Synthesis and Biological Evaluation of Novel FiVe1 Derivatives as Potent and Selective Agents for the Treatment of Mesenchymal Cancers

Abstract

Epithelial-mesenchymal transition (EMT) endows stem cell-like properties to cancer cells. Targeting this process represents a potential therapeutic approach to overcome cancer metastasis and chemotherapy resistance. FiVe1 was identified from an EMT-based synthetic lethality screen and was found to inhibit the stem cell-like properties and proliferation of not only cancer cells undergoing EMT, but also more broadly in mesenchymal cancers that include therapeutically intractable soft tissue sarcomas. FiVe1 functions by directly binding to the type III intermediate filament protein vimentin (VIM) in a mode that induces hyperphosphorylation of Ser56, which results in selective disruption of mitosis and induced multinucleation in transformed VIM-expressing mesenchymal cancer cell types. Cell-based potency (IC₅₀ = 1.6 μM, HT-1080 fibrosarcoma), poor solubility (< 1μM) and low oral bioavailability limits the direct application of FiVe1 as an in vivo probe or therapeutic agent. To overcome these drawbacks, we performed structure-activity relationship (SAR) studies and synthesized a set of 35 new compounds, consisting of diverse modifications of the FiVe1 scaffold. Among these compounds, 4e showed a marked improvement in potency (IC₅₀ = 44 nM, 35-fold improvement, HT-1080) and cell type selectivity (19-fold improvement), when compared to FiVe1. Improvements in the potency of 4e, in terms of overall cytotoxicity, directly correlate with VIM Ser56 phosphorylation status and the oral bioavailability and pharmacokinetic profiles of 4e in mouse are superior to FiVe1. Successful optimization also resulted in potent and selective derivatives 11a, 11j and 11k, which exhibited superior pharmacological profiles, in terms of metabolic stability and aqueous solubility. Collectively, these optimization efforts have resulted in the development of promising FiVe1 analogs with potential applications in the treatment of mesenchymal cancers, as well as in the study of VIM-related biology.

Graphical Abstract

INTRODUCTION

Metastasis is responsible for about 90% of cancer-associated mortality.¹ A fundamental biological event that has been associated with cancer metastasis is the epithelial-mesenchymal transition (EMT) program, which modifies the adhesion molecules expressed by epithelial cells, allowing them to adopt a mesenchymal phenotype with migratory and metastatic behavior.^2–4 The intermediate filament (IF) protein vimentin (VIM) is a canonical marker of EMT and a requisite regulator of mesenchymal cell migration.^{5, 6} VIM is a type III-IF protein that maintains cell integrity in mesenchymal cell types and plays essential roles in cell migration, motility and adhesion.⁷ It is expressed abundantly in cells of mesenchymal origin, and overexpressed in many tumor types, where expression levels correlate with aggressiveness and poor clinical outcomes.⁸ Vimentin overexpression in different mesenchymal cancer cell lines, its importance in EMT, as well as its correlation with increased cancer cell growth, invasion, and migration provide rationale for VIM as a potential therapeutic target for the treatment of diverse mesenchymal cancer types. Soft-tissue sarcoma (STS) constitutes a group of more than 50 different subtypes of rare tumors, comprising less than 1% of adult cancers.⁹ Examples of malignant mesenchymal-derived cell lines from STSs are HT-1080 (fibrosarcoma), RD (rhabdomyosarcoma) or GCT (fibrous histiocytoma) cells, where HT-1080 have been widely used to investigate the role of vimentin in mesenchymal cancer cells.^{10, 11} STSs are derived from mesenchymal cells and consequently express high levels of VIM.¹² As such, it is reasonable to hypothesize that therapeutic strategies that target vimentin could stimulate anticancer effects in a broad range of STSs.

Despite their therapeutic potential in the context of diverse disease states, small molecule probes or drugs that specifically target intermediate filament proteins are lacking. An even fewer number of small molecules have been reported that directly and selectively bind to VIM and modulate its function (Figure S1). The non-selective natural product withaferin A (WFA) was shown to bind vimentin through covalent modification of a cysteine residue present in the highly conserved ɑ-helical coiled coil 2B domain.¹³ WFA induced anti-metastatic features in breast cancer cells by inducing vimentin disassembly and serine 56 phosphorylation.¹⁴ A limited number of additional small molecules have been reported to bind to vimentin, albeit with varying degrees of evidence for selectivity.^15–19 Previously,¹² we discovered a cinnoline-based compound from a synthetic lethality-based high throughput screening (HTS) campaign, termed FiVe1, which selectively and irreversibly inhibited the growth of mesenchymal cancer cells by binding to VIM. Mechanism of actions studies and in vitro labeling experiments suggested that FiVe1 binds specifically to the rod domain of VIM, in a mode that promotes vimentin disorganization and hyperphosphorylation leading to mitotic catastrophe, multinucleation and the loss of stemness. Notably, FiVe1 was not found to be an inhibitor of any cell cycle or cytokinesis-related kinase when profiled against a panel of ~100 kinases at concentrations up to 10 μM.¹² While profound cell type selective biological profiles were observed in vitro, including irreversible activity in transformed and reversible activity in non-transformed mesenchymal cell types, several properties of FiVe1 preclude its in vivo characterization in preclinical models. These include low metabolic stability and low aqueous solubility (<1 μM), which predicted poor pharmacokinetics properties incompatible with the relative poor, albeit selective, cell-based potency properties of the compound.

In order to address these shortcomings and enable assessment of the translational potential of this unique cinnoline-containing chemical series as a therapy for mesenchymal cancers, in this work we completed a medicinal chemistry campaign aimed at improving the potency and pharmacological properties of FiVe1. Herein, we report the design, synthesis, as well as the biological and pharmacological evaluation of novel FiVe1 derivatives that possess improved cell type selective antiproliferative properties and physicochemical profiles suitable for potential preclinical applications.

RESULTS AND DISCUSSION

Lead Optimization Process

To optimize FiVe1, we performed a multidimensional optimization campaign, surveying a wide range of chemical diversity. We used a systematic optimization approach to explore the three different ring systems that constitute the FiVe1 structure (A, B, and C) (Figure 1).

Figure 1.

FiVe1 ring systems examined as part of multi-dimensional SAR and optimization studies.

The challenge and objective was to identify tolerant sites for chemical modifications that retain or improve bioactivity and improve physicochemical and metabolic stability properties. First, we investigated the in vitro metabolism products of FiVe1, to help predict sites that represent potential metabolic liabilities and inform optimization of the FiVe1 structure. FiVe1 was incubated with mouse hepatocytes, and the metabolites were identified by liquid chromatography mass spectrometry (LC-MS/MS) with relative concentrations being estimated based on peak area in the corresponding chromatograms (λ = 252–420 nm) (Table 1).

Table 1.

Metabolites of Compound FiVe1 Identified in Incubations with Mouse Hepatocytes.

Metabolite	Biotransformation(s)	% Peak Area
M1	Dealkylation	33
M2	Oxygenation/Dealkylation	15
M3	Di-Oxygenation	1
M4	Di-Oxygenation	1
M5	Oxygenation	28
M6	Oxygenation	14
M7	Oxygenation	2
FiVe1		6

Not surprisingly, based on its flat highly lipophilic character, most identified metabolites (45% of total species) corresponded to species that were metabolized via oxygenations (M3, M4, M5, M6, and M7), with 33% of the species corresponding to metabolites obtained via dealkylation (M1). An oxygenation/dealkylation transformation afforded metabolite M2 (15%). The remaining 6% of the species corresponded to the parent molecule, FiVe1. These biotransformations did not occur in a specific region in the molecule but similar ratios were observed around the three moieties of FiVe1. FiVe1 selectively inhibits the growth of VIM-expressing mesenchymal cancer cell lines through a VIM-dependent mechanism, with a cell-based therapeutic index of >10 being observed when comparing anti-proliferative effects in cells that lack VIM expression.¹² To evaluate the therapeutic potential of this compound series in the context of STSs, the impact of all target compounds on cell viability was evaluated using the VIM-expressing sarcoma cell lines HT-1080, RD, GCT and a VIM non-expressing epithelial breast cancer cell line (MCF-7). The selectivity index (SI), which indicates the cytotoxic selectivity, was calculated from the ratio of the IC₅₀ values obtained from HT-1080 cells against the IC₅₀ values from MCF-7 cells. Doxorubicin was used as positive control.

SAR Analysis of the A Ring System

Initial studies suggested that the meta C-3 and C-5 positions of phenyl ring A are tolerant to substitution (Figure S2).^10,20Additionally, as shown for compounds S13, S14, S15 in Figure S2, replacement of the phenyl system with a more electron-deficient heterocyclic ring system, to address this potential metabolically labile cytochrome P450 oxidation-prone feature, while conserving structural characteristics of the pharmacophore,²¹ results in compounds that do not inhibit mesenchymal cancer cell growth at concentrations <20 μM. As such, we evaluated a range of substitutions to the C-3 (R¹) and C-5 (R²) positions of the 3-chlorophenyl region A system, while maintaining the piperazine moiety and 8-chlorocinnoline core in place. Compounds 4a–j, containing electron-withdrawing and -donating substituents, including hydrogen, chlorine, nitrile, hydroxy, methoxy, pyrrolidine, amine, and amide, were evaluated. Bioactivities of the resultant compounds are shown in Table 2.

Table 2.

Anti-Proliferation Activities of Compound Series 4 against HT-1080, RD, GCT, MCF-7 cells, and Selectivity Index (SI) of HT-1080 vs MCF-7.

Compound	IC50 (nM)				SI (HT-1080 vs MCF-7)
	HT-1080	RD	GCT	MCF-7
4a	412 ± 64	623 ± 97	315 ± 43	> 20000	>49
4b	378 ± 39	402 ± 62	189 ± 16	> 20000	>53
4c	37 ± 2	33 ± 3	33 ± 3	15510 ± 7800	419
4d	598 ± 28	884 ± 149	402 ± 34	> 20000	>33
4e	44 ± 1	61 ± 9	49 ± 5	11150 ± 4766	253
4f	> 20000	> 20000	> 20000	> 20000	n.d.
4g	86 ± 2	83 ± 14	57 ± 7	12070 ± 5488	140
4h	956 ± 148	1280 ± 631	656 ± 71	> 20000	>21
4i	647 ± 95	1322 ± 227	498 ± 53	> 20000	>31
4j	10810 ± 2912	7407 ± 1159	10750 ± 1147	> 20000	>2
FiVe1	1564 ± 288	3095 ± 555	1686 ± 349	> 20000	>13
Dox	24 ± 5	170 ± 12	137 ± 33	139 ± 40	6

n.d.: not determined

With the exception of 4f and 4j, all of these compounds showed similar or superior potency to that of FiVe1 when evaluated in VIM-expressing sarcoma cell lines and were all more all found to be more selective when evaluated in MCF-7 (VIM non-expressing control cell line). Compounds 4a–b, 4e, 4g, and 4i corresponded to replacement of the hydrogen atom at the C-5 position by chlorine, nitrile, methoxy, hydroxy, and amino substituents, respectively, while holding a chloro- substituent in the C-3 position (R¹). Compounds 4a and 4b showed superior activity and selectivity, with an ~4-fold improvement in potency, when chlorine or nitrile were introduced on the phenyl ring at C-5 (Table 2). When hydrogen at the C-5 position was replaced by methoxy or hydroxy groups (4e and 4g, respectively), the resulting compounds were found to be not only >20-fold more potent when compared to FiVe1, but also >10-fold more selective (Table 2).

