Orally Bioavailable 4-Phenoxy-quinoline Compound as a Potent Aurora Kinase B Relocation Blocker for Cancer Treatment

Abstract

We investigated a novel 4-phenoxy-quinoline-based scaffold that mislocalizes the essential mitotic kinase, Aurora kinase B (AURKB). Here, we evaluated the impact of halogen substitutions (F, Cl, Br, and I) on this scaffold with respect to various drug parameters. Br-substituted LXY18 was found to be a potent and orally bioavailable disruptor of cell division, at sub-nanomolar concentrations. LXY18 prevents cytokinesis by blocking AURKB relocalization in mitosis and exhibits broad-spectrum antimitotic activity in vitro. With a favorable pharmacokinetic profile, it shows widespread tissue distribution including the blood–brain barrier penetrance and effective accumulation in tumor tissues. More importantly, it markedly suppresses tumor growth. The novel mode of action of LXY18 may eliminate some drawbacks of direct catalytic inhibition of Aurora kinases. Successful development of LXY18 as a clinical candidate for cancer treatment could enable a new, less toxic means of antimitotic attack that avoids drug resistance mechanisms.

The Aurora kinase (AURK) family consists of three homologous serine/threonine kinases, Aurora kinase A (AURKA), Aurora kinase B (AURKB), and Aurora kinase C (AURKC).¹ They are key regulators of cell division and attractive oncology targets. Small molecule inhibitors of AURKs do not directly interfere with microtubules, and a review of clinical studies suggests that these inhibitors may avoid the peripheral neurotoxicity observed with spindle toxins.² Targeting these key mitotic kinases may, therefore, have advantages over standard chemotherapeutic approaches.

The AURKs play distinct roles in cell division. While AURKA is essential for the proper assembly of a bipolar mitotic spindle,² AURKB and AURKC are catalytic subunits of the chromosomal passenger protein (CPP) complex, which coordinates karyokinesis with the completion of cytokinesis.³ AURKC, however, is a meiotic rather than mitotic kinase, so AURKB is the catalytic subunit in somatic cells. The localization of the CPP complex is highly coordinated during mitosis. For example, the CPP complex is dynamically relocalized from chromosomes to the spindle midzone with the onset of anaphase. Preventing relocalization may just as effectively compromise AURKB functions as catalytic inhibition but with the added bonus of not altering kinase functions during interphase or in nondividing cells. Despite this, no therapeutic is currently known to block the mitotic localization of AURKB for cancer treatment. Instead, various catalytic inhibitors of AURKs have been clinically tested. The AURK inhibitors in clinical trials often inhibit all three AURKs without much discrimination,⁴ and several trials have been terminated partly due to adverse effects that stemmed from off-target interactions with other kinase families. Also, drug resistance due to compensating AURK mutations has been suggested to contribute to a lack of durable efficacy in clinical trials.⁵

We previously described a mechanism-informed, phenotypic screening (MIPS) assay of a synthetic compound library^6,7 for compounds that disrupt the localization, but not catalytic activity, of the CPP complex. Such compounds prevent cytokinesis, induce polyploidy, and trigger cell death in model rodent and human-derived cancer cell lines. We incorporated phenotypic screening into an optimization protocol and derived a scaffold where compounds shared the ability to disrupt CPP complex localization to varying degrees. These 4-phenoxy-quinoline derivatives exhibited anticancer activity in a variety of human cancer cell lines in vitro.⁷

We chose one compound, LG182, which demonstrated potent in vitro activity and limited in vivo activity,⁷ for further optimization, with the goal of finding a compound with adequate pharmacokinetic (PK) and pharmacodynamic (PD) properties for development as a new drug candidate. This starting compound was subjected to iterative rounds of structural modification coupled with phenotypic activity screening. Halogen analogues of LG182 were found to improve the compound. Halogenation is often used in late-stage lead optimization, improving drug metrics⁸ and sometimes even increasing binding affinity through the formation of an additional halogen bond.⁹ Here, we present studies on a panel of compounds with halogen substitutions in place of an OMe functional group. Bromine substitution produced a promising compound, which we characterize here as LXY18.

