Structure-based development of novel spiro-piperidine ASH1L inhibitors

Abstract

The absent, small, or homeotic-like 1 (ASH1L) protein is a histone lysine methyltransferase that plays a crucial role in various cancers, including leukemia. Despite representing an attractive therapeutic target, only one class of ASH1L inhibitors was identified to date. Herein, we report development of advanced ASH1L inhibitors targeting the catalytic SET domain, which were designed to access previously unexplored binding pocket on ASH1L. Extensive medicinal chemistry combined with structure-based design led to identification of 66s (AS-254s), a highly potent and selective ASH1L inhibitor (IC₅₀ = 94 nM), representing substantially improved inhibitory activity over previously reported compounds targeting ASH1L. Furthermore, 66s effectively blocked cell proliferation and induced apoptosis and differentiation in leukemia cells harboring MLL1 translocations. Overall, this work provides a high-quality chemical probe targeting the catalytic SET domain of ASH1L with increased inhibitory activity and cellular efficacy to study biological functions of ASH1L and potentially to develop novel anti-cancer therapeutics.

Graphical Abstract

INTRODUCTION

Modifications of chromatin, including histone lysine methylation, play essential roles in physiological and disease states (1, 2). The ASH1L (absent, small, or homeotic-like 1) protein belongs to the family of histone lysine methyltransferases, which catalyzes the mono- and demethylation of histone 3 lysine 36 (H3K36), representing an activating chromatin mark (3–6). ASH1L is a large protein (>3,000 amino acids) that contains multiple structural domains, including a catalytic SET domain and three chromatin reader domains (bromodomain, PHD and BAH domains) in addition to a long unstructured region at the N-terminal portion of the protein (3). The SET domain of ASH1L catalyzes the transfer of a methyl group from an S-adenosyl methionine (SAM) cofactor to the lysine substrate (H3K36) (3, 7).

ASH1L was shown to regulate the expression of HOXA genes and plays an important role in development of acute leukemia with translocations of the Mixed Lineage Leukemia 1 (MLL1) gene (also known as KMT2A) (4, 8–11). MLL1 translocations are found in ~5–10% of acute leukemia patients, leading to a poor clinical outcome with only 35% 5-year survival (12–14), highlighting the need for new therapeutics. ASH1L knockdown was shown to induce cell growth arrest, apoptosis, and differentiation, associated with downregulation of HOXA9 genes in leukemia cells with MLL1 translocations, and it also abrogates development of leukemia in mice (8). We have previously shown the importance of the catalytic SET domain of ASH1L in leukemogenesis mediated by MLL1 fusion proteins (15), supporting that small molecule inhibitors of the ASH1L SET domain could result in new anti-leukemic agents. Furthermore, ASH1L overexpression was found in different solid tumors, including thyroid, breast, gastric and liver cancers, and was linked to cancer cell growth and aggressive disease (16–20). All these findings underscore ASH1L as an attractive therapeutic target in hematologic and solid tumors.

We have recently published the first-in-class small molecule inhibitors targeting the catalytic SET domain of ASH1L (15), which constitute the only ASH1L inhibitors reported to date. These compounds, represented by AS-85 (compound 1) and AS-99 (compound 2), bind to the SET domain of ASH1L with sub-micromolar binding affinities and selectively inhibit its catalytic activity with the IC₅₀ values of 0.6 μM and 0.79 μM, respectively (Figure 1A), without blocking other histone methyltransferases (HMTs) (15). AS-99 also demonstrated anti-leukemic activity in leukemic cells with MLL1 translocations, reinforcing that blocking the catalytic SET domain of ASH1L leads to anti-leukemic effects. Here, we present the structure-based development of new ASH1L inhibitors derived from AS-85 and AS-99 to occupy an additional pocket on the SET domain of ASH1L. Additionally, we developed a fluorescence polarization (FP) assay to effectively assess the activity of new ASH1L inhibitors and to determine a systematic structure-activity relationship (SAR) for these compounds. The most potent ASH1L inhibitor we developed, 66s (AS-254s), demonstrated much more pronounced in vitro inhibitory activity and stronger anti-leukemic effect in blocking cell proliferation, inducing apoptosis and differentiation in MLL1-rearranged leukemia cells when compared to the previous ASH1L inhibitors (15). Overall, this work provides new valuable chemical probes targeting the ASH1L SET domain with substantially improved inhibitory activity and cellular efficacy to study biological functions of ASH1L and to potentially develop new anti-cancer therapeutics.

Figure 1. Known ASH1L inhibitors.

A. Chemical structures and activities of known ASH1L inhibitors. IC₅₀ values were measured using HMT assay and K_d values were assessed using ITC (15). B. Binding mode of AS-85 to ASH1L based on the crystal structure of the complex (6WZW in PDB). Color coding: carbon atoms are in gray (for ASH1L) or green (for AS-85), nitrogen atoms are in blue, oxygens in red, sulfur in yellow and fluorine atoms in light blue.

