A Threonine Turnstile Defines a Dynamic Amphiphilic Binding Motif in the AAA ATPase p97 Allosteric Binding Site

Abstract

The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cryo-EM structures of the AAA ATPase p97 was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand substituents. Conversely, diametric rotation of the χ₁ torsion by 150–180° orients the side chain β-methyl groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set of inhibitors of p97 covering a >2,000-fold variation in potencies, with a preference for either small-highly polar or small-apolar groups. The threonine turnstile motif was further validated by a PDB search that identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR structures demonstrating additional gear-like interactions between adjacent Thr pairs. Combined, these data suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets.

Graphical Abstract

The turnstile motion of two neighboring threonines accommodates both polar and apolar ligands in an allosteric binding site.

Introduction

The hexameric AAA+ (ATPases Associated with diverse cellular Activities) p97 serves as a major regulator of protein homeostasis. p97 assists in chromatin- and mitochondria-associated degradation, endoplasmic reticulum (ER)-associated degradation (ERAD), unfolded protein response (UPR), proteasome degradation, Golgi reassembly, endosomal tracking, protein aggregate processing, and autophagy.¹ Key to p97’s function is a remarkable conformational mobility in its 6×3 multi-domain subunits. Binding to nucleotides during the ATPase cycle repositions these domains around the ring-shaped core, transducing phosphate hydrolysis into mechanical force exerted on protein substrates.² The specific elements that control the coordinated ratchet motion within the D1, D2, and N domains in the 550 kDa hexameric complex are still under investigation.³ p97 has been recognized as a potential target for treating cancer as well as neurodegeneration,4^,5 thus triggering drug discovery efforts in both industry and academia.^{6,7,8, 9,10}

Results and discussion

In a recent series of indole-based allosteric inhibitors of p97, we were unable to completely rationalize the structure-activity relationship (SAR) of CF₃-bioisosteres at the indole C-5 position (R¹) of our lead structure (Figure 1).¹¹ Specifically, the analysis of substituent effects revealed a remarkably divergent electronic trend since both the 5-nitro and the 5-methyl substituent showed superior binding affinities versus the corresponding pentafluorosulfanyl-, methoxy-, trifluoromethoxy-, and trifluoromethyl-substituted analogs.¹¹ Even after the subsequent elucidation of a cryo-EM structure with a potent (IC₅₀ = 55 nM) 5-fluoro-substituted inhibitor of the same structural series bound to p97 (PDB entry 5FTJ),³ the inhibitor binding data could not be fully explained by their steric, polar, or electronic properties. Therefore, in the present study, we computationally refined a model of this p97 allosteric site based on the SAR of additional 5-substituted indoles and select arene and heteroarene analogs, which together span a range of more than 3 orders of magnitude in a biochemical assay (Figure 1 and Table 1). Examination of these small molecule probes revealed an unprecedented turnstile motion of two neighboring but discontinuous threonine (Thr) residues, setting up a dynamic side chain - heterocyclic ligand interaction that can accommodate both polar and apolar moieties in the allosteric binding pocket.

Fig. 1.

Structures of inhibitors used for comparing substituent effects at the 5-position of the indole or analogous heterocyclic scaffolds. R¹ see Table 1; R² = H or Me. Additional indole replacements include 5- and 7-azaindoles, naphthalene, benzimidazole, and benzofuran.

Table 1.

Biochemical Activities of p97 Inhibitors^a

Entry	Compound ID	R1 Indole Substitution (R2)	ADPGlo IC50 [μM]	ADPGlo Std. Dev.
1	UPCDC30283 (1)	5-CN (H)	0.044	0.045
2	UPCDC30287 (2)	5-NO2 (H)	0.047	0.040
3	UPCDC30346 (3)	5-F (Me)	0.050	0.044
4	UPCDC30245 (4)	5-F (H)	0.055	0.087
5	UPCDC30361 (5)	5-CN (Me)	0.087	0.047
6	UPCDC30310 (6)	5-CONH2 (H)	0.10	0.032
7	UPCDC30256 (7)	5-OH (H)	0.12	0.073
8	UPCDC30341 (8)	5-CO2Me (H)	0.13	0.076
9	UPCDC30083 (9)	5-H (H)	0.16	0.10
10	UPCDC30317 (10)	5-Cl (H)	0.19	0.14
11	UPCDC30288 (11)	5-N3 (H)	0.23	0.18
12	UPCDC30318 (12)	5-CH3 (H)	0.24	0.11
13	UPCDC30206 (13)	benzo[α]carbazole	0.39	0.069
14	UPCDC30367 (14)	5-CONHMe (H)	0.45	0.12
15	UPCDC30238 (15)	5-OCH3 (H)	0.71	0.22
16	UPCDC30257 (16)	5-OCF3 (H)	3.8	0.8
17	UPCDC30345 (17)	5-azaindole (H)	4.0	1.7
18	UPCDC30297 (18)	5-CF3 (H)	4.67	2.0
19	UPCDC30381 (19)	5-F,7-azaindole (H)	4.74	1.3
20	UPCDC30222 (20)	naphthalene (H)	5.2	1.1
21	UPCDC30221 (21)	benzofuran (H)	20.1	4.60
22	UPCDC30277 (22)	5-SF5 (H)	21.5	0.40
23	UPCDC30368 (23)	5-CONMe2 (H)	31.4	1.60
24	UPCDC30250 (24)	benzimidazole (H)	38.5	6.60
25	UPCDC30201 (25)	N-Me indole (H)	>50	-

^aAssay conditions: ADPGlo with 20 nM p97 ATPase WT in the presence of 100 μM ATP.¹² Assays were repeated multiple times (see Table S1 for an extended overview).

Central to developing a protein binding model capable of rationalizing our p97 SAR was that all inhibitors considered for the refinement contained an identical tetramine side chain, thereby creating a structurally homologous set of compounds differing only in the substitution pattern in the indole region. Semiempirical calculations initially suggested a correlation between the assay data (Table 1) and surface area calculations of the 5-substituted indoles.¹³ Specifically, van der Waals surface areas of the phenyl indole moiety of compounds 1, 2, 4, 6–12, 14–16, 18, 22 and 23 formed a least-squares linear fit with the log IC₅₀ data (R=0.72, df = 14, p <0.01). Upon selecting only examples with nonpolar 5-substitutions (9–12, 15, 16, 18 and 22), the linear fit slightly improved (R=0.91, df = 6, p <0.01). Conversely, the correlation for analogs with polar substitutions (1, 2, 4, 6–8, 14, and 23) dropped to R=0.81 (df = 6, p >0.01) (Figures S1–S3). Among several possible correlations of physicochemical parameters with activity, only the van der Waals surface areas provided R > 0.5. This suggested that for inhibitors with nonpolar indole 5-substituents, the main factor determining potent activity was the size of the substituent, with a preference for smaller groups. Polar substituents formed an independent cohort, and hydrogen bonding capabilities influenced the biological activity in addition to substituent size. Taken together, these data indicated that the protein binding subsite at the indole 5-position was sterically constrained and amphiphilic, accommodating hydrogen bond donors and acceptors, as well as small hydrophobic groups.

The cryo-EM co-structure of 5-F derivative 4 (UPCDC30245) bound to p97 showed how this specific inhibitor molecule engaged in multiple favorable contacts in the protein’s allosteric binding site, including a multipolar bond between the fluorine and a backbone carbonyl carbon (see Figure 2D in ref 3). However, the docking of other derivatives possessing polar and apolar 5-position substituents in the structure failed to provide a rationale for why substituents of divergent polarity should be equally active (Table 1). For example, the structure provided no explanation why a 5-NO₂ substitution would result in greater affinity than a 5-amide substituent, or why a hydrophobic 5-CH₃ substituent would be significantly more potent than a 5-OCF₃ derivative (Figure S4). Therefore, to gain a better understanding of potential conformational variabilities in amino acid backbone and side chain arrangements surrounding this allosteric binding site in the highly dynamic protein, we examined all available hexameric p97 structures in the protein data bank (PDB), including both cryo-EM and X-ray structures (PDB entries 3CF1, 3CF2, 3CF3, 5C1A, 5C18, 5C19, 5FTJ, 5FTK, 5FTL, 5FTM, and 5FTN).^3,14,15 Interestingly, we found that two separate loops (composed of residues 507–511 and 611–616), which border the site where the inhibitor indole 5-position is localized in the cryo-EM co-structure, displayed a high degree of conformational flexibility. In these different structures, the distances between the β-carbons of the amphiphilic sec-propanol side chains of residues Thr 509 and Thr 613 ranged from approx. 3.2 Å to 7.8 Å (Figure 2). Furthermore, we observed that at least one of the side chains of the Thr pair could form van der Waals contacts with substituents at the 5-position of the indole in some conformations of the canonical ensemble. Given the combined steric restriction and divergent SAR of the 5-position substituents, along with the conformational flexibility of these loops, we hypothesized that, upon inhibitor binding and concomitant hydrophobic collapse with the binding site loops, the amphiphilic Thr side chain pair could form a subsite in agreement with the sterically restricted, amphiphilic SAR of the indole series.

Fig. 2.

Variable orientations of loops 507–511 and 611–616 near the allosteric binding site of UPCDC30245 (4). Shown is the distance range between the side chain β-carbons of loop residues Thr 509 and Thr 613 observed in ADP-bound X-ray crystal structure 3CF3 (cyan) and ADP-bound cryo-EM structures 5FTJ (magenta, with inhibitor complex) and 5FTK (green), and ATPγS-bound cryo-EM structures 5FTL (yellow) and 5FTM (orange).

Using cryo-EM co-structure 5FTJ as a starting point, we employed constrained molecular mechanics and dynamics protocols to anneal the bis-Thr side chain pair to form a subsite for the 5-polar substitutions (e.g., the 5-OH of UPCDC30256, 7). This subsite is characterized by a water-coordinated, stabilized hydrogen bonding network with the polar inhibitors (Figure 3a). When the Thr side chain χ₁ torsions are rotated in a turnstile mode (150–180°), a subsite formed by the Thr side chain methyl groups accommodates nonpolar 5-substitutions (e.g., 5-methyl indole inhibitor UPCDC30318, 12, Figure 3b). Moreover, χ rotations of the Thr pair are facilitated by the ATP binding cavity of the D2 domain, which is located on the opposite side of the loops bordering the allosteric site. Specifically, when the γ-hydroxyl groups rotate out of the binding pocket, the water molecule shown in Figure 3a is extruded into the ATP binding cavity (as shown in Figure 3b), and is accommodated, along with the γ-hydroxyls, by bulk solvent in the cavity (Figure 3b). Conversely, in the polar conformation, the γ-methyls of the Thr pair, which have very low solvent accessible surface areas, are oriented toward the ATP binding cavity, and the bulk solvent in the cavity is not significantly affected.

Fig. 3.

Upon inhibitor binding, the loops containing the Thr 509-Thr 613 pair collapse to form an amphiphilic binding site at the indole 5-position. (a) Polar subsite: the Thr γ-hydroxyls engage in a network of hydrogen bonds with polar inhibitor substituents. (b) Hydrophobic subsite: rotation of the Thr χ₁ torsions by 150–180° results in γmethyl group interactions with small hydrophobic moieties on the indole 5-position. In this conformation, the bridging water molecule has been displaced from the binding pocket.

These alternate binding modes incorporate key inhibitor contacts observed in the cryo-EM co-structure (Figure 4), including the critical hydrogen bond between the backbone amide carbonyl of Val 493 and the inhibitor indole NH, π-stacking between the Phe 618 side chain phenyl and the inhibitor indole, favorable hydrophobic collapse between the inhibitor phenyl-indole and the side chains of residues Pro 510, Ala 537, Pro 571, and Lys 614 (i.e., the residue’s side chain methylenes), a favorable dipole-dipole interaction between the Cys 535 thiol and the inhibitor piperidine N, and a hydrogen bond between the carboxylate of Glu 534 and the piperidine-4-amino substituent. In total, these interactions result in a tightly bound phenyl indole scaffold that facilitates inhibitor tetramine side chain interactions with residues of helices 16 and 17 (Figure 4). Specifically, piperazine N1 is predicted to hydrogen bond with the side chain amide of Gln 494, while the substituent’s N4 hydrogen bonds with the carboxylate of Glu 534, and the isopropyl moiety desolvates via favorable hydrophobic collapse with the side chains of Trp 476 and Ile 531 (Figure 4).

Fig. 4.

Schematic of polar (top) and apolar (or hydrophobic) (bottom) inhibitor binding modes exemplified by 5-hydroxyl analog UPCDC30256 (7, top) and 5-methyl analog UPCDC30318 (12, bottom). In both binding modes, the yellow shaded region represents the amphiphilic bis-Thr subsite. Blue dashes indicate hydrogen bonds/dipole-dipole interactions, green dashes indicate hydrophobic contacts, and green lines depict the hydrophobic and steric continuity of the binding site. Pink shading indicates a critical hydrogen bond for optimal inhibitor activity, while green circles indicate π-stacking.

Molecular docking studies employing hydropathic scoring (Table S2 and Figure S5) ^{16, 17} with all of the inhibitors listed in Table 1 were used to further refine the allosteric site on p97. Several key SAR aspects were investigated. Binding modes for high affinity (IC₅₀ <150 nM) polar 5-substitutions (entries 1–8; 5-CN, 5-NO₂, 5-F, 5-CN, 5-CONH₂, 5-OH, and 5-CO₂Me) were distinguished by favorable hydrogen bonding networks similar to those shown in Figures 3a and 5a. For these derivatives, the polar 5-moieties had good complementary fits for the bis-Thr polar subsite that were characterized by either the formation of direct hydrogen bonds with a Thr side chain hydroxyl group, or via water mediation, or both. Similarly, good complementarity was achieved when docking potent (IC₅₀ <500 nM) 5-substituted apolar moieties (entries 9–13; 5-H, 5-Cl, 5-N₃, 5-CH₃, and benzo[α]carbazole). These binding modes were characterized by favorable hydrophobic interactions with the side chain methyl groups of the bis-Thr, as depicted in Figure 3b.

Fig. 5.

Comparison of the binding modes of the 5-substituted amide series comprised of 6, 14, and 23, and for contrast, ester derivative 8. Yellow dashes = hydrogen bonds; red dashes = unfavorable contacts. (a) The potent binding affinity of UPCDC30310 (6, IC₅₀ = 100 nM) is typical of the polar binding mode, with the two Thr side chain hydroxyl groups and the indole 5-position amide nitrogen coordinating a water molecule in a hydrogen bonding network. Additionally, the 5-position amide nitrogen forms a hydrogen bond with the backbone carbonyl carbon of Ser 511. (b) For UPCDC30367 (14), substitution of a single methyl group on the amide nitrogen necessitates the engagement of the Thr 613 methyl group to accommodate the hydrophobic addition, while the polar amide carbonyl group hydrogen bonds with the Thr 509 hydroxyl group. In this suboptimal binding mode (IC₅₀ = 450 nM), the water-mediated hydrogen bond observed for 6 is lost, and the amide methyl moiety assumes an unfavorable, partially solvent-exposed orientation (red sphere). (c) Dimethylation of the amine in the C(5)-amide (UPCDC30368, 23) is sterically prohibited (red sphere), resulting in a near loss of all potency. (d) For comparison with methyl amide 14, the binding model of the more potent methyl ester UPCDC30341 (8, IC₅₀ = 130 nM) indicates that this substituent’s methyl group, unlike the conformationally restricted N-methyl of 14, can avoid solvent exposure by orienting toward the bottom edge of the binding pocket, while the methoxy oxygen engages in a weak hydrogen bond with the coordinated water molecule.

Disruption of the indole NH to Val 493 hydrogen bond (Figure 4) by N-methylation in UPCDC30201 (25) or by replacement of the indole with a naphthalene isostere in UPCDC30222 (20) ablates or greatly diminishes activity, respectively. Introduction of a nitrogen atom at the indole 3-position in benzimidazole UPCDC30250 (24) reduces binding affinity by >2 orders of magnitude. The lone pair of the imine N of the benzimidazole presents an incompatible negative electrostatic potential near the hydrophobic α–, β–, and aromatic carbons of Phe 618. Furthermore, if this N atom is complexed to a water molecule (via hydrogen bonding) during the binding event, an even greater hydrophobic-polar incompatibility ensues.

The 5-azaindole nitrogen of UPCDC30345 (17) has an electrostatic potential similar to the benzimidazole N, and is also suboptimal for this binding pocket. Its negative electrostatic potential or coordination with water places it too close to the hydrophobic side chain components of Pro 510 and the hydrocarbon component of Lys 614. Similarly, other derivatives, such as 5-fluoro-7-azaindole UPCDC30381 (19) introduce a destabilizing polar mismatch between the 7-position N and the backbone carbonyl group of Leu 492.

