Synthesis and identification of GZD856 as an orally bioavailable Bcr-AblT315I inhibitor overcoming acquired imatinib resistance

Abstract Bcr-AblT315I induced drug resistance remains a major challenge to chronic myelogenous leukemia (CML) treatment. Herein, we reported GZD856 as a novel orally bioavailable Bcr-AblT315I inhibitor, which strongly suppressed the kinase activities of both native Bcr-Abl and the T315I mutant with IC50 values of 19.9 and 15.4 nM, and potently inhibited proliferation of corresponding K562, Ba/F3WT and Ba/F3T315I cells with IC50 values of 2.2, 0.64 and 10.8 nM. Furthermore, GZD856 potently suppressed tumor growth in mouse bearing xenograft K562 and Ba/F3 cells expressing Bcr-AblT315I. Thus, GZD856 may serve as a promising lead for the development of Bcr-Abl inhibitors overcoming acquired imatinib resistance.


Introduction
Chronic myelogenous leukemia (CML) is a hematological stem cell disorder caused by expansion of lymphocytic or myeloid blasts in the blood or bone marrow, and resulting from a t (9; 22) reciprocal translocation encoding the constitutively active Bcr-Abl tyrosine kinase 1 . Bcr-Abl, a 210-kDa non-receptor protein kinase that catalyzes the transfer of c-phosphoryl group from ATP to the hydroxyl group of specific tyrosine residues in proteins, is a validated target for development of small molecule inhibitors to treat CML 2 . The first generation Bcr-Abl inhibitor imatinib (1, STI1571) has achieved significant clinical benefit and became the first-line drug for conventional treatment of CML (Figure 1(A)) [3][4][5] . However, many patients developed emerging acquired resistance to imatinib with its widespread used in clinic 6 . The primary mechanism of acquired imatinib resistance is the occurrence of point mutations in the Abl kinase domain, which effects the binding of imatinib in the ATP-binding site 7 . To date, more than 100 different point mutations have been identified in clinic.
To overcome imatinib resistance, several classes of second generation of Bcr-Abl inhibitors have been developed 8 . Nilotinib (2) 9,10 , dasatinib (3) 11,12 and bosutinib (4) 13 have been approved for the treatment of adults in all phases of CML with resistance to imatinib ( Figure 1(A)). However, these second-generation inhibitors are not capable of inhibiting all the kinase mutants identified in patients, especially the most notably Bcr-Abl T315I gatekeeper mutant that represents 15-20% of all clinical acquired resistances 14 . Thus, CML containing the T315I mutation remains a serious medical problem in clinic.
To address this unmet need, considerable efforts have been made to develop the third generation Bcr-Abl inhibitors which can override the T315I mutation [15][16][17][18] , especially for AP24534 (5) 19,20 , GNF-7 21 and GZD824 22 . Among them, only AP24534 (ponatinib, Iclusig V R ) has been approved for the treatment of resistant or intolerant CML and Ph þ ALL patients against imatinib, especially those harboring Bcr-Abl T315I mutation 19,20 . However, FDA suspended the sale of ponatinib due to the increasing numbers of blood clots observed in ponatinib-treated patients after 10 months 23 . Ponatinib was then reauthorized for sale a black-box warning and revised indication statement for concerns over risks of its usage 24 .
As part of our continuous efforts to identify new molecules that could target Bcr-Abl T315I mutant 22,25 , here we report the design, synthesis and biological evaluation of GZD856 (6) 26 as a potent Bcr-Abl T315I inhibitor by substituting imidazo [1,2-b]pyridazine (five-fused-six membered ring) of ponatinib with pyrazole[1,5a]pyrimidine (six-fused-five membered ring) by scaffold hopping strategies (Figure 1(B)), which also keep the hydrogen bond with the residue in kinase hinge region. GZD856 strongly suppressed the kinase activities of both native Bcr-Abl and the T315I mutant, and potently inhibited proliferation of corresponding K562, Ba/ F3 WT and Ba/F3 T315I cells with low nM IC 50 s. It also displayed promising in vivo antitumor efficacy in mouse bearing xenograft K562 and Ba/F3 cells expressing Bcr-Abl T315I .