RESULTS AND DISCUSSION

Development of an FP assay to assess ASH1L inhibitors

The inhibitory activity of our first-in-class ASH1L inhibitors (e.g. AS-85 and AS-99, Figure 1A) was assessed using the histone methyltransferase (HMT) assay (15). The HMT assay is laborious, expensive, and requires radioactive SAM as a cofactor as well as a nucleosome substrate. To efficiently characterize the activity of ASH1L inhibitors, we developed an FP assay, utilizing a fluorescein-labeled ASH1L ligand. Based on the crystal structure of ASH1L with bound AS-85 (Figure 1B), we replaced the azetidine moiety with an aliphatic linker harboring terminal fluorescein, resulting in compound 3 (Figure 2A), representing the FP probe. We also introduced a 7-chloro substituent at the indole ring of 3 to form additional interactions with the backbone amide of G2280 (Figure 1B) for further potency improvement of the FP probe. In addition, we utilized a methyl sulfonamide moiety in compound 3 over the trifluoromethyl group present in AS-85 (Figure 2A) to improve aqueous solubility of the FP probe. Subsequently, we assessed the binding affinity (K_d) of the fluorescein-labeled compound 3 to ASH1L by FP, resulting in K_d = 197 nM and high dynamic range (~120 mP), Figure 2B. We then evaluated this new FP assay in competition experiments with our earlier ASH1L inhibitors AS-99 and AS-6 (15), resulting in the IC₅₀ values of 833 nM and 439 nM, respectively (Figure 2C and Figure S1). Importantly, inhibitory activities obtained for these compounds in the FP assay are very similar to the IC₅₀ values from the HMT assay (IC₅₀ = 790 nM and 520 nM for AS-99 and AS-6, respectively (15)), Figure 1A and Figure S1. These results validate our new FP assay for testing novel ASH1L inhibitors reported here.

Figure 2. Development of FP assay for ASH1L.

A. Chemical structure of fluorescein-labeled probe 3. B. Titration curve for K_d determination for binding of 3 to ASH1L SET using FP assay. C. Titration curve and IC₅₀ value for AS-99 from FP assay with ASH1L SET.

Structure-based design of new ASH1L inhibitors to occupy unexplored hydrophobic pocket

We previously reported first-in-class ASH1L inhibitors, including AS-85 and AS-99, which inhibit ASH1L enzymatic activity at sub-micromolar concentrations (Figure 1A) without affecting other HMTs (15). AS-99, with optimized cell permeability, also demonstrated anti-proliferative activity in the MLL1-rearranged leukemia cells through an on-target mechanism of action (15). Based on promising activity and selectivity of AS-99, here we focused on further optimization of this class of ASH1L inhibitors to improve their activity and drug-like properties. Analysis of the crystal structure of ASH1L in complex with AS-85 (a close structural analog of AS-99, Figure 1A,B) revealed the presence of a hydrophobic pocket on ASH1L, which is adjacent to the azetidine ring of AS-85 (Figure 3A,B).

Figure 3. LMI pocket on ASH1L.

A. Crystal structure of ASH1L-AS-85 reveals LMI pocket on ASH1L (marked in salmon color). ASH1L is shown in surface representation. AS-85 is shown in stick representation (green carbons) and the N-terminal fragment of ASH1L SET crystallization construct forming the crystal contacts with LMI pocket is also shown (yellow carbons, residues A(-3), M(-4) and A(-5)). Coordinates from 6WZW structure in PDB. B. Crystal structure of ASH1L-AS-85 complex (6WZW in PDB) showing LMI pocket is stick representation (carbon atoms of residues forming the LMI pocket are shown in pink).

Interestingly, analysis of the crystal packing contacts revealed that this pocket is occupied by the side chain of a methionine residue (M(–4)) from the N-terminal fragment of the ASH1L crystallization construct (Figure 3A). Since this pocket is predominantly lined by the hydrophobic side chains of L2196, M2226 and I2237, we named it the LMI pocket. Additional residues in this pocket include the aliphatic part of K2228 as well as the side chain of H2258 and the backbone of N2197 and L2198 (Figure 3B). To develop ASH1L inhibitors engaging the LMI pocket, we modified AS-85 compound by replacing the azetidine moiety with a longer spiro-azetidine ring, which yielded compound 4 with an IC₅₀ value of 1.7 μM as assessed by FP (Table 1). Since we planned to introduce larger hydrophobic substituents into compound 4 to reach the LMI pocket, in the subsequent analogues we simplified a relatively large methyl-sulfonyl piperidine moiety at indole nitrogen of 4 into a smaller and more compact tetrahydro-2H-pyran. Further change was made through the introduction of a 7-chloro substituent at the indole ring to interact with the backbone amide of G2280 (Figure 1B). These modifications resulted in compound 22 with ~2.7-fold improved inhibitory activity (IC₅₀ = 0.64 μM, Table 1) and reduced M_w, representing a more favorable scaffold for further substitutions to identify cell permeable ASH1L inhibitors.

Table 1.

Structures and activity of ASH1L inhibitors bearing a spiro-azetidine moiety.

