Design, Structure-Activity Relationship, and in Vivo Characterization of the Development Candidate NVP-HSP990

Christopher M. McBride, Barry Levine, Yi Xia, Cornelia Bellamacina, Timothy Machajewski, Zhenhai Gao, Paul Renhowe, William Antonios-McCrea, Paul Barsanti, Kristin Brinner, Abran Costales,
Brandon Doughan, Xiaodong Lin, Alicia Louie, Maureen McKenna, Kris Mendenhall, Daniel Poon, Alice Rico, Michael Wang, Teresa E. Williams, Tinya Abrams, Susan Fong, Thomas Hendrickson, Dachuan Lei, Julie Lin, Daniel Menezes, Nancy Pryer, Pietro Taverna, Yongjin Xu, Yasheen Zhou, and Cynthia M. Shafer*
Global Discovery Chemistry/Oncology & Exploratory Chemistry, Novartis Institutes for Biomedical Research, 5300 Chiron Way, Emeryville, California 94608, United States
S* Supporting Information

ABSTRACT: Utilizing structure-based drug design, a novel dihydropyridopyrimidinone series which exhibited potent Hsp90 inhibition, good pharmacokinetics upon oral administration, and an excellent pharmacokinetic/pharmacodynamic relationship in vivo was developed from a commercial hit. The exploration of this series led to the selection of NVP-HSP990 as a development candidate.

Hsp90 is ubiquitously expressed in eukaryotic cells and plays an important role in maintaining protein homeostasis and protecting cells from harmful effects caused by stress or heat.1 Hsp90 ensures the conformational and functional stability of a large spectrum of cellular client proteins. The intrinsic ATPase activity is crucial for driving the conforma- tional cycles of Hsp90 and the proper folding of its client proteins. Impairment of Hsp90 ATPase activity disrupts the dynamic ATP-coupled chaperone cycle and alters its interaction with client proteins, resulting in client protein destabilization and eventual degradation via the ubiquitin-proteasome pathway.2
The intense interest in Hsp90 as a cancer target stems from the serendipitous discovery in 1994 that the antitumor agent geldanamycin acts as a bona fide ATP-competitive Hsp90 inhibitor which interrupts the formation of an Hsp90-Src kinase complex.3 Hsp90 is frequently overexpressed in tumor cells, and many of its client proteins are oncogenic drivers (e.g., BCR-ABL, ERBB2, and EML4-ALK). These findings provide a strong rationale for targeting Hsp90 for cancer treatment.4 Hsp90 inhibitors allow the simultaneous inhibition of multiple oncoproteins and signaling pathways that are essential in maintaining the malignant phenotype of tumors. This is a particularly attractive feature given the extraordinary ability of tumor cells to rapidly evolve resistance through mutations or activation of alternative signaling pathways to therapeutics targeting a single oncogene.5 The search for Hsp90 inhibitors superior to geldanamycin has led to clinical development of numerous small molecules at various stages of development.6,7 At the outset of our program, we envisioned an orally available

Hsp90 inhibitor which could provide dosing fl exibility compared to inhibitors such as geldanamycin and radicicol which could only be dosed intravenously. Furthermore, geldanamycin and its analogues suffered preclinically from hepatoxicity due to the quinone moiety. Thus, our efforts focused on finding compounds potent against Hsp90 which lacked pharmacophores with toxicity flags.8
1).9 A three-step sequence beginning with the commercially available 2-bromo-4-fl uorobenzaldehyde (2) using chemistry developed by Ellman and incorporating a key Reformatsky reaction provides ester 4 in good yield and diastereomeric purity (de 96:4).10,11 Removal of the sulfi namide group with acid yielded the amino ester 5 in >95% ee. Nucleophilic ring opening of diketene followed by a Dieckmann condensation provided the tricarbonyl 7 in reasonable yields. The final steps involve a condensation with acetylguanidine to form the key aminopyrimdine intermediate 8, followed by a Suzuki-Miyaura coupling with a boronic ester to yield 9. Compounds 15-22 were obtained in a similar fashion by reaction of the advanced intermediate 8 and the appropriate boronic acid or ester, while compounds 10-14 were obtained through the same synthetic route beginning with the commercially available bromoade- hyde.