As shown in Figure 2, especially remarkable was the observed IC₅₀ value for 4e when compared with FiVe1 (44 nM vs. 1564 nM in HT-1080 cells), with an observed cell-based selectivity index (SI) of 253 (vs. MCF-7 cells). A notable trend was observed for methoxy-substituted compounds. Replacement of the 3-chloro group (R¹) of FiVe1 with a methoxy group, 4d, showed an improvement of >2-fold in cytotoxicity and >2-fold in selectivity. Further, introduction of a methoxy group in both the C-3 and C-5 positions, compound 4c, resulted in a compound with potency in the double digit nanomolar range (IC₅₀ = 37 nM in HT-1080 cells; 419-fold selective vs. MCF-7 cells). Substitution of C3- and/or C5- positions with hydroxy groups (4g–h) provided less potent compounds when compared with their methoxy counterparts 4e and 4c, respectively, while still demonstrating a better activity and selectivity profile when compared to FiVe1. Aniline derivative 4i was demonstrated to have an ~2-fold improvement in potency and selectivity. However, substitution of the aromatic amine group to afford an amide, compound 4j, was found to be detrimental to activity (IC₅₀ values ~10 μM). This observation suggested the functionalities such as amides containing a carbonyl group at the alpha-carbon position of the R² substituent are not favorable, which might be attributed to inadequate or disruptive interactions with VIM. These results are in accordance with the activity observed for compound 4f, which displayed a total loss in activity, suggesting again that bulky substituents in that position are detrimental to the desired activity profile. Collectively, analysis of this data shows a clear trend when comparing how different electron-withdrawing and -donating substituents at the C-3 and C-5 positions of this phenyl ring system serve to significantly modulate cell type-selective antiproliferative activity in mesenchymal cancer cell lines.

Figure 2.

Logarithmic dose-response curves and IC₅₀ values for FiVe1 and 4e in a VIM-expressing mesenchymal cell line (HT-1080) and a non-VIM-expressing cell line (MCF-7). Cells were treated with FiVe1 and 4e at different concentrations for 72 h, followed by the CellTiter-Glo proliferation assay. Values shown are mean ± SD.

SAR Analysis of region B

To continue SAR studies, in the second-round of modifications, we investigated replacement of the piperazine linker moiety (region B), while maintaining the 8-chlorocinnoline and 3-chloro phenyl ring systems of FiVe1

These modifications were introduced not only to assess impact on cell type selective antiproliferative activity, but also to try to enable the disruption of potential tight crystal packing interactions in order to bring about improvements in solubility. As shown in Table 3, compounds 7a–f were synthesized and their in vitro activities and selectivity profiles were determined. Compounds 7a–c, with substitutions on or bridging of the piperazine core displayed moderate loss in activity, with potency IC₅₀ values within 5-fold of that observed for FiVe1 (Table 3). Methyl substitution on the 2-position of the piperazine (7a) led to an ~2-fold loss in antiproliferative potency, when compared with its analog 4a, while maintain equivalent selectivity. Introduction of a cyclic constraint (7b and 7c) was found to be tolerated, although a small loss in potency was observed when compared to FiVe1 (Table 3). In contrast, compounds 7d–f, containing a replacement of the piperazine core structure, were found to be inactive at concentrations <20 μM. When the piperazine was expanded to diazepane (7d), replaced by a classic piperazine bioisostere [3.3.0] system (7e), or replaced by piperidine (7f), the resulting FiVe1 analogs loss appreciable desired biological activity. These results demonstrate that the distances between the piperazine nitrogen atoms, as well as the presence of heteroatoms at both positions, appears to be essential to retain activity. Additionally, these findings demonstrate that the introduction of substituents around the piperazine core can be tolerated, albeit it with a resulting decrease in observed potency.

Table 3.

Anti-Proliferation Activities of Compound Series 7 against HT-1080, RD, GCT, and MCF-7 cells and Selectivity Index (SI) of HT-1080 vs MCF-7.

Compound	IC50 (nM)				SI (HT-1080 vs MCF-7)
	HT-1080	RD	GCT	MCF-7
7a	1148 ± 90	1072 ± 80	1274 ± 180	> 20000	>17
7b	3228 ± 854	5600 ± 898	8862 ± 1487	> 20000	>6
7c	5566 ± 443	6152 ± 535	4034 ± 482	> 20000	>4
7d	> 20000	> 20000	> 20000	> 20000	n.d.
7e	> 20000	> 20000	> 20000	> 20000	n.d.
7f	> 20000	> 20000	> 20000	> 20000	n.d.
FiVe1	1564 ± 288	3095 ± 555	1686 ± 349	> 20000	>13

n.d.: not determined

SAR Analysis of region C

To examine the SAR for region C of the FiVe1 structure, we designed compounds 10a–h to investigate alternatives to the 8-chlorocinnoline core, while maintaining regions A and B constant. Table 4 summarizes the results of the SAR we surveyed.

Table 4.

Anti-Proliferation Activities of Compound Series 10 against HT-1080, RD, GCT, and MCF-7 cells.

Compound	IC50 (nM)
	HT-1080	RD	GCT	MCF-7
10a	> 20000	> 20000	> 20000	> 20000
10b	> 20000	> 20000	> 20000	> 20000
10c	> 20000	> 20000	> 20000	> 20000
10d	> 20000	> 20000	> 20000	> 20000
10e	> 20000	> 20000	> 20000	> 20000
10f	> 20000	> 20000	> 20000	> 20000
10g	> 20000	> 20000	> 20000	> 20000
10h	> 20000	> 20000	> 20000	> 20000
FiVe1	1564 ± 288	3095 ± 555	1686 ± 349	> 20000

Compounds 10a–c contained different substituents around the bicyclic heterocycle. Two quinolines derivatives (10d–e) were prepared to compare the pharmacophore systems of cinnoline and quinoline. Compound 10f corresponded to the isomeric naphthyridine, quinazoline. To compare the biological activities of the bicyclic heterocycles with their analogs without a benzene ring fused, compounds 10g (4-phenylpyrimidine) and 10h (pyridazine) were also tested. Unfortunately, all of these compounds were found to be completely inactive, suggesting a very limited tolerance to change in this region. These results strongly suggest that the electronics, dipole and/or hydrogen bond accepting properties of the bicyclic 8-chloro substituted cinnoline heterocyclic system represents a key feature required for productive engagement with VIM.

Analysis of Vimentin Ser56 Phosphorylation Status

Target identification studies, involving a photo-activatable affinity probe FiVe1 derivative and mass spectrometry-based proteomics, revealed that FiVe1 functions by binding to VIM to induce hyperphosphorylation at Ser56, which, based on mutagenesis data involving S56E phospho-mimetic substitution, was demonstrated to be sufficient to phenocopy FiVe1 anti-mitotic activity.¹² To assess whether observed improvements in potency with respect to overall impact on cell viability and proliferation track directly with VIM S56 phosphorylation status (i.e., changes in overall potency track with on-target activity), we evaluated VIM S56 phosphorylation status in all three sarcoma cell lines (i.e., HT-1080, RD, and GCT) following treatment with FiVe1 (IC₅₀ = 1.6 μM), 4e (IC₅₀ = 44 nM) or the inactive control 10d (IC₅₀ >20 μM). Notably, for compound 4e because of its low IC₅₀ with respect to overall cytotoxicity, VIM S56 phosphorylation could only evaluated at low concentration (i.e., 100 nM). Cells were incubated with or without compound for 24 h. Vimentin phosphorylation at Ser56 was assessed by Western immunoblot analysis using a vimentin phosphorylation site-specific antibody. As shown in Figure 3, consistent with observed differences in overall cell-based potency for compounds within this series, whereas 10d was found to be inactive and FiVe1 treatment induced Ser56 phosphorylation at 1 μM but not at 100 nM, compound 4e was found to induce VIM Ser56 phosphorylation at 10-fold lower concentration (100 nM) when compared to FiVe1 in all tested sarcoma cell lines (i.e., changes in cell-based potency directly track with on-target activity). Analogs presented in this work function via the same mechanism of action as previously reported for FiVe1.

Figure 3.

Western blotting analysis of phosphorylated VIM protein content from HT-1080 (A), RD (B), and GCT (C) sarcoma cells treated for 24 h with FiVe1, 10d, and 4e. The immunoblots illustrate the phosphorylation of VIM at Ser56 by Five1 (1 μM) and 4e (0.1 μM).

Pharmacokinetic properties

Based on its potent and selective cell-based activity profile, the mouse pharmacokinetics properties of compound 4e was investigated and compared to that of FiVe1. Pharmacokinetic curves and the associated parameters are shown in Figure 4 and Table 5, respectively.

Figure 4.

Pharmacokinetic studies of mouse oral administration for 4e and FiVe1.

Table 5.

Mouse Pharmacokinetics of Compounds 4e and FiVe1.

	4e		FiVe1
parameter	po 10 mg/kg	ip 1 mg/kg	po 25 mg/kg
AUC0-last (ng.h/mL)	371.33 ± 68.08	208.33 ± 32.50	309.78 ± 44.41
AUC0-inf (ng.h/mL)	534.33 ± 152.23	211.33 ± 34.56	339.21 ± 151.72
T1/2 (h)	4.68 ± 3.73	0.59 ± 0.09	4.57 ± 1.61
Tmax (h)	0.67 ± 0.29	0.25 ± 0.00	0.5 ± 0.00
Tlast (h)	8	4	18
Cmax (ng/mL)	154.67 ± 20.60	197.00 ± 44.84	110.43 ± 41.91

A 1.0 mg/kg intraperitoneal (I.P.) or 10 mg/kg oral (P.O.) dose of 4e resulted observed maximum concentrations (C_max) of 197.0 ng/mL or 154.67 ng/mL, respectively. Oral administration of 4e was associated with a reasonable elimination half-life (t_1/2 = 4.68 hours), while I.P. administration resulted in rapid drug clearance. Figure 4 shows the comparison in observed systemic concentrations of FiVe1 and 4e (dosed at 25 mg/kg or 10 mg/kg, respectively), measured following oral administration in mice. Although the oral bioavailability for 4e (25%) was modest, the oral pharmacokinetic properties of 4e in mouse are improved. Notably, the maximum concentration (C_max) observed following oral administration FiVe1 at 25 mg/kg was lower (110.43 ng/mL), when compared to 4e. Assuming dose proportional absorption, this could suggest an ~4-fold improvement in bioavailability for 4e, which could be significant in light of the observed differences in cell-based potencies for these two compounds. Based on these pharmacokinetic results, we can conclude that the properties of 4e are compatible with its oral administration facilitating sufficient “time-over-target” to achieve predicted efficacy in mouse xenograft studies. Further, observed differences in bioavailability suggest that more favorable exposure properties are achievable.

Development of Compounds with Improved Solubility

A major liability of FiVe1 is low aqueous solubility (<1 μM in DPBS, Table 6). As such, in parallel to evaluation of biological activity, we characterized the kinetic solubility of all analogs prepared as part of series 4, 7 and 10 described above using an HPLC-based kinetic solubility assay. Unfortunately, the kinetic solubility of compounds within these series (4, 7 and 10) were similarly low (<1 μM) and comparable to the FiVe1 parent compound. We then focused our efforts on further exploring the SAR around the 3-chlorophenyl ring with the objective of introducing solubilizing substituents to generate analogs that retained desirable selective cell-based activity profiles, while demonstrating improvements in aqueous solubility and potential by extension, metabolic stability and absorption properties. We reasoned that we could achieve better compounds, in terms of balanced activity and drug-like properties, via compounds consisting of introduced O-alkylated solubilizing groups or chains in place of the methoxy group of compound 4e. Table 6 summarizes the in vitro activities and solubility profiles of compounds 11a–k, which consist of different substituents at the C-5 position of the 3-chlorosubstituted phenyl ring of system A, while maintaining regions B and C of the FiVe1 core scaffold. Compounds 11a–c were designed to contain basic side chains with primary or tertiary amines. Compound 11a (ethyl amine), 11b (N,N-dimethylethylamine), and 11c (N,N-dimethylpropylamine) retained a moderate and similar level of potency, when compared to FiVe1 in HT-1080, RD, and GCT cells (Table 6). Additionally, they showed a desirable > 40-fold improvement in solubility, when compared to FiVe1 (68, 131, and 48 μM vs <1 uM, respectively, Table 6). Notably, however, 11b and 11c suffered from a loss in cell type selectivity, with both compounds demonstrating toxicity in MCF-7 cells. Interestingly, in contrast, the N-demethylated analog 11a was found to retain a cell type selective activity (Table 6). These results suggest that elongation of the alkyl chain (11b vs 11c) did not provide an improvement in activity and that the presence of a tertiary amine results in non-selective cytotoxicity.

