CB-5339

Discovery of a new class of valosine containing protein (VCP/P97) inhibitors for the treatment of non-small cell lung cancer

Xueyuan Wanga, Enhe Baia, Hui Zhoua, Sijia Shab, Hang Miaob, Yanru Qind, Zhaogang Liuc, Jia Wangc, Haoyang Zhanga, Meng Leib,⁎, Jia Liuc, Ou Haic, Yongqiang Zhua,⁎

A B S T R A C T

Valosine containing protein (VCP/p97) is a member of the AAA ATPase family involved in several essential cellular functions and plays an important role in the ubiquitin-mediated degradation of misfolded proteins. P97 has a significant role in maintaining the cellular protein homeostasis for tumor cell growth and survival and has been found overexpressed in many tumor types. No new molecule entities based on p97 target were approved in clinic. Herein, a series of novel pyrimidine structures as p97 inhibitors were designed and synthesized. After enzymatic evaluations, structure-activity relationships (SAR) were discussed in detailed. Among the screened compounds, derivative 35 showed excellent enzymatic inhibitory activity (IC50, 36 nM). The cellular inhibition results showed that compound 35 had good antiproliferative activity against the non-small cell lung cancer A549 cells (IC50, 1.61 μM). Liver microsome stability showed that the half-life of compound 35 in human liver mi- crosome was 42.3 min, which was more stable than the control CB-5083 (25.8 min). The in vivo pharmacokinetic results showed that the elimination phase half-lives of compound 35 were 4.57 h for ig and 3.64 h for iv, respectively and the oral bioavailability was only 4.5%. These results indicated that compound 35 could be effective for intravenous treatment of non-small cell lung cancer.

Keywords:
Valosine containing protein Non-small cell lung cancer Structure-activity relationships Pyrimidine derivative Pharmacokinetic

1. Introduction

Valosin-containing protein (VCP/p97) is a member of the AAA (ATPase associated with various cellular activities) ATPase family. P97 promotes several biological processes, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD), nuclear membrane fusion after completion of mitosis, Golgi reassembly, activation of transcription factors and autophagy, where it supplies the mechanical force required for extracting proteins by ATP hydrolysis.1–4 As most AAA-ATPases, p97 structurally adopts a ring- shaped homohexamer structure comprising siX identical 90 kDa sub- units arranged in a ring, with each protomer containing three domains: two ATPase domains (D1 and D2) and one N-terminal domain.5–10 The D1 domain has low basal hydrolytic activity, due in part to a very low off rate of ADP.1 The D2 domain is responsible for the major ATPase activity of p97 under physiological conditions. The D2 ATPase region has been displayed to have both a higher Km for ATP and a faster hy- drolysis of ATP to ADP.10 The N-terminal domain binds various cofac- tors that interact with a variety of substrate proteins. Many studies have revealed p97′s role in promoting ERAD in cooperation with the ubi- quitin-proteasome system (UPS). P97 serves as a force-generating ma- chine to remove misfolded poly-ubiquitinated proteins from the ER into the cytosol and then transports them to the proteasome for degrada- tion11,12.
P97 plays an important role in maintaining the cellular protein homeostasis for tumor cell growth and survival, which was found to be overexpressed in several malignancies including non-small cell lung cancer and associated with malignancy.13–18 For instance, it had been shown that RNA interference or overexpression of ATPase deficient protein in tumor cell lines could disrupt the function of p97 to cause cell death.19,20 Besides, gene knockout of p97 in mice was found to be embryonic lethal,21 while mutations of the p97 gene were thought to be associated with the neurodegenerative diseases.22 So in recent years, p97 had drawn great attention as a potential drug target for developing small molecules for cancer therapy. Up to now, a number of p97 in- hibitors had been reported (Fig. 1).23–30 Among them, compound CB- 5083 was the first selective p97 inhibitor with the requisite pharma- cological properties that showed promising preclinical activities.31 Be- sides, two phase I clinical trials (NCT02243917 and NCT02223598) of CB-5083 had been completed in 2017. However, due to the toXicities of CB-5083, the clinical trial was discontinued. So there is a necessity to develop new compounds to meet the unmet medical need. Herein, we described the design, synthesis and structure-activity relationships (SAR) of a series of novel p97 inhibitors. Among the inhibitors, com- pound 35 exhibited good in vitro activities and microsomal stabilities.
Furthermore, compound 35 showed good pharmacokinetic results, which indicated that compound 35 might be effective for the treatment of non-small cell lung cancer.