While previous work had indicated that an aromatic heterocycle attached at the ortho position of ring C was benefi cial for in vitro and cellular potency,12 an evaluation of the effect of further substitution on the C ring was undertaken. Replacement of the hydrogen (Table 1, 10) with a p-fl uoro (9) did not
dramatically affect the biochemical potency; however, the target modulation of two downstream markers, c-Met and Hsp70, and inhibition of cellular proliferation (GTL-16) was improved 4- 6-fold. It is likely that the effect observed on cells was due to an increase in target potency that was not measurable due to assay limitations as the lower limit of the biochemical assay was 10 nM. Later 9 was tested in an assay format using full-length Hsp90 with a lower concentration of Hsp90, which produced an IC50 of 1 nM.13 However, due to technical challenges with this assay format, it was not routinely utilized. Other substituents such as methyl and methoxy (11 and 12), were also tolerated biochemically; however, the target modulation and cellular inhibition readouts resembled those of 10, where R = H. At the C-5 position, small substituents such as fl uoro (13) lost 3-fold potency biochemically and 10-fold potency in cellular readouts, while larger groups such as methoxy (14) were inactive.
With a p-fluoro moiety determined to be the optimal substituent for ring C, a survey of various heterocycles at the ortho position was undertaken. First, varying the methoxy substituent on the pyridyl ring system to larger (15) or smaller (16) moieties was investigated.

removal of the methyl group to give 16 led to nearly a 1000- fold loss of biochemical potency (Table 2).
Pyrazine (17) was equipotent to 9 as were substituted pyrazines 18 and 19. The similarity in biochemical potencies of 18 and 19 suggests that the amino group in 19 does not form additional hydrogen bond contacts with the protein. Com- pound 19, however, exhibited 5-fold improved solubility compared to 18. Five-membered aromatic heterocycles were also explored. Heterocyclic ring systems such as 2,5- disubstituted thiophene 20 and 2,5-disubstituted thiazole 21 were 10-15-fold less potent than 9, while the 2,4-disubstituted thiazole 22 had a biochemical potency similar to that of 9.
The cocrystal structure of compound 9 in the ATP binding pocket of Hsp90 was solved to 1.55 Å resolution (Figure 2). Like other Hsp90 structures, there are four highly conserved water molecules found in the active site which have several strong hydrogen bonds to the protein, around and especially to Asp93. Upon binding of the inhibitor 9, these water molecules form an extensive hydrogen-bonding network linking the aminopyrimidine of 9 to the protein. The methoxypyridyl group of 9 points toward the β sheets and the aminopyrimidine near residue Asp93.
As previously pointed out, only small para substitutions on the C ring were tolerated. A small hydrophobic indentation
formed by backbone and side chain residues Ala111, Val136, and Tyr139 accommodates fluorine (9) and methyl (11) well, but larger substitutions, such as methoxy (12), although still tolerated, clash with the residues that form the hydrophobic indentation. The methyl from the methoxy in 12 is in plane with the C ring13 and extends toward the 5-position where the pocket expands, suggesting that substitutions at the 5-position of ring C would be possible. Indeed, fl uorine (13) was tolerated, whereas methoxy (14) was not tolerated due to both steric and electronic clash of the oxygen with the carbonyl group of Gly135.

Compound 16 was less active than 9 presumably due to the presence of the polar hydroxyl group within the hydrophobic space around residues Val 150, Typ162, and Val186. Both the p-methyl (18) and amine (19) substitutions were equipotent to 9 as they were sterically tolerated. Compound 19 was not improved in potency presumably because the amine failed to make any polar interactions.
