Luminespib

Discovery of novel heat shock protein (Hsp90) inhibitors based on luminespib with potent antitumor activity

Juyoung Junga,c, Jinsun Kwonb, Soojung Honga, An-Na Moona, Jinah Jeonga, Sungwook Kwona,
Jeong-ah Kima, Myoungjae Leea, Hongsub Leea, Jin Hee Leea, Jeewoo Leec,⁎
a Research Laboratories, Ildong Pharmaceutical Co., Hwaseong-si, Gyeonggi-do 18449, South Korea
b AIMS BioScience Co., 2, Baumoe-ro 27-gil, Seocho-gu, Seoul 06752, South Korea
c Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul 08826, South Korea

Abstract

A series of isosteric surrogates of the 4-phenyl group in luminespib were investigated as new scaffolds of the Hsp90 inhibitor for the discovery of novel antitumor agents. Among the synthesized surrogates of isoXazole and pyrazole, compounds 4a, 5e and 12b exhibited potent Hsp90 inhibition in ATPase activity and Her2 degradation assays and significant antitumor activity in A2780 and HCT116 cell lines. Animal studies indicated that com- pared to luminespib, their activities were superior in A2780 or NCI-H1975 tumor Xenograft models. A molecular modeling study demonstrated that compound 4a could fit nicely into the N-terminal ATP binding pocket.

An ATP-dependent molecular chaperone, heat shock protein 90 (Hsp90), regulates the stability and activity of client proteins that play critical roles in proliferation, survival, transformation and apoptosis.1–4 A significant number of these client proteins, including protein kinases such as HER2, C-RAF, B-RAF, and AKT, have been implicated in on- cogenesis. Simultaneous degradation of multiple oncogenic client pro- teins is caused by inhibition of Hsp90 in the ubiquitin-proteasome pathway.5–8 Due to its the selectivity in inhibiting the growth of cancer cells over nontransformed cells, Hsp90 inhibition has emerged as a promising mode of action for anticancer chemotherapy.9–10 Geldanamycin (GD) is a natural 1,4-benzoquinone ansamycin that inhibits Hsp90 by binding to its N-terminal ATP-binding pocket11 and has provided an important opportunity for understanding the role of Hsp90 in tumor growth.12–14 Tanespimycin (17-AAG), a derivative of geldanamycin, as a single or combination drug in clinical trials, has shown broad activity and reduces drug resistance. However, 17-AAG has limited applications due to its poor solubility, bioavailability, hepatotoXicity and extensive metabolism by polymorphic enzymes.

In our program to discover Hsp90 inhibitors as novel antitumor agents, we investigated novel scaffolds based on luminespib in which the phenyl group was substituted with isoXazole or 2,3- or 3,4-pyrazole moieties (Fig. 1). The synthesized compounds were tested for Hsp90 inhibition by measuring their ATPase activity and ability to degrade a client protein and were evaluated for their in vitro cytotoXicity against cancer cell lines. The selected compounds were further evaluated for their antitumor activities in in vivo tumor Xenograft models. The mo- lecular modeling of representative inhibitors was performed to identify the binding mode in the N-terminal ATP binding pocket.

The synthesis of R1-CH2 substituted isoXazole derivatives (4a–x) is described in Scheme 1. The known iodide key intermediate 1 was prepared18 and coupled with ethyl 5-(tributylstannyl)isoXazole-3-carboXylate21 in the presence of tetrakis(triphenylphosphine)palladium(0) to provide the isoXazole compound 2a. Reduction of carboXylate 2a afforded the alcohol intermediate 3a, which was subsequently con- verted into the mesylate intermediate 3e. Alkylation of the alcohol intermediate 3a with methylbromoacetate afforded the ethylcarbamoylether compound 3b. Treatment of the ester 3b with 2 N LiOH or 7 N NH3 provided the acid 3c or amide 3d, respectively. Next, fluoro or cyano substitution reactions from the mesylate intermediate 3e readily afforded the fluoromethyl 3f and cyanomethyl 3g isoXazoles. In addi- tion, aminomethyl 3i was obtained by thermal treatment of the po- tassium phthalimide. The others 3h–x were obtained from the mesylate intermediate 3e by a similar substitution reaction. Finally, removal of the dibenzyl protecting group in 3a–x using boron trichloride provided the final compounds 4a–x.