Computational study
The structure of Bcr-Abl T315I protein was retrieved from the Protein Data Bank (PDB code: 3IK3), which was published by O'Hare et al. 20 The protein was processed using protein preparation wizard, which assigns bond orders and adds hydrogen and missing atoms. GZD856 was built by in LigPrep (LigPrep, version 2.5, Schr€ odinger, LLC, New York, NY, 2011) module using OPLS-2005 force field. Molecular docking was performed in Glide module (Glide, version 5.7, Schr€ odinger, LLC, New York, NY, 2011) with standard precision scoring function.
FRET-based Z 0 -lyte assay detecting peptide substrate phosphorylation The effects of GZD856 on the kinase activity of Bcr-Abl and its mutants were assessed in 384-well plates using the FRET-based Z 0 -Lyte assay system according to manufacturer's instructions (Invitrogen, Carlsbad, CA). Briefly, 10 mL per well reactions contained ATP concentration at 10 mM (for Bcr-Abl wildtype) or 5 mM (for T315I mutant), 2 mM Tyr2 peptide substrate in 50 mM HEPES (pH 7.5), 0.01% BRIJ-35, 10 mM MgCl 2 , 1 mM EGTA, 0.0247 mg/mL Bcr-Abl, and inhibitors as appropriate. The reaction was performed at room temperature for 2.0 h, and then 5 mL of development reagent was added for a further 2 h room temperature incubation followed by the addition of 5 mL of stop solution. Fluorescence signal ratio of 445 nm (coumarin)/520 nm (fluorescein) was examined on EnVision Multilabel Reader (Perkin-Elmer, Inc., Waltham, MA). The data were analyzed using Graphpad Prism5 (Graphpad Software, Inc., La Jolla, CA). The data were the mean values of three experiments.

Stably transformed Ba/F3 cells
The Ba/F3 cell lines stably Bcr-Abl WT and Bcr-Abl T315I mutant were self-established by following procedures similar to those described by von Bubnoff 27 . Briefly, wild-type Bcr-Abl p210 was cloned into pcDNA3.1(þ) (Invitrogen, Carlsbad, CA). Point mutations were introduced to pcDNA3.1(þ) Bcr-Abl using the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Ba/F3 cells were transfected with the constructs using Amaxa Cell Line Nucleofector Kit V (Lonza, Cologne, Germany) by electroporation. Stable lines were selected using Transfected Cells Cloning Kit (Stem Cell Technologies, Vancouver, Canada) with G418 (Merck, Whitehouse Station, NJ) and withdrawal of interleukin-3 (IL-3, R&D). Ba/F3 stable cell lines were verified by monitoring both DNA sequences through DNA sequencing and protein expression levels of the corresponding Bcr-Abl mutants through Western blotting analysis. Their responses to the imatinib, nilotinib and dasatinib were also hired for selecting the right clones. Parent Ba/F3 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and IL-3 (10 ng/mL), while all Bcr-Abl-transformed Ba/F3 stable cell lines were cultured in the similar medium except without IL-3. Stably K562R (Q252H) cells Imatinib-resistant K562 cells which expressed Bcr-Abl Q252H were self-established. Briefly, K562 cells were treated with a range of concentrations of imatinib (from 0.1 mM to 5 mM) over a 3 month period. Single clones were then selected and identified through DNA sequencing, and their response to imatinib, nilotinib and dasatinib were monitored as an internal reference.
Cellular antiproliferation assay using cell counting kit (CCK-8) Cells in the logarithmic phase were plated in 96-well culture dishes ($3000 cells/well). Twenty-four hours later, cells were treated with the corresponding compounds or vehicle control at the indicated concentration for 72 h. CCK-8 was added into the 96-well plates (10 mL/well) and incubated with the cells for 3 h. OD450 and OD650 were determined by a microplate reader. Absorbance rate (A) for each well was calculated as OD450-OD650. The cell viability rate for each well was calculated as V% ¼ (As À Ac)/(Ab À Ac) Â 100%, and the data were further analyzed using Graphpad Prism5 (Graphpad Software, Inc., La Jolla, CA) (As, absorbance rate of the test compound well; Ac, absorbance rate of the well without either cell or test compound; Ab, absorbance rate of the well with cell and vehicle control).

Western blot analysis
The Western blot analysis was carried out by following the protocol described previously 22 . Briefly, after the indicated treatment, cell lysates were collected, dissolving cells in 1Â SDS sample lysis buffer. After being sonicated and boiled, the supernatant of the cell lysate was used for Western blot analysis. Cell lysates were loaded to 8-12% SDS-PAGE and separated by electrophoresis. Separated proteins were then electrically transferred to a PVDF film. After being blocked with 1Â TBS containing 0.1% Tween-20, and 5% nonfat milk, the film was incubated with corresponding primary antibody followed by HRP-conjugated secondary antibody. The protein lanes were visualized using ECL Western Blotting Detection Kit (GE Healthcare, Piscataway, NJ).

Animal studies
Male BALB/c or SCID nude mice were purchased from Vital River Laboratory Animal Technology Inc. (Beijing, China). All animal studies were approved by the Institutional Animal Use and Care Committee of Guangzhou Institute of Biomedicine and Health, Chinese Academy of Science.