Table 6.

Anti-Proliferation Activities of Compound Series 11 against HT-1080, RD, GCT, and MCF-7 cells, Selectivity Index (SI) of HT-1080 vs MCF-7, and Solubility of the Compounds in DPBS.

Compound	IC50 (nM)				SI	Solubility in DPBS (μM)
	HT-1080	RD	GCT	MCF-7
11a	3885 ± 834	4575 ± 227	3551 ± 535	> 20000	>5	68
11b	4722 ± 749	7017 ± 1071	6439 ± 1128	15955	3	131
11c	2718 ± 340	2687 ± 207	3719 ± 330	4562 ± 1062	2	48
11d	500 ± 41	407 ± 131	667 ± 45	> 20000	>40	13
11e	1464 ± 96	1309 ± 168	711 ± 102	> 20000	>14	9
11f	1695 ± 222	1350 ± 508	1060 ± 284	> 20000	>12	< 1
11g	13610 ± 1175	19070 ± 4911	2927 ± 597	> 20000	>2	< 1
11h	> 20000	> 20000	> 20000	> 20000	n.d.	< 1
11i	> 20000	> 20000	> 20000	> 20000	n.d.	< 1
11j	979 ± 77	1119 ± 185	1001 ± 329	> 20000	>20	82
11k	1532 ± 240	1118 ± 321	756 ± 279	> 20000	>13	>200
FiVe1	1564 ± 288	3095 ± 555	1686 ± 349	> 20000	13	< 1

We next examined nonionizable moieties, which involved hydroxylated and alkoxy side chains as shown for compounds 11d–g. We observed a >3-fold improvement in potency for compound 11d (ethanol), while its glycolyl analog 11e retained a similar activity when compared to FiVe1. Here, both compounds retained a good cell type selectivity profile and both ompounds were demonstrated to have significantly improved solubility properties, when compared to FiVe1 (13 and 9 μM vs. <1 μM). Compound 11f, the O-methylated analog of 11d maintained selective antiproliferative activity, albeit with a less desirable profile when compared to 11d in terms of potency, selectivity and solubility (<1 μM). The introduction of 1,1-dimethoxyethyl in the structure (compound 11g) led to a decrease in desirable biological activity and was also found to result in decreased solubility (<1 μM). Introduction of bulky solubilizing substituents, such as tetrahydropyranyl (11h) and morpholine (11i), was not well tolerated and resulted in losses in terms of activity with no appreciable improvements in solubility. This could further suggest that VIM engagement and associated cytotoxicity is modulated by steric hindrance at this site. Finally, the ester-containing compound 11j showed a modest improvement in activity, when compared to FiVe1, while displaying a significant increase in kinetic solubility (82 μM vs. <1 μM)(Table 6). The corresponding carboxylic acid counterpart of 11j, compound 11k, showed a similar level of potency than FiVe1. However, although 11k could present permeability issues in vivo studies, it displayed the better improvement in solubility along all compounds (>200 μM). This also suggests that compound 11j could be hydrolyzed by esterases inside the cells to afford its carboxylic acid, which is more soluble. In general, compounds within series 11 had very good rule-of-five compliance, with the exception of 11h (MW higher than 500), and some analogues displayed excellent solubility profiles while maintaining good selective activity, with amine-containing compound 11a, the ester-containing compound 11j, and their corresponding carboxylic acid 11k being the most soluble compounds. In terms of potency, selectivity and solubility, compound 11d displays significant improvements, when compared to FiVe1.

Microsomal stability

To gain additional SAR insight and evaluate the suitability of a candidate for future in vivo testing, we assessed differences in metabolic stability, and by potential extension pharmacokinetic properties, by evaluating the stability and in vitro clearance of compounds that exhibited good activity, and/or improved solubility, using mouse liver microsomes. The in vitro half-life (t_1/2) and intrinsic clearance (Cl_int(mic)) of selected compounds were both determined. Table 7 presents the mouse liver microsome stability properties of representative compounds selected from series 4 (4c and 4e), series 11 (11a, 11d, 11e, and 11j), as well as S10, S12 and FiVe1. FiVe1, 4c, and 4e exhibited similarly poor stability with only 3.2%, 0.1%, and 0.0% remaining after 60 minutes of incubation, respectively. In contrast, introduction of a hydrocarbon spacer bearing a solubilizing moiety (i.e., 11a, 11d, 11e, and 11j) was found to significantly improve metabolic stability. This could be the result of the presence of steric bulk at the C-5 position enabling metabolic switching, by blocking a metabolic soft spot in the phenyl ring. Alternatively, it could result from steric and/or electronic repulsion within CYP active sites or simply decreased affinity for polarized and less lipophilic substrates. Simultaneous substitution at the C-3, C-4 and C-5 positions (e.g., S12) was also found to suppress metabolism, presumably by directly blocking sites of metabolism, albeit at the cost of biological activity (Figure S2).

Table 7.

Microsomal Stability Parameters Obtained for the Selected Compounds.

Compound	Structure	T1/2 (min)	CLint(mic) (uL/min/mg)	Remaining (T= 60 min)
S10		6.7	205.8	0.4%
S12		32.5	42.6	29.6%
4c		2.1	657.4	0.1%
4e		1.7	805.3	0.0%
11a		>145	<9.6	78.7%
11d		19.1	72.5	10.5%
11e		42.7	32.5	35.7%
11j		35.5	39.0	28.9%
FiVe1		11.9	116.1	3.2%

Compound 11a, with an ethyl-amino group attached at C-5 position, showed excellent stability in mouse liver microsomes, with 78.7% remaining following 60 minutes of incubation. Replacement of the amine group by a hydroxy group (11d) decreased the microsomal stability (10.5% remaining). However, its glycolic derivative, 11e, was found to have reasonable microsome stability (35.7% remaining). The ester-containing compound 11j also displayed reasonable stability (28.9% remaining). Overall, we demonstrate how varying functional groups attached to a linker attached at C-5 position of FiVe1 can modulate and fine-tune microsomal stability. Furthermore, these findings enable the identification of several candidates with favorable properties (i.e., cell type selective activity, solubility and reasonable to good metabolic stability).

Chemistry

Distinct syntheses were designed and executed to obtain substitution at different positions over the FiVe1 structure. General synthetic routes of the main compounds are explained below. All the synthesized target compounds were characterized by ¹H NMR, ¹³C NMR, and high-resolution mass spectra (HRMS) and determined to be >95% pure by high-performance liquid chromatography (HPLC) analyses. The characterization data were in agreement with the assigned structures.

Compound Series 4

To investigate the effects of substituents at the C-3 and C-5 positions of the phenyl moiety (region A), we designed the analogues of FiVe1 outlined in Scheme 1 (compounds 4a–j). The synthesis of compounds 4a–f started with a Buchwald−Hartwig amination between the corresponding starting material 1a–f and Boc-protected piperazine to give intermediates 2a–f by using BINAP in combination with Pd₂(dba)₃ with sodium tert-butoxide in toluene at 100°C. It was necessary to reduce the reaction time from overnight to 4 h to minimize the over-reacted byproducts. Subsequent Boc deprotection with HCl in dioxane gave the desired free piperazinamines intermediates 3a–f, which underwent the nucleophilic displacement reaction with 4-bromo-8-chlorocinnoline (14), in DMF in presence of Et₃N at 90°C to furnish compounds 4a–f.

Scheme 1.

Synthesis of Analogs of 4a–j Varying the Region A of FiVe1^a

^aReagents and conditions: (a) tert-butyl piperazine-1-carboxylate, Pd₂(dba)₃, BINAP, NaO^tBu, toluene, 100°C; (b) 4 N HCl/dioxane, DCM, 0°C; (c) 4-bromo-8-chlorocinnoline, Et₃N, DMF, 90°C; (d) BBr₃, DCM, −75°C; (e) tert-butyl piperazine-1-carboxylate, Pd₂(dba)₃, BINAP, NaO^tBu, toluene, 100°C; (f) 4 N HCl/dioxane, DCM, 0°C; (g) 4-bromo-8-chlorocinnoline, Et₃N, DMF, 90°C; (h) SnCl₂, EtOH, 60°C; (i) acetyl chloride, Et₃N, DCM, 0°C.

The molecular structure of 4e, 8-chloro-4-(4-(3-chloro-5-methoxyphenyl)piperazin-1-yl)cinnoline, was further confirmed by X-ray crystal structure analysis, as shown in Scheme 1 and Figure S3. The details of data collection and structure refinement are listed in Table S1. Compounds 4e and 4c were used as precursors for the synthesis of compounds 4g and 4h, respectively, following treatment with BBr₃. Preparation of compound 4i was accomplished following a similar procedure as indicated above. Amination between 1-bromo-3-chloro-5-nitrobenzene (1i) and Boc-protected piperazine afforded the intermediate 2i, which led 3i after Boc deprotection. Reaction between 3i and 4-bromo-8-chlorocinnoline (14) yielded 4i–NO₂. Compound 4i was prepared from 4i-NO₂ via the reduction of the nitro group with SnCl₂ in EtOH. Finally, compound 4j was obtained by the acylation of 4i with acetyl chloride giving the corresponding N-substituted amide. The cinnoline core, was obtained by diazotization of the commercially available 1-(2-amino-3-chlorophenyl)ethan-1-one with sodium nitrite followed by cyclization upon heating to produce 8-chlorocinnolin-4-ol (12, Supporting Information). The resulting phenol was subjected to phosphorus oxybromide to generate 4-bromo-8-chlorocinnoline (14, Supporting Information).

Compound Series 7

To investigate the SAR of the piperazine core (region B), compounds 7a–f were synthesized as shown in Scheme 2. A similar procedure as described above was used to afford compounds 7a–e. The reaction of 1-bromo-3,5-dichlorobenzene (for preparation of 5a) or 1-bromo-3-chlorobenzene (for the synthesis of 5b–e), with the corresponding N-Boc amino cores afforded compounds 5a–e. N-Boc deprotection was followed to form intermediates 6a–e, which were used without further purification, and reacted with 4-bromo-8-chlorocinnoline (14) to afford the final compounds 7a–e. Compound 7f, was obtained after the treatment of the commercially available 4-(3-chlorophenyl)piperidine with 14.

Scheme 2.

Synthesis of Compounds 7a–f with Modifications around the Piperazine Moiety^a

^aReagents and conditions: (a) 1-bromo-3,5-dichlorobenzene or 1-bromo-3-chlorobenzene, Pd₂(dba)₃, BINAP, NaO^tBu, toluene, 100°C; (b) 4 N HCl/dioxane, DCM, 0°C; (c) 4-bromo-8-chlorocinnoline, Et₃N, DMF, 90°C; (d) 4-bromo-8-chlorocinnoline, Et₃N, DMF, 90°C.

Compound Series 10

We also studied the modification around the cinnoline region (region C). The analogs with variations in the cinnoline core were prepared as shown in Scheme 3. The Buchwald−Hartwig amination between 1-bromo-3-chlorobenzene and Boc-protected piperazine gave intermediate 8, which led the common synthetic intermediate 9 after Boc deprotection with HCl. Specifically, treatment of intermediate 9 with the commercially available aryl bromide system furnished compounds 10a–b, and 10d–h with a variety of heterocycle cores. In a similar fashion, the synthesis of analog 10c was accomplished by the reaction between the prepared bromo-cinnoline intermediate 15 (Supplementary Information) and 9.

Scheme 3.

Synthesis of Compounds 10a–h with Modifications around the Cinnoline Core^a

^aReagents and conditions: (a) tert-butyl piperazine-1-carboxylate, Pd₂(dba)₃, BINAP, NaO^tBu, toluene, 100°C; (b) 4 N HCl/dioxane, DCM, 0°C; (c) aryl bromide system, Et₃N, DMF, 90°C.