Compd.	R	R1	R2	ASH1L IC50 (μM)a
4	H		H	1.7 ± 0.17
22	H		Cl	0.64 ± 0.07
23			Cl	0.61 ± 0.10
24			Cl	0.77 ± 0.17
25			Cl	0.49 ± 0.08

^aIC₅₀ values were measured in FP assay (n = 3, mean ± SD).

With an attempt to occupy the LMI pocket on ASH1L, we introduced several hydrophobic substituents at the spiro-azetidine ring of 22, including a linear ether (compound 23) as well as ethers with the terminal saturated ring (2-(cyclobutylmethoxy)ethyl, compound 24) or aromatic group (5-thiazole, compound 25). These modifications, however, had no substantial effect on the activity of ASH1L inhibitors over 22, suggesting that the spiro-azetidine linker might not be optimal to reach the LMI hydrophobic pocket (Table 1).

Development of ASH1L inhibitors bearing spiro-piperidine linker

Since substitutions of the spiro-azetidine ring in 22 did not provide more potent analogs (Table 1), we then replaced this group with a spiro-piperidine moiety, resulting in compound 33 (IC₅₀ = 0.54 μM), Table 2. We then designed a series of analogs with various substitutions at the terminal nitrogen of the spiro-piperidine group in 33 with the goal to reach the LMI pocket on ASH1L. First, we tested several ether substituents harboring terminal aliphatic or small saturated carbocyclic rings (compounds 34-37, Table 2), resulting in the most potent compound 36 with a 2-cyclobutoxyethyl group (IC₅₀ = 0.28 μM). Subsequently, we investigated introduction of 5- or 6-membered aromatic heterocycles to 33 (compounds 38-43, Table 2). Intriguingly, all these compounds showed modestly (up to 2-fold) improved inhibitory activity as compared to the unsubstituted 33 (Table 2). Among analogs with the terminal aromatic group, the thiazole analog 42 demonstrated the most potent inhibitory activity (IC₅₀ = 0.21 μM), representing 2.6-fold improvement over 33 (Table 2). Encouraged by these results, we also synthesized compound 43 with a shorter aliphatic linker and terminal thiazole ring, but this compound was ~2-fold less potent than 42 (Table 2), suggesting that the longer aliphatic chain is needed to increase inhibitory activity.

Table 2.

Structures and activity of ASH1L inhibitors with a spiro-piperidine moiety.

Compd.	R3	ASH1L IC50 (μM)a
33	H	0.54 ± 0.11
34		0.56 ± 0.11
35		0.75 ± 0.08
36		0.28 ± 0.02
37		0.81 ± 0.12
38		0.50 ± 0.10
39		0.31 ± 0.05
40		0.31 ± 0.09
41		0.47 ± 0.08
42		0.21 ± 0.05
43		0.39 ± 0.07

^aIC₅₀ values were measured in FP assay (n = 3, mean ± SD).

Discovery of 66s as the most potent ASH1L inhibitor

Analysis of the crystal structure of the ASH1L-AS-85 complex and subsequent modeling studies supported that introduction of an amide instead of an ether in the linker substituting spiro-piperidine ring could induce hydrogen bond formation with the backbone carbonyl of F2257 (Figure 4A).

Figure 4. Inhibitors bind to ASH1L engaging the LMI pocket.

A. Predicted binding mode of compound 50 (green carbons) to ASH1L SET (gray carbons) based on the ASH1L-AS-85 crystal structure (6WZW in PDB). Residues forming LMI pocket are shown in magenta. Hydrogen bonds are shown as dashed lines. B. Predicted binding mode of 66 to ASH1L based on ASH1L-AS-85 structure (6WZW in PDB). ASH1L is shown in surface representation (with color coded atoms) and 66s is shown in stick representation. C. Superposition of the ¹H-¹⁵N TROSY-HSQC spectra of 100 μM ASH1L with 5% DMSO (black) and with 200 μM (red) of 66s. D. Chemical shift perturbations upon binding of 66s to ASH1L mapped on the crystal structure of ASH1L SET domain (6WZW in PDB) with modeled binding of 66s. Colors reflect the magnitude of chemical shift perturbations upon 66s binding: red (> 120 Hz), orange (40 to 120 Hz), yellow (10 to 40 Hz), and residues not observed on the NMR spectra due to slow dynamics are marked in gray.

Thus, we synthesized and tested compound 50, which showed an IC₅₀ value of 0.22 μM, demonstrating ~2.5-fold improvement over unsubstituted 33 (Tables 1 and 3). However, replacement of the methyl group on an amide with other alkyl substituents, including trifluoromethyl (compound 51), trifluoroethyl (compound 52), 3-propoxypropyl (compound 53) or cyclopentenyl (compound 54), did not result in improvement of inhibitory activity over 50 (Table 3). Furthermore, replacement of the cyclopenthyl in 54 with tetrahydrofuran (compound 55), resulted in ~2-fold reduced activity (Table 3). Importantly, substitutions of the amide with aromatic rings led to significantly improved inhibitory activity for majority of the compounds (Table 3). For example, out of the compounds with 6-membered rings (compounds 56–59, Table 3), introductions of 3-pyridine ring (compound 56) led to the most potent inhibitory activity against ASH1L (IC₅₀ = 0.11 μM). On the other hand, compound 60 with a reversed amide had ~3-fold reduced inhibitory activity over 59, which harbors the same aromatic ring (Table 3).