2. Results and discussion

2.1. Chemistry

The synthesis of key intermediates 5a-5g, 9 and 13a-13j was out- lined in Scheme 1. Compounds 1a-1c reacted with dimethyl carbonate in the presence of NaH to produce methyl esters 2a-2c (yields 29%- 35%). And then the carbonyl groups of intermediates 2a-2c were am- monified by ammonium acetate to give amines 3a-3c (yields 83%- 90%). Subsequently, compounds 3a-3c reacted with 2,2,2-tri- chloroacetyl isocyanate and ammonia to form 2,4-diol pyrimidyl het- erocyclic rings 4a-4c, which reacted with POCl3 and then were am- monified by different amines R1NH2 to produce key intermediates 5a- 5g (yields 19%-26%). 1H-indole-4-carbonitrile 6 reacted with benze- nesulfonyl chloride to afford N-protected intermediate 7 with the yield of 75%. Intermediate 7 reacted with lithium diisopropylamide at −40 °C followed by methyl iodide to yield indole derivative 8 (yield 69%). Then the N-terminal protected group benzenesulfonyl was re- moved with sodium hydroXide solution to form 2-methyl-4-cyano in- dole 9 (yield 90%). 3-Nitroaniline 10 condensed with acetone under strongly basic conditions to give 2-methyl-4-nitro indole 11 (yield 62%). Then Boc-protecting group was introduced to the N atom of in- dole ring of 11 and the nitro group was reduced to amino group by Fe to give amine 12 (yield 71%). Finally, amine 12 reacted with different R2Cl and deprotected the Boc groups to yield 13a-13j (yields 75%- 85%).
The synthesis of target molecules CB-5083 and 17–20 was sum- marized in Scheme 2. Intermediate 5c was coupled with cyanoindole 9 in the presence of Pd2(dba)3 as the catalyst and Cs2CO3 as base to get the compound 16 (yield 74%). Intermediate 16 reacted with acet- aldehyde oXime in the presence of palladium acetate and triphenyl- phosphine to convert the nitrile group into the primary carboXamide to yield target molecule CB-5083 (yield 70%). And then CB-5083 reacted with Lawesson’s reagent to change the O atom into S one to give mo- lecule 17 (yield 81%). Intermediate 5c reacted with commercially available N-(1H-indol-4-yl)acetamide in the presence of Pd2(dba)3, X- Phos and cesium carbonate to give target molecule 18 (yield 72%). Similar reaction between 5c and 3-aminobenzonitrile produced the target molecule 19 (yield 66%).
The synthesis of target molecules 20–25 was illustrated in Scheme 3. Cyanoindole 9 was coupled with intermediates 5a, 5b, 5d-5f, re- spectively and then the nitrile groups were hydrolyzed to the primary carboXamides to give target molecules 20–24 (yields 60%-72%). The target molecule 25 was prepared from commercially available 2,4-di- chloro pyrimidine 14, which firstly reacted with benzylamine to form compound 15 (yield 85%). Intermediate 15 was coupled with 9 and then the nitrile substituent was converted into the carboXamide to get target molecule 25 (yield 60%). Similar substitution reactions were performed between 5c and indole derivatives 13a-13j to give the target molecules 26–31 and 35–38 (yields 60%–78%) and intermediate 13c reacted with 5d, 5f and 5g to produce target molecules 32–34 (yields 64%-74%) as showed in Scheme 4.

2.2. Biological evaluation

The kinase inhibitory activities of the target compounds were evaluated via ADP-Glo assay (Promega) against purified human p97 enzyme. Cell-based assay included a 72 h Cell Counting Kit-8 (CCK8) viability assay. Metabolic stabilities were investigated in five liver mi- crosomes, mouse, rat, dog, monkey and human. Absolute bioavail- ability (F %) was determined by the pharmacokinetic assessment of areas under the plasma concentration versus time curves following iv and ig administrations.