Three compounds (9, 17, and 22) exhibiting potent Hsp90 biochemical and target modulation activity were chosen for single-dose plasma pharmacokinetics in female CD1 mice. Following iv administration, 9 and 22 displayed moderately low clearance (19-20 mL/min/kg) and plasma half-lives (t1/2) of 146 and 62 min, respectively. The long half-life of compound 9 in mice is consistent with that observed in human liver microsomes in vitro, suggesting that 9 would likely display a longer half-life in humans. Compound 17 exhibited a t1/2 of 96 min and relatively higher clearance (144 mL/min/kg). The volume of distribution (Vss) was high for all three compounds and exceeded the total body blood volume, suggesting extensive tissue drug distribution. Following oral dosing, 9, 17, and 22 were absorbed rapidly with peak plasma concentrations observed by 2 h (Tmax) and Cmax plasma levels of 1280, 271, and 1675 ng/mL, respectively. The oral bioavailabilities of 9 and 22 were 76% and 95%, and the observed plasma exposures (AUC(0,last)) were 6566 and 5783 ng·h/mL, respectively. In comparison, compound 17 had a lower bioavailability of 39% and an approximately 13-15-fold lower exposure (AUC- (0,last)) of 441 ng·h/mL in vivo.
Compounds 9, 17, and 22 were selected for evaluation of anticancer activity in mice on the basis of biochemical and cellular potencies and pharmacokinetic (PK) properties. The GTL-16 human gastric carcinoma line was used for the in vivo pharmacodynamic (PD) and effi cacy assessment of these compounds due to overexpression and constitutive activation of the Hsp90 client protein c-Met, arising from gene amplifi cation;14 thus, c-Met levels were used as a PD marker even though numerous Hsp90 client proteins may be altered
aAbbreviations: ND, not done; qd, daily; q3d, every 3 days, q6d, every
However, only compound 9 maintained prolonged suppression of c-Met levels with 30% and 50% reduction after one week in mice treated with a single dose of 5 and 15 mg/kg, respectively.
The long half-life of compound 9 correlated with the longer duration of reduced c-Met levels in tumors. The longer c-Met suppression after a single dose of 9 conferred significant antitumor efficacy with less frequent dosing and at lower dose levels, for example, 5 mg/kg q3d and 15 mg/kg q6d treatments, resulting in 76% and 69% tumor growth inhibition. A detailed pharmacological evaluation of compound 9 is described elsewhere.5
In conclusion, from a commercially available, highly ligand efficient hit, the novel, orally available (R)-dihydropyridopyr- imidinone series was developed utilizing structure-based drug design. Optimization of this series yielded three potent analogues with complementary pharmacokinetic profi les which allowed alternate dosing regimens in efficacy studies. Although all three compounds exhibited efficacious tumor growth inhibition, 9 (NVP-HSP990) was shown to have superior efficacy and was selected for clinical evaluation. Compound 9 advanced to phase I clinical trials.

General Methods. All reagents and solvents were of commercial quality and were used without further purifi cation. Column chromatography was performed using Merck silica gel 60 (230-400 mesh). The purity of the final compounds and/or intermediates was characterized by high-performance liquid chromatography (HPLC) using a Waters Millenium chromatography system with a 2695 separation module (Milford, MA). The analytical column was reversed-phase Phenomenex Luna C18 5 μm, 4.6 × 50 mm, from Alltech (Deerfield, IL). A gradient elution was used (flow 2.5 mL/
min), typically starting with 5% acetonitrile/95% water and progressing to 100% acetonitrile over a period of 10 min. All solvents contained 0.1% trifluoroacetic acid (TFA). All compounds where biological data are presented have >95% purity as determined by HPLC. Mass spectrometric analysis was performed according to the following liquid chromatography/mass spectrometry (LC/MS) method: Waters ACQUITY UPLC system equipped with a ZQ 2000 MS system; column, Kinetex by Phenomenex, 2.6 μm, 2.1 × 50 mm; column temperature 50 °C; gradient 2-88% (or 0-45% or 65- 95%) solvent B over a 1.29 min period; flow rate 1.2 mL/min. Compounds were detected by a Waters photodiode array detector. All masses were reported as those of the protonated parent ions, molecular weight range 150-850, cone voltage 20 V. 1H and 13C NMR spectra of all compounds were recorded at 300 and 75 MHz, 400 and 101 MHz, or 500 and 125 MHz, respectively. 1H shifts are referenced to the residual protonated solvent signal (e.g., δ 2.50 for d6- DMSO), and 13C shifts are referenced to the deuterated solvent signal (δ 39.5 for d6-DMSO).