Table 3.

Structures and activity of ASH1L inhibitors bearing a spiro-piperidine moiety.

Compd.	R4	ASH1L IC50 (μM)a
50		0.22 ± 0.04
51		0.32 ± 0.08
52		0.26 ± 0.04
53		0.31 ± 0.004
54		0.31 ± 0.05
55		0.55 ± 0.07
56		0.11 ± 0.01
57		0.17 ± 0.03
58		0.18 ± 0.06
59		0.18 ± 0.04
60		0.53 ± 0.08
61		0.11 ± 0.02
62		0.19 ± 0.03
63		0.14 ± 0.01
64		0.16 ± 0.01
65		0.11 ± 0.02
66 (AS-254)		0.12 ± 0.02
66s (AS-254s)		0.094 ± 0.02

^aIC₅₀ values were measured in FP assay (n = 3, mean ± SD).

Among the analogs containing 5-membered heterocycles (compounds 61-66), compound 61 harboring a furan ring demonstrated ~5-fold improved inhibitory activity (IC₅₀ = 0.11 μM) over the unsubstituted 33 (Tables 2 and 3). Introduction of other 5-member heterocycles, such as oxazole (62) or thiophene (63), resulted in slightly weaker inhibitory activity against ASH1L over 61 (Table 3). Finally, the analogs bearing thiazole rings, such as compounds 65, 66 (AS-254), and the salt variant of 66 (66s, AS-254s), showed the most pronounced inhibitory activity against ASH1L, with 66s being the most potent ASH1L inhibitor (IC₅₀ = 0.094 μM), Table 3. Notably, the HCl salt of 66 (compound 66s) was prepared to improve solubility of this compound in biochemical and cellular assays.

We have also prepared an analog of 66 lacking the 7-chloro substituent at the indole ring and confirmed that this modification substantially reduces inhibitory activity (IC₅₀ = 380 nM for 81), Table S1, validating favorable interactions of ASH1L with this substituent. Furthermore, replacement of the thioamide group in 66 with amide (compound 80, IC₅₀ = 8.6 μM) or nitrile (compound 79, IC₅₀ > 10 μM), led to a substantially reduced or loss of inhibitory activity, Table S1. This demonstrates the importance of thioamide for effective binding of this class of compounds to ASH1L and remains consistent with our previous results for AS-99 (15). Notably, the class of ASH1L inhibitors reported here does not compete with the SAM cofactor as increasing concentrations of SAM did not impact inhibitory activity of 66s (Figure S2), consistent with the binding mode of AS-85 (the precursor of 66s) to ASH1L, Figure 1A.

To gain an insight into the binding of 66 with ASH1L, we modeled its binding mode using the ASH1L-AS-85 derived model of 50 and found that the thiazole ring is in a favorable position to interact with the hydrophobic LMI pocket (Figure 4B). We failed to co-crystallize the complex of the ASH1L SET domain bound to 66s, most likely due to interference with the crystal packing when the LMI pocket is occupied by the inhibitor. Therefore, we performed NMR binding experiments in solution and found several chemical shift perturbations in the vicinity of the LMI pocket (Figure 4C,D). Of note, we have previously found that due to slow dynamics, multiple residues are not observed in the NMR spectra of the ASH1L SET domain (7), including restudies in the vicinity of the binding site for 66. However, chemical shift perturbation of several assigned amides, including L2198, W2138 and Q2225, are consistent with the predicted binding mode of 66 (Figure 4B–D), supporting the proposed model of the ASH1L-66 complex. Overall, these results indicate that introduction of aromatic rings, such as pyridine or thiazole, as terminal substituents on the spiro group can lead to 6–8-fold improved inhibitory activity over the previous generation of ASH1L inhibitors, with the most potent compound 66s demonstrating IC₅₀ better than 100 nM.

Compound 66s is a potent ASH1L inhibitor with favorable drug-like properties

Our best compounds (e.g. with IC₅₀ < 0.15 μM in the FP competition assay) were then tested for their effect on inhibition of ASH1L enzymatic activity in the HMT assay using nucleosomes as a substrate, Table 4 (15). Out of the tested compounds, 65 and 66s exhibited the most pronounced ASH1L inhibition (IC₅₀ = 0.15 μM for both compounds in HMT assay), Figure 5 and Table 4. Importantly, these compounds demonstrate over 5-fold improved inhibitory activity over AS-99 in blocking ASH1L catalytic activity, Table 4. Of note, 66s was selected for more extensive in vitro and cellular characterization over 65 due to its better selectivity towards the ASH1L-dependent leukemia cells.

