The way to attack the star inhibitor of KRAS G12C (Part 2) - amg510
Last Update: 2020-06-19
Search more information of high quality chemicals, good prices and reliable suppliers, visit
After a lot of optimization work, the early hits determined by shokat team was modified by Matthew RJanes and others to provide a compound, ars-1620, which is suitable for in vivo applicationAs early as the initial disclosure of research results by shokat laboratory, Amgen company has started to determine the covalent inhibitor of KRAS G12C, and obtained compound 1 by using carmot's technical platform, as shown in Figure 1    < br / > the co crystallization of compound 1 and gdp-kras G12C reveals that compound 1 uses a new binding mode relative to ars-1620The tetrahydroisoquinoline ring of compound 1 occupies a previously undeveloped hidden pocket on the surface of KRAS, which is revealed by the rotation of histidine-95 side chain and contains parts H95, Y96 and Q99As shown in Figure 1 below, although the binding of this concealed pocket results in a multiple increase in cell potency compared to ars-1620, compound 1 has a very high clearance rate and low oral bioavailability in the rodent model system, making it unsuitable for use in vivoTo find an alternative way to use H95 / Y96 / Q99 hidden pocket, which may provide clues with better medicinal properties < br / > < br / > < br / > at the same time, although ars-1620 showed good anti proliferation activity in vitro and in vivo for the tumor mutated by KRAS G12C, in order to get useful molecules in treatment, it seems necessary to further improve the cell efficacy< br / > the superposition of binding modes of compound 1 and ars-1620 indicates that quinozolinone nitrogen (N1) substituted for ars-1620 may provide an alternative means to enter the hidden bag of H95, thus producing a new and enhanced KRAS G12C inhibitor < br / > < br / > < br / / > a series of Phthalazines analogues were prepared to verify the hypothesis, in which the C4 position (y) of the core (x = n, y = C) of the Phthalazines was replaced by a series of arylsThe inhibition of GDP / GTP exchange catalyzed by Sos1 and the reduction of downstream ERK phosphorylation signal were measuredIt was found that the potency of C4 (2) was about 10 times lower than that of ars-1620 (IC50 = 20.1 μ m), and 20 times lower than that of ars-1620 (IC50 = 58 μ m) in p-ERK cell experiment, as shown in Figure 3 The Cocrystallization of < br / > < br / > < br / / > compound 2 and gdp-kras G12C confirmed that compound 2 adopted a binding mode similar to ars-1620, but the newly introduced C4 phenyl substituent occupied the designed H95 / Y96 / Q99 hidden bag, as shown in Figure 4A belowHowever, C4 phenyl does not have many noncovalent contacts with cryptobag residues (e.gY96), which leads to the study of whether further benzene ring substitution will have additional contact with cryptobag residues, thus improving the effectivenessA series of increasing ortho substituents were added to the benzene ring of 2 (see compound 3-8 above)Compound 8 has been shown to be optimal, however, it shows biochemical and cellular IC50 values comparable to ars-1620 In order to further improve the effect, we also tried to change the core of compound 8 It is worth noting that compared with 8, quinazolinone 9 has significant improvement in biochemical and cellular potency, and the potency of the compound obtained in vitro is ~ 3-9 times that of ars-1620 The co crystallization of compound 9 and gdp-kras G12C confirmed that 9 adopted the binding mode of close contact between isopropyl phenyl and Y96, H95 and Q99 residues of the hidden bag, as well as the almost orthogonal orientation of isopropyl benzene ring and phthalazine ring, as shown in Figure 4B  < br / > < br / > < br / > at the same time, the separation of R - and s-atropine by chiral chromatography confirmed that the effect of (R) - 9 was significantly higher than that of the corresponding (s) - 9 atropine, as shown in Figure 5 below Although (R) - 9 has been proved to be a particularly effective inhibitor of KRAS G12C signal (p-erkic50 = 0.130 μ m), the eutectic structure of gdp-kras G12C presents several prospects for further optimization: (1) the substitution of piperazine C2 seems to provide an opportunity to enhance the activity through more contact with C12, E62 and Y96, And (2) (R) - 9 fluorophenol "tail" substitution seems to provide an opportunity to form more