Finally, some polar 5-substituents (14 and 23 (Figure 5b and c, respectively)) and hydrophobic substituents (15, 16, 18, and 22) are either sterically less favorable (e.g., 14) or simply too large to fit into either binding mode. For example, the increased steric bulk of dimethylamide 23 results in the loss of a water-mediated hydrogen bond, solvent exposure, and unfavorable steric contacts (i.e., versus potent amide analog 6) (Figure 5a–c). Interestingly, unlike methylamide 14 (IC₅₀ = 450 nM), which is sterically less favorable than amide 6, methyl ester 8 is significantly more potent (IC₅₀ = 130 nM), as the more flexible methoxy group can rotate slightly to avoid solvent exposure. This orientation also directs the methoxy oxygen of the ester to form a hydrogen bond with a water molecule (Figure 5d), thereby rationalizing its similar potency to C(5)-amide 6.

Based on the importance of the bis-Thr pair in rationalizing the amphiphilic SAR of our allosteric inhibitors of p97, we conducted a search of the PDB to examine the generality of this motif at small molecule ligand binding sites. All PDB entries with ligands having >200 residues in the longest chain and a resolution ≤2.5 Å were evaluated. Figure 6a illustrates the search criteria used for this study. Among the 19,881 entries meeting the criteria, 840 (4.2%) were found to possess discontinuous (>5 residues apart in loop sequence) Thr pairs for which the distance between the residues’ β-carbons was ≤5 Å (similar to our model), and where the Thr β-carbons were in the vicinity of a ligand (Figure 6a). Interestingly, we found an active site with a bis-Thr pair occurring on separate loops in protein kinase B (ATK1) (see Supporting Information and Figure S6 for additional details). Similar to our model, the Thr pair could also adopt conformations to accommodate either a polar or a hydrophobic contact in the same binding pose. In the co-crystal structure (PDB entry 2UVY) depicted in Figure 6b,¹⁸ the bis-Thr pair in the polar binding mode is positioned for hydrogen bonding directly to the 9H-nitrogen of the inhibitor, and when the 9H-nitrogen is replaced with a carbon (PDB entry 2X39, Figure 6c),¹⁹ the bis-Thr χ₁ torsion changes to form an apolar conformation, with the Thr β-methyl groups closing off the hydrophobic pocket. The specific compounds from the indicated PDB entries possess different side chain substituents on the respective positions of their purine and pyrrolo-pyrimidine cores, and thus a comparison of their inhibitory activity is not straightforward. However, similar potencies are observed for analogous pairs with identical substitutions (see core pairs 14 and 15, 17 and 18, 20 and 21, 22 and 23, and 24 and 25 in Table 2 in reference 20).

Figure 6.

(a) Schematic of the criteria used to search the PDB for bis-Thr pairs in the vicinity of ligands. (b) Protein kinase B inhibitor with a 9H-purine component near the bis-Thr pair in the polar conformation. In this structure, the purine N9 engages in a hydrogen bond (yellow dash) with one of the Thr side chain hydroxyls. (c) Protein kinase B inhibitor with a 7H-pyrrolo[2,3-d]pyrimidine moiety near the bis-Thr pair in the apolar conformation. In this inhibitor, N9 is replaced with a carbon, which consequently engages in favorable hydrophobic contacts (orange dash) with a Thr methyl group.

To further broaden the scope of the PDB analysis, the initial search criteria were relaxed to allow for the identification of bis-Thr pairs (within 7 Å of a ligand) occurring in any secondary structural feature (i.e., helices, sheets, and loops). From this search we found additional X-ray co-crystal examples of ligands interacting with at least one Thr of a bis-Thr pair. Examples include heat shock protein 90 (HSP90), β-lactamases (CTX-M-9 and BEL-1), and AMPA glutamate receptor 2 (GluR2) (Figure S7). The HSP90 threonines both occur in β sheets, the β-lactamase threonines occur on a β sheet and a loop, and the GluR2 threonines occur on a β sheet and an α helix. While in these additional examples the bis-Thr pairs did not display the χ rotations observed in PKB inhibitor co-crystal structures, it is interesting that they support a consistently polar binding mode in the solid state (i.e., X-ray structures), as the interacting ligand moieties in these examples were always polar and interacted with the γ-hydroxyl of the Thr side chain. Consequently, we considered the possibility that the bis-Thr turnstile movement may not be readily observable in solid state structures. Therefore, we further expanded our search to examine solution NMR structures for paired Thr χ rotations, under the assumption that the cooperative motion between paired Thr residues’ side chains would be more readily detected in solution. Accordingly, 8,687 non-redundant NMR entries meeting the search criteria described above were identified. Of these, a subset of 271 structures contained Thr pairs where both side chain χ torsions rotated simultaneously by >50°. Although this subset did not contain ligands interacting with bis-Thr pairs, the finding that 271 structures did display full and simultaneous χ rotations occurring in Thr pairs signifies that any subsite created by these residues can be both polar and hydrophobic. It is noteworthy, yet not surprising, that such bis-Thr turnstile motion is more prominent in solution structures versus X-ray structures.

For example, Figure 7a shows the 15 conformers deposited with NMR PDB entry 2FIN. In Figures 7b and 7c, the Thr pair in the polar subsite conformation (conformer 12) and the hydrophobic subsite conformation (conformer 1) are shown, respectively. Movie S1, which was created directly from the 15 conformers deposited for the protein entry, clearly displays the residues simultaneously rotating in a turnstile manner to gear between polar and hydrophobic subsite orientations.

Fig. 7.

The bis-Thr pair in NMR structure, PDB entry, 2FIN. (a) The PDB entry contains 15 conformers, with a bis-Thr pair formed by residues Thr 78 and Thr 95. Over the 15 conformers of the protein, the two Thr residues display a range of χ torsion rotations. (b) An example of a polar bis-Thr conformation as seen in conformer 12 of the protein ensemble. In this example, the hydroxyl hydrogen atom of Thr 78 engages in a hydrogen bond (yellow dash) with the hydroxyl oxygen atom of Thr 95. (c) An example of a hydrophobic (or apolar) bis-Thr conformation as seen in conformer 1 of the protein ensemble. In this example, the methyl groups of Thr 78 and Thr 95 engage in a favorable hydrophobic contact (orange dash). Movie S1 shows the rotations observed for the 15 conformers in the PDB entry.

Conclusions

We utilized a series of focused small molecule probes and extensive structural information from the PDB to develop a unified computational model that is able to rationalize the divergent potency in a series of phenyl indole-based p97 inhibitors. The SAR of all 25 probe molecules can be comprehensively explained by an allosteric binding site model, with the dynamic turnstile nature of two amphiphilic Thr side chains providing a compelling rationalization for the observation that inhibitors possessing either polar or hydrophobic substituents at the same position are simultaneously accommodated in the binding site. Furthermore, the results from our PDB search identified a similar amphiphilic bis-Thr turnstile in ligand complexes of protein kinase B, suggesting this previously unrecognized feature may frequently occur in small molecule protein binding sites. In support of this hypothesis, an analysis of NMR/solution structures provided further evidence for the concept that side chains of Thr pairs are able to interact in a dynamic, gear-like manner in protein structures. We can speculate that the function of such Thr pairs is to position water molecules in protein allosteric sites, as well as other locations that are either functionally important, and/or structurally significant. For example, we suggest that, in the absence of an inhibitor, the function of the bis-Thr pair in the p97 allosteric site identified in the cryo-EM co-structure³ is to shuttle water molecules out of the structurally dynamic interface between the D1 and D2 domains. Future studies will investigate the functional role of this bis-Thr pair in p97 protein dynamics.

Experimental section

Materials and Methods

Synthetic summary schemes, computational methods and any associated references and additional discussion as well as a summary SAR table are available in the online version in the Supplementary Information of this paper.

Synthesis

General Synthetic Methods

All non-aqueous reactions were carried out under a nitrogen atmosphere in oven- or flame-dried glassware unless otherwise noted. Anhydrous tetrahydrofuran and diethyl ether were distilled from sodium benzophenone ketyl; anhydrous dichloromethane and toluene were distilled from CaH₂; alternatively, the same solvents were obtained from a solvent purification system using alumina columns. All other solvents and reagents were used as obtained from commercial sources without further purification unless noted. Reactions were monitored via TLC using 250 μm pre-coated silica gel 60 F₂₅₄ plates, which were visualized with 254 nm and/or 365 nm UV light and by staining with KMnO₄ (1.5 g KMnO₄, 10 g K₂CO₃, and 1.25 mL 10% NaOH in 200 mL water), cerium molybdate (0.5 g Ce(NH₄)₂(NO₃)₆, 12 g (NH₄)₆Mo₇O₂₄•4H₂O, and 28 mL conc. H₂SO₄ in 235 mL water), or vanillin (6 g vanillin and 1.5 mL conc. H₂SO₄ in 100 mL EtOH). Flash chromatography was performed with SiliCycle silica gel 60 (230–400 mesh) or with ISCO MPLC. Microwave reactions were performed using a Biotage Initiator in glass microwave vials (cap sealed) with continuous magnetic stirring and an external surface temperature sensor. ¹H and ³¹C NMR spectra were recorded on Bruker Avance 300, 400, or 500 MHz spectrometers, using the residual solvent as an internal standard. ¹⁹F NMR spectra were obtained using a proton-decoupled pulse sequence without internal standard. IR spectra were obtained on a Smiths IdentifyIR or PerkinElmer Spectrum 100. HRMS data were obtained on a Thermo Scientific Exactive HRMS coupled to a Thermo Scientific Accela HPLC system using a 2.1 × 50 mm 3.5 μm Waters XTerra C₁₈ column eluting with MeCN/H₂O containing 0.1% formic acid. Purity of compounds was assessed using the same HPLC system with either the PDA or an Agilent 385 ELSD. All final screening samples passed QC based on >95% purity by LC/MS/ELSD analysis.

Experimental Procedures

2-(3-Bromophenyl)-1H-indole-5-carbonitrile (26)

A solution of 3-bromo acetophenone (4.6 g, 23 mmol), 4-aminobenzonitrile (2.5 g, 21 mmol), and TsOH•H₂O (36 mg, 0.20 mmol) in toluene (100 mL) was heated overnight under Dean-Stark conditions. The reaction mixture was cooled to room temperature, concentrated, and purified by chromatography on SiO₂ (0 to 100% EtOAc/hexanes) to provide (E)-4-((1-(3-bromophenyl)ethylidene)amino)-benzonitrile (3.6 g, 12 mmol, 57%) as a yellowish oil that was used without further purification.

A solution of (E)-4-((1-(3-bromophenyl)ethylidene)amino)benzonitrile (3.0 g, 10 mmol), Pd(OAc)₂ (225 mg, 1.0 mmol), and Cu(OAc)₂ (5.58 g, 30 mmol) in DMSO (100 mL) was heated at 40 °C for 24 h, cooled to room temperature, diluted with EtOAc (150 mL), and filtered through Celite. The filtrate was washed with sat. NH₄Cl and brine. The organic layer was dried (MgSO₄), filtered, concentrated, and purified by chromatography on SiO₂ (20% EtOAc/hexanes) followed by trituration with EtOAc and hexanes to give 2-(3-bromophenyl)-1H-indole-5-carbonitrile (26, 1.56 g, 5.25 mmol, 52%) as a yellow solid: IR (ATR) 3301, 2216, 1599, 1584, 1560, 1512, 1457, 1441, 1437, 1340, 1322, 1247, 1170, 876, 825, 800, 792, 774, 673 cm^{−1; 1}H NMR (400 MHz, DMSO-d₆) δ 12.22 (s, 1 H), 8.13 (br s, 1 H), 8.09 (br s, 1 H), 7.91 (d, J = 7.6 Hz, 1 H), 7.56 (d, J = 8.4 Hz, 2 H), 7.48-7.43 (m, 2 H), 7.16 (s, 1 H); ³¹C NMR (100 MHz, DMSO-d₆) δ 138.9, 138.5, 133.5, 131.1, 130.8, 128.1, 127.7, 125.8, 124.7, 124.4, 122.5, 120.5, 112.6, 101.7, 100.5; HRMS (ESI⁺) m/z calcd for C₁₅H₁₀BrN₂ 297.0022 (M+H), found 297.0022.

2-(3-(4-((2-(4-Isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carbonitrile (1, UPCDC30283)

A solution of 2-(3-bromophenyl)-1H-indole-5-carbonitrile (26, 0.15 g, 0.50 mmol), LiHMDS (0.20 g, 1.2 mmol), Pd₂(dba)₃ (9.2 mg, 0.010 mmol), and CyJohnPhos (14.0 mg, 0.040 mmol) in anhydrous THF was treated with tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(piperidin-4-yl)carbamate^3,11 (A, 0.213 g, 0.600 mmol). The reaction mixture was heated at 75 °C overnight, cooled to room temperature, diluted with sat. NaHCO₃, and extracted with CH₂Cl₂ (3×). The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (2% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (CH₂Cl₂) to provide tert-butyl (1-(3-(5-cyano-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (0.16 g, 0.28 mmol, 56%) as a yellow foamy solid: IR (ATR) 2965, 2932, 2809, 2216, 1685, 1653, 1599, 1448, 1411, 1362, 1320, 1245, 1172, 1146, 1010, 971, 898, 805, 775 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 9.60 (br s, 1 H), 7.92 (s, 1 H), 7.44 (d, J = 8.4 Hz, 1 H), 7.34 (dd, J = 8.4, 1.6 Hz, 1 H), 7.29 (t, J = 8.0 Hz, 1 H), 7.20 (br s, 1 H), 7.15 (d, J = 7.6 Hz, 1 H), 6.88 (br d, J = 7.2 Hz, 1 H), 6.81 (s, 1 H), 4.05 (br s, 1 H), 3.74 (br s, 2 H), 3.22 (br s, 2 H), 2.66-2.44 (m, 13 H), 1.85-1.64 (m, 4 H), 1.48 (s, 9 H), 1.03 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, CDCl₃) δ 155.6, 151.9, 141.2, 138.7, 132.4, 129.9, 129.1, 125.9, 124.9, 121.0, 116.91, 116.85, 113.8, 111.9, 103.0, 100.0, 80.1, 54.6, 53.90, 53.88, 49.5, 48.7, 30.14, 30.14, 28.6, 18.6; HRMS (ESI⁺) m/z calcd for C₃₄H₄₇O₂N₆ 571.3755 (M+H), found 571.3759.

A solution of TFA (0.53 mL, 7.0 mmol) and triethylsilane (0.11 mL, 0.70 mmol) in CH₂Cl₂ (1 mL) was added to a solution of tert-butyl (1-(3-(5-cyano-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (40 mg, 0.07 mmol) in CH₂Cl₂ (0.5 mL). After 1 h, the reaction mixture was concentrated, diluted with sat. NaHCO₃, and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (8 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 10% MeOH/CH₂Cl₂) to provide 2-(3-(4-((2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carbonitrile 1 (UPCDC30283, 21 mg, 0.045 mmol, 65%) as a yellow oil: IR (ATR) 3334, 2936, 2930, 2872, 2809, 2214, 1599, 1577, 1461, 1381, 1361, 1241, 1176, 1144, 1124, 982, 859, 803, 775, 734 cm^{−1; 1}H NMR (500 MHz, acetone-d₆) δ 11.28 (br s, 1 H), 8.00 (s, 1 H), 7.56 (d, J = 8.5 Hz, 1 H), 7.47 (br s, 1 H), 7.40 (dd, J = 8.3, 1.3 Hz, 1 H), 7.32-7.28 (m, 2 H), 7.02 (s, 1 H), 6.97 (d, J = 7.5 Hz, 1 H), 3.78-3.74 (m, 2 H), 2.90-2.84 (m, 2 H), 2.72 (t, J = 6.3 Hz, 2 H), 2.66-2.55 (m, 4 H), 2.46-2.40 (m, 8 H), 1.98-1.95 (m, 2 H), 1.48 (qd, J = 13.5, 3.5 Hz, 2 H), 0.97 (d, J = 7.0 Hz, 6 H); ³¹C NMR (125 MHz, acetone-d₆) δ 153.2, 142.4, 139.8, 133.1, 130.5, 130.0, 126.2, 125.1, 121.2, 116.9, 116.8, 113.8, 113.1, 103.5, 100.2, 59.0, 55.6, 54.9, 54.6, 49.4, 48.6, 44.4, 33.2, 18.8; HRMS (ESI⁺) m/z calcd for C₂₉H₃₈N₆ 471.3231 (M+H), found 471.3231.

2-(5-Bromo-2-methylphenyl)-5-fluoro-1H-indole (27)

A solution of 1-(5-bromo-2-methylphenyl)ethanone²¹ (5.00 g, 23.5 mmol), 4-fluoroaniline (2.77 g, 24.6 mmol), and TsOH•H₂O (91 mg, 0.47 mmol) in toluene (150 mL) was heated for 40 h under Dean-Stark conditions. The reaction mixture was cooled to room temperature, concentrated, and purified by chromatography on SiO₂ (100% hexanes followed by 5% Et₂O/hexanes with 1% TEA) to provide (E)-1-(5-bromo-2-methylphenyl)-N-(4-fluorophenyl)ethan-1-imine (7.03 g, 23.0 mmol, 98%) as a yellow oil that was used without further purification.