Therefore, novel small molecule Hsp90 inhibitors have been studied to overcome this issue. Among them, resorcinol scaffolds have emerged as a new class of Hsp90 inhibitor. This class of compounds, such as lumine- spib (NVP-AUY922) and ganetespib (STA-9090), exhibit potent and se- lective Hsp90 inhibition and display significant oral activities in cellular and animal models of cancer.18,19 In addition, purine scaffold compounds (e.g., PU-H71, BIIB021) have also been reported as Hsp90 inhibitors.20

Fig. 1. Design of new scaffold Hsp90 inhibitors based on luminespib.

The synthesis of R2-CO substituted isoXazole derivatives 5a–g is il- lustrated in Scheme 2. Acid hydrolysis of the ethylcarboXylate 2a furnished the acid 2b. Amination of the ester 2a with commercially available various amines afforded the requisite amide derivatives 2c–g. Final products 5a–g were obtained after the deprotection of the di- benzyl group.

The synthesis of 1,3-disubstituted pyrazole derivatives is shown in Scheme 3. The iodide intermediate 1 was converted into the alkyne 6 by ethnyltributyltin via the Stille reaction and as then converted into the acetyl 7. The acetyl 7 was condensed with dimethylformamide di- methylacetal to give the dimethylaminoacryloyl 8, which was quanti- tatively cyclized into the unsubstituted C3-linked pyrazole 9a using hydrazine hydrate. Alkyl substituted pyrazoles (9b–e) were obtained by using appropriate alkylation reagents, followed by the deprotection of benzyl groups to produce the desired product 10a–e.

The synthesis of 1,4-disubstituted pyrazole derivatives is presented in Scheme 4. Stille coupling of the iodide 1 with trityl pyrazole tribu- tylstannane using Pd(0) afforded the protected C4-linked pyrazole 11a almost quantitatively, which was deprotected to furnish C4-linked 11b. The 1-methyl, 1-ethyl and 1-isopropyl substituted pyrazoles 11c–e were also obtained from the iodide 1 by direct coupling with the corre- sponding substituted pyrazoles. The final deprotection readily provided the C4-linked pyrazoles 12b–e.

All the synthesized compounds were tested for Hsp90 inhibition by the measurement of the ATPase activity of Hsp90 as well as by the cell- based Her2 degradation assay. For antitumor activity, their cytotoXi- cities were evaluated against the A2780 (human ovarian cancer) and HCT116 (human colon cancer) cell lines. The results are summarized in Tables 1–3, together with the values of luminespib (NVP) for compar-

Scheme 1. Synthesis of isoxazole derivatives. Reagents and conditions: (a) ethyl 5-(tributylstannyl)isoXazole-3-carboXylate, Pd(PPh3)4, toluene, refluX, 2.5 h, 78%; (b) LAH, THF, 0 °C to r.t., 2 h, 61%; (c) BrCH2CO2Me, Cs2CO3, CH3CN, r.t., 24 h, 61%; (d) 2 N LiOH, THF/water, 0 °C, 1 h, 63%; (e) 7 N NH3/ MeOH, KCN, 50 °C, 12 h, 89%; (f) MsCl, Et3N, 0 °C, 3 h, 61%; (g) KF, 18-crown- 6, r.t., 18 h, 78%; (h) KCN, 18-crown-6, r.t., 12 h, 98%; (i) phthalimide po- tassium salt, CH3CN, refluX, 24 h, 95% and then MeNH2, EtOH, refluX, 6.5 h, 99%; (j)-(y) R1NH2 or R1NH, CH2Cl2, 8 h, 60–99%; (z) BCl3, CH2Cl2, 0 °C, 0.5 h, 45–99%.