Table 4.

Activity and physicochemical properties of the most potent ASH1L inhibitors.

Compd.	IC50 (μM)		T1/2 (min)a	cLogPb	tPSAc
	FP	HMT
56	0.11 ± 0.01	0.17 ± 0.02	4.0	4.74	112.29
61	0.11 ± 0.02	0.16 ± 0.04	3.2	4.78	109.16
63	0.14 ± 0.01	0.22 ± 0.02	3.3	5.51	99.93
65	0.11 ± 0.02	0.15 ± 0.01	27.3	4.87	112.29
66s	0.094 ± 0.02	0.15 ± 0.02	29.9	2.54	112.29
AS-99	0.83 ± 0.03	0.79 ± 0.13	6.6	3.29	98.98

^aValues determined using mouse liver microsomes.

^bValues calculated by SwissAMDE (21).

^cValues calculated by ChemDraw 20.1.1.

Figure 5. Inhibition of ASH1L HMT activity and binding affinity of 66s.

A, B. Titration curves and IC₅₀ values from the HMT assay for compounds 66s (A) and 65 (B). Data are mean ± SD from two independent experiments. C. Selectivity of 66s (at 5 μM) against a panel of histone methyltransferases. Data represent two independent experiments each performed in duplicates. D. Binding isotherm from the ITC experiment performed for the binding of 66s to ASH1L. Data are mean ± SD from two independent experiments. A representative binding isotherm is shown.

We then tested 66s in a panel of 15 other histone methyltransferases, including closely related NSD family of HMTs, and did not observe any substantial inhibitory activity against these proteins (Figure 5C), strongly supporting high selectivity of this compound to ASH1L. Finally, we utilized the Isothermal Titration Calorimetry (ITC) to assess binding affinity of 66s to the catalytic SET domain of ASH1L, resulting in K_d = 179 nM and 1:1 binding stoichiometry, Figure 5D. This demonstrates ~5-fold improved binding affinity of 66s to ASH1L over previously reported AS-99 (15).

To further evaluate drug-like properties of our new ASH1L inhibitors, we assessed their microsomal stability in mouse liver microsomes. We found that compounds bearing pyridine (56), furan (61) or thiophene (63) revealed a short half-life (T_1/2 < 5 min), Table 4. In contrast, ASH1L inhibitors containing a thiazole ring (65 and 66s) displayed a much longer half-life in mouse liver microsomes (27.3 min and 29.9 min, respectively, Table 4), demonstrating significant improvement over our earlier generation of ASH1L inhibitors, including AS-99 (T_1/2 = 6.6 min), Table 4. Moreover, the calculated clogP and tPSA values suggest favorable drug-like properties of 66s (Table 4). Overall, our new ASH1L inhibitors have improved activities and drug-like properties over our previously reported compounds, therefore serving as better chemical probes and for their further therapeutic development.

Compound 66s shows potent activity in ASH1L-dependent leukemic cells

Subsequently, we selected our most potent ASH1L inhibitor, 66s, for functional studies in leukemia cells. Since ASH1L catalyzes mono- and di-methylation of H3K36, we tested whether 66s can affect the H3K36me2 mark in the ASH1L-dependent leukemic cells, such as the MLL1-r leukemia cells. Specifically, we treated the MV4;11 cells (harboring MLL-AF4 translocation) and KOPN8 cells (harboring MLL-ENL translocation) with 66s and tested the effect of the compound on the global level of H3K36me2 by Western Blot (WB). Importantly, 66s markedly reduced the H3K36me2 level in a dose-dependent manner in both cell lines with no effect on the global level of H3, which was included as a control (Figure 6A). As expected, reduction of H3K36me2 levels is not complete (Figure 6A) as 66s is not inhibiting the NSD family of H3K36 histone methyltransferases (Figure 5C). Furthermore, 66s lacks inhibition of SETD2 (Figure 5C), which is the only histone methyltransferase introducing H3K36me3, and minor reduction of this mark by 66s most likely results from the depletion of H3K36me2 serving as a substrate for SETD2 to induce H3K36me3. Finally, we independently validated the effect of 66s on H3K36me2 level in the MV4;11 leukemia cells using the AlphaLISA assay, which demonstrated dose-dependent reduction of H3K36me2 in these cells (Figure S3). Overall, all these results support the on-target mechanism of action of 66s in leukemia cells via blocking the catalytic activity of ASH1L.

Figure 6. Activity of 66s in leukemia cells.

A. Assessment of epigenetic marks by WB in MV4;11 and KOPN8 cells treated with 66s for 8d. H2AUb mark has been included as a control. Representative gel of two independent experiments is shown. B. Titration curves from the MTT cell viability assay performed at different time points (days 4, 7, 11 and 14) for 66s in MV4;11 leukemia cell line. n = 4. C. Titration curves from the MTT cell viability assay performed after 14 days of treatment of human MLL1 rearranged (MLL1-r) leukemia cell lines (MV4;11, MOLM13, KOPN8) and control leukemia cell line (K562) with 66s, n = 4. D. Flow cytometry analysis of apoptosis induced by 66s in MV4;11 and KOPN8 cells after 10 days of treatment. Mean ± SD, n= 3 biological replicates. Representative graphs are shown from 2–3 independent experiments in panels B-D. P values were calculated using unpaired 2-tailed t test (^**P < 0.01 and ^****P < 0.0001).