2.3. Discussion

Enzymatic. The enzymatic results of compounds 17–25 and CB-5083 were showed in Table 1. While the O atom (compound CB-5083, IC50 27.0 nM) on the formamide was substituted by S one (17, IC50 192.6 nM), it led to the activity decrease by more than seven folds. When the 4-indol-formamide group of CB-5083 was changed to acetyl substituted 4-indol-amine and 2-methyl was removed at the same time, the activity of compound 18 (IC50 922.1 nM) was greatly reduced. Replacement of indol ring with benzyl one resulted in an inactive compound 19. Subsequently, the replacement of the phenyl ring on benzylamino group by aromatic heterocycle (pyridyl 20), aliphatic heterocycle (morpholinyl 21), aliphatic ring (cyclopropyl 22) led to the complete loss of p97 activities. In addition, the replacement of pyran ring (CB-5083) with oXepane (23) showed potent p97 activity (IC50 82.8 nM). However, when the pyran ring (CB-5083) was replaced by oXecane (24, 119.7 nM) or was removed (25, 119.7 nM), all failed to increase the p97 potencies.
Attention was next focused on the modification of the R2 groups of 2-methylindole and R1 groups of pyrimidin-4-amine in CB-5083 as il- lustrated in Table 2. When the formamide of CB-5083 was changed to acetyl substituted 4-indol-amine, the activity of compound 26 (129.4 nM) was reduced greatly. The introduction of 2-chloroacetyl group at the R2 position (27, 308.1 nM) also failed to increase the p97 with different sulfamide groups to give compounds 35–38 (Table 3). Compared with acrylamide substituent (28, IC50 58.3 nM), methane- sulfonamide (35, 36.4 nM) and cyclopropyl (37, 34.6 nM) substituents showed better activities. However, benzenesulfonamide (36, 134.9 nM) and trifluoromethanesulfonamide groups (38, 100.9 nM) resulted in decreased potencies in a certain extent.
Cellular. The compounds with the IC50 values of enzymatic inhibi- tion less than 0.1 μM were further evaluated their potential antitumor effects in A549 cells. The biological results were listed in Table 4. These compounds showed significant inhibitory potency against A549 cell lines with the IC50 values less than 5 μM. Especially compound 35 in- hibited the cell proliferation at 1 μM level, which was nearly as active as CB-5083. Therefore, compound 35 was used for further evaluation
Microsome stabilities. The metabolic stabilities of the promising compound 35 were determined with various species of liver micro- somes, such as human, mouse, rat, dog and monkey. And the compound CB-5083 was selected as the standard. The half-life (T1/2) and intrinsic clearance (CLint) parameters were used to evaluate their metabolic stabilities. It could give a good indication of the in vivo hepatic clear- ance when the overall clearance mechanism was metabolic and when potency. Conversely, vinyl group at the R2 position (28, 58.3 nM) sig- nificantly increased the activity. Furthermore, compared with com- pounds 26–28, larger sterically hindered groups (29–31) at R2 position led to the decreased activities, which indicated that smaller groups at the R2 position were more favorable for the improvement of activity. Since the introduction of vinyl substituent to compound 28 showed good activity, we changed the benzylamine moiety at the R1 position into pyridyl (32), cyclopropyl (33) and naphthyl (34) groups. However, the biological results indicated that the generated compounds were inactive. Subsequently, we replaced the acrylamide of compound 28 both compounds 35 and CB-5083 displayed good metabolic stabilities in human, mouse and dog species. While for the rat and money liver microsomes, the two compounds were metabolized too rapidly. The half-life of compound 35 in human liver microsome was 42.3 min, which was more stable than the control CB-5083 (25.8 min).
Pharmacokinetic. With these encouraging in vitro data, candidate 35 was further investigated by profiling ig and iv PK in male Sprague- Dawley (SD) rats. The results were illustrated in Table 6. It indicated that the elimination phase half-lives of compound 35 were 4.57 h for ig and 3.64 h for iv, respectively. The oral bioavailability of compound 35 was only 4.2%, which was not suitable for developing an oral drug. However, the data of Cmax, T1/2 and AUC reflected that compound 35 could be a good candidate for iv injection. Nowadays, the in vivo ef- ficacy of this compound was being carried out to evaluate the drug- availability.