1 Next, we explored the isoXazole surrogates (5a–g) with a COR2

First, we investigated the isoXazole surrogates bearing a CH2R group (4a–x) at the C3 position (Table 1). The hydroXylmethyl iso- Xazole analogue 4a and the O-alkyl substituted analogues 4b–d, in which R1 is OCH2CO2Me, OCH2CO2H, and OCH2CONH2, respectively, were examined. Among them, 4a exhibited potent inhibitory activity toward Hsp90 and cancer cells, which was slightly better than that of NVP. The substitution of hydroXyl in 4a with OMs, fluoro and cyano groups did not improve the activity. Mono- or di-substituted amino- methyl isoXazole analogues 4h–s were also explored. Although some of the amine analogues 4j, 4l, 4m, 4q, and 4r demonstrated promising potency, none of compounds were found to be better than NVP for inhibition of Hsp90 and tumors. Cyclicamino, such as morpholinyl, piperidinyl, pyrrolidinyl and N-methylpiperazinyl, analogues 4t-x were also examined. However, this modification resulted in diminished group at the C3 position (Table 2). Whereas the ethyl ester 5a and carboXylic acid 5b showed weak activities, most amide analogues dis- played reasonable inhibition toward Hsp90 and cancer cells. In parti- cular, the ethylamide 5e showed significantly improved activity, in which its Hsp90 inhibition and cytotoXicity in HCT116 were more po- tent than those of NVP.

Finally, we examined N-alkyl pyrazole surrogates in which the two isomers, the 1,3-pyrazole (10a-d) and 1,4-pyrazole (12b-e) analogues, were tested. Compared to NVP, most of the 1,3-pyrazole analogues have similar potencies for Hsp90 inhibition and antitumor activity. The SAR in cell-based assays clearly showed a preference for an alkyl substitu- tion on the pyrazole moiety. The potency was enhanced when alkyl groups, such as methyl, ethyl and isopropyl, were introduced (10b–c,10e). However, the addition of an n-propyl group caused reduced ac- tivity. Compared to NVP and the corresponding 1,3-pyrazoles, the 1,4- pyrazole analogues showed similar potencies. Unsubstituted pyrazole 12b proved to be a potent Hsp90 inhibitor.

Scheme 2. Synthesis of isoxazole-3-carboxylate derivatives. Reagents and conditions: (a) ethyl 5-(tributylstannyl)isoXazole-3-carboXylate, Pd(PPh3)4, to- luene, refluX, 2.5 h, 78%; (b) 2 N LiOH, THF/water, 0 °C, 1 h, 93%; (c) 7 N NH3/ MeOH, KCN, 50 °C, 12 h, 91%; (d) aq. 40% MeNH2, EtOH, refluX, 48 h, 90%; (e) 2 M EtNH2, EtOH, refluX, 1 h, 86%; (f) morpholine, EtOH, refluX, 24 h, 87%; (g) thiomorpholine, EtOH, refluX, 48 h, 67%; (h) BCl3, CH2Cl2, 0 °C, 0.5 h, 56–99%.

Scheme 3. Synthesis of 3-substituted pyrazole derivatives. Reagents and conditions: (a) ethynyltributyltin, Pd(PPh3)4, toluene, refluX, 2 h, 75%; (b) HCOOH, NaHCO3, 95 °C, 1 h, 45%; (c) N,N-dimethylformamide dimethylacetal, EtOH, refluX, 4 h, 76%; (d) NH2NH2, EtOH, r.t., 48 h, 99%; (e)-(h) R3I, K2CO3, CH3CN, 1 h, 78–87%; (i) BCl3, CH2Cl2, 0 °C, 0.5 h, 40–99%.