Similarly to inhibitors targeting other histone methyltransferases (e.g. EZH2 or SETD2 inhibitors (22–24)), ASH1L inhibitors may require prolonged treatment to observe phenotypic changes in cancer cells. Indeed, in the MTT cell viability studies performed with 66s in the ASH1L-dependent MV4;11 leukemia cells we observed time-dependent cell growth inhibition, with GI₅₀ values improving from 6.8 μM to 0.74 μM between day 3 and day 14 of treatment with 66s, Figure 6B. Compound 66s has also demonstrated pronounced anti-proliferative effects in other MLL1-rearranged leukemia cells, MOLM13 and KOPN8, resulting in the GI₅₀ values of 0.72 and 0.79 μM, respectively, after 14 days of treatment (Figure 6C). Importantly, the activity of 66s was >10-fold weaker in K562 leukemia cell line, which lacks a MLL1 translocation and is not dependent on ASH1L (Figure 6C), supporting selectivity of this compound. Notably, the anti-proliferative effect of 66s is several fold stronger than observed for our earlier generation of ASH1L inhibitors represented by AS-99 (15), Figure S4

We then assessed the effect of 66s on apoptosis in leukemia cells with MLL1 translocations and found that it strongly increased the level of Annexin V+ in MV4;11 and KOPN8 cells as measured by flow cytometry, indicating that these cells are undergoing apoptosis (Figure 6D, Figure S4). Furthermore, 66s also induced differentiation of leukemia cells, reflected by a substantial increase in the expression level of CD11b and CD14 differentiation markers as assessed by flow cytometry analysis in MV4;11 and KOPN8 leukemia cells (Figure 7A,B). This was further accompanied by substantial morphological changes of leukemia cells upon treatment with 66s, manifested by increased cell size, higher cytoplasmic/nuclear ratio, multi-lobi nuclei and highly vacuolated cytoplasm (Figure 7C). These effects strongly recapitulate the effects observed upon genetic inactivation of ASH1L or deletion of the SET domain in the MLL1-rearranged leukemia cells (8, 15), suggesting specificity and on-target mechanism of action of the 66s ASH1L inhibitor.

Figure 7. 66s induces differentiation of MLL1-r leukemia cells.

A, B. Quantification of CD11B (A) or CD14 (B) expression in human leukemia cells treated for 10 days with 66s, detected by flow cytometry; mean ± SD, n = 3 biological replicates. Two independent experiments were performed for each cell line in triplicates. Representative graphs are shown. P values were calculated using unpaired 2-tailed t test (*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001). C. Wright-Giemsa–stained cytospins for MV4;11 and KOPN8 cells after 10 days of treatment with DMSO or 66s.

Synthesis

The synthesis of compound 4 is outlined in Scheme 1. Primary amine 5 was prepared as described previously (15). Condensation of amine 5 with 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid yielded spiroazetidine 6, which was converted to thioamide 7 in the presence of sodium hydrosulfide hydrate and magnesium chloride. Deprotection of N-Boc group in compound 7 yielded target compound 4.

Scheme 1. Synthesis of spiro-azetidine analog 4^α.

^αReagents and conditions: (a) 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid, EDCI, DMAP, DCM, r.t., overnight; (b) sodium hydrosulfide hydrate, magnesium chloride, DMF, r.t., overnight; (c) TFA, DCM, 1 h.

The preparation of compound 22 was commenced from 2-chloro-3-nitrobenzoic acid (8) as illustrated in Scheme 2. The Bartoli indole synthesis involved the reaction of nitroarene 8 with a vinyl Grignard reagent, providing access to 7-chloro-1H-indole-6-carboxylic acid (9), which was reacted with CH₃I in the presence of K₂CO₃ to afford methyl ester 10. Iodination of compound 10 followed by N-Boc protection furnished 1-(tert-butyl) 6-methyl 7-chloro-3-iodo-1H-indole-1,6-dicarboxylate (12). Treatment of 12 with (3-cyanophenyl)boronic acid via Suzuki coupling afforded compound 13. The synthesis of compound 16 was achieved by N-Boc deprotection of compound 13 and successive nucleophilic substitution. Subsequent reduction of ester 16 with LiBH₄ gave rise to alcohol 17, which was transformed into azide 18 in the presence of DPPA and DBU. The Staudinger reduction of azide 18 delivered primary amine 19, which was then functionalized through amidation to provide spiro-azetidine analog 20. The target compound 22 was obtained from spiro-azetidine derivative 20, following a similar procedure as described for compound 4.

Scheme 2. Synthesis of spiro-azetidine analog 22^α.