A solution of (E)-1-(5-bromo-2-methylphenyl)-N-(4-fluorophenyl)ethan-1-imine (4.03 g, 13.2 mmol), Pd(OAc)₂ (0.296 g, 1.32 mmol), and Cu(OAc)₂ (7.32 g, 39.5 mmol) in DMSO (130 mL) was heated at 40 °C for 45 h, cooled to room temperature, diluted with EtOAc (500 mL), and filtered through Celite. The filtrate was washed with water (3×) and brine. The organic layer was dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (10% Et₂O/hexanes) followed by trituration with Et₂O and hexanes to give 2-(5-bromo-2-methylphenyl)-5-fluoro-1H-indole (27, 2.01 g, 6.61 mmol, 50%) as a white powder: IR (ATR) 3430, 2993, 2980, 1769, 1758, 1586, 1480, 1372, 1245, 1241, 1055 cm^{−1; 1}H NMR (300 MHz, DMSO-d₆) δ 11.49 (s, 1 H), 7.73 (d, J = 2.1 Hz, 1 H), 7.47 (dd, J = 8.1, 2.1 Hz, 1 H), 7.39 (dd, J = 8.9, 4.7 Hz, 1 H), 7.34-7.30 (m, 2 H), 6.97 (td, J = 9.2, 2.4 Hz, 1 H), 6.66 (d, J = 1.5 Hz, 1 H), 2.44 (s, 3 H); ³¹C NMR (100 MHz, DMSO-d₆) δ 157.1 (d, J_CF = 231.0 Hz), 137.4, 134.9, 134.3, 133.2, 133.1, 130.9, 130.3, 128.4 (d, J_CF = 10.0 Hz), 118.8, 112.3 (d, J_CF = 10.0 Hz), 109.9 (d, J_CF = 25.0 Hz), 104.6 (d, J_CF = 23.0 Hz), 102.8 (d, J_CF = 4.0 Hz), 20.6; ¹⁹F NMR (376 MHz, DMSO-d₆) δ -124.6; HRMS (ESI⁺) m/z calcd for C₁₅H₁₂BrFN 304.0132 (M+H), found 304.0131.

1-(3-(5-Fluoro-1H-indol-2-yl)-4-methylphenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (3, UPCDC30346)

A solution of 2-(5-bromo-2-methylphenyl)-5-fluoro-1H-indole (27, 0.100 g, 0.409 mmol), tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(piperidin-4-yl)carbamate (A, 0.160 g, 0.450 mmol), Pd₂(dba)₃ (8 mg, 0.008 mmol), and CyJohnPhos (12 mg, 0.033 mmol) in anhydrous THF (0.5 mL) in a microwave vial was degassed by bubbling argon for 20–30 min. The reaction mixture was charged with LiHMDS (0.180 g, 1.02 mmol) and the vial was sealed and heated at 80 °C overnight. The reaction mixture was cooled to room temperature, diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (5 to 15% MeOH/CH₂Cl₂) to provide tert-butyl (1-(3-(5-fluoro-1H-indol-2-yl)-4-methylphenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (0.116 g, 0.201 mmol, 49%): ¹H NMR (300 MHz, CDCl₃) δ 8.23 (s, 1 H), 7.34-7.28 (m, 2 H), 7.18 (d, J = 8.4 Hz, 1 H), 7.01 (d, J = 2.1 Hz, 1 H), 6.97-6.86 (m, 2 H), 6.54 (d, J = 0.9 Hz, 1 H), 4.11 (br s, 1 H), 3.72 (d, J = 12.0 Hz, 2 H), 3.24 (br s, 2 H), 2.81-2.48 (m, 13 H), 2.38 (s, 3 H), 1.78 (br s, 4 H), 1.47 (s, 9 H), 1.11 (br s, 6 H); HRMS (ESI⁺) m/z calcd for C₃₄H₄₉FN₅O₂ 578.3845 (M+H), found 578.3864.

To a solution of tert-butyl (1-(3-(5-fluoro-1H-indol-2-yl)-4-methylphenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (0.100 g, 0.173 mmol) in CH₂Cl₂ (2 mL) at 0 °C was added trifluoroacetic acid (0.64 mL, 8.6 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 3 h, treated with sat. NaHCO₃, and extracted with EtOAc (3×). The combined organic layers were washed with brine, dried (MgSO₄), filtered, concentrated, and purified by chromatography on SiO₂ (2 to 5% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 5% MeOH/CH₂Cl₂) to provide 1-(3-(5-fluoro-1H-indol-2-yl)-4-methylphenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine 3 (UPCDC30346, 49 mg, 0.10 mmol, 60%) as an off-white solid: IR (ATR) 3157, 2930, 2807, 1607, 1491, 1448, 1379, 1178, 1123, 1107, 982, 848, 785, 760 cm^{−1; 1}H NMR (400 MHz, CD₃OD) δ 7.33 (dd, J = 8.8, 4.4 Hz, 1 H), 7.19 (dd, J = 10.0, 2.4 Hz, 1 H), 7.16 (d, J = 8.4 Hz, 1 H), 7.11 (d, J = 2.4, 1 H), 6.88 (dd, J = 8.4, 2.8 Hz, 1 H), 6.86 (td, J = 9.2, 2.4 Hz, 1 H), 6.48 (br s, 1 H), 3.69 (app d, J = 12.6 Hz, 2 H), 2.78-2.74 (m, 4 H), 2.65-2.50 (m, 12 H), 2.38 (s, 3 H), 2.00 (app d, J = 12.4 Hz, 2 H), 1.53 (qd, J = 11.9, 3.5 Hz, 2 H), 1.07 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, CD₃OD) δ 159.2 (d, J_CF = 230.3 Hz), 151.0, 141.5, 134.6, 134.4, 132.6, 130.5 (d, J_CF = 10.3 Hz), 128.6, 118.9, 118.0, 112.6 (d, J_CF = 9.8 Hz), 110.3 (d, J_CF = 26.3 Hz), 105.3 (d, J_CF = 23.3 Hz), 102.9 (d, J_CF = 4.9 Hz), 58.4, 56.2, 55.9, 54.1, 50.5, 49.6, 43.8, 32.8, 20.4, 18.7; HRMS (ESI⁺) m/z calcd for C₂₉H₄₁FN₅ 478.3341 (M+H), found 478.3340.

2-(5-Bromo-2-methylphenyl)-1H-indole-5-carbonitrile (28)

Prepared by the same 2 step procedure as for the compound 27 using 1-(5-bromo-2-methylphenyl)ethanone¹⁸ (5.00 g, 23.5 mmol, 1.0 equiv) and 4-aminobenzonitrile (2.97 g, 24.6 mmol, 1.05 equiv). The crude residue was purified by chromatography on SiO₂ (10 to 20% EtOAc/hexanes) followed by trituration with EtOAc and hexanes to give 2-(5-bromo-2-methylphenyl)-1H-indole-5-carbonitrile (28, 1.37 g, 4.39 mmol, step 1, 85% and step 2, 34%) as a yellow solid: IR (ATR) 3308, 2992, 2982, 2220, 1769, 1758, 1471, 1372, 1241, 1094, 1049 cm^{−1; 1}H NMR (400 MHz, DMSO-d₆) δ 12.00 (s, 1 H), 8.10 (s, 1 H), 7.75 (d, J = 2.0 Hz, 1 H), 7.56 (d, J = 8.4 Hz, 1 H), 7.52-7.46 (m, 2 H), 7.32 (d, J = 8.4 Hz, 1 H), 6.81 (d, J = 1.2 Hz, 1 H), 2.43 (s, 3 H); ³¹C NMR (100 MHz, DMSO-d₆) δ 138.13, 138.07, 135.2, 133.5, 133.2, 131.1, 130.7, 127.9, 125.8, 124.4, 120.7, 118.8, 112.5, 103.3, 101.4, 20.5; HRMS (ESI⁺) m/z calcd for C₁₆H₁₂BrN₂ 311.0178 (M+H), found 311.0176.

2-(5-(4-((2-(4-Isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)-2-methylphenyl)-1H-indole-5-carbonitrile (5, UPCDC30361)

A solution of 2-(5-bromo-2-methylphenyl)-1H-indole-5-carbonitrile (28, 0.100 g, 0.397 mmol), tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(piperidin-4-yl)carbamate (A, 0.123 g, 0.437 mmol), Pd₂(dba)₃ (6 mg, 0.006 mmol), and CyJohnPhos (9.0 mg, 0.025 mmol) in anhydrous THF (0.5 mL) in a microwave vial was degassed by bubbling argon for 20–30 min. The reaction mixture was charged with LiHMDS (0.180 g, 1.02 mmol) and the vial was sealed and heated at 80 °C for 12 h. The reaction mixture was cooled to room temperature, diluted with water and extracted with EtOAc (3×). The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (0 to 10% MeOH/CH₂Cl₂) to provide tert-butyl (1-(3-(5-cyano-1H-indol-2-yl)-4-methylphenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (0.093 g, 0.16 mmol, 51%) as a brown solid: IR (ATR) 3301, 2960, 2932, 2890, 2218, 1685, 1659, 1605, 1499, 1465, 1363, 1318, 1299, 1172, 1144, 1010, 898, 803, 749, 728 cm^{−1; 1}H NMR (500 MHz, CDCl₃) δ 8.83 (br s, 1 H), 7.97 (s, 1 H), 7.47 (d, J = 8.0 Hz, 1 H), 7.42 (d, J = 8.5 Hz, 1 H), 7.18 (d, J = 8.0 Hz, 1 H), 7.00 (br s, 1 H), 6.90 (d, J = 8.0 Hz, 1 H), 6.63 (br s, 1 H), 4.08 (br s, 1 H), 3.71 (app d, J = 10.0 Hz, 2 H), 3.23 (br s, 2 H), 2.77-2.48 (m, 13 H), 2.37 (s, 3 H), 1.80-1.75 (m, 4 H), 1.47 (s, 9 H), 1.08 (d, J = 4.5 Hz, 6 H); ³¹C NMR (125 MHz, CDCl₃) δ 155.5, 149.7, 140.6, 137.9, 132.2, 132.0, 128.7, 127.3, 126.0, 124.9, 121.0, 117.5, 117.4, 111.8, 103.17, 103.13, 80.1, 58.4, 55.0, 53.6, 53.1, 50.0, 48.6, 30.2, 28.7, 20.0, 18.5; HRMS (ESI⁺) m/z calcd for C₃₅H₄₉O₂N₆ 585.3912 (M+H), found 585.3911.

To a solution of tert-butyl (1-(3-(5-cyano-1H-indol-2-yl)-4-methylphenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (0.070 g, 0.112 mmol) in CH₂Cl₂ (2 mL) at 0 °C was added trifluoroacetic acid (0.36 mL, 4.8 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 2 h, treated with sat. NaHCO₃, and extracted with CH₂Cl₂ (3×). The combined organic layers were washed with brine, dried (MgSO₄), filtered, concentrated, and purified by chromatography on SiO₂ (0 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 5% MeOH/CH₂Cl₂) to provide 2-(5-(4-((2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)-2-methylphenyl)-1H-indole-5-carbonitrile 5 (UPCDC30361, 38 mg, 0.078 mmol, 66%) as a light yellow solid: IR (ATR) 3293, 3120, 2956, 2924, 2811, 2705, 2214, 1603, 1560, 1500, 1459, 1379, 1333, 1320, 1273, 1232, 1176, 1113, 982, 803 cm^{−1; 1}H NMR (400 MHz, CD₃OD) δ 7.98 (d, J = 0.8 Hz, 1 H), 7.52 (d, J = 8.8 Hz, 1 H), 7.38 (dd, J = 8.4, 1.6 Hz, 1 H), 7.18 (d, J = 8.8 Hz, 1 H), 7.11 (d, J = 2.8 Hz, 1 H), 6.94 (dd, J = 8.4, 2.8 Hz, 1 H), 6.64 (d, J = 0.8 Hz, 1 H), 3.71-3.68 (m, 2 H), 2.78-2.71 (m, 4 H), 2.66-2.50 (m, 12 H), 2.37 (s, 3 H), 2.00 (d, J = 10.8 Hz, 2 H), 1.52 (qd, J = 11.9, 3.6 Hz, 2 H), 1.07 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, CD₃OD) δ 151.1, 142.4, 139.8, 133.5, 132.7, 130.1, 128.6, 126.6, 125.1, 121.9, 118.7, 118.4, 113.0, 103.4, 102.9, 58.2, 56.3, 56.0, 54.1, 50.3, 49.6, 43.7, 32.8, 20.3, 18.7; HRMS (ESI⁺) m/z calcd for C₃₀H₄₁N₆ 485.3387 (M+H), found 485.3388.

2-(3-(4-((2-(4-Isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carboxamide (6, UPCDC30310)

To a solution of tert-butyl (1-(3-(5-cyano-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (62 mg, 0.11 mmol) in DMSO was added K₂CO₃ (15 mg, 0.11 mmol) in water (0.2 mL) at 0 °C then a solution of 30% w/w hydrogen peroxide (1.2 mL) was added dropwise to the mixture. The resulting mixture was stirred at 5 °C for 5 min then the ice bath was removed. After another 5 min, water (10 mL) was added to the reaction mixture and extracted with EtOAc (3×10mL). The combined organic layers were washed with brine, dried (MgSO₄), filtered, concentrated, and purified by chromatography on SiO₂ to provide tert-butyl (1-(3-(5-carbamoyl-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (35 mg, 0.059 mmol, 55%) as a yellow oil: IR (ATR) 3208, 2973, 2962, 1668, 1653, 1586, 1474, 1448, 1431, 1363, 1333, 1245, 1146, 1103, 1049, 1020, 757, 710, 689 cm^{−1; 1}H NMR (500 MHz, CDCl₃) δ 9.55 (br s, 1 H), 8.15 (s, 1 H), 7.66 (d, J = 8.5 Hz, 1 H), 7.44 (d, J = 8.5 Hz, 1 H), 7.30 (t, J = 7.8 Hz, 2 H), 7.18 (d, J = 7.5 Hz, 1 H), 6.88 (d, J = 8.0 Hz, 1 H), 6.83 (s, 1 H), 6.36 (br s, 1 H), 5.74 (br s, 1 H), 4.11 (br s, 1 H), 3.81 (app d, J = 8.1 Hz, 2 H), 3.18 (br s, 2 H), 2.80-2.64 (m, 11 H), 2.45-2.44 (m, 2 H), 1.78-1.73 (m, 4 H), 1.47 (s, 9 H), 1.10 (d, J = 6.0 Hz, 6 H); HRMS (ESI⁺) m/z calcd for C₃₄H₄₈O₃N₆ 589.3861 (M+H), found 589.3859.

A solution of TFA (0.45 mL, 5.9 mmol) and triethylsilane (0.10 mL, 0.59 mmol) in CH₂Cl₂ (1 mL) was added to a solution of tert-butyl (1-(3-(5-carbamoyl-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (35 mg, 0.059 mmol) in CH₂Cl₂ (0.5 mL). After 1 h, the reaction mixture was concentrated, diluted with sat. NaHCO₃, and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (8 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 10% MeOH/CH₂Cl₂) to provide 2-(3-(4-((2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carboxamide 6 (UPCDC30310, 19 mg, 0.039 mmol, 65%) as a yellow foam: IR (ATR) 3302, 2943, 2829, 1637, 1603, 1540, 1474, 1435, 1384, 1338, 1250, 1176, 1146, 1020, 757, 738, 723, 710 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 9.71 (br s, 1 H), 8.10 (s, 1 H), 7.62 (d, J = 8.4 Hz, 1 H), 7.36 (d, J = 8.4 Hz, 1 H), 7.24 (d, J = 8.4 Hz, 2 H), 7.12 (d, J = 7.2 Hz, 1 H), 6.87 (d, J = 8.0 Hz, 1 H), 6.78 (s, 1 H), 6.39 (br s, 1 H), 5.98 (br s, 1 H), 3.69 (d, J = 12.0 Hz, 2 H), 2.80-2.70 (m, 4 H), 2.62-2.45 (m, 12 H), 1.94 (app d, J = 10.8 Hz, 2 H), 1.52-1.44 (m, 2 H), 1.02 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, CDCl₃) δ 171.1, 152.2, 140.6, 139.1, 132.9, 129.9, 129.0, 125.2, 121.6, 120.8, 116.8, 116.4, 113.8, 111.2, 100.5, 58.1, 55.3, 54.6, 53.7, 48.9, 48.7, 43.5, 32.6, 18.9; HRMS (ESI⁺) m/z calcd for C₂₉H₄₁ON₆ 489.3336 (M+H), found 489.3335.