Previous work with these 4-phenoxy-quinoline-based derivatives focused on structure–activity relationships that could be discerned from systemically modifying side groups attached to varying locations of the 3-ring core (Scheme 1).^6,7 Our potent in vitro compound, LG182, harbors an aromatic ring with di-meta substitutions of an OMe group and an acetamide group. Leaving this structural feature alone, we explored substitutions at the position labeled R of the ring most distal to the di-meta ring. In LG182, this site is occupied by an OMe group. However, many other substitutions at R were not well tolerated. Since the presence or absence of the OMe group did impact potency, we surmised substitution at this site was important but maybe size-limited. In addition, positive contribution of fluorine to the potency prompted us to explore the other halogen members at this position. These additional analogues were synthesized as described in Scheme 1.

Scheme 1. Synthesis of 6-Halo, 4-Phenoxy-quinoline Derivatives.

Reagents and conditions are as follows: (a) K₂CO₃, DMF, N₂, 115 °C, 12 h, 50–89%.

Retinal pigment epithelial (RPE) cells expressing the MYC oncoprotein plus the histone 2B protein fused to a green fluorescent protein (H2B-GFP) were used to assess drug potency in vitro. The minimum effective concentration (MEC) in RPE-MYC^H2B-GFP cells that induce phenotypes reminiscent of AURKB inhibition was used to compare compounds, as this was previously found to be an effective metric.^6,7 The most easily discernable outcome from inhibiting the localization of the CPP complex is the induction of polyploid cells, as AURKB localizes during telophase to the midbody and mediates the induction of cytokinesis.¹⁰ However, the MECs for mitotic arrest, polyploidy, and cell death were the same in this assay as we observed before,⁷ and we referred to them as MECs without discrimination (Table 1). Substitution with either of three heavier halogens, Cl, Br, and I, increased the potency to a similar extent, reducing MEC 3- to 4-folds, relative to OMe-substituted LG182 and F-substituted LXY19.

Table 1. MECs of Quinoline Analogues To Induce Polyploidy^a.

^aThe screening cell line RPE-MYC^H2BGFP was treated with the indicated compounds at concentrations from 0.5 nM to 1 μM for 72 h. The MECs that elicit multinucleation were determined. Multinucleation was defined as more than 5% of multinucleated cells in the population after deduction of the basal level of polyploidy in the control group treated with 0.1% of DMSO. The mean percentage of polyploid cells at the MEC was shown. Values in parentheses were derived from three independent experiments. MEC data are presented as mean ± SD from three independent experiments. Scale bars: 10 μm.

Immunofluorescence (IF) analysis with markers that are used as surrogates for the activity of AURKs can be used to assess the catalytic inhibition of these mitotic kinases as we described before.^6,7 For AURKB, the intensity of Histone 3 phosphorylation at Serine 10 (H3Ser10P), a direct target of AURKB in mitosis, is a well-established surrogate of its kinase activity. We have previously combined phenotypic screening in RPE-MYC^H2B-GFP cells with IF for surrogate markers of AURK activity as an MIPS method.^6,7 Here, we continued to assess compounds with this combination approach to ensure the halogenated compounds are not catalytic inhibitors but added an IF assay to ensure they also disrupt AURKB positioning at the spindle midzone during anaphase. Halogenated compounds did so, irrespective of which halogen was substituted at R. IF examination of earlier stages of mitosis suggested that localization of AURKB on prometaphase kinetochores was not affected in two human cancer cell lines NCI-H23 and DU-145 and a model cell line RPE-MYC^Bcl2 (Figure S1). Furthermore, AURKB was still present on anaphase chromosomes in all three cell lines (Figures 1A and S2), suggesting the compounds prevented the release of AURKB from chromosomes at the anaphase onset. Consequently, these compounds prevented repositioning at the spindle midzone, so despite our previous designation of this class of compounds as AURKB localization inhibitors, these compounds may be better described as inhibitors of AURKB relocation, as earlier localization during mitosis does not seem to be altered. Each compound prevented AURKB relocation and induced polyploidy at similar threshold effective concentrations in the RPE-MYC^H2B-GFP cells, indicating that accumulation of polyploid cells resulted from a loss of AURKB functions in cell division. None of these compounds inhibited phosphorylation of the AURKB substrate Histone 3 at Serine 10 (Figures 1B and S3 and Table S5) or AURKA autophosphorylation at Thr288 (Table S5 and Figures S3 and S4), so these compounds do not inhibit the catalytic activity of AURKs.