extensive contact with lipophilic residues In addition, despite the promising prospect of (R) - 9, PK study showed that there was no measurable oral bioavailability in BALB / c mice Low membrane permeability and relatively poor water solubility are considered to be possible pathogenic factors Therefore, C2 and C7 modifications are also considered as potential opportunities to regulate the biopharmaceutical properties of (R) - 9 to improve the bioavailability of analogues in the future, as shown in Figure 5  < br / > < br / > In addition to ERK phosphorylation experiment, KRAS p.g12c (MIA PaCa-2) and KRAS p.g12s (A549) cells were used to screen to confirm that the growth inhibition activity was only for p.g12c mutant cells Naphthol (10) and indazole (11) substitutes do show enhanced activity in ERK phosphorylation and activity determination, but these analogues continue to show poor water solubility and low MDCK permeability, as shown in Figure 5 above  < br / > in order to improve the permeability of MDCK, the effect of removing the phenols from (R) - 9 to get fluorophenyl (12) was studied It was found that the modification had no loss of activity in p-ERK and activity determination, but the permeability of MDCK increased more than three times Good MDCK permeability was also preserved in the analogues with piperazine C2 methyl substituent Methylpiperazine (13) not only improved cell activity (p-erkic50 = 47 nm), but also showed measurable oral bioavailability in mice (12%) Attempts to replace the fluorophenyl tail of 13 with a larger lipophilic substituent (e.g., o-chlorophenyl (14) or o-trifluorophenyl (15)) resulted in decreased cell activity and MDCK permeability Similarly, although the reintroduction of phenols (to provide fluorophenol (16)) in 13 had little effect on cell activity, the permeability of the membrane was significantly reduced  < br / > because the phenolic functional groups of (R) - 9 and (R) - 16 are the key factors for these analogues to reduce the permeability of MDCK, an alternative strategy was studied to reduce this adverse effect Assuming that the desolvation of phenol increases the energy cost of membrane permeation in the process of membrane diffusion, this paper studies whether nitrogen doping at C8 position of quinazolone ring can provide an internal hydrogen bond receptor to meet the adjacent phenol donors in the process of membrane permeation, so as to improve the permeability of MDCK Results compared with their non aza homologues (9 and 16), azoquinolone (17) and (18) not only showed significant permeability of MDCK, but also significantly improved the solubility of water The oral bioavailability of compound 17 was still disappointing (1.3%), while the oral bioavailability of azoquinolone 18 was significantly increased (33%), and the cell activity was significantly increased (p-ERK IC50 = 44 nm; Mia PaCa-2 IC50 = 5 nm) Considering the permeability, bioavailability, in vivo clearance, efficacy, metabolic stability and water solubility of MDCK, 18 was further optimized  < br / > subsequent studies have found a new challenge for compound 18 Although the space crowding environment of isopropylphenyl quinazolinone aromatic bond greatly limits the rotation, resulting in the generation of the detachable atoisomer, it does not completely limit the rotation around the bond The isomer undergoes slow mutual transformation at 25 ° C with a half-life of 8 days and a free energy barrier of 26kcal / mol, which greatly complicates the development of (R) - 18 as a pure substance, as shown in Figure 6 below  < br / > These strategies include: (1) increasing the energy barrier of the conversion of atropine isomers to form a single pure atropine; (2) reducing the potential barrier of the conversion to form a freely convertible mixture of atropine; or (3) making the substituent of the hidden bag symmetrical to avoid the generation of the chiral axis After study (R) - 24 became the main candidate drug for pharmacodynamic and efficacy test in xenotransplantation model  < br / > < br / > < br / / > to detect the effect of (R) - 24 on KRAS signal transduction in vivo, and (R) - 24 was administrated to nude mice subcutaneously with mia-paca-2t2 human tumor cells (pure krasp G12C), and the effect of (R) - 24 on KRAS signal transduction was observed Serum and tumor samples were collected 2 hours after administration, showing that (R) - 24 was exposed in a dose proportion between