2-(3-(4-((2-(4-Isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indol-5-ol (7, UPCDC30256)

To a solution of N-(2-(4-isopropylpiperazin-1-yl)ethyl)-1-(3-(5-methoxy-1H-indol-2-yl)phenyl)piperidin-4-amine 15¹ (UPCDC30238, 60.0 mg, 0.124 mmol) in dry CH₂Cl₂ (15 mL) under nitrogen was added dropwise boron tribromide (1.0 M solution in CH₂Cl₂, 0.49 mL, 0.49 mmol) at room temperature and stirred for 1 h. The reaction was quenched with MeOH, concentrated and purified by chromatography on SiO₂ (8 to 20% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 10% MeOH/CH₂Cl₂) to provide 2-(3-(4-((2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indol-5-ol 7 (UPCDC30256, 55.0 mg, 0.119 mmol, 96%) as a colorless oil: IR (ATR) 3260, 2960, 2932, 2818, 1705, 1623, 1599, 1584, 1489, 1452, 1420, 1381, 1363, 1312, 1295, 1273, 1253, 1221, 1200, 1178, 1146, 1118, 971 cm^{−1; 1}H NMR (500 MHz, acetone-d₆) δ 10.49 (s, 1 H), 7.42 (s, 1 H), 7.26-7.21 (m, 3 H), 6.97 (d, J = 2.5 Hz, 1 H), 6.88-6.86 (m, 1 H), 6.71-6.69 (m, 2 H), 3.76-3.72 (m, 2 H), 2.86-2.81 (m, 2 H), 2.73 (t, J = 6.3 Hz, 2 H), 2.65-2.55 (m, 2 H), 2.48-2.41 (m, 10 H), 1.97-1.94 (m, 2 H), 1.48 (qd, J = 14.0, 3.5 Hz, 2 H), 0.98 (d, J = 6.5 Hz, 6 H); ³¹C NMR (125 MHz, acetone-d₆) δ 153.1, 152.2, 140.0, 134.4, 133.0, 131.0, 130.2, 116.6, 115.9, 113.5, 112.8, 112.3, 105.0, 99.1, 58.9, 55.7, 54.9, 54.5, 49.4, 48.8, 44.2, 33.2, 30.6, 18.8; HRMS (ESI⁺) m/z calcd for C₂₈H₄₀ON₅ 462.3227 (M+H), found 462.3227.

Methyl 2-(3-bromophenyl)-1H-indole-5-carboxylate (29)

A solution of 3-bromo acetophenone (8.3 g, 41 mmol), 4-aminobenzoate (5.7 g, 37 mmol), and 4 Å molecular sieves (24 g) in toluene (180 mL) was refluxed for 28 h under Dean-Stark conditions. A second portion of 4 Å molecular sieves (16 g) was added and refluxed for an additional 95 h. The reaction mixture was filtered through a pad of Celite, concentrated and purified by chromatography on SiO₂ (5 to 10% Et₂O/hexanes with 2% TEA) to give methyl (E)-4-((1-(5-bromo-2-methylphenyl)ethylidene)amino)-benzoate (2.40 g, ca 20%) as a yellowish-green solid.

A solution of methyl (E)-4-((1-(5-bromo-2-methylphenyl)ethylidene)amino)-benzoate (2.34 g, 7.04 mmol), Pd(OAc)₂ (0.158 g, 0.704 mmol), and Cu(OAc)₂ (3.84 g, 21.1 mmol) in DMSO (70 mL) was heated at 40 °C for 40 h, cooled to room temperature, diluted with water (280 mL), filtered through Celite, and extracted with EtOAc (4x). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (6 to 10% Et₂O/hexanes) followed by trituration with EtOAc and hexanes to give methyl 2-(3-bromophenyl)-1H-indole-5-carboxylate (29, 1.6 g, 4.8 mmol, 69%) as a white solid: IR (ATR) 2993, 2911, 2227, 1433, 1404, 1308, 1042, 951, 925, 725, 697, 667 cm^{−1; 1}H NMR (400 MHz, DMSO-d₆) δ 12.02 (s, 1 H), 8.25 (d, J = 1.2 Hz, 1 H), 8.11 (t, J = 1.8 Hz, 1 H), 7.91-7.88 (m, 1 H), 7.76 (dd, J = 8.6, 1.7 Hz, 1 H), 7.54 (ddd, J = 8.0, 1.9, 0.9 Hz, 1 H), 7.46 (dt, J = 15.0, 7.8 Hz, 2 H), 7.17 (s, 1 H), 3.85 (s, 3 H); ³¹C NMR (100 MHz, DMSO-d₆) δ 167.1, 139.8, 137.7, 133.9, 131.1, 130.5, 128.0, 127.5, 124.2, 123.0, 122.8, 122.5, 121.1, 111.4, 101.1, 51.7; HRMS (ESI⁺) m/z calcd for C₁₆H₁₃BrO₂N 330.0124 (M+H), found 330.0123.

Methyl 2-(3-(4-((2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carboxylate (8, UPCDC30341)

A solution of methyl 2-(3-bromophenyl)-1H-indole-5-carboxylate (29, 0.150 g, 0.445 mmol), tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(piperidin-4-yl)carbamate (A, 0.189 g, 0.534 mmol) and K₃PO₄ (0.146 g, 0.668 mmol) in deoxygenated dioxane (1 mL) in a 2–5 mL microwave vial was degassed by bubbling with argon for 20–30 min. The reaction mixture was charged with Pd₂(dba)₃ (8 mg, 0.009 mmol), and CyJohnPhos (13 mg, 0.036 mmol) and the vial was sealed and heated at 110 °C for 18 h. The reaction mixture was cooled to room temperature, diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (0 to 15% MeOH/EtOAc) to provide methyl 2-(3-(4-((tert-butoxycarbonyl)(2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carboxylate (0.18 g, 0.30 mmol, 68%) as a brown solid: IR (ATR) 2958, 2813, 1707, 1685, 1601, 1577, 1446, 1435, 1308, 1247, 1165, 1144, 1124, 1089, 1008, 917, 768 cm^{−1; 1}H NMR (300 MHz, CDCl₃) δ 8.87 (br s, 1 H), 8.38 (s, 1 H), 7.89 (dd, J = 8.6, 1.4 Hz, 1 H), 7.40 (d, J = 8.7 Hz, 1 H), 7.32 (t, J = 8.0 Hz, 1 H), 7.21 (s, 1 H), 7.14 (d, J = 7.5 Hz, 1 H), 6.91 (dd, J = 8.3, 1.4 Hz, 1 H), 6.86 (s, 1 H), 4.13 (br s, 1 H), 3.94 (s, 3 H), 3.81 (d, J = 12.3 Hz, 2 H), 3.25-3.24 (m, 2 H), 2.87-2.47 (m, 13 H), 1.85-1.77 (m, 4 H), 1.48 (s, 9 H), 1.06 (d, J = 6.6 Hz, 6 H); ³¹C NMR (75 MHz, CDCl₃) δ 168.3, 155.5, 151.9, 140.1, 139.5, 133.0, 130.0, 129.0, 123.7, 123.6, 122.4, 116.8, 116.6, 113.7, 110.7, 101.0, 80.0, 54.7, 53.6, 52.0, 49.7, 48.5, 30.2, 28.7, 18.5; HRMS (ESI⁺) m/z calcd for C₃₅H₅₀O₄N₅ 604.3857 (M+H), found 604.3856.

To a solution of methyl 2-(3-(4-((tert-butoxycarbonyl)(2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carboxylate (80 mg, 0.13 mmol) in CH₂Cl₂ (2 mL) at 0 °C was added TFA (0.40 mL, 5.3 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 2 h, treated with sat. NaHCO₃, and extracted with EtOAc (2x). The combined organic layer was washed with brine, dried (MgSO₄), filtered, concentrated, and purified by chromatography on SiO₂ (0 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 5% MeOH/CH₂Cl₂) to provide methyl 2-(3-(4-((2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carboxylate 8 (UPCDC30341, 30 mg, 0.060 mmol, 45%): IR (ATR) 3327, 2958, 2930, 2809, 1707, 1691, 1599, 1577, 1545, 1433, 1379, 1344, 1310, 1249, 1169, 1122, 1088, 982, 768, 690 cm^{−1; 1}H NMR (300 MHz, CD₃OD) δ 8.29 (s, 1 H), 7.79 (dd, J = 8.4, 1.2 Hz, 1 H), 7.43 (app d, J = 9.0 Hz, 2 H), 7.32-7.26 (m, 2 H), 6.96-6.92 (m, 1 H), 6.90 (s, 1 H), 3.90 (s, 3 H), 3.79 (app d, J = 12.3 Hz, 2 H), 2.84-2.73 (m, 4 H), 2.66-2.49 (m, 12 H), 2.01 (app d, J = 11.7 Hz, 2 H), 1.53 (qd, J = 11.8, 3.1 Hz, 2 H), 1.07 (d, J = 6.6 Hz, 6 H); ³¹C NMR (75 MHz, CD₃OD) δ 170.1, 153.4, 141.8, 141.5, 134.2, 130.7, 130.1, 124.1, 123.9, 122.4, 117.9, 117.5, 114.7, 111.8, 100.8, 58.4, 56.2, 55.9, 54.1, 52.3, 49.9, 43.8, 32.8, 18.7; HRMS (ESI⁺) m/z calcd for C₃₀H₄₁O₂N₅ 504.3333 (M+H), found 504.3330.

8-(3-(1H-Indol-2-yl)phenyl)-1,4-dioxa-8-azaspiro[4.5]decane (30)

A suspension of 2-(3-bromophenyl)-1H-indole¹¹ (0.40 g, 1.5 mmol), K₃PO₄ (0.48 g, 2.2 mmol), Pd₂(dba)₃ (32 mg, 0.034 mmol), CyJohnPhos (42 mg, 0.12 mmol) in dry, degassed dioxane (10 mL) was treated with 1,4-dioxa-8-azaspiro[4.5]decane (0.30 mL, 2.4 mmol). The flask was sealed and the reaction mixture was heated at 110 °C for 6 h under microwave irradiation. The reaction mixture was diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered, concentrated and purified by chromatography on SiO₂ (30 to 40% EtOAc/hexanes) to provide 8-(3-(1H-indol-2-yl)phenyl)-1,4-dioxa-8-azaspiro[4.5]decane (30, 0.40 g, 1.2 mmol, 81%) as a pale yellow foam: Mp 142-143 °C; IR (ATR) 3348, 2957, 2927, 2886, 1600, 1484, 1354, 1219, 1096, 776, 746 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1 H), 7.64 (d, J = 7.6 Hz, 1 H), 7.40 (dd, J = 8.0, 0.4 Hz, 1 H), 7.32 (t, J = 8.0 Hz, 1 H), 7.27-7.26 (m, 1 H), 7.20 (td, J = 7.6, 1.2 Hz, 1 H), 7.16-7.12 (m, 2 H), 6.93 (dd, J = 8.4, 2.0 Hz, 1 H), 6.81 (app d, J = 1.2 Hz, 1 H), 4.02 (s, 4 H), 3.42 (t, J = 5.8 Hz, 4 H), 1.90 (t, J = 5.8 Hz, 4 H); ³¹C NMR (100 MHz, CDCl₃) δ 151.5, 138.6, 136.8, 133.4, 129.9, 129.4, 122.3, 120.7, 120.3, 116.7, 116.2, 113.8, 111.0, 107.2, 100.0, 64.5, 48.0, 34.7; HRMS (ESI⁺) m/z calcd for C₂₁H₂₃O₂N₂ 335.1754 (M+H), found 335.1758.

1-(3-(1H-Indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (9, UPCDC30083)

A solution of 8-(3-(1H-indol-2-yl)phenyl)-1,4-dioxa-8-azaspiro[4.5]decane (30, 0.20 g, 0.60 mmol) in acetone (70 mL) and 3.5 M HCl (60 mL) was heated at 80 °C for 3.5 h. The reaction mixture was cooled to 0 °C, quenched with Na₂CO₃ (14 g) and extracted with EtOAc (3×). The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (25 to 30% EtOAc/hexanes) to provide 1-(3-(1H-indol-2-yl)phenyl)piperidin-4-one (0.14 g, 0.48 mmol, 80%) as a pale yellow solid which is not stable under air and was used immediately: ¹H NMR (300 MHz, CDCl₃) δ 8.35 (s, 1 H), 7.63 (d, J = 7.8 Hz, 1 H), 7.42-7.35 (m, 2 H), 7.28-7.27 (m, 1 H), 7.23-7.10 (m, 3 H), 6.95 (app dd, J = 8.0, 2.3 Hz, 1 H), 6.82 (app d, J = 1.5 Hz, 1 H), 3.69 (t, J = 6.0 Hz, 4 H), 2.61 (t, J = 6.2 Hz, 4 H); HRMS (ESI⁺) m/z calcd for C₁₉H₁₉ON₂ 291.1492 (M+H), found 291.1489.

A solution of crude 1-(3-(1H-indol-2-yl)phenyl)piperidin-4-one (ca. 87 mg, 0.30 mmol) in 1,2-DCE (3 mL) was treated with 2-(4-isopropylpiperazin-1-yl)ethanamine²² (B, 0.077 g, 0.45 mmol) followed by Ti(OiPr)₄ (0.10 mL, 0.32 mmol) and stirred at room temperature overnight. The reaction mixture was treated with NaBH(OAc)₃ (130 mg, 0.60 mmol) in a single portion. After 2 h, the reaction mixture was diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, concentrated and purified by chromatography on SiO₂ (5 to 20% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 7% MeOH/CH₂Cl₂) to provide 1-(3-(1H-indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine 9 (UPCDC0083, 95 mg, 0.21 mmol, 71% over 2-steps) as a pale yellow foam: IR (ATR) 3422, 3222, 2957, 2931, 2808, 1600, 1450, 1294, 1144, 776, 746 cm^{−1; 1}H NMR (400 MHz, CD₃OD) δ 7.52 (d, J = 8.0 Hz, 1 H), 7.40-7.37 (m, 2 H), 7.26-7.20 (m, 2 H), 7.11-7.07 (m, 1 H), 7.00 (app t, J = 7.4 Hz, 1 H), 6.82 (dt, J = 7.5, 1.9 Hz, 1 H), 6.77 (s, 1 H), 3.70-3.66 (m, 2 H), 2.66 (td, J = 12.2, 1.5 Hz, 2 H), 2.59-2.36 (m, 14 H), 1.87 (br d, J = 11.2 Hz, 2 H), 1.41 (qd, J = 11.8, 3.5 Hz, 2 H), 1.01 (d, J = 6.8 Hz, 6 H); ³¹C NMR (100 MHz, CD₃OD) δ 153.3, 139.8, 138.7, 134.8, 130.6, 122.6, 121.2, 120.5, 117.8, 116.9, 114.5, 112.1, 99.7, 58.4, 56.0, 55.8, 54.0, 49.9, 49.5, 43.6, 32.9, 18.7; HRMS (ESI⁺) m/z calcd for C₂₈H₄₀N₅ 446.3278 (M+H), found 446.3277.

2-(3-Bromophenyl)-5-chloro-1H-indole (31)

A solution of 3-bromo acetophenone (4.0 g, 20 mmol), 4-chloroaniline (2.6 g, 20 mmol), and TsOH•H₂O (35 mg, 0.20 mmol) in toluene (50 mL) was heated overnight under Dean-Stark conditions. The reaction mixture was cooled to room temperature, concentrated, and purified by chromatography on SiO₂ (0 to 5% EtOAc/hexanes) to provide (E)-1-(3-bromophenyl)-N-(4-chlorophenyl)ethan-1-imine as a yellowish oil (4.6 g, 15 mmol, 74%) that was used without further purification.

A solution of (E)-1-(3-bromophenyl)-N-(4-chlorophenyl)ethan-1-imine (1.2 g, 3.9 mmol), Pd(OAc)₂ (87 mg, 0.39 mmol), and Cu(OAc)₂ (2.2 g, 12 mmol) in DMSO (100 mL) was heated at 40 °C for 24 h, cooled to room temperature, diluted with EtOAc (150 mL), and filtered through Celite. The filtrate was washed with sat. NH₄Cl, and brine. The organic layer was dried (MgSO₄), filtered, concentrated, and purified by chromatography on SiO₂ (20% EtOAc/hexanes) followed by trituration with EtOAc and hexanes to give 2-(3-bromophenyl)-5-chloro-1H-indole (31, 0.76 g, 2.5 mmol, 63%) as a yellow solid: IR (ATR) 3431, 1683, 1564, 1452, 1439, 1305, 1281, 1249, 1057, 922, 865, 803, 772, 680 cm^{−1; 1}H NMR (300 MHz, CD₃OD) δ 7.94 (t, J = 1.7 Hz, 1 H), 7.72 (dt, J = 7.8, 1.4 Hz, 1 H), 7.50 (d, J = 1.8 Hz, 1 H), 7.43 (ddd, J = 8.0, 1.8, 1.1 Hz, 1 H), 7.35-7.29 (m, 2 H), 7.07 (dd, J = 8.7, 2.1 Hz, 1 H), 6.78 (s, 1 H); ³¹C NMR (75 MHz, CD₃OD) δ 139.2, 137.3, 135.9, 131.7, 131.40, 131.38, 129.0, 126.3, 125.0, 124.0, 123.3, 120.6, 113.4, 100.4.