Figure 1.

Effect of quinoline analogues on kinase activity and localization of AURKB. RPE-MYC^BCL2 cells were treated for 6 h with the indicated compounds before staining with DAPI alongside antibodies that recognize H3Ser10P, AURKB, or kinetochore proteins. Cells treated with the vehicle (0.1% of DMSO) were used as a negative control. (A) Effect on AURKB mitotic localization. (B) Effect on phosphorylation of H3 at Ser10. For quantitation in (A and B), the fluorescence intensity along the white dashed line was determined using ImageJ software and was plotted as a relative gray value over distance in pixels. More than 20 mitotic cells in each group were scored, and all displayed similar images as the representative cell presented. Scale bars: 10 μm.

To confirm that these halogenated compounds have a broad anticancer activity, we assayed their bioactivities in a panel of 17 cancer cell lines from various tissue origins. The formation of multinucleation was used as a convenient readout for the disablement of AURKB.⁷ Consistent with the findings in the screening assay, SY142, LXY18, and LC08 had lower MECs in inducing polyploidy relative to LG182 and LXY19 in the majority of the human cancer cell lines (Figure 2), suggesting that substitutions with heavier halogens improved potency in general in human cancer cell lines. We reached the same conclusion when the half-maximal inhibitory concentration (IC₅₀) was evaluated (Figure 2C).

Figure 2.

Induction of multinucleation by quinoline analogues. RPE-MYC^BCL2 cells and 17 human cancer cell lines were treated for 72 h with the indicated compounds at the indicated concentrations (A) or at a concentration range from 0.5 nM to 1 μM (B). Cells were then stained with DAPI to visually assess the MEC using multinucleation as a metric of activity. (A) Induction of multinucleation. Scale bars: 10 μm. (B) Violin plot of the MECs for polyploidy. Each black dot denotes a cancer cell line and the RPE-MYC^H2B-GFP cell line is in purple. The blue dots indicate the mean of MEC for a given compound in 17 cancer cell lines. (C) Violin plot of the half-maximal inhibitory concentration (IC₅₀) for polyploidy. Each black dot denotes a cancer cell line. The blue dots indicate the mean of IC₅₀ for a given compound in 17 cancer cell lines. The MTT assay was used to determine the growth inhibition by the indicated compounds as described in the material and method section included in the supplementary file.

Halogenation of sp² carbons is known to increase lipophilicity and improve membrane permeability and oral absorption.¹¹ We next examined whether halogenation impacted plasma drug exposure. Compounds were delivered to mice orally using three types of delivery vehicles. We tested lipid-soluble corn oil, hydrophilic hydroxypropyl methylcellulose (HPMC), and water-soluble, neutral polyethylene glycol 300 (PEG300), which represent oil, suspension, and aqueous delivery modes, respectively. Plasma samples were collected from 0 to 6 h, and drug concentration was quantified by LC–MS/MS (Figure 3A). Among the compounds tested, LXY18 achieved the highest level of plasma exposure with each of the three formulations, as judged by the dose-normalized area under curve (AUC_0–6h) (Figure 3B) or the AUC_0–6h after further normalization to bioactivity (Figure 3C). Among the three oral delivery formulations, the largest AUC or maximum plasma concentration (C_max) value for LXY18 was obtained with lipid-soluble corn oil (Figure 3B–E).

Figure 3.