(S,E)-N-(2-Bromo-4-fl uorobenzylidene)-2-methylpropane-2- sulfinamide (3). A round-bottom flask was charged with dry THF (50 mL), titanium ethoxide (41 mL), (S)-(-)-tert-butanesulfinamide (12.0 g, 99.1 mmol), and 2-bromo-4-fluorobenzaldehyde (2) (18.2 g, 90.1 mmol). The resulting reaction mixture was stirred under N2 at rt for 4 h. The reaction mixture was diluted with EtOAc, and a mixture of brine with Celite was added with vigorous stirring. The resulting emulsion was filtered through a pad of Celite and washed with EtOAc. The filtrate was transferred to a separatory funnel and the aqueous layer removed. The organics were washed with brine, dried (Na2SO4), and concentrated to afford 3 as a yellow oil that solidified upon standing (26.5 g, 86.3 mmol, 96%): 1H NMR (300 MHz, CDCl3) δ 8.86 (s, 1H), 8.03 (m, 1H), 7.35 (m, 1H), 7.11 (m, 1H), 1.11 (s, 9H); LC/MS m/z 307.9 (MH+), tR = 3.22 min.
( R )-Ethyl 3-(2-Bromo-4- fl uorophenyl)-3-(( S )-(1,1- dimethylethyl)sulfi namido)propanoate (4). An oven-dried three-necked round-bottom fl ask, reflux condensor, and addition funnel were assembled under positive N2 pressure and cooled to room temperature. The flask was charged with Zn dust (21.3 g, 326.0 mmol), CuCl (32.6 g, 32.6.mmol), and dry THF (60 mL). The reaction mixture was heated to refl ux temperature and stirred vigorously with an overhead stirrer for 30 min. The reaction was removed from the oil bath, and the addition funnel was charged with ethyl bromoacetate (3.6 mL, 32.6 mmol) and dry THF (30 mL). The ethyl bromoacetate solution was added slowly so that a gentle refluxing of the reaction mixture was maintained. Once addition was complete, the reaction mixture was stirred for 20 min and then heated to 50 °C for 30 min. The reaction mixture was then cooled to 0 °C and the addition funnel charged with 3 (6.60 g, 21.5 mmol) in dry THF (20 mL). This solution was then added dropwise and the reaction mixture stirred for an additional 4 h at 0 °C. The reaction mixture was filtered through a pad of Celite, and the filter pad was washed twice with Et2O. The filtrate was washed with 0.25 M citric acid, satd NaHCO3(aq), dried (Na2SO4), and concentrated to afford 4 (7.30 g, 18.4 mmol, 86%) as a clear oil: 1H NMR (300 MHz, CDCl3) δ 7.39 (m, 1H), 7.28 (m, 1H), 7.01 (m, 1H), 5.14 (m, 1H), 4.92 (d, J = 5.4, 2H), 4.1 (m, 2H), 2.90 (m, 2H), 1.22 (m, 12H); LC/MS m/z 396.0 (MH+), tR = 2.96 min; HPLC tR = 4.11 min (major diastereomer), tR = 3.88 min (minor diastereomer); de = 96%.
(R)-Ethyl 3-Amino-3-(2-bromo-4-fl uorophenyl)propanoate Hydrochloride (5). Compound 4 (7.30 g, 18.4 mmol) was stirred with Et2O (37 mL), EtOH (1.2 mL), and 4 M HCl in Et2O (37 mL) for 30 min. The resulting suspension was filtered, and the solids were triturated with Et2O and hexanes. The solid was dried under vacuum to afford 5 as a white solid (5.23 g, 15.2 mmol, 83%): 1H NMR (300 MHz, CD3OD) δ 7.61 (m, 2H), 7.33 (m, 1H), 5.18 (m, 1H), 4.85 (br s, 3H), 4.13 (q, J = 7.2, 2H), 3.15 (ddd, 2H), 1.22 (t, J = 7.5, 3H); LC/MS m/z 292.0 (MH+), tR = 1.97 min. To analyze the ee, a portion of the material was freebased by dissolving it in EtOAc and washing the organic layer three times with 10% Na2CO3(aq). The organic layer was dried over Na2SO4 and concentrated to aff ord the freebase. A racemic mixture of 5 was also prepared and analyzed by chiral HPLC to confirm separation of the enantiomers (Chiralpak AD column, 1 mL/min, MeOH): (S)-enantiomer, tR = 5.84 min; (R)-enantiomer, tR = 7.47 min; >95% ee.