Compound Series 11

To convert the water-insoluble FiVe1 compound into a water-soluble one, we hypothesized to attach covalently an appropriate solubilizing side chain in the alkoxy group on C-5 position in the phenyl ring A of the structure. Scheme 4 describes the synthesis of compounds 11a–k with linked substituents in the C-5-aryl position. For the synthesis of these compounds, 3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenol (4g) was used as starting material. In the presence of sodium hydride, the nucleophilic substitution of 4g with the corresponding substituted alkyl bromide afforded the targeted compounds 11a–j. Compound 11k was obtained by the hydrolysis of the ester 11j by adding lithium hydroxide.

Scheme 4.

Synthesis of Compounds 11a–k with the Introduction of Solubilizing Moieties in C-5 Position of FiVe1^a

^aReagents and conditions: (a) Alkyl bromide, NaH, DMF, r.t.; (b) LiOH, THF/H₂O.

CONCLUSIONS

In summary, we have described the systematic optimization of FiVe1, a vimentin-specific anticancer drug that leads to vimentin phosphorylation and disorganization during metaphase, resulting in mitotic catastrophe in mesenchymal cancer cells. Different series of FiVe1 derivatives were designed and synthesized as novel selective anticancer agents to target vimentin-expressing mesenchymal cancer cells. Initial SAR around the phenyl ring proved both C-3 (R¹) and the C-5 (R²) positions as the most favorable for substitutions that maintain antiproliferative activity and facilitate favorable modulation of potency and physical properties. In this series, compounds 4c and 4e displayed low nanomolar IC₅₀ activities for vimentin-expressing sarcoma cell lines, with improvement in potency (> 20-fold) and selectivity (> 15-fold) when compared to FiVe1. Modifications through the piperazine moiety evidenced the importance of this system to maintain activity. Additionally, all modulations we performed around the cinnoline core that were intended to improve activity resulted in dramatic loss of cytotoxicity. Based on its improved cell-based activity profile, 4e was selected to investigate on-target activity, by examining VIM phosphorylation status at Ser56. Improvements in potency in terms of overall cell type selective cytotoxicity directly track with concentration-dependent impact on VIM Ser56 phosphorylation, thereby demonstrating that this novel compound function via the same mechanism of action as FiVe1. Additionally, the pharmacokinetics profile for 4e in mouse demonstrated a superior oral bioavailability, when compared to FiVe1. Finally, attachment of solubilizing groups through alkoxy on the C-5 position of phenyl ring A led to the discovery of compounds 11a, 11j, and 11k among others, which exhibited superior profiles in terms of liver microsome stability and aqueous solubility, while maintaining selective cell-based activity. Collectively, these results show the development of promising FiVe1 analogs with potential preclinical applications in the treatment of mesenchymal cancers and beyond.

EXPERIMENTAL SECTION

Compound Synthesis and Characterization.

All solvents and chemicals were of reagent grade. All air- or moisture-sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Unless otherwise specified, commercially available reagents and solvents were used without further purification. Flash column chromatography was carried out on a Teledyne ISCO CombiFlash Rf system using prepacked columns. Analytical-grade solvents (acetonitrile, dichloromethane [DCM], dimethylformamide [DMF], ethanol 99.8% v/v, ethyl acetate [EtOAc], hexane, methanol [MeOH], toluene) were used without further purification. Anhydrous MgSO₄ was used as a drying agent for the organic phases. Thin-layer chromatography (TLC) was performed on EMD precoated silica gel 60 F254 plates, and spots were visualized with UV light. NMR experiments were performed with Bruker Avance III 500, or 600 MHz spectrometers. ¹H, and ¹³C NMR data are reported as chemical shifts (δ) in parts per million (ppm) and are calibrated using residual undeuterated solvent as an internal reference. Proton spectra are reported as chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad), coupling constant (J) in Hz, and number of protons. Carbon NMR spectra are reported as chemical shift alone. Yields refer to chromatographically and spectroscopically (¹H NMR) homogeneous materials unless otherwise stated. Nominal mass spectra were obtained using an Agilent InfinityLab 1260 mass spectrometer (ESI). Method: A 6 min gradient of 5−95% acetonitrile (containing 0.1% formic acid) in water (containing 0.1% formic acid) was used with a 10 min run time at a flow rate of 1 mL/min. A Zorbax C8 column (5 μm, 4.6 × 50 mm) was used at a temperature of 35 °C. HRMS were obtained using Agilent 6230 TOF LC/MS mass spectrometer. All the synthesized target compounds were characterized by ¹H NMR, ¹³C NMR, and HRMS and determined to be >95% pure by HPLC analyses.

General procedure for the synthesis compounds 4a–f

A mixture of 4-bromo-8-chlorocinnoline (14) (1 equiv), the corresponding aryl-piperazine HCl salt (1 equiv) and Et₃N (3 equiv) in DMF (10 mL) was stirred at 90°C for 4 h. After monitoring the end of the reaction by TLC, the reaction was cooled down at room temperature. Cold water was added to the reaction mixture affording the precipitation of the product, which was collected by centrifugation. The precipitate was purified by flash chromatography (CHCl₃/MeOH) to give the title compound.

8-chloro-4-(4-(3,5-dichlorophenyl)piperazin-1-yl)cinnoline (4a)

The title compound was synthesized according to above general procedure from 3a and 4-bromo-8-chlorocinnoline (14) as an orange solid (126 mg, 82% yield). See Supporting Information for the synthesis of 3a. ¹H NMR (600 MHz, CDCl₃): δ 9.06 (s, 1H), 7.92 (dd, J = 8.5, 1.2 Hz, 1H), 7.89 (dd, J = 7.4, 1.2 Hz, 1H), 7.60 (dd, J = 8.5, 7.4 Hz, 1H), 6.89 (t, J = 1.7 Hz, 1H), 6.84 (d, J = 1.7 Hz, 2H), 3.55 – 3.51 (m, 4H), 3.51 – 3.46 (m, 4H). ¹³C NMR (151 MHz, CDCl₃): δ 152.3, 147.1, 144.6, 136.2 (2C), 135.8, 135.4, 130.6, 129.3, 122.2, 121.7, 120.1, 114.5 (2C), 51.5 (2C), 48.5 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₁₆Cl₃N₄: 393.0; found 393.0; HRMS (m/z): [M + H]⁺ calcd for C₁₈H₁₆Cl₃N₄: 393.0435; found, 393.0433

3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)benzonitrile (4b)

The title compound was synthesized according to above general procedure from 3b and 4-bromo-8-chlorocinnoline (14) as an orange solid (56 mg, 75% yield). See Supporting Information for the synthesis of 3b. ¹H NMR (600 MHz, CDCl₃): δ 9.07 (s, 1H), 7.95 – 7.89 (m, 2H), 7.62 (dd, J = 8.4, 7.5 Hz, 1H), 7.17 – 7.14 (m, 2H), 7.10 – 7.07 (m, 1H), 3.54 (s, 8H). ¹³C NMR (151 MHz, CDCl₃): δ 151.9, 147.1, 144.4, 136.3, 136.2, 135.5, 130.7, 129.5, 122.8, 122.2, 121.6, 120.2, 118.0, 117.2, 114.6, 51.4 (2C), 48.2 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₁₆Cl₂N₅: 384.1; found 384.0; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₁₆Cl₂N₅: 384.0777; found, 384.0766

8-chloro-4-(4-(3,5-dimethoxyphenyl)piperazin-1-yl)cinnoline (4c)

The title compound was synthesized according to above general procedure from 3c and 4-bromo-8-chlorocinnoline (14) as a yellow solid (157 mg, 64% yield). See Supporting Information for the synthesis of 3c. ¹H NMR (600 MHz, CDCl₃): δ 9.05 (s, 1H), 7.93 (dd, J = 8.6, 1.1 Hz, 1H), 7.88 (dd, J = 7.4, 1.2 Hz, 1H), 7.59 (dd, J = 8.5, 7.4 Hz, 1H), 6.18 – 6.16 (m, 2H), 6.09 (t, J = 2.6 Hz, 1H), 3.80 (s, 6H), 3.56 – 3.51 (m, 4H), 3.49 – 3.44 (m, 4H). ¹³C NMR (151 MHz, CDCl₃): δ 161.8 (2C), 152.9, 147.0, 144.8, 136.2, 135.2, 130.5, 129.1, 122.2, 121.9, 95.7 (2C), 92.4, 55.5 (2C), 51.8 (2C), 49.3 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₂₂ClN₄O₂: 385.1; found 385.1; HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₂ClN₄O₂: 385.1426; found, 385.1431

8-chloro-4-(4-(3-methoxyphenyl)piperazin-1-yl)cinnoline (4d)

The title compound was synthesized according to above general procedure from compound 3d and 4-bromo-8-chlorocinnoline (14) as a yellow solid (37 mg, 72% yield). See Supporting Information for the synthesis of 3d. ¹H NMR (600 MHz, CDCl₃): δ 9.06 (s, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 7.3 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.24 (t, J = 8.2 Hz, 1H), 6.62 (dd, J = 8.2, 2.3 Hz, 1H), 6.54 (t, J = 2.6 Hz, 1H), 6.50 (dd, J = 8.1, 2.3 Hz, 1H), 3.82 (s, 3H), 3.57 – 3.52 (m, 4H), 3.49 – 3.44 (m, 4H). ¹³C NMR (151 MHz, CDCl₃): δ 160.7, 152.2, 146.9, 144.7, 136.1, 135.1, 130.3, 130.1, 128.9, 122.1, 121.8, 109.2, 105.3, 103.1, 55.3, 51.7 (2C), 49.2 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₂₀ClN₄O: 355.1; found 355.1; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₂₀ClN₄O: 355.1320; found, 355.1322

8-chloro-4-(4-(3-chloro-5-methoxyphenyl)piperazin-1-yl)cinnoline (4e)

The title compound was synthesized according to above general procedure from compound 3e and 4-bromo-8-chlorocinnoline (14) as a yellow solid (453 mg, 76% yield). See Supporting Information for the synthesis of 3e. ¹H NMR (500 MHz, CDCl₃): δ 9.05 (s, 1H), 7.92 (dd, J = 8.6, 1.2 Hz, 1H), 7.88 (dd, J = 7.4, 1.2 Hz, 1H), 7.59 (dd, J = 8.5, 7.4 Hz, 1H), 6.59 (t, J = 1.9 Hz, 1H), 6.47 (t, J = 1.9 Hz, 1H), 6.39 (t, J = 2.2 Hz, 1H), 3.80 (s, 3H), 3.55 – 3.49 (m, 4H), 3.47 (dd, J = 4.4, 1.6 Hz, 4H). ¹³C NMR (151 MHz, CDCl₃): δ 161.3, 152.6, 147.0, 144.7, 136.1, 135.78, 135.2, 130.5, 129.2, 122.2, 121.9, 109.4, 105.7, 101.3, 55.6, 51.6 (2C), 48.9 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₁₉Cl₂N₄O: 389.1; found 389.1; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₁₉Cl₂N₄O: 389.0930; found, 389.0936. Elemental analysis calculated (%) for C₁₉H₁₈Cl₂N₄O: C 58.62, H 4.66, N 14.39. Found: C 58.37, H 4.63, N 14.07.