Pharmacokinetic parameters of quinoline analogues after oral gavage. Compounds were suspended or dissolved into the indicated formulation and delivered to nude mice (8–11 weeks old female BALB/c). Two mice were used for each formulation. (A) Schematic diagram of timeline for blood collection after a single oral administration. (B) Scatter plot of the dose-normalized AUC_0–6h value. The area under the plasma concentration–time curve from time 0 to 6 h was divided by the dose. (C) Scatter plot of the AUC_0–6h/dose/MEC (dose-normalized AUC_0–6h divided by the mean of MEC). (D) Scatter plot of the activity normalized C_max (C_max/MEC). (E) Scatter plot of the maximum plasma concentration (C_max) after a single dose of 50 mg/kg. Each dot in (B–E) represents a mouse. The color indicates the formulation described in (A).

The above findings suggested that LXY18 had both improved potency and plasma exposure after an oral dose and was a top compound among the halogenated analogues. The pharmacokinetic parameters of LXY18 were studied further in Wistar rats (Table 2). After oral administration of 2 mg/kg of LXY18 in corn oil, we observed a peak absorption (C_max) of 0.46 ± 0.35 μM 1 h post administration and a half-life (T_1/2) of 2.84 ± 1.36 h. Compared with the AUC of the same amount of LXY18 intravenously delivered, the overall oral availability was calculated to be 51.40%.

Table 2. Pharmacokinetic Parameters of LXY18 after a Single Oral or Intravenous Administration in Rats^a.

parameters	unit	PO			IV
		2 mg/kg (n = 2)	10 mg/kg (n = 3)	50 mg/kg (n = 3)	2 mg/kg (n = 3)
Cmax	μM	0.46 ± 0.35	1.37 ± 0.38	3.56 ± 1.11
Cmax/dose	μM	0.23 ± 0.18	0.14 ± 0.04	0.07 ± 0.02
Tmax	h	1.00 ± 0.00	0.67 ± 0.29	0.67 ± 0.29
T1/2	h	2.84 ± 1.36	2.30 ± 1.66	5.26 ± 0.41	3.10 ± 2.41
CL	L/h/kg	3.15 ± 1.43	3.85 ± 1.40	4.43 ± 1.84	1.85 ± 0.93
MRT0–∞	h	4.41 ± 0.80	6.39 ± 2.35	9.80 ± 4.27	2.57 ± 2.74
AUC0–∞	μM·h	1.83 ± 0.83	7.46 ± 3.13	32.10 ± 10.75	3.51 ± 2.20
AUC0–∞/dose	μM·h/(mg/kg)	0.91 ± 0.42	0.75 ± 0.31	0.64 ± 0.22	1.76 ± 1.10
Vz	L/kg	11.50 ± 0.32	12.89 ± 10.45	33.88 ± 15.16	8.53 ± 7.54
Vss	L/kg				4.78 ± 5.69
bioavailability	%	51.40%	41.92%	36.09%

^aEight-week-old female Wistar rats, 180–220 g each, were treated with either 2 mg/kg of LXY18 dissolved in PEG300 at pH 5.63 by tail vein injection (IV) or the indicated dose of LXY18 suspended in corn oil by oral gavage (PO). The number of rats (n) are indicated above. C_max is the maximum concentration; C_max/dose is C_max divided by dose; T_max is the time to maximum concentration; T_1/2 is the terminal half-life; CL is the total body clearance; MRT_0–∞ is the mean residence time extrapolated to infinity; AUC_0–∞ is the area under the plasma concentration–time curve from time zero to infinity; AUC_0–∞/dose is AUC_0–∞ divided by dose; Vz is the volume of distribution based on the terminal phase; Vss is an estimate of the volume of distribution at a steady state based on the last observed concentration; and bioavailability = [AUC_0–∞/dose (PO)]/[AUC_0–∞/dose (IV)] × 100%. Data are presented as mean +/– SD.

