br s, 4H), 3.14–3.01 (m, 4H), 2.39 (d, J = 13.3 Hz, 2H), 2.02–1.92 (m, 2H). ¹³C NMR (100 MHz, MeOD) δ 172.43, 157.08, 155.04, 154.70, 144.94, 133.47, 132.98, 131.14, 130.92, 127.07, 125.18, 99.80, 49.12, 45.46, 42.53, 34.91. HRMS (ESI) m/z calculated for C₂₁H₂₄Cl₂N₇O [M + H]⁺: 460.1414, found 460.1409.

(4-(3-Chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-fluorophenyl)piperidin-4-yl)methanone (10e)

Off-white solid, yield 53%. Mp: 135–138 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (s, 1H), 7.35–7.27 (m, 2H), 7.21 (t, J = 8.7 Hz, 2H), 3.42 (br s, 8H), 2.82 (d, J = 13.2 Hz, 4H), 2.18 (d, J = 12.8 Hz, 2H), 1.84–1.67 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.62, 162.37, 159.95, 158.41, 154.14, 141.96, 129.69, 127.67, 116.29, 116.08, 98.88, 49.51, 48.06, 43.76, 41.09, 37.08. HRMS (ESI) m/z calculated for C₂₁H₂₄ClFN₇O [M + H]⁺: 444.1709, found 444.1710.

(4-(3-Chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-chlorophenyl)piperidin-4-yl)methanone (10f)

White solid, yield 51%. Mp: 184–188 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.23 (s, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H), 3.44 (br s, 8H), 2.82 (d, J = 15.6 Hz, 4H), 2.17 (d, J = 12.9 Hz, 2H), 1.76 (d, J = 7.5 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.52, 158.34, 158.10, 154.50, 144.71, 131.60, 130.10, 129.43, 127.68, 98.94, 49.65, 47.96, 43.70, 41.08, 36.84. HRMS (ESI) m/z calculated for C₂₁H₂₄Cl₂N₇O [M + H]⁺: 460.1414, found 460.1416.

(4-(4-Bromophenyl)piperidin-4-yl)(4-(3-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)methanone (10g)

White solid, yield 56%. Mp: 181–185 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.10 (s, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 3.41 (br s, 8H), 2.82 (br s, 4H), 2.15 (d, J = 12.7 Hz, 2H), 1.73 (d, J = 9.7 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.45, 160.45, 158.41, 152.79, 144.79, 135.71, 132.41, 128.05, 127.84, 98.77, 49.70, 48.26, 43.84, 40.83, 36.99. HRMS (ESI) m/z calculated for C₂₁H₂₄ClBrN₇O [M + H]⁺: 504.0909, found 504.0904.

(4-(3-Chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(3,4-dichlorophenyl)piperidin-4-yl)methanone (10h)

Off-white solid, yield 49%. Mp: 195–199 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.22 (d, J = 15.1 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.51 (s, 1H), 7.27 (d, J = 6.8 Hz, 1H), 3.61 (br s, 8H), 3.08 (br s, 4H), 2.39 (d, J = 13.4 Hz, 2H), 1.96 (br s, 2H). ¹³C NMR (100 MHz, MeOD) δ 172.31, 158.46, 154.59, 144.99, 132.97, 131.17, 131.14, 130.94, 127.14, 125.19, 122.38, 98.78, 49.19, 49.05, 42.55, 42.47, 34.84. HRMS (ESI) m/z calculated for C₂₁H₂₃Cl₃N₇O [M + H]⁺: 494.1024, found 494.1024.

(4-(3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-fluorophenyl)piperidin-4-yl)methanone (10i)

Off-white solid, yield 32%. Mp: 194–196 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.27 (s, 1H), 7.31–7.21 (m, 4H), 3.42 (br s, 8H), 2.87 (br s, 4H), 2.17 (s, 2H), 1.78 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.63, 158.88, 157.61, 154.74, 141.82, 127.65, 118.64, 117.03, 116.23, 101.59, 49.40, 48.75, 43.63, 36.77. HRMS (ESI) m/z calculated for C₂₁H₂₄BrFN₇O [M + H]⁺: 490.1184, found 490.1186.