8-chloro-4-(4-(3-chloro-5-(pyrrolidin-1-yl)phenyl)piperazin-1-yl)cinnoline (4f)

The title compound was synthesized according to above general procedure from compound 3f and 4-bromo-8-chlorocinnoline (14) as an orange solid (32 mg, 58% yield). See Supporting Information for the synthesis of 3f. ¹H NMR (600 MHz, CDCl₃): δ 9.06 (s, 1H), 7.93 (dd, J = 8.6, 1.2 Hz, 1H), 7.88 (dd, J = 7.4, 1.2 Hz, 1H), 7.59 (dd, J = 8.5, 7.4 Hz, 1H), 6.31 (t, J = 1.9 Hz, 1H), 6.17 (t, J = 1.9 Hz, 1H), 6.00 (t, J = 2.1 Hz, 1H), 3.55 – 3.51 (m 4H), 3.48 – 3.44 (m, 4H), 3.35 – 3.23 (m, 4H), 2.06 – 1.96 (m, 4H). ¹³C NMR (151 MHz, CDCl₃): δ 152.2, 148.9, 146.5, 144.2, 135.6, 135.2, 134.6, 129.9, 128.5, 121.6, 121.4, 104.3, 103.9, 97.5, 51.2 (2C), 48.9 (2C), 47.3 (2C), 24.9 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₂H₂₄Cl₂N₅: 428.1; found 428.1; HRMS (m/z): [M + H]⁺ calcd for C₂₂H₂₄Cl₂N₅: 428.1403; found, 428.1410

3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenol (4g)

A solution of BBr₃ (336 uL, 3.5 mmol) in DMC (2 mL) was added dropwise to a solution of compound 4e (272.3 mg, 0.70 mmol) in anhydrous DCM (10 mL) under N₂ atmosphere at −75 °C. The reaction mixture was stirred for 16 h at 0 °C. After this period, the solution was quenched with ice water, and the organic layer washed with water. The organic layer was dried over anhydrous MgSO₄, concentrated by rotary evaporation, and purified by flash chromatography to give 4g (160 mg, 61% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.70 (br, 1H), 9.12 (s, 1H), 8.07 (dd, J = 8.6, 1.2 Hz, 1H), 8.04 (dd, J = 7.4, 1.1 Hz, 1H), 7.72 (dd, J = 8.5, 7.4 Hz, 1H), 6.50 (t, J = 2.1 Hz, 1H), 6.33 (t, J = 2.2 Hz, 1H), 6.27 (t, J = 1.9 Hz, 1H), 3.55 – 3.53 (m, 4H), 3.44 – 3.37 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 159.0, 152.7, 145.8, 144.2, 136.1, 134.1, 133.1, 130.6, 129.1, 122.9, 120.9, 106.3, 106.2, 100.8, 50.7 (2C), 47.5 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₁₇Cl₂N₄O: 375.1; found 375.0; HRMS (m/z): [M + H]⁺ calcd for C₁₈H₁₇Cl₂N₄O: 375.0774; found, 375.0780

5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)benzene-1,3-diol (4h)

A solution of BBr₃ (480 uL, 5 mmol) in DMC (2 mL) was added dropwise to a solution of compound 4e (150 mg, 0.39 mmol) in anhydrous DCM (10 mL) under N₂ atmosphere at −75 °C. The reaction mixture was stirred for 16 h at 0 °C. After this period, the solution was quenched with ice water, and the organic layer washed with water. The organic layer was dried over anhydrous MgSO₄, concentrated by rotary evaporation, and purified by flash chromatography to give 4h (65.4 mg, 47% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.09 (s, 1H), 8.31 – 8.26 (m, 1H), 8.26 – 8.23 (m, 1H), 7.72 (t, J = 8.1 Hz, 1H), 5.90 – 5.87 (m, 2H), 5.83 (t, J = 2.0 Hz, 1H), 4.42 – 4.29 (m, 4H), 4.07 – 3.89 (br, 2H), 3.51 – 3.46 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 159.0 (2C), 150.9, 149.9, 138.6, 135.1, 133.2, 127.3, 125.8, 122.3, 117.5, 95.1, 94.0 (2C), 50.9 (2C), 47.5 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₁₈ClN₄O₂: 357.1; found 357.1; HRMS (m/z): [M + H]⁺ calcd for C₁₈H₁₈ClN₄O₂: 357.1113; found, 357.1120

3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)aniline (4i)

A stirred solution compound 4i-NO₂ (100 mg, 0.25 mmol) and tin (II) chloride dihydrate (142 mg, 0.75 mmol) in ethanol (5 ml) was stirred and heated at 60°C for 6 hours. Upon cooling to room temperature, the solvent was removed in vacuo and the residue partitioned between dichloromethane and 2 M sodium hydroxide. The organic layer was separated and dried over magnesium sulfate. The residue was subjected to flash chromatography to afford compound 4i (45 mg, 48%) as an orange solid. See Supporting Information for the synthesis of 4i-NO₂. ¹H NMR (600 MHz, DMSO-d₆): δ 9.12 (s, 1H), 8.06 (d, J = 8.6 Hz, 1H), 8.03 (d, J = 7.4 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 6.20 (t, J = 2.1 Hz, 1H), 6.13 (t, J = 2.1 Hz, 1H), 6.10 (t, J = 1.8 Hz, 1H), 5.26 (br, 2H), 3.56 – 3.51 (m, 4H), 3.41 – 3.36 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 152.6, 150.7, 145.8, 144.2, 136.1, 134.1, 133.1, 130.6, 129.1, 122.9, 120.9, 105.0, 103.3, 99.2, 50.8 (2C), 47.8 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₁₈Cl₂N₅: 374.1; found 374.1; HRMS (m/z): [M + H]⁺ calcd for C₁₈H₁₈Cl₂N₅: 374.0934; found, 374.0939

N-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenyl)acetamide (4j)

To a stirred solution of 4i (30 mg, 0.08 mmol) and Et₃N (34 μL, 0.24 mmol) in DCM (5 mL) at 0 °C was slowly added acetyl chloride (6 μL, 0.08 mmol). The mixture was stirred at room temperature until completion of the reaction. Cold water was added to the reaction mixture affording the precipitation of the product, which was collected by centrifugation. The precipitate was purified by flash chromatography (CHCl₃/MeOH) to give the title compound as an orange solid (6 mg, 25%). ¹H NMR (600 MHz, CDCl₃): δ 9.05 (s, 1H), 7.92 (dd, J = 8.5, 1.2 Hz, 1H), 7.89 (dd, J = 7.4, 1.2 Hz, 1H), 7.60 (dd, J = 8.5, 7.4 Hz, 1H), 7.41 (s, 1H), 7.27 (br, 1H), 6.85 (t, J = 1.8 Hz, 1H), 6.70 (t, J = 2.1 Hz, 1H), 3.57 – 3.44 (m, 8H), 2.19 (s, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 168.6, 152.4, 147.1, 144.7, 139.9, 136.1, 135.4, 135.3, 130.5, 129.2, 122.2, 121.9, 112.1, 111.4, 105.9, 51.57 (2C), 48.80 (2C), 25.0. MS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₂₀Cl₂N₅O: 416.1; found 416.0; HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₀Cl₂N₅O: 416.1039; found, 416.1044

General procedure for the synthesis of compounds 7a–f

A mixture of 4-bromo-8-chlorocinnoline (14) (1 equiv), the corresponding 3-chlorophenyl alkyl-piperazine HCl salt (1 equiv) and Et₃N (3 equiv) in DMF (10 mL) was stirred at 90°C for 4 h. After monitoring the end of the reaction by TLC, the reaction was cooled down at room temperature. Cold water was added to the reaction mixture affording the precipitation of the product, which was collected by centrifugation. The precipitate was purified by flash chromatography (CHCl₃/MeOH) to give the title compound.

(S)-8-chloro-4-(4-(3,5-dichlorophenyl)-3-methylpiperazin-1-yl)cinnoline (7a)

The title compound was synthesized according to above general procedure from compound 6a and 4-bromo-8-chlorocinnoline (14) as a brown solid (26 mg, 78% yield). See Supporting Information for the synthesis of 6a. ¹H NMR (600 MHz, CDCl₃): δ 9.06 (s, 1H), 7.98 (dd, J = 8.5, 1.2 Hz, 1H), 7.90 (dd, J = 7.4, 1.2 Hz, 1H), 7.61 (dd, J = 8.5, 7.4 Hz, 1H), 6.87 (t, J = 1.7 Hz, 1H), 6.80 (d, J = 1.7 Hz, 2H), 4.17 – 4.09 (m, 1H), 3.77 – 3.69 (m, 1H), 3.62 – 3.55 (m, 1H), 3.52 – 3.40 (m, 3H), 3.34 – 3.24 (m, 1H), 1.37 (d, J = 6.6 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃): δ 151.2, 147.0, 145.1, 136.2, 135.9 (2C), 135.4, 130.6, 129.3, 122.2, 121.5, 119.5, 114.5 (2C), 56.8, 51.6, 51.3, 42.9, 13.4. MS-ESI (m/z): [M + H]+ calcd for C₁₉H₁₈Cl₃N₄: 407.1; found 407.1; HRMS (m/z): [M + H]+ calcd for C₁₉H₁₈Cl₃N₄: 407.0592; found, 407.0602

8-chloro-4-(5-(3-chlorophenyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)cinnoline (7b)

The title compound was synthesized according to above general procedure from compound 6b and 4-bromo-8-chlorocinnoline (14) as a brown solid (13 mg, 49% yield). See Supporting Information for the synthesis of 6b. ¹H NMR (600 MHz, DMSO-d₆): δ 8.88 (s, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 7.4 Hz, 1H), 7.48 (t, J = 8.8, 7.5 Hz, 1H), 7.08 (t, J = 8.1 Hz, 1H), 6.64 (t, J = 2.1 Hz, 1H), 6.58 – 6.52 (m, 2H), 5.15 (s, 1H), 4.77 (s, 1H), 4.34 (dd, J = 9.8, 1.9 Hz, 1H), 3.73 (dd, J = 9.6, 1.9 Hz, 1H), 3.73 (dd, J = 9.6, 1.9 Hz, 1H), 3.34 (d, J = 9.7 Hz, 1H), 2.19 (q, J = 10.0 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 148.2, 145.6, 140.9, 134.4, 133.4, 132.1, 130.9, 130.8, 126.9, 124.1, 118.3, 116.1, 112.3, 111.6, 60.7, 59.5, 56.9, 54.9, 37.5. MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₁₇Cl₂N₄: 371.1; found 371.0; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₁₇Cl₂N₄: 371.0825; found, 371.0838

8-chloro-4-(3-(3-chlorophenyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)cinnoline (7c)

The title compound was synthesized according to above general procedure from compound 6c and 4-bromo-8-chlorocinnoline (14) as a brown solid (10 mg, 57% yield). See Supporting Information for the synthesis of 6c. ¹H NMR (600 MHz, DMSO-d₆): δ 9.19 (s, 1H), 8.13 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 7.5 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.23 (t, J = 8.1 Hz, 1H), 6.94 (t, J = 2.2 Hz, 1H), 6.89 (dd, J = 8.5, 2.4 Hz, 1H), 6.79 (dd, J = 7.8, 1.8 Hz, 1H), 4.66 – 4.58 (m, 2H), 3.69 (dd, J = 11.5, 2.4 Hz, 2H), 3.21 (dd, J = 11.4, 2.1 Hz, 2H), 2.04 – 1.97 (m, 2H), 1.91 – 1.85 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 152.1, 145.7, 141.8, 134.7, 133.8, 132.7, 130.5, 130.4, 128.6, 123.2, 120.5, 117.7, 113.5, 112.5, 58.5 (2C), 52.9 (2C), 26.2 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₁₉Cl₂N₄: 385.1; found 385.1; HRMS (m/z): [M + H]⁺ calcd for C₂₀H₁₉Cl₂N₄: 385.0981; found, 385.0993

8-chloro-4-(4-(3-chlorophenyl)-1,4-diazepan-1-yl)cinnoline (7d)