^αReagents and conditions: (a) vinylmagnesium bromide, THF, −45 °C to r.t., overnight; (b) K₂CO₃, CH₃I, DMF, r.t., 3 h; (c) KOH, I₂, DMF, r.t., 3 h; (d) di-tert-butyl-dicarbonate, Et₃N, DMAP, DCM, r.t., overnight; (e) Pd(dppf)Cl₂^.DCM_, (3-cyanophenyl)boronic acid, NaHCO₃, THF/H₂O, 65 °C, 12 h; (f) TFA, DCM, 3 h; (g) Cs₂CO₃, tetrahydro-2H-pyran-4-yl methanesulfonate 15, 100 °C, 18 h; (h) LiBH₄, THF/MeOH, 65 °C, 25 min; (i) DPPA, DBU, THF, 0 °C to r.t., overnight; (j) PPh₃, THF/H₂O, r.t., 16 h; (k) 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid, EDCI, DMAP, DCM, r.t., overnight; (l) sodium hydrosulfide hydrate, magnesium chloride, DMF, r.t., overnight; (m) TFA, DCM, 1 h.

To target the new hydrophobic pocket, we designed and synthesized spiro-azetidines 23–25, in which a flexible ether-containing moiety was tethered to the azetidine motif (Scheme 3). The carboxylic acids 28a-b and 28i were prepared by esterification of alcohols 26a-b and 26i, followed by cleavage of the tert-butyl group with TFA. Subsequent condensation of carboxylic acids 28a-b and 28i with N,O-dimethylhydroxylamine yielded Weinreb amides 29a-b and 29i, which underwent LiAlH₄ reduction to afford aldehydes 30a-b and 30i. Finally, the synthesis of target compounds 23–25 was accomplished by reacting aldehydes 30a-b and 30i with amine 22 via reductive amination.

Scheme 3. Synthesis of spiro-azetidine analogs 23–25^α.

^αReagents and conditions: (a) tert-butyl 2-bromoacetate, 40 % aq. NaOH, tetrabutylammonium bromide, toluene, r.t., 16 h; (b) TFA, DCM, 8 h; (c) N,O-dimethylhydroxylamine, EDCI, DMAP, DCM, r.t., overnight (d) LiAlH₄, Et₂O, 0 °C, 30 min; (e) 22, sodium triacetoxyborohydride, Et₃N, DCE, 4 h.

Upon preparing the spiro-azetidine analogs, we found that the azetidine moiety was sensitive to both acid and base, leading to ring-opening products. Of particular note, these byproducts did not exert any ASH1L inhibitory activity. Accordingly, we turned our attention to the spiro-piperidine moiety. As depicted in Scheme 4, amine 19 was reacted with 7-(tert-butoxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid under standard conditions to produce spiropiperidine analog 31, which underwent thioamidation to furnish intermediate 32. Afterward, the N-Boc protective group in compound 32 was removed, providing the desired compound 33. The target compounds 34–43 were obtained by reductive amination using aldehydes 30a-i, following procedures similar to those described for compounds 23–25.

Scheme 4. Synthesis of spiro-piperidine analogs 33–43^α.

^αReagents and conditions: (a) 7-(tert-butoxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid, EDCI, DMAP, DCM, r.t., overnight; (b) sodium hydrosulfide hydrate, magnesium chloride, DMF, r.t., overnight; (c) TFA, DCM, 1 h; (d) sodium triacetoxyborohydride, aldehydes 30a-i, Et₃N, DCE, r.t., 4 h; (e) t-BuOK, methoxymethyltriphenylphosphonium chloride, 0 °C to r.t., 6 h; (f) 4N HCl, THF, 60 °C, 3.5 h; (g) 33, sodium triacetoxyborohydride, Et₃N, DCE, r.t., 4 h.

Thiazole-5-carbaldehyde (44) underwent a Witting reaction with methoxymethyltriphenylphosphonium chloride, resulting in alkene 45 (Scheme 4). Subsequent treatment of alkene 45 with 4N HCl led to the formation of 2-(thiazol-5-yl)acetaldehyde (46). In a similar manner, aldehyde 46 was used to synthesize compound 43 through a reductive amination reaction previously described in Scheme 3.

As depicted in Scheme 5, treatment of 2,2-dimethoxyethan-1-amine (47) with appropriate acetic anhydrides, 3,3,3-trifluoropropanoyl chloride or carboxylic acids furnished the aminoacetaldehyde derivatives 48a-b, 48c, and 48d-m, respectively. Cleavage of the acetal protecting group in compounds 48a-m with Amberlyst-15 in acetone/H₂O afforded the desired aldehydes 49a-m. The reductive amination reactions were carried out to provide the amide-containing spiro-piperidine analogs 50–52, 56–59, and 61–66 using corresponding aldehydes 49a-m.

Scheme 5. Synthesis of amide-containing spiro-piperidine analogs 50–52, 56–59, and 61–66^α.