Next, three oral doses 2, 10, and 50 mg/kg of LXY18 were compared. Dose-linear increases in the AUC and C_max were observed. The bioavailability decreased from 51.40 to 41.92 and 36.09% when the dose of LXY18 was elevated from 2 to 10 and 50 mg/kg. Rats that received two lower oral doses of LXY18 had a similar apparent volume of distribution during the terminal phase (Vz) of 11.50–12.89 L/kg and a half-life of 2.30–2.84 h. The half-life increased to 5.26 h and Vz was elevated to 33.88 L/kg in rats that were treated with 50 mg/kg. After the tail vein injection of LXY18, the terminal phase Vz and steady-state volume of distribution (Vss) were determined to be 8.53 ± 7.54 and 4.78 ± 5.69 L/kg, respectively (Table 2). The compound was widely distributed since the Vz or Vss value was more than sixfold larger than the rat’s total body water volume (0.7 L/kg).

Elucidation of drug tissue distribution is usually exploited to confirm suitable exposure for inhibition of the therapeutic target and can also identify potential tissues where toxicity may be problematic. Quantitative tissue distribution studies (Figure 4) were pursued to confirm the wide drug tissue distribution predicted by Vz and Vss values (Table 2). Tissues were dissected at 1, 4, or 8 h after a single oral administration of LXY18 (Figure 4A). A plasma LXY18 concentration of 3.22 ± 0.97 μM was found 1 h after oral administration, and LXY18 was detected in all tissues and organs examined (Figure 4B). The highest LXY18 exposure was present in adipose tissue (AUC_1–8h = 8.28 ± 2.19 μM·h). The liver had the lowest LXY18 exposure (AUC_1–8h = 0.87 ± 0.54 μM·h), likely resulting from metabolic clearance of the compound in the liver. Importantly, LXY18 was detected in brain tissue and the cerebrospinal fluid after 1 h with a concentration of 0.59 ± 0.27 and 0.02 ± 0.00 μM, respectively, suggesting that the compound transits the blood–brain barrier readily.

Figure 4.

LXY18 tissue distribution studies. Wistar rats received a single oral dose of 50 mg/kg LXY18. The peripheral blood was collected at 1, 4, and 8 h after drug administration. The plasma drug concentration at each time point was determined by LC–MS/MS. Three nine-week-old female Wistar rats, 200–230 g each, were used at each time point. (A) Flow chart of the tissue collection scheme for drug distribution studies. (B) Drug concentrations in different tissues.

Plasma concentration has generally been considered a surrogate for the concentration of drug available to reach a tumor. However, intratumoral drug concentrations can vary considerably from plasma levels due to heterogeneity in the tumor microenvironment.¹² Intratumoral concentration of LXY18 was further tested in immunodeficient mice bearing xenografts of two different human cancer cell lines. One was a human gastric cancer cell line NCI-N87 and the other was a human lung cancer cell line NCI-H23. In this experiment, we quantified LXY18 after mice had received repeated treatments (Figure 5A). This design is to examine if LXY18 might induce metabolic enzymes and consequently reduce LXY18’s plasma exposure after multiple treatments. The maximum tolerated dose (MTD) of LXY18 orally delivered in corn oil was determined to be 100 mg/kg/day in BALB/c nude mice under a long-term daily repeat treatment. We chose to perform treatment with 50 mg/kg of LXY18 twice a day for 5 days before LXY18 was quantified in the plasma after the last oral treatment.

Figure 5.

PK properties and drug concentrations in tumor tissues. Female, 12–14 week-old BALB/c nude mice bearing NCI-H23 and NCI-N87 xenografts were treated by oral gavage with 50 mg/kg of LXY18 dissolved in corn oil, twice a day at 9:00 a.m. and 5:00 p.m. for 5 consecutive days. On the sixth day, 100 μL of blood was collected at 0, 0.5, 1, 3, and 6 h after oral administration of LXY18 at 9:00 am. Tumors were also harvested at the last blood collection time to quantify the LXY18 concentration. Three mice were used in each group. (A) Experiment design in tumor-bearing mice to examine plasma LXY18 exposure levels and accumulation in tumor tissues. (B) Plasma LXY18 exposure after repeated administration. Each point, here and in (C), represents the mean ± SD. (C) LXY18 concentration in blood and tumor tissues at 6 h after drug administration.