1-(3-(5-Chloro-1H-indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (10, UPCDC30317)

A solution of 2-(3-bromophenyl)-5-chloro-1H-indole (31, 77 mg, 0.25 mmol), LiHMDS (0.10 g, 0.60 mmol), Pd₂(dba)₃ (4.6 mg, 0.005 mmol), and CyJohnPhos (7.0 mg, 0.020 mmol) in anhydrous THF was treated with tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(piperidin-4-yl)carbamate (A, 0.106 g, 0.300 mmol). The reaction mixture was heated at 75 °C overnight, cooled to room temperature, diluted with sat. NaHCO₃, and extracted with CH₂Cl₂ (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), concentrated, and purified by chromatography on SiO₂ (2 to 10% MeOH/CH₂Cl₂) to give tert-butyl (1-(3-(5-chloro-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (68 mg, 0.12 mmol, 47%) as a foam: IR (ATR) 2962, 2956, 2949, 2932, 2926, 2807, 1685, 1653, 1599, 1575, 1463, 1446, 1411, 1381, 1363, 1329, 1303, 1269, 1245, 1172, 1144, 1103, 1059, 1048, 1010, 993, 982, 971, 915, 900, 861, 772, 755, 734, 718, 692 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 9.13 (br s, 1 H), 7.55 (d, J = 1.6 Hz, 1 H), 7.29-7.25 (m, 2 H), 7.17 (br s, 1 H), 7.13-7.08 (m, 2 H), 6.84 (br d, J = 7.2 Hz, 1 H), 6.69 (s, 1 H), 4.08 (br s, 1 H), 3.71-3.69 (m, 2 H), 3.21 (br s, 2 H), 2.69-2.44 (m, 13 H), 1.71 (s, 4 H), 1.49 (s, 9 H), 1.04 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, CDCl₃) δ 155.6, 151.8, 140.1, 135.3, 133.0, 130.3, 129.8, 125.6, 122.3, 119.8, 118.2, 116.8, 116.5, 113.73, 113.72, 112.0, 99.3, 80.1, 58.4, 54.7, 53.8, 49.5, 48.6, 30.1, 28.6, 18.6; HRMS (ESI⁺) m/z calcd for C₃₃H₄₇ClO₂N₅ 580.3413 (M+H), found 580.3411.

A solution of TFA (0.84 mL, 11.2 mmol) and triethylsilane (0.18 mL, 1.1 mmol) in CH₂Cl₂ (1 mL) was added to a solution of tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(1-(3-(5-(trifluoromethyl)-1H-indol-2-yl)phenyl)piperidin-4-yl)carbamate (65 mg, 0.11 mmol) in CH₂Cl₂ (0.5 mL). After 1 h, the reaction mixture was concentrated, diluted with sat. NaHCO₃, and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (8 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 10% MeOH/CH₂Cl₂) to provide 1-(3-(5-chloro-1H-indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine 10 (UPCDC30317, 41 mg, 0.085 mmol, 76%) as a yellow foam: IR (ATR) 3181, 2960, 2932, 2926, 2814, 1599, 1577, 1461, 1448, 1381, 1359, 1344, 1310, 1294, 1273, 1217, 1176, 1146, 1118, 1059, 917, 861, 790, 775, 755, 738, 690 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 8.75 (s, 1 H), 7.56 (d, J = 2.0 Hz, 1 H), 7.29 (t, J = 8.0 Hz, 2 H), 7.19 (t, J = 2.0 Hz, 1 H), 7.12-7.07 (m, 2 H), 6.90 (dd, J = 8.4, 2.0 Hz, 1 H), 6.70 (d, J = 1.6 Hz, 1 H), 3.73-3.70 (m, 2 H), 2.85-2.75 (m, 4 H), 2.66-2.48 (m, 13 H), 2.01-1.98 (m, 2 H), 1.52 (qd, J = 11.6, 3.0 Hz, 2 H), 1.04 (d, J = 6.8 Hz, 6 H); ³¹C NMR (100 MHz, CDCl₃) δ 152.1, 140.2, 135.2, 132.9, 130.4, 129.8, 125.7, 122.4, 119.9, 116.4, 116.3, 113.6, 111.9, 99.4, 58.1, 55.1, 54.6, 53.6, 48.8, 48.6, 43.5, 32.6, 18.8; HRMS (ESI⁺) m/z calcd for C₂₈H₃₉ClN₅ 480.2889 (M+H), found 480.2887.

tert-Butyl (1-(3-(5-amino-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (32)

A solution of tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(1-(3-(5-nitro-1H-indol-2-yl)phenyl)piperidin-4-yl)carbamate¹¹ (0.102 g, 0.173 mmol) in MeOH (5 mL) was evacuated, flushed with argon (2x) and treated with 10% Pd/C (0.019 g, 0.17 mmol). The reaction mixture was evacuated and subjected to H₂ (1 atm - balloon). After 2 h, the solution was filtered through a plug of Celite, rinsed with MeOH and concentrated to provide tert-butyl (1-(3-(5-amino-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (32, 0.070 g, 0.15 mmol, 72%) as an orange oil that was used without further purification: ¹H NMR (400 MHz, CD₃OD) δ 7.36 (br s, 1 H), 7.26-7.20 (m, 3 H), 6.94 (br s, 1 H), 6.89 (br d, J = 5.2 Hz, 1 H), 6.69 (br d, J = 8.4 Hz, 1 H), 6.61 (br s, 1 H), 4.09-3.81 (m, 3 H), 3.35-3.25 (m, 2 H), 2.85-2.50 (m, 13 H), 2.01-1.74 (m, 4 H), 1.47 (s, 9 H), 1.11 (br s, 6 H); HRMS (ESI⁺) m/z calcd for C₃₃H₄₉O₂N₆ 561.3912 (M+H), found 561.3912.

tert-Butyl (1-(3-(5-azido-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (33)

Into a dried flask under nitrogen was added tert-butyl (1-(3-(5-amino-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (32, 0.085 g, 0.15 mmol) in AcOH (4 mL). The reaction mixture was cooled to 0 °C and protected from light (foil wrap) and treated with a solution of NaNO₂ (0.012 g, 0.17 mmol) in H₂O (0.4 mL). After 10 min, a solution of NaN₃ (0.010 g, 0167 mmol) in H₂O (0.4 mL) was added dropwise. After 45 min, the mixture was slowly poured into H₂O (10 mL) and sat. Na₂CO₃ was added (8 mL) until neutral pH. The mixture was extracted with EtOAc (3×), washed with brine, dried (Na₂SO₄), filtered, and concentrated. The crude product was purified by chromatography on SiO₂ (0 to 10 % MeOH/CH₂Cl₂) to provide tert-butyl (1-(3-(5-azido-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (33, 0.053 g, 0.090 mmol, 60%) as an orange foam: ¹H NMR (400 MHz, acetone-d₆) δ 10.92 (br s, 1 H), 7.47-7.43 (m, 2 H), 7.29-7.26 (m, 3 H), 6.95-6.92 (m, 1 H), 6.87 (br s, 1 H), 6.83 (dd, J = 8.6, 2.2 Hz, 1 H), 4.47 (br s, 1 H), 3.91 (br d, J = 11.6 Hz 2 H), 3.24 (br s, 2 H), 2.80 (t, J = 12.0 Hz, 2 H), 2.67 (dt, J = 13.1, 6.5 Hz, 1 H), 2.53 (br s, 8 H), 2.44 (t, J = 7.4 Hz, 3 H), 1.93 (br s, 2 H), 1.75 (br s, 2 H), 1.45 (s, 9 H), 1.00 (d, J = 6.4 Hz, 6 H); HRMS (ESI⁺) m/z calcd for C₃₃H₄₇O₂N₈ 587.3816 (M+H), found 587.3815.

1-(3-(5-Azido-1H-indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (11, UPCDC30288)

To a cooled solution of tert-butyl (1-(3-(5-azido-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (33, 0.050 g, 0.085 mmol) and lutidine (0.020 mL, 0.17 mmol) in dry CH₂Cl₂ (5 mL) was added dropwise a solution of TMSOTf (0.023 mL, 0.127 mmol) in CH₂Cl₂ (5 mL) at 0 ^°C. After 2 h, the reaction mixture was quenched with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with water followed by brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (8 to 20% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (10% MeOH/CH₂Cl₂) to provide 1-(3-(5-azido-1H-indol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine 11 (UPCDC30288, 0.025 g, 0.051 mmol, 60%) as an orange foam: IR (ATR) 2960, 2937, 2931, 2876, 2811, 1599, 1582, 1577, 1476, 1458, 1452, 1381, 1359, 1342, 1331, 1305, 1266, 1217, 1176, 1146, 1117, 852, 777, 772, 751 cm^{−1; 1}H NMR (400 MHz, acetone-d₆) δ 10.84 (br s, 1 H), 7.44-7.42 (m, 2 H), 7.30-7.24 (m, 3 H), 6.94-6.91 (m, 1 H), 6.87-6.86 (m, 1 H), 6.83 (dd, J = 8.6, 2.2 Hz, 1 H), 3.74 (dt, J = 12.5, 3.4 Hz, 2 H), 2.86 (td, J = 11.9, 2.3 Hz, 2 H), 2.71 (t, J = 6.2 Hz, 2 H), 2.66-2.56 (m, 2 H), 2.47-2.38 (m, 10 H), 1.99-1.94 (m, 2 H), 1.50-1.40 (m, 2 H), 0.98 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, acetone-d₆) δ 153.2, 141.4, 141.3, 136.0, 135.9, 133.72, 133.68, 132.4, 131.1, 131.0, 130.4, 116.7, 116.4, 114.3, 113.6, 113.23, 113.18, 110.3, 99.5, 99.4, 59.0, 55.6, 54.9, 54.6, 49.4, 48.7, 44.3, 33.3, 18.8; HRMS (ESI⁺) m/z calcd for C₂₈H₃₉N₈ 487.3292 (M+H), found 487.3293.

2-Bromo-6,11-dihydro-5H-benzo[a]carbazole (34) ²³

A mixture of phenylhydrazine (0.506 g, 4.44 mmol) and 7-bromo-1-tetralone (1.02 g, 4.44 mmol) in EtOH (10 mL) and conc. HCl (0.5 mL) was heated at reflux for 4 h. The reaction mixture was concentrated and the resulting solid was suspended in hexanes/CH₂Cl₂ (21 mL, 20:1) and stirred for 30 min. The light red solid (34, 0.84 g, 2.8 mmol, 63%) was collected by filtration and dried: ¹H NMR (300 MHz, CDCl₃) δ 8.11 (br s, 1 H), 7.56 (d, J = 7.5 Hz, 1 H), 7.44-7.43 (m, 1 H), 7.38 (d, J = 8.1 Hz, 1 H), 7.29-7.28 (m, 1 H), 7.23-7.22 (m, 1 H), 7.20-7.12 (m, 1 H), 3.02-2.95 (m, 4 H); HRMS (ESI⁺) m/z calcd for C₁₆H₁₃NBr 298.0220 (M+H), found 298.0224.

1-(6,11-Dihydro-5H-benzo[a]carbazol-2-yl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (13, UPCDC30206)

A suspension of 34 (0.079 g, 0.26 mmol), K₃PO₄ (0.087 g, 0.40 mmol), Pd₂(dba)₃ (0.005 g, 0.005 mmol) and CyJohnPhos (0.0076 g, 0.021 mmol) in dry degassed dioxane (1 mL) was treated with A (0.11 g, 0.32 mmol) in dioxane (2.5 mL). The reaction mixture was degassed by bubbling argon for 15 min, sealed and heated at 120 °C for 48 h. The reaction mixture was cooled to room temperature, diluted with sat. NaHCO₃ and extracted with CH₂Cl₂ (3×). The combined organic layer was washed with brine, dried (Na₂SO₄) and concentrated. The crude residue was purified by chromatography on SiO₂ (2% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (CH₂Cl₂) to afford tert-butyl (1-(6,11-dihydro-5H-benzo[a]carbazol-2-yl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (0.050 g, 0.087 mmol, 33%) as a pale yellow amorphous solid: ¹H NMR (300 MHz, CDCl₃) δ 8.28 (br s, 1 H), 7.54 (d, J = 7.5 Hz, 1 H), 7.37 (d, J = 8.1 Hz, 1 H), 7.20-7.09 (m, 3 H), 6.94 (d, J =1.5 Hz, 1 H), 6.75 (dd, J = 1.8, 8.1 Hz, 1 H), 3.75-3.72 (m, 2 H), 3.28-3.24 (m, 2 H), 2.96-2.80 (br s, 4 H), 2.83-2.76 (m, 2 H), 2.56-2.31 (m, 11 H), 1.81-1.72 (m, 6 H), 1.48 (s, 9 H), 1.04 (d, J = 6.3 Hz, 6 H); HRMS (ESI⁺) m/z calcd for C₃₅H₅₀O₂N₅ 572.3959 (M+H), found 572.3956.

A solution of trifluoroacetic acid (1.0 mL, 13.3 mmol) and triethylsilane (0.046 mL, 0.29 mmol) in CH₂Cl₂ (1.0 mL) was added to a solution of tert-butyl (1-(6,11- dihydro-5H-benzo[a]carbazol-2-yl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (0.033 g, 0.058 mmol) in CH₂Cl₂ (1.0 mL). After 2 h, the reaction mixture was concentrated, diluted with sat. NaHCO₃, and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄) and concentrated. The crude residue was purified by chromatography on SiO₂ (5 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 2% MeOH/CH₂Cl₂) to afford 13 (UPCDC30206, 0.015 g, 0.032 mmol, 57%) as a pale yellow oil: IR (ATR) 3218, 2924, 2839, 2181, 1728, 1609, 1465, 1381, 1195, 740 cm^{−1; 1}H NMR (500 MHz, acetone-d₆) δ 10.65 (br s, 1 H), 7.49 (d, J = 8.0 Hz, 1 H), 7.36 (d, J = 8.0 Hz, 1 H), 7.31 (d, J = 1.5 Hz, 1 H), 7.11-7.06 (m, 2 H), 7.03-6.99 (m, 1 H), 6.73 (dd, J = 1.5, 8.0 Hz, 1 H), 3.66 (d, J = 12.5 Hz, 2 H), 2.96-2.91 (m, 4 H), 2.89-2.76 (m, 2 H), 2.72-2.69 (m, 2 H), 2.61-2.56 (m, 3 H), 2.46-2.39 (m, 10 H), 1.96-1.93 (m, 2 H), 1.44 (qd, J = 13.5, 3.5 Hz, 2 H) 0.97 (d, J = 6.5 Hz, 6 H); ³¹C NMR (100 MHz, acetone-d₆) δ 151.8, 138.3, 134.8, 130.5, 129.5, 128.3, 127.5, 122.4, 119.9, 119.1, 115.1, 112.3, 111.9, 110.2, 59.0, 55.7, 54.9, 54.6, 49.4, 49.2, 44.4, 33.4, 20.7, 18.8; HRMS (ESI⁺) m/z calcd for C₃₀H₄₂N₅ 472.3440 (M+H), found 472.3433.

2-(3-(4-((tert-Butoxycarbonyl)(2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1- yl)phenyl)-1H-indole-5-carboxylic acid (35)

To a solution of methyl 2-(3-(4-((tert-butoxycarbonyl)(2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-1H-indole-5-carboxylate (0.650 g, 1.08 mmol) in a mixture of THF (10 mL) and H₂O (5 mL) was added LiOH (0.079 g, 3.23 mmol, portionwise), and the mixture was stirred at 60 °C for 24 h. The reaction mixture was cooled to room temperature and diluted with EtOAc and the phases were separated. The basic aqueous layer was acidified with 1N HCl and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered, concentrated to provide crude 2-(3-(4-((tert-butoxycarbonyl)(2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1- yl)phenyl)-1H-indole-5-carboxylic acid (35, 0.443 g, 0.751 mmol, 70%) as a brown solid, which was used without further purification: HRMS (ESI⁺) m/z calcd for C₃₄H₄₈O₄N₅ 590.3701 (M+H), found 590.3701.