(R)-Ethyl 3-(2-Bromo-4-fl uorophenyl)-3-(3-oxobutanamido)- propanoate (6). To a stirred solution of 5 (20 g, 61.5 mmol) in DCM (400 mL) were added triethylamine (25.7 mL, 184.6 mmol) and diketene (5 mL, 64.6 mmol). The reaction was stirred for 18 h at rt. The mixture was partitioned between DCM and aq NaHCO3 and extracted with DCM. The organic layer was washed with water and brine, dried (Na2SO4), filtered, and evaporated to give a crude oil which was purified on silica gel (hexane/EtOAc) to provide 6 as an oil (18.5 g, 80%): LC/MS m/z 373.8 (MH+), tR = 2.22 min.
(6R)-3-Acetyl-6-(2-bromo-4-fl uorophenyl)-piperidine-2,4- dione (7). To a glass pressure vessel containing 6 (7.1 g, 19 mmol) was added NaOMe in MeOH (0.3 M, 150 mL). The vessel was sealed and the reaction heated in an oil bath at 100 °C for 18 h behind a blast shield. After being cooled to room temperature, the reaction mixture was diluted with aq NH4Cl and extracted with DCM. The organic layer was washed with water and brine, dried (Na2SO4), filtered, and evaporated to give crude 7 (5.7 g, 91%) which was used directly in the next step: LC/MS m/z 329.9 (MH+), tR = 2.48 min.
(R)-2-Amino-7-(2-bromo-4-fl uorophenyl)-4-methyl-7,8- dihydropyrido[4,3-d]pyrimidin-5(6H)-one (8). To the mixture of
7(5.7 g, 17.4 mmol) and acetylguanidine (2.6 g, 26 mmol) in ethanol (170 mL) was added pyrrolidine (1.6 mL, 19.1 mmol). The reaction mixture was heated in an oil bath at 110 °C for 18 h. After being cooled to room temperature, the reaction mixture was further cooled to 0 °C for 1 h. A crystalline material was collected, washed with cold ethanol, and air-dried to aff ord 8 (2.6 g, 29%): LC/MS m/z 374.0 (MH+ + Na+), tR = 2.27 min; 1H NMR (400 MHz, d6-DMSO) δ 7.97 (d, J = 3.13 Hz, 1 H) 7.61 (dd, J = 8.44, 2.57 Hz, 1 H) 7.30-7.37 (m, 1 H) 7.21-7.30 (m, 1 H) 7.11 (br s, 2 H), 4.91-5.00 (m, 1 H) 3.22 (dd, J = 16.31, 6.09 Hz, 1 H) 2.83 (dd, J = 16.29, 5.97 Hz, 1 H) 2.61 (s, 3 H); 13C NMR (101 MHz, d6-DMSO) δ 168.94, 166.97, 164.95, 162.24, 159.77, 136.49, 136.46, 129.28, 129.19, 121.74, 120.10, 119.85, 115.17, 114.96, 109.91, 50.99, 36.87, 24.15.