Both tumor models yielded similar plasma drug concentration vs time curves (Figure 5B). Multiple doses to the xenografted mice decreased C_max 2.4–3.8-folds relative to mice that received a single dose (Figure 5C vs Figure 3E). Likewise, the normalized AUC_0–6h declined 1.6–1.7-folds (Figure 5B vs Figure 3B). The plasma concentrations of LXY18 at 6 h were comparable among two mouse models with different xenografts (0.35 ± 0.18 μM for NCI-H23 versus 1.04 ± 0.64 μM for NCI-N87) (Figure 5B). LXY18 concentrations in the corresponding tumor tissues at 6 h approximated those found in the plasma (Figure 5C).

In addition to the plasma drug concentrations at 1, 4, and 8 h (left y-axis), the area under the concentration–time curve from time 1 to 8 h (AUC_1–8h) is also presented (right y-axis).

Given the favorable oral bioavailability and accumulation in tumor cells observed in vivo, LXY18 was deemed suitable to test for efficacy in murine tumor models. NCI-N87-bearing mice were treated with LXY18 for 33 consecutive days with the control group receiving an equal volume of the vehicle. No obvious adverse effects were observed in the treatment group, and the body weight remained consistent with the control group (Figure 6A). LXY18 significantly reduced the tumor volume, with a tumor growth inhibition of 55.13% ± 29.05% (p < 0.001) (Figure 6B), and tumor weight at the endpoint (Figure 6C,D). Consistent with the observed reduction in xenograft volume, the percentage of cells positive for Ki-67 in the treatment group (33.94% ± 10.06%) was significantly reduced relative to the control group (59.4% ± 6.34%, p = 0.0004 by Student’s t-test) (Figure 6E,F). The blood vessel density in LXY18-treated tumors was reduced relative to the size-matched tumors in the control group (Figure S5). Consistent with the effect of LXY18 on cultured NCI-N87 cells, the compound engendered polyploidy (Figure 7A,B), elevated the mitotic population (Figure 7C,D), and triggered apoptosis (Figure 7E,F) in NCI-N87 tumor tissues. These findings imply that the tumor-suppressive activity of LXY18 is associated with inactivation of AURKB. LXY18 showed no sign of suppressing xenografts of NCI-H23 in NCG mice (Figure S6), indicating the importance of identifying predictive biomarkers for LXY18.

Figure 6.

LXY18 inhibits the growth of NCI-N87 human gastric carcinoma xenografts. Mice bearing xenografts were treated with LXY18 for 33 days (100 mg/kg b.i.d.), monitored twice a week for tumor sizes, and then euthanized to harvest tumor tissues. (A) Mouse body weight. (B) Tumor growth curve. (C) Gross morphology of tumors. (D) Tumor weight. (E and F) Tumor proliferation. Tumor tissues were immunohistochemically stained for the proliferation marker Ki-67 (E). Three randomly chosen fields in each tumor section were scored for Ki-67-positive cells and data from n = 6 mice are presented as mean ± SEM (F). p values were calculated using an unpaired Student’s t-test using GraphPad Prism 8.0.2 software (ns = not significant: *p <0.05%, **p <0.01%, ***p < 0.001%).

Figure 7.

LXY18 elicits mitotic arrest, polyploidy, and apoptosis in vivo. Mice bearing a xenograft of the human NCI-N87 gastric cancer cell line were treated with 100 mg/kg of LXY18 twice a day for 5 days and then euthanized to harvest tumor tissues. The tumor tissues were analyzed for polyploid (A, B) and mitotic population (C, D) by immunohistochemical staining of CD44 plus staining DNA with DAPI (A, B) and phosphorylation of Histone 3 at Ser10 (C, D), respectively. Apoptosis was detected by the TUNEL assay (E, F). Three randomly chosen fields in each tumor section from two or three mice were scored for each phenotype, and data are presented as mean ± SEM. The p value is calculated by unpaired t-test in GraphPad Prism 8.0.2, not significant: p > 0.05%, **p < 0.01%.