(4-(3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-chlorophenyl)piperidin-4-yl)methanone (10j)

Off-white solid, yield 43%. Mp: 180–184 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.27 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 3.96–3.59 (m, 4H), 3.43 (d, J = 12.9 Hz, 2H), 3.36 (d, J = 12.0 Hz, 2H), 3.21 (q, J = 7.3 Hz, 4H), 2.57 (d, J = 13.9 Hz, 2H), 2.17 (t, J = 11.6 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.16, 158.80, 157.01, 155.13, 142.52, 132.36, 129.77, 127.52, 119.08, 101.59, 48.02, 45.91, 41.54, 32.21. HRMS (ESI) m/z calculated for C₂₁H₂₄BrClN₇O [M + H]⁺: 506.0888, found 506.0898.

(4-(3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(4-bromophenyl)piperidin-4-yl)methanone (10k)

White solid, yield 52%. Mp: 175–178 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.26 (s, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 3.96–3.59 (m, 4H), 3.46–3.35 (m, 4H), 3.21 (q, J = 7.3 Hz, 4H), 2.56 (d, J = 13.8 Hz, 2H), 2.19 (t, J = 11.5 Hz, 2H). ¹³C NMR (100 MHz, MeOD) δ 171.48, 158.91, 156.41, 154.62, 141.99, 132.44, 126.96, 121.34, 119.03, 101.53, 47.93, 46.51, 41.70, 38.90, 32.07. HRMS (ESI) m/z calculated for C₂₁H₂₄Br₂N₇O [M + H]⁺: 550.0383, found 550.0393.

(4-(3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-yl)piperazin-1-yl)(4-(3,4-dichlorophenyl)piperidin-4-yl)methanone (10l)

Hoary solid, yield 34%. Mp: 195–198 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.27 (s, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 7.29 (dd, J = 8.5, 2.2 Hz, 1H), 3.66 (br s, 8H), 3.28 (br s, 2H), 3.21 (t, J = 11.8 Hz, 2H), 2.49 (d, J = 13.4 Hz, 2H), 2.08 (t, J = 15.2 Hz, 2H). ¹³C NMR (100 MHz, MeOD) δ 171.49, 158.97, 156.50, 154.56, 144.10, 133.13, 131.31, 127.08, 125.16, 118.95, 101.57, 48.36, 47.05, 45.26, 41.94, 33.24. HRMS (ESI) m/z calculated for C₂₁H₂₃BrCl₂N₇O [M + H]⁺: 540.0499, found 540.0510.

(4-(9H-Purin-6-yl)piperazin-1-yl)(4-(4-fluorophenyl)piperidin-4-yl)methanone (14a)

White solid, yield 59%. Mp: 173–175 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (s, 1H), 8.11 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 3.90 (br s, 4H), 3.39 (br s, 4H), 3.10 (d, J = 12.5 Hz, 2H), 2.96 (t, J = 11.7 Hz, 2H), 2.29 (d, J = 13.3 Hz, 2H), 1.99 (t, J = 11.0 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.63, 153.55, 152.07, 143.98, 138.97, 132.51, 127.97, 120.46, 119.26, 48.77, 44.51, 44.42, 44.18, 42.47, 34.17. HRMS (ESI) m/z calculated for C₂₁H₂₅FN₇O [M + H]⁺: 410.2099, found 410.2097.