Mice xenograft models
K562 and Bcr-Abl T315I cells were re-suspended in normal saline (NS) solution (2.5 Â 10 7 and 1 Â 10 7 cell/mL respectively). A 0.2 mL amount of cell suspension was injected subcutaneously into the right flank of each mouse. Mice were randomly grouped based on the tumor volume when the mean tumor volume reached 100-200 mm 3 . GZD856 and imatinib were dissolved in a vehicle containing 1% DMSO, 22.5% Cremophor, 7.5% ethanol and 69% NS. Mice were treated for the 16 consecutive days once daily by oral gavage with GZD856 (10 mg/kg), imatinib (50 mg/kg) and vehicle, respectively. Tumor volume and body weight were monitored once every 2 days. Tumor volume was calculated as the LÂW 2 /2 (L and W are the length and width of the tumor, respectively). Tumor volume data were analyzed with the one-way ANOVA method using software SPSS 17.0 (SPSS Inc., Chicago, IL).

Synthesis of GZD856
Reagents and solvents were obtained from commercial suppliers and used without further purification. Flash chromatography was performed using silica gel (300-400 mesh). All reactions were monitored by TLC, silica gel plates with fluorescence F254 were used and visualized with UV light. 1H and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer at 400 MHz and Bruker AV-500 spectrometer at 125 MHz, respectively (Bruker, Billerica, MA). Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (d) of NMR are reported in parts per million (ppm) units relative to internal control (TMS). The low or high resolution of ESI-MS was recorded on an Agilent 1200 HPLC-MSD mass spectrometer (Santa Clara, CA) or Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer (Foster City, CA), respectively.

Methyl 3-ethynyl-4-methylbenzoate (9)
To a solution of methyl 3-iodo-4-methylbenzoate (7) (2.76 g, 10 mmol) and trimethylsilyl acetylene (0.98 g, 10 mmol) in acetonitrile (20 mL) were added Pd(dppf)Cl 2 (0.07 g, 0.1 mmol), CuI (0.19 g, 1 mmol) and triethylamine (10 mL). The mixture was stirred at 80 C for 2 h under argon atmosphere. The reaction mixture was filtered through a pad of Celite. The filtrate was concentrated, and the resulting residue was solved in CH 3 OH (25 mL) and treated with K 2 CO 3 (9.8 g, 60 mmol) solution, and the mixture was stirred at room temperature for 3 h. The reaction solution was concentrated, and EtOAc and H 2 O were added to the residue. The organic layer was separated, and the aqueous layer was exacted with EtOAc (3 Â 50 mL). The combined layers were dried over Na 2 SO 4 and concentrated. The resulting crude was further purified by flash chromatography to give the product as a white solid (1.56 g, 90.0%). 1

Kinase activities evaluation
The kinase inhibitory activities of GZD856 against Bcr-Abl WT and Bcr-Abl T315I were evaluated by using the well established FRETbased Z'-Lyte assay 22,28 . Imatinib and nilotinib were used as positive controls to validate the screening conditions. Under the screening conditions, imatinib and nolitinib potently inhibited the enzymatic activity of Bcr-Abl WT with IC 50 values of 98.2 and 43.5 nM, which were highly consistent to the reported data 9,29 . As shown in Table 1, the designed GZD856 potently inhibited Bcr-Abl WT and Bcr-Abl T315I with IC 50 values of 19.9 and 15.4 nM, respectively, which displayed similar activity to ponatinib (19), suggested that GZD856 can be a potent Bcr-Abl inhibitor overcoming acquired imatinib resistance. However, imatinib and nilotinib were both significantly less potent for Bcr-Abl T315I mutation.

Molecular docking studies
Molecular docking study was performed to investigate the binding mode of compound GZD856 with Bcr-Abl T315I (PDB code: 3IK3) using Glide module (Glide, version 5.7 Schr€ odinger, LLC, New York, NY) with standard precision scoring function. GZD856 bound to the ATP binding site of the DFG-out conformation of Bcr-Abl T315I (Figure 2), which was similar to that of ponatinib 19,20 . The pyrazolo[1,5-a]pyrimidine of GZD856 formed an essential hydrogen bond with the NH of Met318 in the hinge region of Bcr-Abl T315I . The amide formed two hydrogen bonds with Glu286 and Asp381, and the trifluoromethylphenyl group bound deeply into the hydrophobic pocket. The alkynyl linker made favorable van der Waals interactions with the gatekeeper Ile315 avoiding steric clash.