Discussion and Conclusions

Quinoline-based derivatives have been developed that inhibit the catalytic activity of several mitotic kinases, including AURKs.¹³ We explored noncatalytic inhibitory compounds from this class, which work by mislocalizing AURKB and other CPP complex components.⁷ Here, we have further characterized this activity for an optimized group of halogenated compounds, of which Br-substituted LXY18 and other heavy halogen-substituted analogues are shown to act as potent AURKB relocation blockers.

LXY18 can be delivered as an effective oral oncology agent, with a potent and broad spectrum of antimitotic activity in cancer cell lines, widespread tissue distribution, and the ability to cross the blood–brain barrier. The first generation of quinoline-based AURKB mislocalizers had limited activity in vivo and failed to suppress tumorigenesis in our recently published study.⁷ In the present study, LXY18 becomes the first reported quinoline-based compound that suppresses tumorigenesis by preventing the mitotic relocation of AURKB.

LXY18 might prevent the AURKB repositioning by binding and interfering with any component of the CPP complex. Furthermore, a variety of mitotic regulators are essential for the timely positioning of AURKB at the spindle midzone.¹⁴ Disabling any of these proteins or blocking their interaction with the CPP complex might hinder the relocation of AURKB at anaphase onset. Testing the physical interaction between LXY18 and these regulators individually in a label-free assay might provide insight into the mode of action of LXY18. Alternatively, pulldown assays followed by LC–MS/MS analysis of bound protein might reveal a mitotic target for LXY18.

In summary, we have found that LXY18 possesses potent in vitro and in vivo anticancer activity that can be delivered effectively using an oral formulation. Further characterization of metabolism, toxicity profile, and relevant targets of LXY18 and the identification of biomarkers that predict treatment response will support continued advancement toward clinical applications.

Safety Statement

No unexpected or unusually high safety hazards were encountered.

Glossary

Abbreviations Used

AURKA

Aurora kinase A

AURKB

Aurora kinase B

AURKC

Aurora kinase C

cLogP

calculated octanol/water partition coefficient

CPP

chromosomal passenger protein

DAPI

4′,6-diamidino-2-phenylindole

DMF

N,N-dimethylformamide

DMEM

Dulbecco’s modified Eagle’s medium

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

DMF

N,N-dimethylformamide

DMAP

4-dimethylamino pyridine

MEC

minimum effective concentration

MIPS

mechanism-informed phenotypic screening

NaH

sodium hydride

OD

optical density

PE

petroleum ether

PK

pharmacokinetic

PD

pharmacodynamics

RPE

retinal pigment epithelium

rt

room temperature

SARs

structure–activity relationships

THF

tetrahydrofuran

TLC

thin layer chromatography

Data Availability Statement

All data analyzed during this study are included within the manuscript (and its Supporting Information files). Data deposition does not apply to the current study.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00054.

• Additional figures and tables of bioassays, analytic data (¹H NMR), general experimental procedure, starting material preparation, and biology experimental methods (PDF)

Author Contributions

^# Lead Contact: dun.yang@mbicr.org (D.Y.).

Author Contributions

G.L. synthesized all compounds. Q.S., C.Y., J.K., Y.L., and H.L. performed all biology experiments. T.Z. and X.Z. performed PD/PK experiments. J.L. performed HPLC and LC–MS/MS analyses. Q.S., T.D.A., N.C, J.Z., and D.Y. participated in the biological data analysis. G.L., J.L., and N.N. analyzed NMR and LC–MS/MS data. All participated in writing the manuscript. D.Y. finalized the manuscript. All authors are employed by the J. Michael Bishop Institute of Cancer Research and/or Anticancer Bioscience, Chengdu, China. J.L., T.Z., and Q.S. contributed equally to this paper.

The J. Michael Bishop Institute of Cancer Research receives funding through an endowment from Anticancer Bioscience, a company actively engaged in the commercial development of cancer therapeutics.

The authors declare the following competing financial interest(s): Anticancer Bioscience has submitted intellectual property filings (PCT/CN2021/091425 and PCT/CN2021/127309) that cover compounds described in this study. Dr. D.Y. and J.Z. are stockholders of Anticancer Bioscience.