3. Conclusion

A novel series of p97 inhibitors were designed and synthesized. The structure activity relationship (SAR) was discussed in detail and the results demonstrated that the benzylamine linked to the pyrimidine structure and the pyran ring were necessary for maintaining good ac- tivities. Besides, hydrophilic and smaller groups in the indole structure showed better cellular activities. From the optimized results, compound 35 was screened and showed nanomolar level in the inhibition of p97 activity. Further cellular assay indicated that compound 35 could in- hibit the proliferation of non-small cell lung cancer lines A549 with the IC50 value of 1.61 μM. Candidate 35 exhibited good liver microsomal stabilities in mice, dog and human. For human liver microsome, the half-life of compound 35 was 42.3 min, which was more stable than the control CB-5083 (25.8 min). The in vivo pharmacokinetic results showed that the elimination phase half-lives of compound 35 were 4.57 h for ig and 3.64 h for iv, respectively. However, the oral bioa-vailability was only 4.5%, which needs to be improved in the next work. These results showed that compound 35 might be an effective candidate for intravenous treatment of non-small cell lung cancer. The in vivo efficacy of this compound was being performed to evaluate the drug-availability.

4. Experimental section

4.1. General methods

Unless otherwise indicated, chemicals, solvents and reagents were purchased from commercial suppliers and they were used without any purification. Absolutely anhydrous solvents (CH2Cl2, THF, DMF, etc.) were purchased from Energy packaged under nitrogen in Sure/Seal bottles. All reactions involving air or moisture-sensitive reagents were performed under an argon atmosphere. All reactions were detected by thin layer chromatography on silica gel 60 plate coated with 0.25 mm layer and spotted with UV light or iodine. All final products were purified to > 95% purity. The purity of the final products was determined by HPLC (Thermo) on an Agilent Poroshell 120 EC-C18 column (50 mm × 4.6 mm, 2.7 μm) with 0.1% FA/ACN (gradient eluted pro- gram: 0–5 min 90/10–5/95 v/v; 5–11.9 min 5/95 v/ v; 11.9–12.1 min 5/95–90/10 v/v; 12.1–15 min 90/10 v/ v) at 0.3 mL/min flow rate and 254 nm detector wavelength. 1H and 13C spectra were acquired in CDCl3, DMSO‑d6 or CD3OD at room temperature on a Bruker Avance 400 spectrometer with chemical shift (δ, ppm) reported relative to TMS as an internal standard. High-resolution mass spectra (HRMS) were recorded on a ZAB-HS instrument using an electrospray source (ESI). To a 0 °C tetrahydro-4H- pyran-4-one (1c) (10 g, 100 mmol) in THF (300 mL) was added NaH (6 g, 250 mmol). The miXture was stirred for 30 min, and dimethyl carbonate (23 g, 250 mmol) was then added and stirred for an additional 30 min. The resulting miXture was then stirred at 45 °C for 12 h and then poured into a 0 °C HCl solution (0.4 N, 300 mL). The aqueous phase was separated and extracted with ethyl acetate (100 mL × 3); the combined organic layers were washed with evaporated in vacuo. The residue was purified by column chromatography (petroleum ether/EtOAc = 25:1) to give methyl-4-oXote- methyl 4-amino-5,6-dihydro-2H-pyran-3-carboXylate 3c was then distrahydro-2H-pyran-3- carboXylate (2c) (5.5 g, yield 35%, purity 98%) as a colorless liquid. MS (ESI) m/z 159.1 [M+H]+. 2c (5.5 g, 34.97 mmol) and ammonium acetate (8.1 g, 104.9 mmol) in methanol (100 mL) were stirred at room temperature overnight. The solved in acetonitrile (50 mL), and 2,2,2-trichloroacetyl isocyanate (7.2 g, 38.46 mmol) was added. The resulting miXture was stirred for 30 min, and the precipitated solids were collected and dissolved in a solution of ammonia in methanol (10 mL, 7 N). Then the resulting miXture was heated at 70 °C for 1 h. The reaction was cooled down and the precipitated solids were collected and dried to afford the diol (4c) (3.8 g, yield 65%, purity 97%) as a white solid. MS (ESI) m/z 169.2 [M
+H]+.A solution of 4c (3.8 g, 22.7 mmol) in POCl3 (20 mL) was refluXed and stirred for 3 h. After being cooled to room temperature, the miXture was concentrated in vacuo. The residue was diluted with water (100 mL) and extracted with DCM (50 mL × 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromato- graphy (petroleum ether/EtOAc = 25:1) to give intermediate 2,4-di- chloro-7,8-dihydro-5H-pyrano[4,3-d]pyrimidine (1.4 g, yield 32%, purity 97%). MS (ESI) m/z 203.4 [M+H]+. (9) (2.6 g, yield 90%, purity 98%) as a yellow solid. MS (ESI) m/z 157.3 [M+H]+; 1H NMR (400 MHz, DMSO‑d6) δ 7.61 (m, 1H, Ph), 7.43 (m, 1H, Ph), 7.13 (m, 1H, Ph), 6.30 (m, 1H, 3-H of indole), 2.44 (s, 3H, CH3).