(4-(9H-Purin-6-yl)piperazin-1-yl)(4-(4-chlorophenyl)piperidin-4-yl)methanone (14b)

White solid, yield 63%. Mp: 186–188 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (s, 1H), 8.10 (s, 1H), 7.34 (dd, J = 8.8, 5.3 Hz, 2H), 7.26 (t, J = 8.8 Hz, 2H), 4.28–3.69 (m, 6H), 3.58–3.41 (m, 2H), 3.21 (d, J = 12.8 Hz, 2H), 3.03 (t, J = 11.7 Hz, 2H), 2.36 (d, J = 13.7 Hz, 2H), 2.09 (t, J = 12.3 Hz, 2H). ¹³C NMR (100 MHz, DMSO) δ 171.52, 162.65, 160.22, 153.48, 152.14, 140.24, 138.96, 127.66, 119.26, 116.59, 116.38, 48.15, 46.08, 44.59, 44.40, 43.12, 41.96, 33.25. HRMS (ESI) m/z calculated for C₂₁H₂₅ClN₇O [M + H]⁺: 426.1804, found 426.1806.

(4-(9H-Purin-6-yl)piperazin-1-yl)(4-(4-bromophenyl)piperidin-4-yl)methanone (14c)

White solid, yield 67%. Mp: 234–236 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (s, 1H), 8.10 (s, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 3.88 (br s, 4H), 3.39 (br s, 4H), 3.01–2.83 (m, 4H), 2.23 (d, J = 13.1 Hz, 2H), 1.86 (t, J = 10.0 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.08, 153.48, 152.07, 144.18, 138.97, 131.75, 129.50, 127.67, 119.25, 49.18, 44.82, 44.49, 44.28, 43.12, 35.66. HRMS (ESI) m/z calculated for C₂₁H₂₅BrN₇O [M + H]⁺: 472.1278, found 472.1279.

(4-(9H-Purin-6-yl)piperazin-1-yl)(4-(3,4-dichlorophenyl)piperidin-4-yl)methanone (14d)

White solid, yield 69%. Mp: 203–205 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (s, 1H), 8.11 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.50 (s, 1H), 7.24 (d, J = 9.9 Hz, 1H), 3.93 (br s, 8H), 2.95–2.78 (m, 4H), 2.20 (d, J = 13.0 Hz, 2H), 1.79 (d, J = 13.4 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.84, 153.46, 152.08, 146.64, 139.03, 132.14, 131.63, 129.72, 127.66, 126.52, 119.27, 49.58, 46.15, 44.61, 43.44, 36.39. HRMS (ESI) m/z calculated for C₂₁H₂₄Cl₂N₇O [M + H]⁺: 460.1414, found 460.1411.

(4-(4-Fluorophenyl)piperidin-4-yl)(4-(quinazolin-4-yl)piperazin-1-yl)methanone (18a)

White solid, yield 67%. Mp: 145–147 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.81 (s, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.53 (s, 1H), 7.24 (d, J = 8.4 Hz, 2H), 3.91–3.45 (m, 6H), 3.41–3.35 (m, 2H), 3.25 (d, J = 12.6 Hz, 2H), 3.05 (t, J = 12.1 Hz, 2H), 2.34 (d, J = 13.6 Hz, 2H), 2.14 (t, J = 11.7 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.08, 164.10, 153.95, 151.69, 143.15, 133.31, 132.74, 128.50, 127.71, 126.28, 125.54, 120.72, 116.06, 49.09, 48.06, 41.66, 32.56. HRMS (ESI) m/z calculated for C₂₄H₂₇FN₅O [M + H]⁺: 420.2194, found 420.2189.

(4-(4-Chlorophenyl)piperidin-4-yl)(4-(quinazolin-4-yl)piperazin-1-yl)methanone (18b)

Off-white solid, yield 64%. Mp: 106–108 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (s, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.80 (s, 2H), 7.52 (s, 1H), 7.38–7.16 (m, 4H), 3.80–3.41 (m, 6H), 3.26–3.17 (m, 4H), 3.03 (t, J = 11.9 Hz, 2H), 2.34 (d, J = 13.3 Hz, 2H), 2.08 (t, J = 11.8 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.53, 164.11, 153.95, 151.65, 140.21, 133.30, 128.50, 127.54, 126.27, 125.69, 116.65, 116.44, 116.29, 48.99, 48.83, 48.20, 41.97, 33.27. HRMS (ESI) m/z calculated for C₂₄H₂₇ClN₅O [M + H]⁺: 436.1899, found 436.1902.