2-(3-(4-((2-(4-Isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-N-methyl-1H-indole-5-carboxamide (14, UPCDC30367)

To a solution of 2-(3-(4-((tert-butoxycarbonyl)(2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1- yl)phenyl)-1H-indole-5-carboxylic acid (35, 0.100 g, 0.169 mmol), methylamine hydrochloride (17.5 mg, 0.254 mmol) and TEA (0.191 mL, 1.36 mmol) in dry acetonitrile (0.5 mL) was slowly added T3P (0.107 g, 0.144 mmol; 50% EtOAc solution) at 0 °C. After 1 h, another portion of T3P (0.107 g, 0.144 mmol; 50% EtOAc solution) was added and the reaction mixture was allowed to warm to room temperature overnight. The reaction mixture was quenched with slow addition of 0.5M NaOH and the mixture was stirred at room temperature for 1 h. The crude product was extracted with EtOAc (3×) and the combined organic layer was washed with water followed by brine, dried (Na₂SO₄), filtered and concentrated to provide crude tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(1-(3-(5-(methylcarbamoyl)-1H-indol-2-yl)phenyl)piperidin-4-yl)carbamate (77.5 mg, 0.129 mmol, 76%), which was used without further purification: ¹H NMR (500 MHz, CD₃OD) δ 8.07 (s, 1 H), 7.60 (d, J = 8.5 Hz, 1 H), 7.43 (d, J = 9.0 Hz, 2 H), 7.32-7.27 (m, 2 H), 6.96 (d, J = 7.5 Hz, 1 H), 6.89 (s, 1 H), 4.04 (br s, 1 H), 3.93-3.91 (m, 2 H), 3.27 (br s, 2 H), 2.95 (s, 3 H), 2.86 (t, J = 12.3 Hz, 2 H), 2.69-2.50 (m, 11 H), 1.98-1.93 (m, 2 H), 1.77-1.76 (m, 2 H), 1.47 (s, 9 H), 1.10 (d, J = 6.0 Hz, 6 H); HRMS (ESI⁺) m/z calcd for C₃₅H₅₁O₃N₆ 603.4017 (M+H), found 603.4013.

To a solution of tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(1-(3-(5-(methylcarbamoyl)-1H-indol-2-yl)phenyl)piperidin-4-yl)carbamate (72.5 mg, 0.120 mmol) in CH₂Cl₂ (2 mL) at 0 °C was added trifluoroacetic acid (0.36 mL, 4.8 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 2 h, treated with sat. NaHCO₃ and extracted with CH₂Cl₂ (3×). The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (0 to 5% MeOH/CH₂Cl₂ to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 5% MeOH/CH₂Cl₂) to provide 2-(3-(4-((2-(4-Isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-N-methyl-1H-indole-5-carboxamide 14 (UPCDC30367, 30 mg, 0.059 mmol, 49%): IR (ATR) 3275, 2923, 2813, 1627, 1599, 1547, 1458, 1407, 1176, 1148, 1117, 980, 805, 777, 764 cm^{−1; 1}H NMR (500 MHz, CD₃OD) δ 8.07 (s, 1 H), 7.60 (d, J = 8.5 Hz, 1 H), 7.43 (d, J = 8.5 Hz, 2 H), 7.32-7.27 (m, 2 H), 6.96 (app d, J = 7.0 Hz, 1 H), 6.88 (s, 1 H), 3.82 (app d, J = 12.5 Hz, 2 H), 2.95 (s, 3 H), 2.85-2.79 (m, 4 H), 2.72-2.53 (m, 12 H), 2.05 (app d, J = 11.5 Hz, 2 H), 1.56 (qd, J = 11.8, 3.3 Hz, 2 H), 1.09 (d, J = 6.5 Hz, 6 H); ³¹C NMR (125 MHz, CD₃OD) δ 172.3, 153.4, 141.6, 140.6, 134.4, 130.6, 130.1, 126.8, 121.8, 121.0, 118.0, 117.4, 114.7, 111.8, 100.6, 58.3, 56.3, 55.9, 54.1, 50.0, 43.8, 32.7, 27.0, 18.7; HRMS (ESI⁺) m/z calcd for C₃₀H₄₃ON₆ 503.3493 (M+H), found 503.3492.

((3-Bromophenyl)ethynyl)trimethylsilane (36) ²⁴

To a solution of 1-bromo-3-iodobenzene (2.00 g, 6.72 mmol) in dry, degassed acetonitrile (20.0 mL) was added PdCl₂(PPh₃)₂ (0.24 g, 0.34 mmol) and CuI (0.065 g, 0.34 mmol). The mixture was degassed and backfilled with argon and charged with Et₃N (0.95 mL, 6.7 mmol) and trimethylsilylacetylene (1.94 mL, 13.4 mmol). The mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated and the crude product was extracted with EtOAc, washed with water, brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (100% hexanes) to obtain 36 as an orange oil (1.7 g, 6.6 mmol, 99%): IR (ATR) 2956, 2160, 1582, 1588, 1470, 1450, 1260, 1245, 1079 cm^{−1; 1}H NMR (500 MHz, CDCl₃) 7.62 (t, J = 2.0 Hz, 1 H), 7.45-7.43 (m, 1 H), 7.16 (app t, J = 7.5 Hz, 1 H), 0.25 (s, 9 H); ³¹C NMR (CDCl₃, 125 MHz) 134.8, 131.7, 130.6, 129.7, 125.3 122.2, 103.4, 96.0, 0.00.

8-(3-((Trimethylsilyl)ethynyl)phenyl)-1,4-dioxa-8-azaspiro[4.5]decane (37)

A solution of 36 (200 mg, 0.790 mmol), 1,4-dioxa-8-azaspiro[4.5]decane (127 mg, 0.87 mmol), Pd₂(dba)₃ (36 mg, 0.039 mmol), DavePhos (16 mg, 0.039 mmol) in dry THF (5.0 mL) was degassed by bubbling argon and backfilled with argon three times. To this solution was added LiHMDS (340 mg, 1.97 mmol). The reaction mixture was degassed and the reaction vial was sealed and heated at 70 °C for 3 h. The reaction mixture was cooled to room temperature, diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (10 to 30% EtOAc/hexanes) to provide 37 (0.17 g, 0.54 mmol, 68%) as an off-white sticky solid: IR (ATR) 2957, 2879, 2829, 2143, 1589, 1478, 1246, 1101, 833 cm^{−1; 1}H NMR (300 MHz, CDCl₃) δ 7.16 (t, J = 7.8 Hz, 1 H), 7.03 (br s, 1 H), 6.94-6.88 (m, 2 H), 3.99 (s, 4 H), 3.32 (t, J = 5.7 Hz, 4 H), 1.82 (t, J = 5.7 Hz, 4 H), 0.24 (s, 9 H); HRMS (ESI⁺) m/z calcd for C₁₈H₂₆NO₂Si 316.1733 (M+H), found 316.1725.

8-(3-Ethynylphenyl)-1,4-dioxa-8-azaspiro[4.5]decane (38)

A solution of 37 (820 mg, 2.47 mmol) in THF (10.0 mL) was treated with TBAF (0.95 mL, 75 wt % H₂O) at 0 °C. The reaction mixture was warmed to room temperature. After 20 min, the reaction mixture was extracted with EtOAc (2x), washed with H₂O, brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (10 to 30% EtOAc/hexanes) to give 38 (0.508 g, 2.08 mmol, 85%) as a yellow oil: IR (ATR) 3282, 2956, 2926, 2881, 1664, 1591, 1569, 1235, 1097 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 7.18 (t, J = 8.0 Hz, 1 H), 7.06 (dd, J = 1.6, 2.4 Hz, 1 H), 6.96-6.92 (m, 2 H), 3.99 (s, 4 H), 3.33 (t, J = 5.6 Hz, 4 H), 3.02 (s, 1 H), 1.82 (t, J = 5.6 Hz, 4 H); ³¹C NMR (100 MHz, CDCl₃) 150.7, 129.2, 123.1, 122.8, 119.9, 117.3, 107.2, 84.4, 76.5, 64.5, 47.5, 34.5; HRMS (ESI⁺) m/z calcd for C₁₅H₁₈O₂N 244.1332 (M+H), found 244.1331.

3-Iodopyridin-4-amine (39) ²⁵

To a refluxing solution of 4-aminopyridine (2.00 g, 21.3 mmol) and Na₂CO₃ (1.35 g, 12.8 mmol) in H₂O (7.6 mL) was slowly added a solution of KI (3.99 g, 23.8 mmol) and I₂ (4.09 g, 15.9 mmol) in H₂O (16.8 mL) and the mixture was heated at reflux for 22 h. The reaction mixture was cooled to room temperature and extracted with EtOAc (2x). The combined organic layer was washed with satd Na₂S₂O₃, followed by brine, dried (MgSO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (50 to 75% EtOAc/hexanes) to give 39 as an off-white solid (1.66 g, 7.54 mmol, 36%): ¹H NMR (300 MHz, CDCl₃) δ 8.55 (s, 1 H), 8.08 (d, J = 5.4 Hz, 1 H), 6.57 (d, J = 5.4 Hz, 1 H), 4.72 (br s, 2 H).

N-(3-Iodopyridin-4-yl)acetamide (40) ²⁶

A solution of 39 (1.65 g, 7.50 mmol) in CH₂Cl₂ (15 mL) was treated with acetic anhydride (0.71 mL, 7.5 mmol) and TEA (1.58 mL, 11.3 mmol) at room temperature. After 17 h, the solution was concentrated. The residue was diluted with THF/H₂O (6.0 mL, 1/1) and treated with LiOH (0.20 g, 1.1 mmol) at room temperature. After 30 min, the reaction mixture was extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (MgSO₄), filtered and concentrated to give 40 (1.68 g, 6.40 mmol, 85%, containing approx. 5% residual solvent) as a light orange solid: ¹H NMR (400 MHz, CDCl₃) δ 8.81 (s, 1 H), 8.39 (d, J = 5.6 Hz, 1 H), 8.32 (d, J = 5.6 Hz, 1 H), 7.61 (br s, 1 H), 2.28 (s, 3 H).

8-(3-(1H-Pyrrolo[3,2-c]pyridin-2-yl)phenyl)-1,4-dioxa-8-azaspiro[4.5]decane (41)

To a microwave vial equipped with a magnetic stir bar was added 38 (200 mg, 0.822 mmol), 40 (323 mg, 1.23 mmol), PdCl₂(PPh₃)₂ (29 mg, 0.041 mmol), CuI (8.0 mg, 0.04 mmol), 1,1,3,3-tetra-methylguanidine (0.31 mL, 2.5 mmol) and DMF (1.0 mL). The mixture was degassed by bubbling argon for 15 min, sealed, and then heated at 80 °C for 1 h under microwave irradiation. The reaction mixture was diluted with H₂O and extracted with EtOAc (2x). The combined organic layer was washed with H₂O, brine, dried (Na₂SO₄), filtered and concentrated. The solid was precipitated from a solution of MeOH/CH₂Cl₂/hexanes to afford 41 as a white powder (89 mg, 0.27 mmol, 33%): IR (ATR) 3092, 2956, 2829, 2726, 1595, 1573, 1541, 1494, 1494, 1224, 1095 cm^{−1; 1}H NMR (300 MHz, DMSO-d₆) δ 11.88 (s, 1 H), 8.83 (br s, 1 H), 8.19 (br s, 1 H), 7.46 (s, 1 H), 7.38 (br s, 1 H), 7.29-7.28 (m, 2 H), 7.02 (s, 1 H), 6.94 (s, 1 H), 3.93 (s, 4 H), 3.39 (t, J = 5.4 Hz, 4 H), 1.75 (t, J = 5.1 Hz, 4 H); ³¹C NMR (75 MHz, DMSO-d₆) δ 150.8, 140.3, 140.1, 139.5, 132.0, 129.6, 115.8, 115.3, 112.5, 106.4, 97.3, 63.7, 46.6, 33.9; HRMS (ESI⁺) m/z calcd for C₂₀H₂₂N₃O₂ 336.1707 (M+H), found 336.1704.

1-(3-(1H-Pyrrolo[3,2-c]pyridin-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (17, UPCDC30345)

To a solution of 41 (0.080 g, 0.24 mmol) in acetone (15 mL) was added 3M HCl (10 mL) and the reaction mixture was heated at 80 °C for 6 h. The solution was cooled to room temperature and kept at this temperature overnight. The solution was cooled to 0 °C, neutralized with solid Na₂CO₃ and extracted with EtOAc (2x). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered and concentrated to afford 1-(3-(1H-pyrrolo[3,2-c]pyridin-2-yl)phenyl)piperidin-4-one as a yellow solid that was used without further purification: HRMS (ESI⁺) m/z calcd for C₁₈H₁₈ON₃ 292.1444 (M+H), found 292.1443.

To a suspension of 1-(3-(1H-pyrrolo[3,2-c]pyridin-2-yl)phenyl)piperidin-4-one (0.070 g, 0.24 mmol), B (0.045 g, 0.26 mmol) in a mixture of 1,2-DCE and THF (2:1, 1.5 mL) was added Ti(OiPr)₄ (0.080 mL, 0.26 mmol). After 1.5 h, the reaction mixture was treated with NaBH(OAc)₃ (0.031 g, 0.14 mmol). After 2 h, a second portion of NaBH(OAc)₃ (0.025 g, 0.012 mmol) was added. After 1 h, the solution was treated with 0.5 M NaOH (aq) and the resulting suspension was extracted with EtOAc (2x). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (0 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 5% MeOH/CH₂Cl₂) to give 17 as an off-white solid (UPCDC30345, 47 mg, 0.11 mmol, 42% - 2 steps): IR (ATR) 3081, 2943, 2924, 2808, 1772, 1601, 1575, 1541, 1496 cm^{−1; 1}H NMR (400 MHz, CD₃OD) δ 8.75 (s, 1 H), 8.11 (d, J = 5.6 Hz, 1 H), 7.43-7.41 (m, 2 H), 7.34-7.28 (m, 2 H), 7.00 (d, J = 7.2 Hz, 1 H), 6.96 (s, 1 H), 3.82 (d, J = 12.8 Hz, 2 H), 2.86-2.78 (m, 4 H), 2.70-2.52 (m, 12 H), 2.05-2.01 (m, 2 H), 1.54 (qd, J = 12.0, 3.5 Hz, 2 H), 1.08 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, CD₃OD) δ 153.4, 143.3, 142.5, 142.2, 140.3, 133.6, 130.8, 127.7, 118.1, 117.8, 114.8, 107.9, 98.8, 58.4, 56.3, 55.9, 54.1, 49.9, 49.5, 43.8, 32.8, 18.7; HRMS (ESI⁺) m/z calcd for C₂₇H₃₉N₆ 447.3231 (M+H), found 447.3229.

5-Fluoro-3-iodopyridin-2-amine (42) ²⁷

To a stirring solution of 5-fluoropyridin-2-amine (1.5 g, 13.4 mmol) in H₂SO₄ (2M, 20.0 mL) was slowly added KIO₃ (1.4 g, 6.7 mmol). The reaction mixture was heated at 100 °C and a solution of KI (2.24 g, 13.4 mmol) in H₂O (10.0 mL) was added. After 30 min, the reaction mixture was cooled to room temperature and the solution was treated with NaHCO₃ until pH 8–9. The reaction mixture was extracted with EtOAc (3×). The combined organic layers were washed with sat. NaHSO₃, H₂O, brine, dried (Na₂SO₄), filtered and concentrated to afford 42 (2.29 g, 9.62 mmol, 72%) that was used without further purification.

N-(5-Fluoro-3-iodopyridin-2-yl)acetamide (43)

A solution of 42 (1.20 g, 5.04 mmol) in AcOH (10.0 mL) was treated with acetic anhydride (0.47 mL, 5.0 mmol) and the mixture was heated at 100 °C for 4 h. The reaction mixture was cooled to room temperature and carefully treated with sat. K₂CO₃. The reaction mixture was extracted with EtOAc (3×). The combined organic layer was washed with H₂O, brine, dried (Na₂SO₄), filtered and concentrated. The product was purified by chromatography on SiO₂ (0 to 70% EtOAc/hexanes) to obtain 43 as a white powder (926 mg, 3.31 mmol, 66%): M.p. 148–150 °C; IR (ATR) 3223, 3189, 3029, 3001, 1688, 1655, 1569, 1515, 1431, 1262 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 2.4 Hz, 1 H), 7.89 (dd, J = 7.2, 2.8 Hz, 1 H), 7.66 (br s, 1 H), 2.33 (s, 3 H); ³¹C NMR (100 MHz, CDCl₃) δ 170.1, 155.6 (d, J_CF = 259 Hz), 147.6, 135.6 (d, J_CF = 24 Hz), 135.1 (d, J_CF = 21 Hz), 24.0; HRMS (ESI⁺) m/z calcd for C₇H₇FIN₂O 280.9587 (M+H), found 280.9579.