4.2.3. N-(2-methyl-1H-indol-4-yl)acetamide (13a)

At room temperature, to a solution of 3-nitroaniline (1 g, 7.25 mmol) in DMSO (20 mL) was added acetone (0.8 g, 14.5 mmol). The miXture was stirred for 5 min, and t-BuOK (1.2 g, 10.87 mmol) was then added. The reaction miXture was stirred at room temperature for 24 h and then was added water (50 mL) and the pH was adjusted to 4 and extracted with DCM (50 mL × 3); the combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na SO ,
To a solution of 2,4-dichloro-7,8-dihydro-5H-pyrano[4,3-d]pyr- 2 4 imidine (1.4 g, 6.83 mmol) in acetonitrile (50 mL) was added phe- nylmethanamine (2.2 g, 20.5 mmol) and triethylamine (2.1 g, 20.5 mmol). The resulting solution was then stirred at room tempera- ture for 12 h and concentrated in vacuo. The residue was purified by column chromatography (petroleum ether/EtOAc = 5:1) to afford compound 5c (1.6 g, yield 86%, purity 98%). MS (ESI) m/z 276.6 [M + H]+; 1H NMR (400 MHz, DMSO‑d6) δ 7.39–7.18 (m, 5H, Ph), 4.55 (d, J = 5.9 Hz, 2H, OCH2), 4.44 (s, 2H, CH2Ph), 3.88 (t, J = 5.6 Hz, 2H, OCH2CH2), 2.62 (t, J = 5.0 Hz, 2H, OCH2CH2).

4.2.2. 2-methyl-1H-indole-4-carbonitrile (9).