The title compound was synthesized according to above general procedure from compound 6d and 4-bromo-8-chlorocinnoline (14) as a brown solid (15 mg, 65% yield). See Supporting Information for the synthesis of 6d. ¹H NMR (600 MHz, DMSO-d₆): δ 8.98 (s, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.91 (d, J = 7.4 Hz, 1H), 7.55 (t, J = 8.7, 7.4 Hz, 1H), 7.10 (t, J = 8.1 Hz, 1H), 6.77 (t, J = 2.2 Hz, 1H), 6.71 (dd, J = 8.5, 2.5 Hz, 1H), 6.55 (dd, J = 7.8, 1.8 Hz, 1H), 3.86 – 3.78 (m, 4H), 3.70 – 3.65 (m, 2H), 3.60 (t, J = 6.0 Hz, 2H), 2.17 – 2.11 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 148.8, 145.8, 144.2, 134.4, 134.2, 132.5, 130.6, 130.1, 127.3, 123.4, 119.4, 115.3, 111.1, 110.4, 51.7, 51.3, 48.5, 47.9, 26.2. MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₁₉Cl₂N₄: 373.1; found 373.0; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₁₉Cl₂N₄: 373.0981; found, 373.0998

8-chloro-4-((3aR,6aS)-5-(3-chlorophenyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)cinnoline (7e)

The title compound was synthesized according to above general procedure from compound 6e and 4-bromo-8-chlorocinnoline (14) as a yellow solid (22 mg, 58% yield). See Supporting Information for the synthesis of 6e. ¹H NMR (600 MHz, DMSO-d₆): δ 8.70 (s, 1H), 8.34 (d, J = 8.9 Hz, 1H), 7.91 (d, J = 7.4 Hz, 1H), 7.46 (t, J = 8.3 Hz, 1H), 7.16 (t, J = 8.2 Hz, 1H), 6.61 (d, J = 7.9 Hz, 1H), 6.56 – 6.47 (m, 2H), 4.11 (t, J = 8.4 Hz, 2H), 3.79 (d, J = 10.9 Hz, 2H), 3.54 (t, J = 8.0 Hz, 2H), 3.32 (d, J = 11.3 Hz, 2H), 3.21 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 148.7, 145.3, 140.6, 133.7, 132.5, 131.7, 130.3, 130.1, 125.9, 124.4, 117.7, 114.9, 111.1, 110.5, 55.4 (2C), 51.2 (2C), 41.3 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₁₉Cl₂N₄: 385.1; found 385.1; HRMS (m/z): [M + H]⁺ calcd for C₂₀H₁₉Cl₂N₄: 385.0981; found, 385.0994

8-chloro-4-(4-(3-chlorophenyl)piperidin-1-yl)cinnoline (7f)

The title compound was synthesized according to above general procedure from the commercially available 4-(3-chlorophenyl)piperidine and 4-bromo-8-chlorocinnoline (14) as an orange solid (7.9 mg, 40% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.08 (s, 1H), 7.99 – 7.96 (m, 2H), 7.66 (t, J = 7.9 Hz, 1H), 7.39 (s, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.30 – 7.26 (m 2H), 3.82 (d, J = 12.5 Hz, 2H), 3.13 (t, J = 12.0 Hz, 2H), 2.83 (td, J = 11.8, 5.9 Hz, 1H), 2.03 – 1.85 (m, 4H). ¹³C NMR (151MHz, DMSO-d₆): δ 148.1, 145.8, 144.7, 136.1, 133.1, 132.9, 130.4, 130.4, 128.7, 126.8, 126.2, 125.6, 122.9, 121.1, 51.8 (2C), 41.0, 32.4 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₁₈Cl₂N₃: 358.1; found 358.0; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₁₈Cl₂N₃: 358.0872; found, 358.0881

tert-butyl 4-(3-chlorophenyl)piperazine-1-carboxylate (8)

To a solution of 1-bromo-3-chlorobenzene (500 mg, 2.6 mmol) in toluene (20 mL) was added tert-butyl piperazine-1-carboxilate (581 mg, 3.1 mmol), Pd₂(dba)₃ (119 mg, 0.13 mmol), BINAP (162 mg, 0.26 mmol), and NaO^tBu (350 mg, 3.6 mmol). The resulting mixture was stirred for 4 h at 100°C. After cooling down, the reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated. The reside was purified by flash chromatography (hexane/ethyl acetate) to provide compound 8 as a yellow solid (555 mg, 72% yield). ¹H NMR (500 MHz, CDCl₃): δ 7.17 (t, J = 8.1 Hz, 1H), 6.87 (t, J = 2.2 Hz, 1H), 6.84 (dd, J = 8.0, 2.8 Hz, 1H), 6.78 (dd, J = 8.1, 2.4 Hz, 1H), 3.62 – 3.51 (m, 4H), 3.14 – 3.13 (m, 4H), 1.48 (s, 9H). MS-ESI (m/z): [M – C₄H₈ + H]⁺ calcd for C₁₁H₁₄ClN₂O₂: 241.0; found: 241.0.

1-(3-chlorophenyl)piperazine (9)

To a solution of the compound 8 (500 mg, 1.68 mmol) in dichloromethane (10 mL) at 0°C was added 4 N HCl in dioxane (5 mL). The reaction mixture was stirred for 3 h and then solvent was evaporated to give crude as HCl salt after removal of all solvent. This crude was used for the next reaction without further purification.

General procedure for the synthesis of compounds 10a–h

A mixture of the corresponding aryl bromide system (1 equiv), compound 9 (1 equiv) and Et₃N (3 equiv) in DMF (10 mL) was stirred at 90°C for 16 h. After monitoring the end of the reaction by TLC, the reaction was cooled down at room temperature. Cold water was added to the reaction mixture affording the precipitation of the product, which was collected by centrifugation. The precipitate was purified by flash chromatography (CHCl₃/MeOH) to give the title compound.

4-(4-(3-chlorophenyl)piperazin-1-yl)cinnoline (10a)

The title compound was synthesized according to above general procedure from compound 9 and the commercially available 4-bromocinnoline as a yellow solid (22 mg, 58% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.02 (s, 1H), 8.35 (d, J = 8.5 Hz, 1H), 8.11 (d, J = 8.5 Hz, 1H), 7.89 (ddd, J = 8.4, 6.8, 1.3 Hz, 1H), 7.79 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.27 (t, J = 8.1 Hz, 1H), 7.05 (t, J = 2.2 Hz, 1H), 7.00 (dd, J = 8.4, 2.4 Hz, 1H), 6.84 (dd, J = 7.8, 1.9 Hz, 1H), 3.56 – 3.44 (m, 8H).¹³C NMR (151 MHz, DMSO-d₆): δ 152.0, 149.9, 144.0, 135.1, 133.9, 130.5, 130.3, 129.3, 129.3, 123.1, 119.4, 118.5, 114.8, 113.9, 50.6 (2C), 47.6 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₁₈ClN₄: 325.1; found 325.1; HRMS (m/z): [M + H]⁺ calcd for C₁₈H₁₈ClN₄: 325.1215; found, 325.1219

4-(4-(3-chlorophenyl)piperazin-1-yl)-8-methoxycinnoline (10b)

The title compound was synthesized according to above general procedure from compound 9 and the commercially available 4-bromo-8-methoxycinnoline as a yellow solid (15 mg, 51% yield). See Supporting Information for the synthesis of 14. ¹H NMR (600 MHz, DMSO-d₆): δ 9.03 (s, 1H), 7.70 (t, J = 8.1 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.29 – 7.23 (m, 2H), 7.04 (t, J = 2.2 Hz, 1H), 6.99 (dd, J = 8.4, 2.4 Hz, 1H), 6.84 (dd, J = 7.8, 1.9 Hz, 1H), 4.04 (s, 3H), 3.46 (s, 8H). ¹³C NMR (151 MHz, DMSO-d₆): δ 155.9, 152.0, 143.8, 142.6, 135.8, 133.9, 130.5, 130.1, 120.7, 118.5, 114.8, 113.9, 113.9, 108.6, 56.0, 50.6(2C), 47.6 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₂₀ClN₄O: 355.1; found 355.1; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₂₀ClN₄O: 355.1320; found, 355.1335

8-chloro-4-(4-(3-chlorophenyl)piperazin-1-yl)-7-methoxycinnoline (10c)

The title compound was synthesized according to above general procedure from compound 9 and compound 15 as a yellow solid (21 mg, 65% yield). See Supporting Information for the synthesis of 15. ¹H NMR (600 MHz, DMSO-d₆): δ 9.03 (s, 1H), 8.10 (d, J = 9.4 Hz, 1H), 7.78 (d, J = 9.5 Hz, 1H), 7.27 (t, J = 8.1 Hz, 1H), 7.04 (t, J = 2.2 Hz, 1H), 6.99 (dd, J = 8.4, 2.5 Hz, 1H), 6.84 (dd, J = 7.9, 1.9 Hz, 1H), 4.09 (s, 3H), 3.59 – 3.43 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆): δ 155.3, 151.9, 146.8, 144.1, 134.7, 133.9, 130.5, 123.7, 118.5, 117.2, 116.2, 114.7, 115.2, 113.8, 57.0, 50.7 (2C), 47.4 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₁₉Cl₂N₄O: 389.1; found 389.1; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₁₉Cl₂N₄O: 389.0930; found, 389.0944

8-chloro-4-(4-(3-chlorophenyl)piperazin-1-yl)quinoline (10d)

The title compound was synthesized according to above general procedure from compound 9 and the commercially available 4-bromo-8-chloroquinoline as a yellow solid (8 mg, 47% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 8.79 (d, J = 5.2 Hz, 1H), 8.10 (dd, J = 8.5, 1.3 Hz, 1H), 7.95 (dd, J = 7.5, 1.2 Hz, 1H), 7.56 (dd, J = 8.5, 7.5 Hz, 1H), 7.27 (t, J = 8.1 Hz, 1H), 7.18 (d, J = 5.2 Hz, 1H), 7.04 (t, J = 2.2 Hz, 1H), 6.99 (dd, J = 8.3, 2.4 Hz, 1H), 6.84 (dd, J = 7.8, 1.9 Hz, 1H), 3.55 – 3.43 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆): δ 156.7, 152.1 (2C), 145.3, 133.9 (2C), 130.7, 130.0, 125.5, 123.8, 123.6, 118.4, 114.7, 113.8, 109.7, 51.6 (2C), 47.6 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₁₈Cl₂N₃: 358.1; found 358.1; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₁₈Cl₂N₃: 358.0872; found, 358.0874

4-(4-(3-chlorophenyl)piperazin-1-yl)quinoline (10e)

The title compound was synthesized according to above general procedure from compound 9 and the commercially available 4-bromoquinoline as a yellow solid (12 mg, 61% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 8.72 (d, J = 4.9 Hz, 1H), 8.09 (dd, J = 8.4, 1.4 Hz, 1H), 7.97 (dd, J = 8.3, 1.2 Hz, 1H), 7.72 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H), 7.57 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 7.26 (t, J = 8.1 Hz, 1H), 7.07 – 7.02 (m, 2H), 7.02 – 6.97 (m, 1H), 6.87 – 6.81 (m, 1H), 3.51 – 3.46 (m, 4H), 3.33 – 3.28 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 155.9, 152.2, 150.9, 149.1, 133.9, 130.5, 129.6, 129.1, 125.5, 123.7, 122.7, 118.4, 114.8, 113.9, 109.1, 51.5 (2C), 47.8 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₉H₁₉ClN₃: 324.1; found 324.1; HRMS (m/z): [M + H]⁺ calcd for C₁₉H₁₉ClN₃: 324.1262; found, 324.1252

4-(4-(3-chlorophenyl)piperazin-1-yl)quinazoline (10f)

The title compound was synthesized according to above general procedure from compound 9 and the commercially available 4-bromoquinazoline as a yellow solid (30 mg, 67% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 8.66 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.85 – 7.83 (m, 2H), 7.57 (dt, J = 8.3, 4.1 Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H), 6.99 (t, J = 2.1 Hz, 1H), 6.93 (dd, J = 8.5, 2.4 Hz, 1H), 6.81 (dd, J = 7.9, 2.0 Hz, 1H), 3.90 – 3.84 (m, 4H), 3.46 – 3.40 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 163.6, 153.6, 151.9, 151.3, 133.9, 132.8, 130.5, 128.1, 125.7, 125.3, 118.2, 115.8, 114.5, 113.5, 48.8 (2C), 47.3 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₁₈ClN₄: 325.1; found 325.1; HRMS (m/z): [M + H]⁺ calcd for C₁₈H₁₈ClN₄: 325.1215; found, 325.1229