8-(3-(5-Fluoro-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-1,4-dioxa-8-azaspiro[4.5]decane (44) ²⁸

To a microwave vial equipped with a magnetic stir bar was added 38 (300 mg, 1.23 mmol), 43 (432 mg, 1.47 mmol), PdCl₂(PPh₃)₂ (44 mg, 0.062 mmol), CuI (19.0 mg, 0.098 mmol), 1,1,3,3-tetra-methylguanidine (0.47 mL, 3.7 mmol) and DMF (1.0 mL). The reaction mixture was degassed for 10 min. by argon bubbling and the vial was sealed and heated at 80 °C under microwave irradiation. The reaction mixture was diluted with H₂O and extracted with EtOAc (2x). The combined organic layer was washed with H₂O, brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (30 to 50% EtOAc/hexanes) to obtain 44 as a light yellow solid (145 mg, 0.41 mmol, 33%): IR (ATR) 3211, 2872, 2821, 1614, 1576, 1489, 1446, 1287, 1196, 1131, 1110, 876, 758 cm^{−1; 1}H NMR (500 MHz, DMSO-d₆) δ 12.22 (s, 1 H), 8.17 (s, 1 H), 7.78 (d, J = 9.5 Hz, 1 H), 7.52 (s, 1 H), 7.33-7.27 (m, 2 H), 6.95 (d, J = 8.0 Hz, 1 H), 6.91 (s, 1 H), 3.93 (s, 4 H), 3.39 (t, J = 5.0 Hz, 4 H), 1.74 (t, J = 5.0 Hz, 4 H).

1-(3-(5-Fluoro-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)piperidin-4-one (45)

To a solution of 44 (0.135 g, 0.382 mmol) in acetone (20 mL) was added 3M HCl (15 mL). The reaction mixture was heated at 80 °C for 6 h. The solution was cooled to 0 °C, neutralized with Na₂CO₃, and extracted with EtOAc (2x). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered and concentrated. The solid that formed upon concentration was collected by filtration, washed with hexanes and dried in vacuo to give 45 as a brown solid (82 mg, 0.26 mmol, 69%, approx. 90% purity): ¹H NMR (300 MHz, CDCl₃) δ 11.51 (s, 1 H), 8.19 (s, 1 H), 7.64 (dd, J = 2.7, 9.0 Hz, 1 H), 7.46 (app t, J = 7.8 Hz, 1 H), 7.40 (s, 1 H), 7.32 (d, J = 7.5 Hz, 1 H), 7.03 (dd, J = 2.1, 8.1 Hz, 1 H), 6.75 (d, J = 2.1 Hz, 1 H), 3.69 (t, J = 6.3 Hz, 4 H), 2.62 (t, J = 6.0 Hz, 4 H); HRMS (ESI⁺) m/z calcd for C₁₈H₁₇FN₃O 310.1356 (M+H), found 310.1348.

1-(3-(5-Fluoro-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (19, UPCDC30381)

To a suspension of 45 (0.070 g, 0.23 mmol) and B (0.046 g, 0.27 mmol) in THF/1,2-dichloroethane (1/2, 1.5 mL) was added Ti(OiPr)₄ (0.083 mL, 0.27 mmol) at room temperature. After 30 min, NaBH(OAc)₃ (0.030 g, 0.13 mmol) was added. After 2 h, a second portion of NaBH(OAc)₃ (0.031 g, 0.14 mmol) was added. After 1 h, additional NaBH(OAc)₃ (0.030 g, 0.13 mmol) was added and the reaction mixture was stirred overnight. The reaction mixture was diluted with sat. NaHCO₃ and EtOAc/MeOH. The suspension was filtered through Celite and the filtrate was concentrated. The residue was extracted with EtOAc, washed with H₂O, brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (5 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (5% MeOH/CH₂Cl₂) to give 19 as an off-white foamy solid (UPCDC30381, 0.051 g, 0.11 mmol, 49%): IR (ATR) 3170, 2930, 2807, 1610, 1586, 1489, 1446, 1343, 1176, 980, 758 cm^{−1; 1}H NMR (500 MHz, CD₃OD) δ 8.06 (s, 1 H), 7.68 (dd, J = 2.5, 9.5 Hz, 1 H), 7.45 (s, 1 H), 7.34-7.31 (m, 2 H), 7.01-6.99 (m, 1 H), 6.81 (s, 1 H), 3.85 (d, J = 12.5 Hz, 2 H), 2.90 (t, J = 6.5 Hz, 2 H), 2.87-2.82 (m, 3 H), 2.73-2.57 (m, 11 H), 2.09 (d, J= 12.0 Hz, 2 H), 1.62 (qd, J = 11.5, 3.0 Hz, 2 H), 1.10 (d, J = 6.5 Hz, 6 H); ³¹C NMR (125 MHz, CD₃OD) δ 157.2 (d, J_CF = 238 Hz), 153.2, 147.6, 143.4, 133.7, 131.3 (d, J_CF = 30 Hz), 130.7, 123.5 (d, J_CF = 8 Hz), 118.0 (d, J_CF = 39 Hz), 114.9, 114.8, 114.6, 98.3 (d, J_CF = 4 Hz), 57.5, 56.4, 56.1, 53.8, 49.7, 43.5, 31.9, 18.6; ¹⁹F NMR (376 MHz, CD₃OD) δ -141.4; HRMS (ESI+) m/z calcd for C₂₇H₃₈FN₆ 465.3136 (M+H), found 465.3133.

2-(3-Bromophenyl)naphthalene (46)²⁹

To a flask containing 1-bromo-3-iodobenzene (0.28 mL, 2.2 mmol), 2-naphthalene boronic acid (0.26 g, 1.5 mmol), Pd(PPh₃)₂Cl₂ (0.023 g, 0.033 mmol) and K₂CO₃ (0.405 g, 2.93 mmol) was added degassed DMF (4.0 mL). The reaction mixture was heated at 80 °C overnight. The reaction mixture was cooled to room temperature, diluted with H₂O, and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (0 to 10% EtOAc/hexanes) to give 46 (0.265 g, 0.94 mmol, 64%) as an off-white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.01 (d, J = 1.2 Hz, 1 H), 7.94-7.90 (m, 2 H), 7.89-7.86 (m, 2 H), 7.70 (dd, J = 2.0, 8.4 Hz, 1 H), 7.66-7.63 (m, 1 H), 7.55-7.48 (m, 3 H), 7.35 (app t, J = 8.0 Hz, 1 H); ³¹C NMR (100 MHz, CDCl₃) δ 143.5, 130.6, 130.5, 130.4, 128.8, 128.4, 127.8, 126.6, 126.4, 126.2, 126.1, 125.4; HRMS (ESI+) m/z calcd for C₁₆H₁₁Br 282.0044 (M+), found 282.0062.

N-(2-(4-Isopropylpiperazin-1-yl)ethyl)-1-(3-(naphthalen-2-yl)phenyl)piperidin-4-amine (20, UPCDC30222)

A suspension of 46 (0.100 g, 0.353 mmol), A (0.150 g, 0.424 mmol), K₃PO₄ (0.116 g, 0.529 mmol), Pd₂(dba)₃ (0.006 g, 0.007 mmol) and CyJohnPhos (0.010 g, 0.028 mmol) in dry degassed dioxane (5 mL) was heated at 110 °C overnight. The reaction mixture was cooled to room temperature, diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄) and concentrated. The crude residue was purified by chromatography on SiO₂ (10 to 30% EtOAc/CH₂Cl₂) to give tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(1-(3-(naphthalen-2-yl)phenyl)piperidin-4-yl)carbamate (0.101 g, 0.18 mmol, 51%) as a light yellow oil: IR (ATR) 2959, 2928, 2807, 1679, 1592, 1448, 1410, 1383, 1362, 1178, 1146, 1010, 982, 907, 767 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 8.01 (d, J = 0.8 Hz, 1 H), 7.91-7.85 (m, 3 H), 7.72 (dd, J = 2.0, 8.8 Hz, 1 H), 7.52-7.45 (m, 2 H), 7.36 (app t, J = 8.0 Hz, 1 H), 7.25-7.23 (m, 1 H), 7.19 (d, J = 7.6 Hz, 1 H), 6.95 (dd, J = 2.0, 8.0 Hz, 1 H), 4.17 (br s, 1 H), 3.85 (d, J = 12.4 Hz, 2 H), 3.25 (br s, 2 H), 2.88-2.84 (m, 2 H), 2.69-2.47 (m, 11 H), 1.86-1.79 (m, 4 H), 1.47 (s, 9 H), 1.05 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, CDCl₃) δ 155.4, 151.8, 142.3, 139.3, 133.7, 132.7, 129.7, 128.4, 128.3, 127.8, 126.4, 126.0, 125.92, 125.91, 119.1, 116.1, 115.8, 79.9, 54.7, 53.9, 49.9, 48.7, 30.2, 28.7, 18.7; HRMS (ESI+) m/z calcd for C₃₅H₄₉N₄O₂ 557.3850 (M+H), found 557.3849.

A solution of trifluoroacetic acid (0.83 mL, 11.0 mmol) and triethylsilane (0.09 mL, 0.52 mmol) in CH₂Cl₂ (1.5 mL) was added to a solution of tert-butyl (2-(4-isopropylpiperazin-1-yl)ethyl)(1-(3-(naphthalen-2-yl)phenyl)piperidin-4-yl)carbamate (0.055 g, 0.099 mmol) in CH₂Cl₂ (1.5 mL). After 1 h, the reaction mixture was concentrated, diluted with sat. NaHCO₃, and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄) and concentrated. The crude residue was purified by chromatography on SiO₂ (8 to 20% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 10% MeOH/CH₂Cl₂) to afford 20 (UPCDC30222, 0.030 g, 0.066 mmol, 67%, containing approx. 5% residual solvents) as a light yellow oil: IR (ATR) 2957, 2932, 2805, 1702, 1593, 1461, 1379, 1232, 1176, 1129, 982, 854, 779 cm^{−1; 1}H NMR (500 MHz, acetone-d₆) δ 8.15 (d, J = 1.0 Hz, 1 H), 7.98-7.96 (m, 2 H), 7.91 (d, J = 8.0 Hz, 1 H), 7.82 (dd, J = 2.0, 8.5 Hz, 1 H), 7.54-7.48 (m, 2 H), 7.35-7.32 (m, 2 H), 7.19 (dd, J = 0.5, 7.5 Hz, 1 H), 6.99 (dd, J = 2.5, 8.5 Hz, 1 H), 3.77 (dt, J = 3.0, 12.5 Hz, 2 H), 2.90-2.85 (m, 2 H), 2.72 (t, J = 6.0 Hz, 2 H), 2.65-2.55 (m, 3 H), 2.47-2.40 (m, 10 H), 1.98-1.95 (m, 2 H), 1.47 (qd, J = 13.0, 4.0, 2 H), 0.98 (d, J = 6.5 Hz, 6 H); ³¹C NMR (125 MHz, acetone-d₆) δ 153.3, 142.5, 140.1, 134.7, 133.6, 130.3, 129.1, 129.0, 128.4, 127.1, 126.7, 126.4, 126.3, 118.6, 116.2, 115.9, 59.0, 55.7, 54.9, 54.6, 49.4, 48.9, 44.4, 33.4, 18.8; HRMS (ESI+) m/z calcd for C₃₀H₄₁N₄ 457.3331 (M+H), found 457.3322.

2-(3-Bromophenyl)benzofuran (47)³⁰

Benzofuran-2-ylboronic acid (0.408 g, 2.47 mmol), 1-bromo-3-iodobenzene (0.482 mL, 3.70 mmol), PdCl₂(PPh₃)₂ (0.088 g, 0.12 mmol), K₂CO₃ (0.682 g, 4.94 mmol) was treated with DMF (4.0 mL) and heated at 80 °C overnight. The reaction mixture was diluted with H₂O and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (0 to 10% EtOAc/hexanes) to give 47 as an off-white solid (0.55 g, 2.0 mmol, 82%): ¹H NMR (400 MHz, CDCl₃) δ 8.02 (t, J = 1.2 Hz, 1 H), 7.80-7.77 (m, 1 H), 7.61-7.58 (m, 1 H), 7.54-7.51 (m, 1 H), 7.49-7.46 (m, 1 H), 7.34-7.29 (m, 2 H), 7.27-7.23 (m, 1 H), 7.05 (d, J = 0.8 Hz, 1 H); ³¹C NMR (100 MHz, CDCl₃) δ 155.1, 154.3, 132.6, 131.5, 130.5, 129.0, 128.0, 124.9, 123.6, 123.3, 123.1, 121.3, 111.4, 102.6; HRMS (ESI+) m/z calcd for C₁₄H₉OBr 271.9837 (M+), found 271.9858.

1-(3-(Benzofuran-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (21, UPCDC30221)

A suspension of A (187 mg, 0.527 mmol), 2-(3-bromophenyl)benzofuran (47, 120 mg, 0.44 mmol) and K₃PO₄ (144 mg, 0.66 mmol) in dry degassed dioxane (1.5 mL) was degassed for 5 min with argon. To this mixture was added Pd₂(dba)₃ (8 mg, 0.009 mmol) and CyJohnPhos (12 mg, 0.035 mmol). The reaction vial was sealed and the mixture was heated at 110 °C for 12 h. The reaction mixture was diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (10 to 30% EtOAc/CH₂Cl₂) to give tert-butyl (1-(3-(benzofuran-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)-carbamate (0.19 g, 0.36 mmol, 82%) as a light yellow oil: IR (ATR) 2962, 2930, 2807, 1685, 1601, 1450, 162, 1143, 1008, 775, 749 cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 7.50 (dd, J = 0.8, 7.6 Hz, 1 H), 7.46 (d, J = 8.0 Hz, 1 H), 7.38 (s, 1 H), 7.28-7.14 (m, 4 H), 6.93 (d, J = 0.8 Hz, 1 H), 6.65 (dt, J = 7.2, 1.6 Hz, 1 H), 4.11 (br s, 1 H), 3.76 (d, J = 12.0 Hz, 2 H), 3.19 (br s, 2 H), 2.86-2.75 (m, 2 H), 2.63-2.42 (m, 11 H), 1.80-1.76 (m, 4 H), 1.43 (s, 9 H), 0.99 (d, J = 6.8 Hz, 6 H); ³¹C NMR (100 MHz, CDCl₃) δ 156.3, 155.4, 154.8, 151.5, 131.2, 129.5, 129.3, 124.2, 122.9, 120.8, 116.8, 116.4, 112.9, 111.1, 101.3, 79.8, 58.4, 54.6, 53.8, 53.0, 49.6, 48.6, 39.9, 30.1, 28.5, 18.6; HRMS (ESI+) m/z calcd for C₃₃H₄₇O₃N₄ 547.3643 (M+H), found 547.3643.

A solution of tert-butyl (1-(3-(benzofuran-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)-carbamate (0.100 g, 0.18 mmol) in CH₂Cl₂ (1.5 mL) was treated with trifluoroacetic acid (1.5 mL) and triethylsilane (0.15 mL, 0.95 mmol) in CH₂Cl₂ (1.5 mL). After 1 h, the solution was concentrated, diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄) and concentrated. The crude residue was purified by chromatography on SiO₂ (0 to 10% MeOH/CH₂Cl₂) to afford 21 as a yellow foam (UPCDC30221, 0.060 g, 0.13 mmol, 73%): IR (ATR) 2957, 2928, 2807, 1599, 1567, 1489, 1450, 1254, 1220, 1176, 1144, 1122, 982, 747 cm^{−1; 1}H NMR (500 MHz, acetone-d₆) δ 7.62 (d, J = 7.5 Hz, 1 H), 7.55 (d, J = 8.0 Hz, 1 H), 7.52-7.51 (m, 1 H), 7.35-7.28 (m, 3 H), 7.26-7.22 (m, 1 H), 7.00 (dt, J = 7.5, 2.0 Hz, 1 H), 3.75 (dt, J = 12.5, 3.0 Hz, 2 H), 2.92-2.87 (m, 3 H), 2.73 (app t, J = 6.0 Hz, 3 H), 2.67-2.56 (m, 3 H), 2.48-2.41 (m, 9 H), 2.00-1.96 (m, 2 H); 1.46 (qd, J = 13.5, 4.0 Hz, 2 H), 0.98 (d, J = 6.5 Hz, 6 H); ³¹C NMR (125 MHz, acetone-d₆) δ 157.4, 155.6, 153.0, 131.8, 130.3, 130.2, 125.1, 123.9, 121.8, 117.4, 116.1, 112.9, 111.8, 102.2, 59.0, 55.6, 54.9, 54.6, 49.4, 48.6, 44.4, 32.3, 18.8; HRMS (ESI+) m/z calcd for C₂₈H₃₉N₄O 447.3118 (M+H), found 447.3118.