different doses (10-100 mg / kg), and the phosphorylation of downstream ERK was maximally inhibited at low doses (30 mg / kg) (Fig 8a) Free plasma and total tumor exposure (3 and 11 nm, respectively) were significantly lower in the 30 mg / kg group than in vitro p-ERK IC50 (28 nm; measured after incubation for 2 hours), which provided a preliminary indication that the covalent inactivation of KRAS G12C may have a long-lasting downstream effect, even in the absence of circulating drugs Then (R) - 24 was analyzed by the same method in the study of the effectiveness of transplanted tumor in nude mice (R) - 24 take orally once a day (10-100 mg / kg) within two weeks after tumor establishment, completely inhibit tumor growth at a dose of 30 mg / kg, and cause tumor regression at a dose of 60 mg / kg (Fig 8b) (R) The results of the early PK / PD study were confirmed again by the time course PK study (30mg / kg, PO) of-24 in nude mice, which showed that the treatment of 2H p-ERK IC50 in vitro < 2-3H was enough to achieve tumor growth arrest (Figure 8C)  < br / > Because of the decrease of bioavailability and the decrease of solubility in biological suspension medium, the (R) - 24 analogues with excellent water solubility are preferred Two strategies are adopted: (1) reducing the lipophilicity of (R) - 24 by changing the quinozolinone C6 halogen substituent; (2) increasing the polar surface area of (R) - 24 by introducing additional nitrogen atoms into the hidden bag ring, as shown in Figure 9  < br / > Using nitrogen atom (see compounds 34 and 35) to replace the carbon atom near the isopropyl or methyl substituent significantly improves the solubility of water, but does not significantly change the activity of KRAS However, this substitution also significantly reduced the permeability of MDCK, providing molecules without measurable oral bioavailability (BALB / c mice) Although dinitrogen (36) also improved the solubility of water, the obtained pyrimidine analogues also had low permeability and no measurable oral bioavailability Next, we studied whether reducing the lipophilicity of C6 substituent of quinazolinone could improve the solubility of water while maintaining cell activity Although C6 chloro substituent (compound 37) with fluoro substituent (R) - 24 resulted in a slight decrease in activity in cell analysis (e.g., p-ERK IC50 = 90 nm), this change also resulted in a slight increase in water solubility (~ 3 times) and had no adverse effect on membrane permeability But (R) - 37 did not show any measurable oral bioavailability (% f < 0.5) However, the combination of C6 fluoro substitution and nitrogen incorporation in concealed pocket aromatic rings (compounds 38-40) overcomes the low bioavailability of the previous analogues Although the addition of nitrogen atoms again resulted in a significant decrease in the MDCK permeability of all three compounds, the combination of this property significantly increased the solubility of water (greater than 423 μ m in all biological suspension media) From these studies, (R) - 38 has become a prominent molecule, showing good activity (p-ERK IC50 = 68 nm), medium permeability (PAB = 6 μ cm / s), and excellent oral bioavailability (crystal state: 22-40%), as shown in Figure 9  < br / > it is satisfactory that, (R) - 38 is exposed to plasma in a dose proportion when administered orally (0.3100 mg / kg) in a xenotransplantation model of miapaca-2 in nude mice, and significantly inhibits ERK phosphorylation at a dose ≥ 10 mg / kg (Fig 10a) The PK / PD relationship of (R) - 38 was further studied, and the time course pharmacodynamics was studied in the xenotransplantation model (Fig 10b) The study supports the early results of (R) - 24 and (R) - 38, indicating that although ERK phosphorylation occurs 60-120 minutes after administration
This article is an English version of an article which is originally in the Chinese language on echemi.com and is provided for information purposes only.
This website makes no representation or warranty of any kind, either expressed or implied, as to the accuracy, completeness ownership or reliability of
the article or any translations thereof. If you have any concerns or complaints relating to the article, please send an email, providing a detailed
description of the concern or complaint, to email@example.com
. A staff member will contact you within 5 working days. Once verified, infringing content
will be removed immediately.