Preclinical Development of PQR514, a Highly Potent PI3K Inhibitor Bearing a Difluoromethyl-Pyrimidine Moiety

Abstract

The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is a critical regulator of cell growth and is frequently hyperactivated in cancer. Therefore, PI3K inhibitors represent a valuable asset in cancer therapy. Herein we have developed a novel anticancer agent, the potent pan-PI3K inhibitor PQR514 (4), which is a follow-up compound for the phase-II clinical compound PQR309 (1). Compound 4 has an improved potency both in vitro and in cellular assays with respect to its predecessor compounds. It shows superiority in the suppression of cancer cell proliferation and demonstrates significant antitumor activity in an OVCAR-3 xenograft model at concentrations approximately eight times lower than PQR309 (1). The favorable pharmacokinetic profile and a minimal brain penetration promote PQR514 (4) as an optimized candidate for the treatment of systemic tumors.

Figure 1

(A) Chemical modifications in the heteroaromatic moiety. Chemical structures of PQR309 (1),2,3, and PQR514 (4). (B) Docking of PQR514 (4) (magenta) into PI3Kγ (gray) starting from PDB 5OQ4 (from ref (11)). The structural water molecule is shown as a red ball. Water-mediated H bonds are depicted as dashed black lines. The Val882 backbone nitrogen involved in a H bond with the morpholine in the hinge region is shown as a blue ball.

Scheme 1. Synthesis of Compounds 4–8

Synthesis of compound 5 is reported in ref (16). Reagents and conditions: (i) morpholine derivative (M_n–H), CH₂Cl₂, 0 °C → rt, o/n. (ii) (1) RBpin generated in situ (see the Supporting Information), Pd(OAc)₂/PPh₃ (cat.), K₂CO₃, THF, 60 °C, 2–4 h; (2) HCl, H₂O, 60 °C, 2 h. (iii) M_n–H, DIPEA, EtOH, 0 °C → rt, o/n. (iv) M_n–H, DIPEA, dioxane, 70 °C, o/n as reported in the literature.

Figure 2

(A) Waterfall plot of compound PQR514 (4) in a full NTRC (Netherlands Translational Research Center BV) Oncolines cell panel of 66 cancer cell lines. Concentrations of half-maximal growth inhibition (GI₅₀) for 4 were obtained from dose–response growth curves, and individual GC₅₀ values of a cell line were related to the mean GI₅₀ of all cell lines; cell lines were sorted by lowest to highest sensitivity for 4. Individual cell line names and GI₅₀ values are reported in Table S7. (B) Cellular inhibition profile of PQR514 (4), PQR309 (1), and PQR530 (16). The correlation of log-(IC₅₀) in nanomoles for the inhibition of cell proliferation in 44 tumor cell lines is depicted. R² and p values are from the nonparametric Spearman’s correlation. Dashed line: perfect correlation with identical IC₅₀ values. Blue line: linear regression for PQR514 (4) versus PQR530 (16). Gray line: linear regression for PQR514 (4) versus PQR309 (1). LogIC₅₀ data of PQR309 (1) and PQR530 (16) were extracted from ref (16).

Figure 3

PK/PD assessment of PQR514 (4). Compound 4 was administered to male C57BL/6J mice as a single dose per os (p.o., 10 mg/kg). (A) Levels of 4 in male C57BL/6J mice tissues: plasma, brain, and thigh muscle (n = 3). Compound 4 was extracted from each tissue at the indicated time points and quantified using LC–MS. (B) Glucose and (C) insulin plasma levels after the oral administration of 4 in male C57BL/6J mice. All values: mean ± SEM. Error bars not shown are smaller than the symbols. Values are reported in Tables S11 and S12.

Figure 4

OVCAR-3 human ovarian cancer xenograft model in BALB/c nude mice: Tumor cells were subcutaneously inoculated at day 0, and the daily oral application of the indicated agents was started at day 17 (28 × QD). (A) Tumor weight was determined at day 45. (B) Tumor size was measured and calculated as described in the text. (C) Body weight was determined at the depicted time points. Statistics: *p < 0.05, **p < 0.001, ^$p < 0.0001, n = 8. (A) One-way ANOVA with Tukey’s correction for multicomparisons, mean ± SEM. PQR309 (1) and vehicle data were reprinted from ref (11). (B,C) Two-way ANOVA with Bonferroni or Dunnett’s correction for multicomparisons, mean ± SEM. (B) Statistic symbols: above, PQR309 (1); below, PQR514 (4).