2-(3-(4-((2-(4-Isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-N,N-dimethyl-1H-indole-5-carboxamide (23, UPCDC30368)

Prepared according to the procedure for 14 using dimethylamine hydrochloride instead of methylamine hydrochloride to provide crude tert-butyl (1-(3-(5-(dimethylcarbamoyl)-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (77 mg, 0.13 mmol, 74%), which was used for the next reaction without further purification: ¹H NMR (500 MHz, CD₃OD) δ 7.65 (s, 1 H), 7.45 (d, J = 8.0 Hz, 1 H), 7.42 (br s, 1 H), 7.32-7.28 (m, 2 H), 7.19 (d, J = 8.5 Hz, 1 H), 6.96 (app d, J = 7.0 Hz, 1 H), 6.87 (s, 1 H), 4.05 (br s, 1 H), 3.93-3.91 (m, 2 H), 3.28 (br s, 2 H), 3.11 (s, 6 H), 2.87 (t, J = 12.0 Hz, 2 H), 2.71-2.51 (m, 11 H), 1.97-1.91 (m, 2 H), 1.78-1.76 (m, 2 H), 1.48 (s, 9 H), 1.08 (d, J = 6.5 Hz, 6 H); HRMS (ESI⁺) m/z calcd for C₃₆H₅₃O₃N₆ 617.4174 (M+H), found 617.4172.

To a solution of tert-butyl (1-(3-(5-(dimethylcarbamoyl)-1H-indol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropylpiperazin-1-yl)ethyl)carbamate (82.3 mg, 0.133 mmol) in CH₂Cl₂ (2 mL) at 0 °C was added trifluoroacetic acid (0.39 mL, 5.3 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 2 h, treated with sat. NaHCO₃, and extracted with CH₂Cl₂ (3×). The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, concentrated, and purified by chromatography on SiO₂ (0 to 10% MeOH/CH₂Cl₂ with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 5 % MeOH/CH₂Cl₂) to provide 2-(3-(4-((2-(4-isopropylpiperazin-1-yl)ethyl)amino)piperidin-1-yl)phenyl)-N,N-dimethyl-1H-indole-5-carboxamide 23 (UPCDC30368, 36 mg, 0.069 mmol, 52%): IR (ATR) 3225, 2930, 2808, 1599, 1541, 1499, 1444, 1383, 1318, 1176, 1146, 1071, 982, 803, 779, 764 cm^{−1; 1}H NMR (400 MHz, CD₃OD) δ 7.65 (s, 1 H), 7.45 (d, J = 8.4 Hz, 1 H), 7.42 (br s, 1 H), 7.32-7.27 (m, 2 H), 7.19 (dd, J = 8.2, 1.4 Hz, 1 H), 6.96-6.94 (m, 1 H), 6.86 (s, 1 H), 3.81 (app d, J = 12.4 Hz, 2 H), 3.11 (s, 6 H), 2.85-2.78 (m, 4 H), 2.70-2.52 (m, 12 H), 2.04 (app d, J = 12.0 Hz, 2 H), 1.55 (qd, J = 11.9, 3.2 Hz, 2 H), 1.08 (d, J = 6.4 Hz, 6 H); ³¹C NMR (100 MHz, CD₃OD) δ 175.6, 153.4, 141.5, 139.4, 134.4, 130.6, 129.9, 128.0, 121.8, 120.7, 118.2, 117.4, 114.7, 111.9, 100.2, 58.4, 56.3, 55.9, 54.1, 50.0, 43.8, 32.8, 18.7; HRMS (ESI⁺) m/z calcd for C₃₁H₄₅ON₆ 517.3649 (M+H), found 517.3649.

1-Benzhydryl-2-(3-bromophenyl)-1H-benzo[d]imidazole (48)

A mixture of 3-bromobenzaldehyde (0.216 g, 2.00 mmol), o-phenylenediamine (0.388, 2.10 mmol), boric acid (0.006 g, 0.09 mmol) and glycerin (1 drop) in H₂O (3 mL) was heated at 80 °C until disappearance of starting material. The water was decanted off and MeOH (ca 10 mL) was added. The mixture was stirred for 2 h at room temperature. The formed precipitate was collected by filtration and washed with cold MeOH to afford 2-(3-bromophenyl)-1H-benzo[d]imidazole³¹ as a white solid (0.32 g, 1.2 mmol, 59%): Mp. 264–265 °C; IR (ATR) 3038, 2957, 2799, 1562, 1436, 1398, 1357, 742, 727 cm^{−1; 1}H NMR (500 MHz, DMSO-d₆) δ 13.04 (s, 1 H), 8.37 (t, J = 2.0 Hz, 1 H), 8.20-8.18 (m, 1 H), 7.69 (ddd, J = 8.0, 1.0, 1.0 Hz, 2 H), 7.56-7.50 (m, 2 H), 7.27-7.19 (m, 2 H); ³¹C NMR (125 MHz, DMSO-d₆) δ 150.1, 144.1, 135.5, 132.9, 132.8, 131.6, 129.3, 125.8, 123.4, 122.7, 122.4, 119.5, 112.0; HRMS (ESI⁺) m/z calcd for C₁₃H₁₀N₂Br 273.0027 (M+H), found 273.0023. To a solution of 2-(3-bromophenyl)-1H-benzo[d]imidazole (0.273 g, 0.99 mmol) in anhydrous THF (8 mL) was added NaH (0.048 g, 1.2 mmol, 60% dispersion). The reaction mixture was stirred at room temperature for 20 min then diphenylmethyl chloride (0.41 g, 1.9 mmol) was added. The mixture was stirred at room temperature for 30 min and then heated to 70 °C. After 24 h, the reaction mixture was cooled to room temperature, diluted with sat. NaHCO₃, extracted with EtOAc, dried (Na₂SO₄), and concentrated. The crude residue was purified by chromatography on SiO₂ (10 to 20% EtOAc/hexanes) to afford 48 as a white foam (0.50 g, 1.1 mmol, 89%): ¹H NMR (500 MHz, CDCl₃) δ 7.85-7.82 (m, 2 H), 7.66-7.64 (m, 1 H), 7.51 (d, J = 7.5 Hz, 1 H), 7.36 (app q, J = 3.5 Hz, 7 H), 7.33 (t, J = 8.0 Hz, 2 H), 7.27-7.24 (m, 2 H), 7.18-7.15 (m, 5 H), 7.06-7.03 (m, 1 H), 6.96 (s, 1 H), 6.82 (d, J = 8.5 Hz, 1 H).

1-(3-(1H-Benzo[d]imidazol-2-yl)phenyl)-N-(2-(4-isopropylpiperazin-1-yl)ethyl)piperidin-4-amine (24, UPCDC30250)

A suspension of A (87 mg, 0.24 mmol), 1-benzhydryl-2-(3-bromophenyl)-1H-benzo[d]imidazole (48, 90 mg, 0.21 mmol) and K₃PO₄ (67 mg, 0.31 mmol) in dry degassed dioxane (1.5 mL) was degassed for 20 min with argon. To this mixture was added Pd₂(dba)₃ (4 mg, 0.004 mmol) and CyJohnPhos (6 mg, 0.02 mmol). The reaction vial was sealed and the mixture was heated at 110 °C for 12 h. The reaction mixture was diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), concentrated and purified by chromatography on SiO₂ (10 to 20% MeOH/CH₂Cl₂) to afford tert-butyl (1-(3-(1-benzhydryl-1H-benzo[d]imidazol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropyl-piperazin-1-yl)ethyl)carbamate as a pale yellow solid (89 mg, 0.12 mmol, 61%): ¹H NMR (500 MHz, CDCl₃) δ 7.82 (d, J = 8.0 Hz, 1 H), 7.32 (app t, J = 3.5 Hz, 6 H), 7.21 (t, J = 7.5 Hz, 2 H), 7.16 (app t, J = 3.5 Hz, 4 H), 7.10 (d, J = 7.5 Hz, 1 H), 7.04-6.97 (m, 4 H), 6.82 (d, J = 8.5 Hz, 1 H), 4.10 (br s, 1 H), 3.57 (d, J = 11.5 Hz, 2 H), 3.28-3.20 (m, 2 H), 2.67-2.47 (m, 13 H), 1.69 (br s, 5 H), 1.49 (s, 9 H), 1.10 (d, J = 6.5 Hz, 6 H); HRMS (ESI+) m/z calcd for C₄₅H₅₇N₆O₂ 713.4538 (M+H), found 713.4531.

A solution of tert-butyl (1-(3-(1-benzhydryl-1H-benzo[d]imidazol-2-yl)phenyl)piperidin-4-yl)(2-(4-isopropyl-piperazin-1-yl)ethyl)carbamate (0.089 g, 0.13 mmol) in CH₂Cl₂ (2 mL) was treated with trifluoroacetic acid (2.0 mL) followed by triethylsilane (0.40 mL, 2.5 mmol). The reaction was stirred at room temperature overnight. The solution was then heated at 70 °C overnight, concentrated, diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄) and concentrated. The crude residue was purified by chromatography on SiO₂ (10 to 25% MeOH/CH₂Cl₂) to afford a pale yellow foam which was filtered through basic Al₂O₃ (0 to 10% MeOH/CH₂Cl₂) to afford 24 as a pale yellow foam (UPCDC30250, 0.023 g, 0.051 mmol, 41%): IR (ATR) 3069, 2938, 2912, 2811, 1600, 1450, 1357, 1176, 1115, 776, 736 cm^{−1; 1}H NMR (500 MHz, CD₃OD) δ 7.74 (t, J = 2.0 Hz, 1 H), 7.61 (dd, J = 6.0, 3.5 Hz, 2 H), 7.53 (d, J = 7.5 Hz, 1 H), 7.38 (t, J = 8.0 Hz, 1 H), 7.27 (dd, J = 6.0, 3.5 Hz, 2 H), 7.11 (dd, J = 8.5, 2.5 Hz, 1 H), 3.85 (d, J = 12.5 Hz, 2 H), 2.84 (t, J = 12.0 Hz, 2 H), 2.77 (t, J = 7.0 Hz, 2 H), 2.68-2.51 (m, 11 H), 2.02 (d, J = 11.5 Hz, 2 H), 1.53 (qd, J = 12.0, 3.5 Hz, 2 H), 1.09 (d, J = 6.5 Hz, 6 H); ³¹C NMR (125 MHz, CD₃OD, several signals missing) δ 152.5, 151.9, 130.2, 129.4, 122.5, 118.1, 117.1, 114.2, 57.0, 54.8, 54.5, 52.7, 48.2, 48.1, 42.4, 31.3, 17.3; HRMS (ESI⁺) m/z calcd for C₂₇H₃₉N₆ 447.3231 (M+H), found 447.3230.

2-(3-Bromophenyl)-1-methyl-1H-indole (49)

To a solution of 2-(3-bromophenyl)-1H-indole¹¹ (0.499 g, 1.83 mmol) in DMF (9 mL) was added NaH (0.110 g, 2.75 mmol, 60% dispersion) at 0 °C. The reaction mixture was warmed to room temperature. After 45 min, the reaction mixture was cooled to 0 °C and treated with iodomethane (0.12 mL, 1.9 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The reaction mixture was diluted with H₂O and extracted with EtOAc (3×). The combined organic layer was washed with brine, dried (Na₂SO₄), filtered and concentrated. The residue was purified by chromatography on SiO₂ (40% hexanes/CH₂Cl₂) to give 49 (0.52 g, 1.8 mmol, 99%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 7.68 (s, 1 H), 7.65 (d, J = 8.0 Hz, 1 H), 7.54 (d, J = 8.0 Hz, 1 H), 7.44 (d, J = 8.0 Hz, 1 H), 7.38 (d, J = 8.8 Hz, 1 H), 7.33 (d, J = 8.0 Hz, 1 H), 7.28 (d, J = 7.6 Hz, 1 H), 7.16 (t, J = 7.6 Hz, 1 H), 6.59 (s, 1 H), 3.75 (s, 3 H); HRMS (ESI⁺) m/z calcd for C₁₅H₁₃NBr 286.0226 (M+H), found 286.0225.

N-(2-(4-Isopropylpiperazin-1-yl)ethyl)-1-(3-(1-methyl-1H-indol-2-yl)phenyl)piperidin-4-amine (25, UPCDC30201)

A suspension of A (105 mg, 0.297 mmol), 2-(3-bromophenyl)-1-methyl-1H-indole (49, 71 mg, 0.25 mmol) and K₃PO₄ (81 mg, 0.37 mmol) in dry degassed dioxane (2.0 mL) was treated with Pd₂(dba)₃ (5 mg, 0.005 mmol) and CyJohnPhos (7 mg, 0.02 mmol). The reaction vial was sealed and the mixture was heated at 110 °C for 11 h under microwave irradiation conditions. The reaction mixture was diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layers were washed with brine, dried (Na₂SO₄), concentrated and purified by chromatography on SiO₂ (10 to 20%, MeOH/CH₂Cl₂) to afford tert-butyl(2-(4-isopropylpiperazin-1-yl)ethyl)(1-(3-(1-methyl-1H-indol-2-yl)phenyl)-piperidin-4-yl)carbamate as a pale yellow oil (100 mg, 0.17 mmol, 72%): ¹H NMR (500 MHz, CDCl₃) δ 7.63 (d, J = 7.5 Hz, 1 H), 7.36-7.32 (m, 2 H), 7.26-7.22 (m, 1 H), 7.15-7.12 (m, 1 H), 7.05 (d, J = 1.5 Hz, 1 H), 6.96 (d, J = 8.5 Hz, 2 H), 6.55 (s, 1 H), 4.15 (br s, 1 H), 3.81 (d, J = 12.5 Hz, 2 H), 3.75 (s, 3 H), 3.25 (br s, 2 H), 2.84-2.82 (m, 2 H), 2.71-2.48 (m, 11 H), 1.85 (br s, 4 H), 1.48 (s, 9 H), 1.06 (d, J = 6.5 Hz, 6 H); ³¹C NMR (125 MHz, CDCl₃) δ 155.4, 151.2, 142.1, 138.3, 133.7, 129.2, 128.0, 121.6, 120.5, 120.4, 119.8, 117.6, 116.0, 109.6, 101.4, 79.8, 58.5, 54.6, 53.8, 53.1, 49.5, 48.6, 40.0, 31.2, 30.1, 28.6, 18.6; HRMS (ESI⁺) m/z calcd for C₃₄H₅₀O₂N₅ 560.3965 (M+H), found 560.3959.

A solution of trifluoroacetic acid (1.5 mL) and triethylsilane (0.15 mL, 0.93 mmol) in CH₂Cl₂ (1.5 mL) was added to a solution of tert-butyl(2-(4-isopropylpiperazin-1-yl)ethyl)(1-(3-(1-methyl-1H-indol-2-yl)phenyl)-piperidin-4-yl)carbamate (0.10 g, 0.18 mmol) in CH₂Cl₂ (1.5 mL). The reaction mixture was stirred at room temperature for 1 h, concentrated, diluted with sat. NaHCO₃ and extracted with EtOAc (3×). The combined organic layers were washed with brine, dried (Na₂SO₄) and concentrated. The crude residue was purified by chromatography on SiO₂ (8 to 20% CH₂Cl₂/MeOH with 1% TEA) followed by filtration through basic Al₂O₃ (0 to 10% MeOH/CH₂Cl₂) to afford 25 as a pale yellow solid (UPCDC30201, 0.061 g, 0.13 mmol, 74%): IR (ATR) 2981, 2808, 1596, 1462, 1339, 1178, 1145, 984, 776 cm^{−1; 1}H NMR (400 MHz, acetone-d₆) δ 7.56 (d, J = 7.6 Hz, 1 H), 7.40 (dd, J = 8.4, 0.8 Hz, 1 H), 7.34 (t, J = 8.0 Hz, 1 H), 7.19 (td, J = 8.0, 0.8 Hz, 1 H), 7.11-7.07 (m, 2 H), 7.03 (dd, J = 8.0, 2.0 Hz, 1 H), 6.94 (d, J = 7.6 Hz, 1 H), 6.52 (d, J = 0.8 Hz, 1 H), 3.75-3.71 (m, 5 H), 2.86 (td, J = 12.4, 2.8 Hz, 2 H), 2.71 (t, J = 6.0 Hz, 2 H), 2.69-2.62 (m, 2 H), 2.61-2.39 (m, 10 H), 1.96-1.92 (m, 2 H), 1.45 (qd, J = 13.5, 4.0 Hz, 2 H), 0.98 (d, J = 6.5 Hz, 6 H); ³¹C NMR (100 MHz, acetone-d₆) δ 152.6, 143.0, 139.3, 134.3, 129.9, 129.0, 122.1, 120.9, 120.3, 120.2, 117.6, 116.4, 110.5, 101.8, 59.0, 55.6, 54.9, 54.6, 49.4, 48.6, 44.4, 33.2, 31.5, 18.8; HRMS (ESI⁺) m/z calcd for C₂₉H₄₂N₅ 460.3440 (M+H), found 460.3443.