Scheme 4. Synthesis of 4-substituted pyrazole derivatives. Reagents and conditions: (a) 4-(tributylstannyl)-1-R4-1H-pyrazole, Pd(PPh3)4, toluene, refluX, 12 h, 60–99%; (b) CF3CO2H, CH2Cl2/MeOH, 75 °C, 3 h, 99%; (c) BCl3, CH2Cl2, 0 °C, 0.5 h, 34–87%.

The eight compounds selected from the in vitro study were evaluated for in vivo antitumor activity in a mouse A2780 tumor Xenograft model.
a BALB/c-nu/nude mice (n = 6) transplanted with tumor cells were treated with resorcinol compounds. b Tumor growth inhibition: [1-(mean volume of treated tumors)/(mean volume of control tumors)] × 100%.

For the tumor model, A2780 human ovarian adenocarcinoma cells were cultured and implanted into nude mice, and 5 or 6 days later, tumor size and volume were measured. The compounds were suspended in 1% crystal ligand (green) in the ATP-binding site of Hsp90. Hydrogen bonds are shown as yellow dashed lines. (B) Ligand interaction diagram. Hydrogen bonds are depicted by magenta lines, and the corresponding π-cation interaction is depicted by the red line.

carboXymethyl cellulose (CMC) and were administered intravenously (iv) at a dose of 50 mg/kg three times weekly for two or three weeks. Luminespib (NVP) was used as a reference for comparison. As shown in Table 4, all the tested compounds except 4r sig- nificantly inhibited tumor growth with a range of TGI = 40.3–53.0% without any death or body weight loss. In particular, compounds 4m, 5e and 10e showed higher inhibitions compared to that of NVP.

A further in vivo study was conducted in another tumor model (See Table 5). The three compounds (4a, 5e, 12b) were administered i.v. at 200 mg/kg once weekly for three weeks to athymic mice bearing NCI-H1975 human lung carcinoma xenografts. While compound 5e ex- hibited comparable activity to NVP, compounds 4a and 12b displayed higher antitumor growth inhibition than NVP without any death. This dose schedule was well tolerated, and body weight loss was not observed.

To understand the binding mode of 4a, we performed a docking study of 4a with an Hsp90 X-ray crystal structure (2VCI.pdb) using Glide SP.22 As shown in Figure 2, the binding mode of 4a was similar to that of luminespib (green), which binds to the Hsp90 ATP binding site. The resorcinol moiety of 4a was located deep in the pocket and had key hydrogen bonding interactions with Asn51, Asp93 and water mole- cules. Furthermore, the amide group of 4a formed hydrogen bonds with Gly97 and Lys58. The isoXazole ring made a π-cation interaction with Lys58, and its hydroXymethyl group showed hydrogen bonding with Asp54, features that are not shown with luminespib.

In summary, the molecular chaperone Hsp90 regulates the stability and activity of oncogenic client proteins that play critical roles in tumor proliferation, survival, and transformation, demonstrating that Hsp90 inhibition is a promising strategy for anticancer chemotherapy. As part of our research program to discover novel Hsp90 inhibitors, we in- vestigated isosteric surrogates of the 4-phenyl group appended to the isoXazole in luminespib in which the 4-substituted phenyl group was modified with 3,5-substituted isoXazole and 1,3 (or 1,4)-substituted pyrazole groups as new scaffolds. Among them, compounds 4a, 5e and 12b exhibited not only potent Hsp90 inhibition in ATPase activity and Her2 degradation assays but also significant antitumor activity in the A2780 and HCT116 cell lines. Animal studies indicated that compounds 4a, 5e and 12b exhibited slightly better or comparable activity than luminespib in A2780 or NCI-H1975 tumor Xenograft models. The re- sults indicated that isoXazole and pyrazole surrogates of luminespib potentially possess promising features as drug candidates for anticancer therapy. Further studies in animals will be reported in due course.

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

This work was supported by a grant (HA17C0053, 1720340) from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea.

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