(4-(4-Bromophenyl)piperidin-4-yl)(4-(quinazolin-4-yl)piperazin-1-yl)methanone (18c)

White solid, yield 72%. Mp: 189–191 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.84–7.78 (m, 2H), 7.57–7.45 (m, 3H), 7.31 (d, J = 8.6 Hz, 2H), 3.92–3.38 (m, 6H), 3.26 (br s, 4H), 3.07 (t, J = 12.0 Hz, 2H), 2.36 (d, J = 13.8 Hz, 2H), 2.17 (t, J = 11.8 Hz, 2H). ¹³C NMR (100 MHz, DMSO) δ 171.15, 164.09, 153.94, 151.66, 142.67, 133.29, 132.29, 129.77, 128.50, 127.44, 126.27, 125.69, 116.29, 48.92, 48.11, 41.58, 32.38. HRMS (ESI) m/z calculated for C₂₄H₂₇BrN₅O [M + H]⁺: 482.1373, found 482.1374.

(4-(3,4-Dichlorophenyl)piperidin-4-yl)(4-(quinazolin-4-yl)piperazin-1-yl)methanone (18d)

White solid, yield 76%. Mp: 254–256 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.83–7.79 (m, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.56–7.48 (m, 2H), 7.25 (dd, J = 8.5, 2.2 Hz, 1H), 3.94–3.31 (m, 7H), 3.27 (br s, 3H), 3.06 (t, J = 12.0 Hz, 2H), 2.37 (d, J = 13.8 Hz, 2H), 2.18 (t, J = 11.6 Hz, 2H). ¹³C NMR (100 MHz, DMSO) δ 170.67, 164.09, 153.94, 151.68, 144.67, 133.29, 132.44, 131.96, 130.47, 128.51, 127.43, 126.32, 126.29, 125.69, 116.31, 49.00, 48.98, 48.17, 41.56, 32.31. HRMS (ESI) m/z calculated for C₂₄H₂₆Cl₂N₅O [M + H]⁺: 470.1509, found 470.1506.

4.2. In vitro kinase assay

In vitro AKT1 kinase inhibitory activity was evaluated via a homogeneous time-resolved fluorescence (HTRF) assay (LANCE) using an AKT kinase kit (Cisbio) in a 384-well plate. To each well was added subsequently AKT1, STK substrate-biotin, the tested compounds and ATP in kinase buffer (50 mM HEPES, 5 mM MgCl₂, 1 mM DTT, 0.02% NaN₃ and 0.01% BSA, pH 7.0) and then the samples were incubated for 45 minutes at 25 °C. Finally, Sa-XL665 and STK Ab-Cryptate were added to stop the enzymatic step and further incubation for 2 hours was carried out to finish the detection process. The fluorescence ratio (665 nm/620 nm) was obtained using a microplate reader (Perkin Elmer, USA).

4.3. Cell proliferation assays (MTT)

Human prostate cancer cell line PC-3 was purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium containing 10% fetal bovine serum (Gibco, USA) and 100 IU mL^–1 penicillin and 100 μg mL^–1 streptomycin in a humidified atmosphere with 5% CO₂ at 37 °C. All the compounds were evaluated for their anti-proliferative activity against PC-3 cells using an MTT assay. PC-3 cells were seeded in a 96-well plate and cultured for 10 hours at 37 °C. Thereafter, cells were treated with the tested compounds at serial concentrations and DMSO (control) and incubated for 72 hours. MTT (5 mg mL^–1, 10 μL) was added to the culture which was incubated for another 4 hours. Subsequently, the formazan crystals were extracted with DMSO and the absorbance (OD₅₇₀) was detected with a microplate reader (Bio-Rad 680, USA). The IC₅₀ values were calculated with GraphPad Prism (version 5.0).