4-(4-(3-chlorophenyl)piperazin-1-yl)-6-phenylpyrimidine (10g)

The title compound was synthesized according to above general procedure from compound 9 and the commercially available 4-bromo-6-phenylpyrimidine as a yellow solid (16 mg, 51% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 8.63 (s, 1H), 8.21 – 8.16 (m, 2H), 7.51 – 7.45 (m, 3H), 7.36 (d, J = 1.3 Hz, 1H), 7.22 (t, J = 8.1 Hz, 1H), 7.00 (t, J = 2.2 Hz, 1H), 6.92 (dd, J = 8.5, 2.4 Hz, 1H), 6.81 (dd, J = 7.9, 1.9 Hz, 1H), 3.91 – 3.81 (m, 4H), 3.32 – 3.24 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 162.1, 161.8, 157.9, 151.9, 137.4, 133.9, 130.4, 130.1, 128.5 (2C), 126.8 (2C), 118.3, 114.8, 113.8, 98.5, 47.3 (2C), 43.1 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₂₀ClN₄: 351.1; found 351.1; HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₀ClN₄: 351.1371; found, 351.1368

4-(4-(3-chlorophenyl)piperazin-1-yl)pyridazine (10h)

The title compound was synthesized according to above general procedure from compound 9 and the commercially available 4-bromopyridazine as a yellow solid (11 mg, 48% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.02 (d, J = 3.3 Hz, 1H), 8.66 (d, J = 6.3 Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H), 7.03 – 6.97 (m, 2H), 6.95 (dd, J = 8.4, 2.4 Hz, 1H), 6.82 (dd, J = 7.8, 1.9 Hz, 1H), 3.61 – 3.53 (m, 4H), 3.37 – 3.28 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 151.8, 149.9, 145.6, 140.1, 133.9, 130.5, 118.4, 114.7, 113.9, 107.5, 46.9 (2C), 44.2 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₁₄H₁₆ClN₄: 275.1; found 275.1; HRMS (m/z): [M + H]⁺ calcd for C₁₄H₁₆ClN₄: 275.1058; found, 275.1051

General procedure for the synthesis of compounds 11a–j

The corresponding bromide (3 equiv) was added dropwise to a mixture of 4g (1 equiv) and sodium hydride (4 equiv, in 60% mineral oil) in DMF. The reaction mixture was stirred at room temperature for at least 8 h until the reaction was completed as determined by TLC. After removing the solvent under reduced pressure, water was added, and mixture was extracted with DCM. The crude product was purified by flash chromatography eluting with DCM/methanol.

2-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)ethan-1-amine (11a)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 2-bromoethan-1-amine as a yellow solid (10.2 mg, 39% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.14 (s, 1H), 8.08 (dd, J = 8.6, 1.2 Hz, 1H), 8.04 (dd, J = 7.4, 1.1 Hz, 1H), 7.73 (dd, J = 8.5, 7.4 Hz, 1H), 6.70 (t, J = 2.0 Hz, 1H), 6.55 (d, J = 2.2 Hz, 1H), 6.51 (t, J = 1.9 Hz, 1H), 4.21 (t, J = 5.1 Hz, 2H), 3.58 – 3.52 (m, 4H), 3.51 – 3.45 (m, 4H), 3.44 – 3.35 (br, 2H), 3.20 (t, J = 5.1 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 159.5, 152.6, 145.8, 144.1, 136.1, 134.4, 133.0, 130.6, 129.0, 122.9, 120.9, 108.2, 105.3, 100.7, 64.7, 50.6 (2C), 47.4 (2C), 38.3. MS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₂₂Cl₂N₅O: 418.1; found 418.1; HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₂Cl₂N₅O: 418.1196; found, 418.1204

2-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)-N,N-dimethylethan-1-amine (11b)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 2-bromo-N,N-dimethylethan-1-amine as a yellow solid (14.3 mg, 43% yield). ¹H NMR (600 MHz, CDCl₃): δ 9.04 (s, 1H), 7.91 (dd, J = 8.5, 1.2 Hz, 1H), 7.88 (dd, J = 7.4, 1.2 Hz, 1H), 7.59 (dd, J = 8.5, 7.4 Hz, 1H), 6.59 (t, J = 1.9 Hz, 1H), 6.47 (t, J = 1.9 Hz, 1H), 6.44 (t, J = 2.2 Hz, 1H), 4.06 (t, J = 5.6 Hz, 2H), 3.54 – 3.49 (m, 4H), 3.48 – 3.42 (m, 4H), 2.75 (t, J = 5.6 Hz, 2H), 2.35 (s, 6H). ¹³C NMR (151 MHz, CDCl₃): δ 160.4, 152.6, 147.0, 144.7, 136.2, 135.7, 135.3, 130.5, 129.2, 122.2, 121.9, 109.5, 106.2, 102.0, 66.2, 58.2, 51.6 (2C), 48.8 (2C), 45.8 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₂H₂₆Cl₂N₅O: 446.1; found 446.1; HRMS (m/z): [M + H]⁺ calcd for C₂₂H₂₆Cl₂N₅O: 446.1509; found, 446.1510

3-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)-N,N-dimethylpropan-1-amine (11c)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 3-bromo-N,N-dimethylpropan-1-amine as a yellow solid (9.7 mg, 50% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.13 (s, 1H), 8.06 (dd, J = 8.5, 1.2 Hz, 1H), 8.03 (dd, J = 7.4, 1.1 Hz, 1H), 7.72 (dd, J = 8.5, 7.4 Hz, 1H), 6.64 (t, J = 1.9 Hz, 1H), 6.48 (t, J = 2.2 Hz, 1H), 6.44 (t, J = 1.9 Hz, 1H), 3.99 (t, J = 6.4 Hz, 2H), 3.58 – 3.45 (m, 8H), 2.34 (t, J = 7.1 Hz, 2H), 2.15 (s, 6H), 1.82 (p, J = 6.7 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 160.3, 152.6, 145.8, 144.1, 136.1, 134.4, 133.1, 130.5, 129.1, 122.9, 120.9, 107.7, 104.9, 100.4, 66.0, 55.6, 50.6 (2C), 47.4 (2C), 45.2 (2C), 26.8. MS-ESI (m/z): [M + H]⁺ calcd for C₂₃H₂₈Cl₂N₅O: 460.2; found 460.1; HRMS (m/z): [M + H]⁺ calcd for C₂₃H₂₈Cl₂N₅O: 460.1665; found, 460.1671

2-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)ethan-1-ol (11d)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 2-bromoethan-1-ol as an orange solid (21.3 mg, 47% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.13 (s, 1H), 8.11 – 7.95 (m, 2H), 7.72 (t, J = 8.0 Hz, 1H), 6.64 (s, 1H), 6.50 (s, 1H), 6.46 (s, 1H), 4.86 (t, J = 5.7 Hz, 1H), 4.00 (t, J = 5.0 Hz, 2H), 3.74 – 3.66 (m, 2H), 3.59 – 3.40 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆): δ 160.4, 152.6, 145.8, 144.1, 136.1, 134.4, 133.1, 130.5, 129.1, 122.9, 120.9, 107.7, 105.0, 100.5, 69.8, 59.5, 50.6 (2C), 47.5 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₀H₂₁Cl₂N₄O₂: 419.1; found 419.0; HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₁Cl₂N₄O₂: 419.1036; found, 419.1044

3-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)propane-1,2-diol (11e)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 3-bromopropane-1,2-diol as an orange solid (7.9 mg, 35% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.13 (s, 1H), 8.10 – 8.06 (m, 1H), 8.05 – 8.02 (m, 1H), 7.72 (t, J = 7.6 Hz, 1H), 6.64 (t, J = 2.0 Hz, 1H), 6.50 (t, J = 2.1 Hz, 1H), 6.45 (t, J = 1.9 Hz, 1H), 4.96 (br, 1H), 4.68 (br, 1H), 4.01 (dd, J = 10.0, 4.1 Hz, 1H), 3.87 (dd, J = 10.0, 6.2 Hz, 1H), 3.81 – 3.73 (m, 1H), 3.56 – 3.51 (m, 4H), 3.50 – 3.45 (m, 4H), 3.45 – 3.41 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 160.5, 152.6, 145.8, 144.2, 136.1, 134.3, 133.1, 130.5, 129.1, 122.9, 120.9, 107.7, 105.1, 100.5, 69.9, 69.8, 62.6, 50.6 (2C), 47.5 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₁H₂₃Cl₂N₄O₃: 449.1; found 449.1; HRMS (m/z): [M + H]⁺ calcd for C₂₁H₂₃Cl₂N₄O₃: 449.1142; found, 449.1145

8-chloro-4-(4-(3-chloro-5-(2-methoxyethoxy)phenyl)piperazin-1-yl)cinnoline (11f)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 1-bromo-2-methoxyethane as an orange solid (17.0 mg, 52% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.13 (s, 1H), 8.07 (dd, J = 8.6, 1.2 Hz, 1H), 8.04 (dd, J = 7.4, 1.1 Hz, 1H), 7.72 (dd, J = 8.5, 7.4 Hz, 1H), 6.65 (t, J = 2.0 Hz, 1H), 6.51 (t, J = 2.2 Hz, 1H), 6.47 (t, J = 2.0 Hz, 1H), 4.12 – 4.08 (m, 2H), 3.66 – 3.61 (m, 2H), 3.57 – 3.51 (m, 4H), 3.51 – 3.45 (m, 4H), 3.30 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 160.1, 152.6, 145.8, 144.2, 136.1, 134.4, 133.0, 130.6, 129.1, 122.9, 120.9, 107.8, 104.9, 100.5, 70.3, 67.2, 58.2, 50.6 (2C), 47.4 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₁H₂₃Cl₂N₄O₂: 433.1; found 433.0; HRMS (m/z): [M + H]⁺ calcd for C₂₁H₂₃Cl₂N₄O₂: 433.1193; found, 433.1196

8-chloro-4-(4-(3-chloro-5-(2,2-dimethoxyethoxy)phenyl)piperazin-1-yl)cinnoline (11g)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 2-bromo-1,1-dimethoxyethane as an orange solid (11.2 mg, 38% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.13 (s, 1H), 8.07 (dd, J = 8.5, 1.2 Hz, 1H), 8.04 (dd, J = 7.5, 1.2 Hz, 1H), 7.72 (t, J = 8.5, 7.4 Hz, 1H), 6.66 (t, J = 1.9 Hz, 1H), 6.52 (t, J = 2.2 Hz, 1H), 6.49 (t, J = 1.9 Hz, 1H), 4.67 (t, J = 5.2 Hz, 1H), 3.99 (d, J = 5.2 Hz, 2H), 3.57 – 3.50 (m, 4H), 3.52 – 3.46 (m, 4H), 3.34 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆): δ 159.8, 152.6, 145.7, 144.2, 136.1, 134.4, 133.0, 130.6, 129.1, 122.9, 120.9, 107.9, 105.0, 101.6, 100.5, 67.4, 53.6 (2C), 50.6 (2C), 47.4 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₂H₂₅Cl₂N₄O₃: 463.1; found 463.1; HRMS (m/z): [M + H]⁺ calcd for C₂₂H₂₅Cl₂N₄O₃: 463.1298; found, 463.1313