To a solution of 1H-indole-4-carbonitrile (6) (5 g, 35.17 mmol) in THF (100 mL) was added NaH (1.3 g, 52.75 mmol) at 0 °C. The miXture was stirred for 5 min and benzenesulfonyl chloride (6.5 g, 42.21 mmol) was then added. The reaction was allowed to room temperature and stirred for an additional 30 min and then poured into a precooled satu- rated aqueous NH4Cl solution (300 mL). The aqueous phase was sepa- rated and extracted with ethyl acetate (100 mL × 3); the combined or- ganic layers were washed with water (100 mL) and brine (100 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was re- crystallized (EtOAc) to give the 1-(phenylsulfonyl)-1H-indole-4-carboni- trile (7) (7.4 g, yield 75%, purity 98%). MS (ESI) m/z 283.1 [M+H]+.
At −40 °C, to the solution of 7 (7.4 g, 26.38 mmol) in THF (100 mL) was slowly added LDA (2.0 M in THF, 26.4 mL, 52.8 mmol). The miX- ture was stirred for an additional 1 h and then MeI (7.5 g, 52.76 mmol) was added. The resulting miXture was then allowed to warm to room temperature and stirred for an extra 12 h. The miXture was poured into a precooled saturated aqueous NH4Cl solution (200 mL) and extracted with ethyl acetate (100 mL × 3); the combined organic layers were washed with water (100 mL) and brine (100 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by column chromatography (petroleum ether/EtOAc = 10:1) to give 2-methyl-4- nitro-1H-indole (11) (0.8 g, yield 62%, purity 98%) as yellow solid. MS (ESI) m/z 175.4 [M−H]−.
At room temperature to a solution of 11 (0.8 g, 4.48 mmol) in DCM (30 mL) were added (Boc)2O (1.5 g, 6.72 mmol) and DMAP (109 mg,
0.89 mmol). Then the miXture was stirred for 3 h. The reaction was washed with water (50 mL) and brine (50 mL). The separated organic layer was concentrated in vacuo to give the crude tert-butyl 2-methyl-4- nitro-1H-indole-1-carboXylate (1.1 g, yield 91%, purity 95%) as yellow solid, which was used in the next step without further purification. MS (ESI) m/z 275.3 [M−H]−, 1H NMR (400 MHz, DMSO‑d6) δ 8.47 (d, J = 8.3 Hz, 1H, Ph), 8.14 (dd, J1 = 0.8 Hz, J2 = 8.2 Hz, 1H, Ph), 7.44 (t, J = 8.2 Hz, 1H, Ph), 7.09 (s, 1H, 3-H of indole), 2.64 (s, 3H, CH3), 1.65 (s, 9H, CH3).
At room temperature, to a solution of tert-butyl 2-methyl-4-nitro- 1H-indole-1-carboXylate (1.1 g, 4.05 mmol) in ethanol (40 mL) was added Fe (1.1 g, 20.26 mmol) and saturated ammonium chloride solu- tion (8 mL). Then the miXture was stirred at 60 °C for 3 h. The resulting solution was filtered and concentrated in vacuo and then the resulting solid was diluted with water (50 mL) and extracted with DCM (50 mL × 3); the combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by column chromatography (pet- roleum ether/EtOAc = 10:1) to give tert-butyl 4-amino-2-methyl-1H- indole-1-carboXylate (12) as yellow solid (0.8 g, yield 78%, purity 98%). MS (ESI) m/z 247.2 [M+H]+.
At room temperature, to a solution of 12 (0.8 g, 3.17 mmol) in DCM (30 mL) were added acetyl chloride (298 mg, 3.8 mmol) and Et3N (0.96 g, 9.51 mmol). Then the miXture was stirred for 3 h. The reaction solution was concentrated in vacuo to give the crude tert-butyl 4- filtered and evaporated in vacuo. The residue was purified by column acetamido-2-methyl-1H-indole-1-carboXylate (813 mg, yield 89%, chromatography (petroleum ether/EtOAc = 20:1) to give 2-methyl-1- (phenylsulfonyl)-1H-indole-4-carbonitrile (8) (5.4 g, yield 69%, purity 98%) as a white solid. MS (ESI) m/z 297.4 [M+H]+. purity 95%), which was used in the next step without further pur- ification. MS (ESI) m/z 289.5 [M+H]+.

4.3. Biological assay

4.3.1. Kinase inhibition assay

The ATPase assay was performed according to the following pro- tocol: compounds were diluted in DMSO with a 3-fold 10-point serial dilution starting at 10 μM. The tenth concentration point was solvent control group (no drug). The assay was done in a 384-well plate with each row as a single dilution series with duplicate of each compound concentration point. In 4 μL total volume, 60 μg/mL p97 hexameric enzyme and 100 μM ATP were added to start the reaction. The plate was sealed and incubated at 25 °C for 60 min after miXing thoroughly in an orbital shaker. ADP Glo reagents 1 and 2 were added according to the manufacturer’s protocol (Promega, Madison, WI). The luminescence was measured by CLARIO Star Plate Reader as the end point of the reaction. The program Graphpad Prism 6 was used to fit nonlinear curve and calculate IC50 of each compounds.31

4.3.2. Cell culture and inhibition of cell proliferation

A549 cell lines were cultured according to ATCC guidelines. Cells were plated (2000 cells/well) in a volume of 90 μL/well of complete media in 96-well cell culture plates and cultured at 37 °C with 5% CO2 for 24 h. Inhibitors were dissolved in DMSO (less than 0.1%) and tested in duplicate utilizing 3-fold serial dilutions with the highest con- centration at 10 μM. Inhibitors were incubated with cells at 37 °C with 5% CO2. After 72 h treatment, Cell Counting Kit-8 (CCK-8) was added to the plates to measure cell viabilities. Absorbance at 450 nm was measured and using GraphPad software to fit sigmoidal curve to determine IC50 value.