Cellular antiproliferation assay
The antiproliferative activity of GZD856 was also examined against a panel of leukemia cells with differing Bcr-Abl status (Table 2). Imatinib, nilotinib, ponatinib and taxol were utilized as positive controls to validate the screening conditions. As shown in Table 2, imatinib and nilotinib were both significantly less potent for K562R (Q252H) and Ba/F3 T315I cells. GZD856 strongly suppressed the proliferation of K562, K562R (Q252H) and murine Ba/F3 cells ectopically expressing Bcr-Abl WT and Bcr-Abl T315I , with IC 50 values of 2.2, 67.0, 0.64 and 10.8 nM, respectively, which were equivalent to the potency of ponatinib and much higher than the potency of imatinib and nilotinib ( Table 2). As a confirmation of GZD856's Bcr-Abl-selective effect, GZD856 was significantly less potent against the proliferation of MOLT4 and U937 leukemia cells  (negative for Bcr-Abl expression), which are much selective than ponatinib. Further, GZD856 also displayed less potent antiproliferative activity against non-cancer cells HFL-1 (human embryonic lung fibroblasts) with IC 50 value of 6.78 mM 26 . The studies suggested that GZD856 could be a selective anti-cancer drug. However, the cytotoxic agent taxol inhibited the growth of all tested cells with similar IC 50 values.

Western blot study
To further validate the cellular kinase inhibitory activity of GZD856, we examined its effects on the activation of Bcr-Abl and its downstream signals in Bcr-Abl positive K562 CML cells and stably transformed Ba/F 3 cells expressing Bcr-Abl WT and Bcr-Abl T315I by Western blot analysis ( Figure 3). GZD856 efficiently suppressed the activation of both Bcr-Abl and downstream Crkl and STAT5 in dose-dependent manners in K562 CML cells. Similar inhibition was also observed in the Ba/F3 cells expressing Bcr-Abl WT and Bcr-Abl T315I . Not surprisingly, none of the three FDA approved drugs (imatinib, nilotinib, dasatinib) showed obviously suppression on the activation of Bcr-Abl T315I 22 .

In vivo antitumor studies
Giving its highly promising antiproliferative activity in vitro and pharmacokinetic (PK) properties in vivo in rats 26 , GZD856 may possess good in vivo efficacy when orally administered. Thus, the in vivo antitumor effect of GZD856 was first evaluated in subcutaneous K562 xenograft model of human CML. Imatinib was used as a positive control at dose of 50 mg/kg/day to validate the animal models. The animals were dosed orally with GZD856 at 10 mg/kg/day. As shown in Figure 4, GZD856 almost completely eradicated the tumor at doses of 10 mg/kg/day after 8 days of treatment, whereas imatinib only induced tumor stasis at a dose of 50 mg/kg/day. Notably, after the cessation of treatment (16 days of dosing), there was no sign of tumor recurrence in the following 7 days. Also, at the dose of 10 mg/kg/day, GZD856 was well tolerated with no mortality or significant body loss (<5% relative to vehicle-matched controls) during the treatment.
Similar to that of the K562 xenograft studies, the antitumor efficacy of GZD856 was further evaluated in a xenograft model using Ba/F3 cell expressing Bcr-Abl T315I . The animals were administered GZD856 at doses of 20 and 50 mg/kg via oral gavage once daily for 16 days. Imatinib was again used as a reference compound and administered orally at dose of 100 mg/kg/day for 16 days. As shown in Figure 5, GZD856 dose-dependently inhibited tumor growth in the xenograft model bearing Bcr-Abl T315I . Although GZD856 did not show obvious inhibition of tumor growth at dose of 20 mg/kg/day, it induced almost about 90% tumor regression at a dose of 50 mg/kg/day after a 16 days consecutive treatment. In contrast, 100 mg/kg/day of imatinib failed to exhibit inhibition of tumor growth in the Bcr-Abl T315I model.

Conclusions
In summary, we have successfully discovered GZD856 as a new Bcr-Abl inhibitor which maintains significant inhibition against Bcr-Abl gatekeeper T315I mutant. GZD856 strongly suppressed both native Bcr-Abl and the T315I mutant with IC 50 values of 19.9 and 15.4 nM, and potently inhibited proliferation of corresponding K562 and Ba/F3 T315I cells with IC 50 values of 2.2 and 10.8 nM. Western blot analysis further supported its kinase inhibition    against Bcr-Abl and Bcr-Abl T315I . In vivo efficacy studies in mouse xenograft models of human leukemia demonstrated that GZD856 potently suppresses growth of tumors driven by native Bcr-Abl and Bcr-Abl T315I , which indicated that GZD856 is a promising anticancer lead for further development.