8-chloro-4-(4-(3-chloro-5-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)phenyl)piperazin-1-yl)cinnoline (11h)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 2-(2-bromoethoxy)tetrahydro-2H-pyran as an orange solid (5.8 mg, 31% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.13 (s, 1H), 8.05 (d, J = 8.6 Hz, 1H), 8.03 (d, J = 7.4 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H), 6.65 (t, J = 1.9 Hz, 1H), 6.51 (t, J = 2.1 Hz, 1H), 6.48 (t, J = 1.8 Hz, 1H), 4.67 – 1.62 (m, 1H), 4.19 – 4.10 (m, 2H), 3.90 (dt, J = 11.5, 4.0 Hz, 1H), 3.77 (dt, J = 11.4, 8.2, 3.1 Hz, 1H), 3.69 (dt, J = 11.6, 4.5 Hz, 1H), 3.57 – 3.60 (m, 4H), 3.51 – 1.46 (m, 4H), 3.44 (dt, J = 11.0, 4.9 Hz, 1H), 1.77 – 1.65 (m, 1H), 1.62 (td, J = 9.1, 4.3 Hz, 1H), 1.51 – 1.42 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 160.2, 152.6, 145.8, 144.1, 136.1, 134.4, 133.1, 130.5, 129.1, 122.9, 120.9, 107.8, 105.0, 100.1, 98.0, 67.4, 65.2, 61.3, 50.6 (2C), 47.4 (2C), 30.2, 24.9, 19.0. MS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₉Cl₂N₄O₃: 503.2; found 503.1; HRMS (m/z): [M + H]⁺ calcd for C₂₅H₂₉Cl₂N₄O₃: 503.1611; found, 503.1621

4-(2-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)ethyl)morpholine (11i)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available 4-(2-bromoethyl)morpholine as an orange solid (15.2 mg, 39% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.12 (s, 1H), 8.06 (d, J = 8.5 Hz, 1H), 8.03 (d, J = 7.4 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H), 6.50 (t, J = 2.2 Hz, 1H), 6.47 (d, J = 1.9 Hz, 1H), 4.09 (t, J = 5.7 Hz, 2H), 3.61 – 3.47 (m, 12H), 2.69 (s, 2H), 2.48 (s, 4H). ¹³C NMR (151 MHz, DMSO): δ 160.6, 153.1, 146.3, 144.6, 136.6, 134.9, 133.5, 131.0, 129.6, 123.4, 121.4, 108.2, 105.5, 100.9, 66.6 (2C), 65.9, 57.4, 54.0 (2C), 51.1 (2C), 47.9 (2C). MS-ESI (m/z): [M + H]⁺ calcd for C₂₄H₂₈Cl₂N₅O₂: 488.2; found 488.2. HRMS (m/z): [M + H]⁺ calcd for C₂₄H₂₈Cl₂N₅O₂: 488.1615; found, 488.1621

methyl 3-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)propanoate (11j)

The title compound was synthesized according to above general procedure from compound 4g and the commercially available methyl 3-bromopropanoate as an orange solid (6.3 mg, 25% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.57 (s, 1H), 8.31 – 8.26 (m, 2H), 7.90 (t, J = 8.7 Hz, 1H), 6.47 (t, J = 2.0 Hz, 1H), 6.29– 6.24 (m, 2H), 5.13 (t, J = 6.5 Hz, 2H), 4.08 – 4.02 (m, 4H), 3.66 (s, 3H), 3.54 – 3.47 (m, 4H), 3.25 (t, J = 6.5 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 170.5, 159.1, 151.9, 151.9, 145.5, 134.4, 134.1, 131.7, 131.5, 131.5, 124.2, 119.9, 105.9, 105.6, 100.1, 60.7, 52.1, 50.8 (2C), 46.6 (2C), 32.9. MS-ESI (m/z): [M + H]⁺ calcd for C₂₂H₂₃Cl₂N₄O₃: 461.1; found 461.1; HRMS (m/z): [M + H]⁺ calcd for C₂₂H₂₃Cl₂N₄O₃: 461.1142; found, 461.1147

3-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)propanoic acid (11k)

LiOH (3.4 mg, 0.14 mmol) was added to a stirred solution of compound 11j (13 mg, 0.028 mmol) in THF (1 mL) and water (1 mL) at 0 °C. The reaction mixture stirred for 8 h at room temperature. Then, it was acidified to pH=3 by the addition of 1 M aqueous solution of HCl. A brown precipitate appeared which was collected by filtration and dried in vacuum (6.7 mg, 53% yield). ¹H NMR (600 MHz, DMSO-d₆): δ 9.76 (br, 1H), 9.59 (s, 1H), 8.30 – 8.26 (m, 2H), 7.90 (t, J = 7.2 Hz, 1H), 6.46 (t, J = 2.3 Hz, 1H), 6.30– 6.27 (m, 2H), 5.09 (t, J = 6.6 Hz, 2H), 4.08 – 4.02 (m, 4H), 3.52 – 3.47 (m, 4H), 3.18 (t, J = 6.5 Hz, 2H). MS-ESI (m/z): [M + H]⁺ calcd for C₂₁H₂₁Cl₂N₄O₃: 477.1; found 477.0; HRMS (m/z): [M + H]⁺ calcd for C₂₁H₂₁Cl₂N₄O₃: 477.0985; found, 477.1001

Cell Lines.

HT-1080, RD, and MCF-7 cells were obtained from ATCC and maintained in DMEM (Corning) supplemented with 10% FBS (Corning) and Anti-anti (Gibco) at 37°C in 5% CO2 in a humidified incubator. GCT cells were maintained in McCoy media. Before the screens, the cell lines were passaged at least twice after thawing. Cultures were confirmed to be free of mycoplasma infection using the MycoAlert Mycoplasma Detection Kit (Lonza).

Cell Proliferation Assay.

All compounds were pre-diluted in DMSO to a 10 mM stock concentration. Compounds were plated as a dose-response in a serial 1:3 dilutions of each agent. A set of control wells with DMSO was included on all plates as negative control. To ensure reproducibility and comparability with the subsequent combination studies, the IC₅₀ of Doxorubicin was used as reference in a dose response format in each plate as positive (total killing) control. Cells were seeded in white 384-well plates (Greiner) at 1000 cells/well in 50 ul of media using a Multidrop dispenser and allowed to attach for 2 h. Compounds were transferred to each well using a 100 nL head affixed to an Agilent Bravo automated liquid handling platform, and plates were incubated at 37°C in 5% CO₂ for an additional 72 hours. To measure the cell viability, CellTiter-Glo reagent (diluted 1:6 in water, Promega) was dispensed into the wells (30 uL), incubated for 3 minutes, and luminescence was read on a Envision plate reader (Perkin-Elmer). Final DMSO concentration in assay wells was 0.2%. The assay was performed with three biological replicates.

Kinetic Solubility Determination.

A solution containing 2% DMSO was prepared for each compound in DPBS (pH 7.4) by performing dilution using a 10 mM DMSO stock solution of each compound. This solution was sonicated for 30 min and shaken at for 16 h at 37°C, filtered and injected into the HPLC to compare the area found at wavelength 254 nm. Compounds which showed concentration of the soluble fraction higher than the LOD value (1 uM) were subjected for a calibration curve by plotting the area under the curve at 254 nm (UV by HPLC) against the concentration of the compounds injected after performing a serial dilution (0 μM–500 μM in DMSO) to calculate the exact concentration.

Metabolic Stability Study in Liver Microsomes.

In vitro metabolism was evaluated in liver microsomes (Xenotech) from mouse (CD-1 Mouse) by Wuxi AppTec. The incubation mixture was prepared in 100 mM potassium phosphate buffer containing 0.56 mg/mL microsomal protein, 1 mM NADPH, and 100 μM of compound. Reactions were initiated with the addition of NADPH. Samples were incubated at 37 °C and aliquots were sampled at 0, 5, 15, 30, 45, and 60 min. Reactions were quenched with acetonitrile at each time point. Samples were shaken for 10 min and centrifuged at 4000 rpm for 20 minutes at 4°C. Supernatant was transferred (80 μL) into 240 μL HPLC water, and mixed by plate shaker for 10 min. Each bioanalysis plate was sealed and shaken for 10 minutes prior to LC-MS/MS analysis.

Pharmacokinetic Study in Mouse.

All animal related procedures were conducted by Wuxi AppTec under their protocol in compliance with Animal Welfare Act regulations and the Guide for the Care and Use of Laboratory Animals. The formulation of FiVe1 and 4e was a suspension in 10% DMSO, 10% solutol, and 80% water. Following ip and po administration, whole blood was collected and transferred to an Eppendorf microcentrifuge tube containing EDTA. The blood was centrifuged, and plasma was transferred to a 96-well plate. Samples (5 μL) were precipitated and diluted with acetonitrile (200 μL) containing internal standard, and then the mixture was vortex-mixed for 10 min at 800 rpm and centrifuged for 15 min at 3220g at 4 °C. An aliquot of 50 μL supernatant was transferred to another clean 96-well plate and centrifuged for 5 min at 3220g at 4 °C. Then the supernatant was directly injected into an LC-MS/MS-AK_Q-Trap 6500 system for analysis. The pharmacokinetics parameters were calculated by analyzing the compound concentration in plasma samples using the pharmacokinetic software Phoenix WinNonlin 6.3.

Western Blot Analysis.

Total protein was harvested by lysing cells in RIPA Lysis and Extraction Buffer (Thermo Fisher, 89900) and Halt protease and phosphatase inhibitors (Thermo Fisher 78440). Lysates were incubated on a rocker at 4°C for 30 min and insoluble protein content was eliminated following centrifugation at 13,000 x g for 15 min. Protein concentrations were quantified with the Pierce BCA Protein Assay kit (Thermo Fisher, 23225). Proteins were then mixed with 10X Bolt^™ Sample Reducing Agent (Thermo Fisher, B0009) and 4X Bolt^™ LDS Sample Buffer (Thermo Fisher, B0007) and exposed to 95°C for 5 min. SDS-PAGE was performed using Bolt^™ 4 – 12% Bis-Tris protein gels (Thermo Fisher, NW04122BOX) and protein was transferred to PVDF membranes using wet transfer. Membranes were blocked with 5% nonfat dry milk in TBST (Tris buffered saline with 0.1% Tween 20) for an hour at room temperature and then probed with primary antibodies against VIM (Abcam, ab8069), S56-VIM (Cell Signaling Technology, 7391), and β-Tubulin (Sigma-Aldrich, T8328). After incubating with 800CW/680RD IRDye-linked secondary antibodies (LI-COR), membranes were imaged on an Odyssey^® CLx (LI-COR).

Crystal Structure of 4e.

The single crystal X-ray diffraction studies were carried out on a Bruker APEX II Ultra diffractometer equipped with Mo K_ɑ radiation (λ =0.71073 Å). Crystals of 4e were used as received (grown from MeOH). A 0.320 × 0.120 × 0.060 mm colorless crystal was mounted on a Cryoloop with Paratone oil. Data were collected in a nitrogen gas stream at 100(2) K using ϕ and ω scans. Crystal-to-detector distance was 50 mm and exposure time was 4.0 seconds (depending on the 2θ range) per frame using a scan width of 0.65°. Data collection was 100.0% complete to 25.242° in θ. A total of 35760 reflections were collected covering the indices, −9<=h<=9, −37<=k<=37, −9<=l<=8. 3511 reflections were found to be symmetry independent, with a R_int of 0.0298. Indexing and unit cell refinement indicated a Primitive, Monoclinic lattice. The space group was found to be P2₁/c. The data were integrated using the Bruker SAINT Software program and scaled using the SADABS software program. Solution by direct methods (SHELXT) produced a complete phasing model consistent with the proposed structure. All nonhydrogen atoms were refined anisotropically by full-matrix least-squares (SHELXL-2014). All carbon bonded hydrogen atoms were placed using a riding model. Their positions were constrained relative to their parent atom using the appropriate HFIX command in SHELXL-2014. Crystallographic data are summarized in Table S1.

Highlights.

• Novel potent and selective FiVe1 derivatives were synthesized.

• Structure-activity relationship studies yielded a more potent compound 4e, with potency in the double digit nanomolar range.

• Compound 4e was found to induce VIM Ser56 phosphorylation at 10-fold lower concentration (100 nM) when compared to FiVe1

• The pharmacokinetics profile for 4e in mouse demonstrated a superior oral bioavailability than FiVe1

• Discovery of new compounds derived from 4e exhibited superior profiles in terms of liver microsome stability and aqueous solubility.