4.4. Kinase selectivity assay

The compounds were prepared to 50× final assay concentration in 100% DMSO, and the working stock of each compound was added to the assay well as the first component in the reaction followed by the remaining components. Kinases were diluted in specific buffer prior to addition to the reaction mix. There was no pre-incubation step between the compound and the kinase prior to initiation of the reaction. The reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of phosphoric acid to a concentration of 0.5%. 10 μL of the reaction mixture was then spotted onto a P30 filtermat and washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting.

The positive control wells contained all components of the reaction, except the compound of interest, and DMSO (at a final concentration of 2%) was included in these wells to control for solvent effects. The blank wells contained all components of the reaction with a reference inhibitor replacing the compound of interest. This abolished kinase activity and established the baseline (0% kinase activity remaining).

4.5. Western blotting

Western blot analysis was carried out using a whole-cell lysate. PC-3 cells were treated with DMSO, 10h (1, 5 and 10 μM) or GSK690693 (1, 5 and 10 μM) for 4 hours, respectively. Afterward, the cells were rinsed twice with ice-cold PBS and lysed in a cell lysis buffer (50 mM Tris-HCl, pH 8.0; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS; 150 mM NaCl; 1 mM PMSF; and a protease inhibitor cocktail). The protein concentration of each sample was measured by using bicinchoninic acid (BCA) protein assay reagent (Thermo Pierce, IL). 20 μg of lysate was loaded into each lane. Antibodies for GSK3β and AKT (1 : 1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). The secondary antibodies were horseradish peroxidase (HRP)-linked goat-anti rabbit IgG (Santa Cruz Biotechnology, USA). After SDS–PAGE, proteins were electroblotted onto a PVDF membrane. The blots were blocked with 5% BSA/TBST for 1 hour at room temperature, probed with specific ABS overnight at 4 °C, washed, incubated with linked goat-anti rabbit IgG (Santa Cruz, USA), and probed with ECL-plus (GE Healthcare, Sweden). The immunoreactive bands were detected using an image analyzer (LAS1000; Fuji Photo Film, Japan).

4.6. Cell apoptosis assay

Apoptosis was quantified by an annexin V-FITC/propidium iodide (PI) binding assay. Cells were seeded in a 6-well plate with 1 μM, 5 μM or 10 μM 10h for 48 hours. The treated cells were washed twice with cold phosphate-buffered saline (PBS) and re-suspended in binding buffer (100 μL) to which 2 μL of annexin V-FITC and 5 μL of PI were added. The samples were gently vortexed and incubated for 15 minutes at room temperature in the dark. After addition of 200 μL of binding buffer, samples were immediately analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson, CA). The percentage of apoptotic cells was analysed using Flowjo software.

4.7. Human liver microsome metabolic stability assay

Testosterone, diclofenac and propafenone were tested as control drugs in this assay as all of them are substrates of cytochrome P450 enzymes. A DMSO solution of the tested compound (10 mM, 10 μl per well) and a solution of microsome (80 μl per well) were added to a 96-well plate and the mixture was first incubated at 37 °C for 10 min. Then, potassium phosphate buffer (100 mM, 10 μl per well) was added and no co-factor (NCF) remaining was evaluated after a further incubation for 60 min. After this pre-warming process, an NADPH regenerating system (10 μl per well) was added, and each compound was tested at 5 time points (5 min, 10 min, 20 min, 30 min and 60 min). At each time point, stop solution (including 100 ng ml^–1 tolbutamide and 100 ng ml^–1 labetalol, cold in 4 °C, 300 μl per well) was added to terminate the reaction. The sampling plates were shaken for approximately 10 min, and then the samples were centrifuged at 4000 rpm for 20 min at 4 °C to afford the supernatant (100 μl) for LC/MS test. Intrinsic clearance (CL_int) and half-life (T_1/2) values were then calculated: CL_int(mic) = 0.693/half-life/mg microsome protein per ml; CL_int(liver) = CL_int(mic) × (45 mg microsomal protein per g liver weight) × (20 g liver weight per kg body weight).

Conflicts of interest

The authors declare no conflict of interest.