4.3.3. In vitro liver microsome stabilities

The liver microsomal incubations consisted of PBS (pH 7.4) con- taining 1 μM compounds 35 and CB-5083, 40 mM NADP, 80 mM G6P, 100 U/mL G6PDH, 120 mM MgCl2 and 0.5 mg/mL liver microsomes (mouse, rat, dog, monkey and human, respectively). The reaction miXtures were pre-incubated for 3 min at 37 °C before the addition of corresponding liver microsomes, then terminated at 0, 5, 10, 20, 30, 60 min by adding equal volume of ice-cold acetonitrile. The final con- centration of organic solvents was < 0.1% in all incubations. The samples were centrifuged at 4000 rpm for 20 min at 4 °C and then analyzed by liquid chromatography-mass spectrometry (ABSciex API4000). 4.3.4. In vivo pharmacokinetics Male Sprague-Dawley rats (200 g) were administrated with the test compounds intravenously (iv) at 5 mg/kg or orally (ig) at 30 mg/kg. Compound 35 was dissolved in miXture of 40% PEG400, 10% Castor oil, 5% EtOH and 45% buffered saline for tail-vein or oral administra- tion. Blood samples (0.1 mL) were then obtained via orbital sinus puncture at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h time points and collected into heparinized tubes. Heparinized blood samples collected for PK analyses were centrifuged at 4000 rpm for 10 min at 4 °C. LC/MS/MS analysis of compound 35 was performed under opti- mized conditions to obtain the best sensitivity and selectivity of the analyte in selected reaction monitoring mode (SRM). Plasma con- centration-time data were analyzed by a non-compartment model using the software Kinetica 5.1. References 1. Meyer H, Bug M, Bremer S. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat Cell Biol. 2012;14(2):117–123. 2. Chou TF, Deshaies RJ. Development of p97 AAA ATPase inhibitors. Autophagy. 2014;7(9):1091–1092. 3. Dargemont C, Ossareh-Nazari B. Cdc48/p97, a key actor in the interplay between autophagy and ubiquitin/proteasome catabolic pathways. Biochim Biophys Acta. 2012;1823(1):138–144. 4. Orme CM, Bogan JS. The ubiquitin regulatory X (UBX) domain-containing protein TUG regulates the p97 ATPase and resides at the endoplasmic reticulum-golgi in- termediate compartment. J Biol Chem. 2012;287(9):6679–6692. 5. Brunger AT, DeLaBarre B, Davies JM, et al. X-ray structure determination at low resolution. Acta Crystallogr D Biol Crystallogr. 2009;65(Pt 2):128–133. 6. Dreveny I, Kondo H, Uchiyama K, et al. Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47. EMBO J. 2004;23(5):1030–1039. 7. Huyton T, Pye VE, Briggs LC, et al. The crystal structure of murine p97/VCP at 3.6 Å. J Struct Biol. 2003;144(3):337–348. 2004;146(1–2):44–57. 8. Peters JM, Harris JR, Lustig A, et al. Ubiquitous soluble Mg2+-ATPase complex: a structural study. J Mol Biol. 1992;223(2):557–571. 9. Rouiller I, Butel VM, Latterich M, et al. A major conformational change in p97 AAA ATPase upon ATP binding. Mol Cell. 2000;6(6):1485–1490. 10. Wang Q, Song C, Li CC. Molecular perspectives on p97-VCP: progress in under- standing its structure and diverse biological functions. J Struct Biol. 11. Raasi S, Wolf DH. Ubiquitin receptors and ERAD: a network of pathways to the proteasome. Semin Cell Dev Biol. 2007;18(6):780–791. 12. Wolf DH, Stolz A. The Cdc48 machine in endoplasmic reticulum associated protein degradation. Biochim Biophys Acta. 2012;1823(1):117–124. 13. Zhu WW, Kang L, Gao YP, et al. EXpression level of valosin containing protein is associated with prognosis of primary orbital MALT lymphoma. Asian Pac J Cancer Prev. 2013;14(11):6439–6443. 14. Tsujimoto Y, Tomita Y, Hoshida Y, et al. Elevated expression of valosin-containing protein (p97) is associated with poor prognosis of prostate cancer. Clin Cancer Res. 2004;10(9):3007–3012. 15. Yamamoto S, Tomita Y, Hoshida Y, et al. EXpression CB-5339 level of valosin-containing protein (p97) is correlated with progression and prognosis of non-small-cell lung carcinoma. Ann Surg Oncol. 2004;11(7):697–704.
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