(R)-2-Amino-7-(4-fl uoro-2-(6-methoxypyridin-2-yl)phenyl)- 4-methyl-7,8-dihydropyrido[4,3-d]pyrimidin-5(6H)-one (9). To a small glass pressure vessel containing a mixture of 8 (226 mg, 0.65 mmol) and 2-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)- pyridine (395 mg, 2.58 mmol) in DME (4 mL) were added Pd(dppf)Cl2/CH2Cl2 (53 mg, 0.064 mmol) and 2 M Na2CO3(aq) (2 mL, 4 mmol). The reaction mixture was degassed with argon and sealed. The mixture was then heated to 100 °C in an aluminum block for 2 h. Additional 2-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxabor- olan-2-yl)pyridine (198 mg, 1.29 mmol) and Pd(dppf)Cl2/CH2Cl2 (53 mg, 0.064 mmol) were added. The reaction mixture was again heated to 100 °C for 17 h. After being cooled to room temperature, the mixture was diluted with EtOAc, washed with 2 M Na2CO3(aq) and brine, dried (Na2SO4), filtered, and evaporated to give the crude product, which was purified by reversed-phase preparative HPLC and freebased to give 9 (112 mg, 46%): 1H NMR (400 MHz, CDCl3) δ 7.65 (dd, J = 8.4, 7.2 Hz, 1 H), 7.60 (dd, J = 8.6, 5.5 Hz, 1 H), 7.09- 7.18 (m, 2 H), 6.96 (dd, J = 7.4, 0.8 Hz, 1 H), 6.76 (dd, J = 8.2, 0.8 Hz, 1 H), 6.14 (s, 1 H), 5.48 (s, 2 H), 5.11 (ddd, J = 10.8, 4.7, 1.0 Hz, 1 H), 3.93 (s, 3 H), 3.19-3.26 (m, 1 H), 3.02-3.10 (m, 1 H), 2.72 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 171.13, 168.89, 165.44, 163.22, 162.76, 162.30, 160.78, 155.12, 141.81, 139.28, 134.38, 128.46, 117.16 (d, J = 21 Hz), 116.98, 116.82 (d, J = 21 Hz), 111.30, 53.71, 49.52, 39.40, 24.47; LC/MS m/z 380.1 (MH+), tR = 0.67 min; HRMS found m/z 380.1525, C20H19FN5O2 requires m/z 380.1523.
(R)-2-Amino-7-(4-fl uoro-2-(6-methoxypyrazin-2-yl)phenyl)- 4-methyl-7,8-dihydropyrido[4,3-d]pyrimidin-5(6H)-one (17). 17 was synthesized by the same method as 9: 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 6.3 Hz, 1 H), 7.69 (dd, J = 8.6, 5.5 Hz, 1 H), 7.21-7.26 (m, 1 H), 7.18 (dd, J = 9.0, 2.7 Hz, 1 H), 5.87 (s, 1 H), 5.29 (s, 2 H), 5.04 (dd, J = 11.2, 4.9 Hz, 1 H), 3.99 (s, 3 H), 3.24-3.30 (m,1H), 3.10-3.20 (m, 1 H), 2.75 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 170.75, 168.30, 165.49, 164.78, 162.37, 159.34, 149.43, 138.36, 136.10, 135.22, 129.11, 117.58 (d, J = 22.5 Hz), 117.24 (d, J = 22.5 Hz), 111.58, 54.28, 49.92, 39.93, 24.73; LC/MS m/z 381.0 (MH+), tR = 0.59 min; HRMS found m/z 381.1475, C19H18FN6O2 requires m/z 381.1475
(R)-2-Amino-7-(4-fl uoro-2-(2-methoxythiazol-4-yl)phenyl)-4- methyl-7,8-dihydropyrido[4,3-d]pyrimidin-5(6H)-one (22). 22 was synthesized by the same method as 9: 1H NMR (400 MHz, CDCl3) δ 7.48 (dd, J = 8.8, 5.7 Hz, 1 H), 7.16 (dd, J = 9.4, 2.7 Hz, 1 H), 7.09 (td, J = 8.4, 2.7 Hz, 1 H), 6.72 (s, 1 H), 6.23 (s, 1 H), 5.38 (s,
2H), 5.19 (ddd, J = 9.4, 5.1, 2.0 Hz, 1 H), 4.08-4.12 (m, 3 H), 3.32 (ddd, J = 16.6, 5.3, 1.2 Hz, 1 H), 3.07 (dd, J = 16.4, 9.4 Hz, 1 H), 2.76 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 174.54, 171.14, 168.99, 165.31, 162.91, 162.22, 160.82, 147.35, 136.27 (d, J = 204 Hz), 128.14 (d, J = 7.5 Hz), 116.83 (d, J = 22.5 Hz), 115.83(d, J = 22.5 Hz), 111.47, 109.40, 58.74, 49.67, 38.74, 24.53; LC/MS m/z 386.0 (MH+), tR = 0.66 min; HRMS found m/z 386.1094, C18H17FN5O2S requires m/z 386.1087.

S* Supporting Information
General methods, additional high-resolution mass spectrometry data, details for the biochemical and cellular assays, in vivo experiments, and structural biology methods. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author
*Phone: 510-879-9421. E-mail: [email protected].
The authors declare no competing fi nancial interest.

We thank Weiping Jia for his analytical support.

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