^αReagents and conditions: (a) for 48a: acetic anhydride, Et₃N, DCM, r.t., 2 h; for 48b: 2,2,2-trifluoroacetic anhydride, Et₃N, DCM, r.t., 2 h; for 48c: 3,3,3-trifluoropropanoyl chloride, Et₃N, DCM, 0 °C, 10 min; for 48d-m: carboxylic acids, EDCI, DMAP, DCM, r.t., overnight; (b) Amberlyst-15, acetone/H₂O, r.t., overnight; (c) 33, sodium triacetoxyborohydride, Et₃N, DCE, r.t., 4 h.

An alternative synthetic approach was adopted to prepare spiro-piperidine analogs 53–55 and 60 (Scheme 6). Briefly, deprotection of the Boc group in compound 32 followed by alkylation of the corresponding amine with tert-butyl (2-bromoethyl)carbamate yielded spiropiperidine analog 67, which was subjected to thioamidation with sodium hydrosulfide hydrate to generate compound 68. Cleavage of the N-Boc group and successive acylation led to target compounds 53–55. The synthesis of target compound 60 was achieved through the acylation of 4-fluoroaniline (69) followed by N-alkylation (Scheme 6).

Scheme 6. Synthesis of amide-containing spiro-piperidine analogs 53–55 and 60^α.

^αReagents and conditions: (a) i. TFA, DCM, 1 h; ii. tert-butyl (2-bromoethyl)carbamate, K₂CO₃, KI, Et₃N, 60 °C, 16 h; (b) sodium hydrosulfide hydrate, magnesium chloride, DMF, r.t., overnight; (c) i. TFA, DCM, r.t., 1 h; ii. carboxylic acids, HATU, DIPEA, 0 °C to r.t., 1.5 h; (d) 3-bromopropanoyl chloride, Et₃N, 0 °C, 50 min; (e) 33, K₂CO₃, KI, Et₃N, DMF, 60 °C, 8 h.

CONCLUSIONS

ASH1L greatly contributes to the pathogenesis of acute leukemia with MLL1 translocations (8), which still represents an unmet medical need. To date, only one class of ASH1L inhibitors has been reported, namely thioamide compounds that we recently developed (15). In this study we focused on the development of potent ASH1L inhibitors derived from our recently reported AS-99, which blocks the catalytic SET domain of ASH1L, with the goal to improve the activity and drug-like properties (15). Our new inhibitors were developed to occupy the unexplored hydrophobic pocket on ASH1L (Figure 3), which we identified by analyzing the crystal structure of ASH1L in complex with inhibitor AS-85. To occupy this pocket, named LMI, we utilized a spiro-piperidine moiety substituted with various hydrophobic groups, which were introduced to the core structures of ASH1L inhibitors. By combining structure-based design and medicinal chemistry, we developed nanomolar ASH1L inhibitors, including the most potent compound 66s (IC₅₀ = 94 nM in FP and 150 nM in HMT assay), representing 6–8-fold improved inhibitory activity over the previous ASH1L inhibitors (15). Our lead compound 66s also demonstrates high selectivity for ASH1L over 15 other histone methyltransferases assessed here. When tested in leukemia cells with MLL1 translocations, 66s reduced the level of H3K36me2 mark, supporting the on-target mechanism of action. The 66s compound also revealed pronounced anti-leukemic activity in blocking proliferation (GI₅₀ ~ 0.7 μM in a panel of the MLL1-rearranged leukemia cell lines) and inducing apoptosis and differentiation of these cells, outperforming our previous ASH1L inhibitors (15). Importantly, the effects induced by 66s recapitulate the effects observed upon genetic inactivation of ASH1L (8) or deletion of the ASH1L SET domain (15), supporting on-target activity of our new ASH1L inhibitors. This newly developed ASH1L inhibitor also demonstrates strong selectivity (>10-fold) towards the MLL1-rearranged leukemia cells over control cells. Ultimately, 66s reported here represents a new selective ASH1L inhibitor with nanomolar in vitro activity and sub-micromolar activity in the MLL1-r leukemia cells, establishing it as the most potent ASH1L inhibitor reported to date. This compound could serve as a valuable chemical probe to further assess biological functions of ASH1L and to explore its role in human diseases. The outcome of this work should also pave the way towards identification of novel therapeutic agents for leukemia and other cancers.

EXPERIMENTAL SECTION

General Chemistry

All reagents were commercially available and used without further purification. Nuclear magnetic resonance spectra were recorded in CDCl₃, CD₃OD and DMSO solutions. ¹H and ¹³C NMR were recorded on Bruker spectrometers operating at 600 MHz. Data are reported as follows: chemical shift (δ), multiplicity, integrated intensity, and coupling constant (J) in hertz. Low-resolution mass spectroscopy (LRMS) images were obtained on Shimadzu LC-2020 system (DUIS-ESI). The purity of the final compounds was assessed on a Shimadzu Prominence high-performance liquid chromatography (HPLC) system. High-performance liquid chromatography (HPLC) analysis was performed on Shimadzu Prominence system (20 series: binary pump, UV/vis at 254 nm, heated column compartment 28 °C) using a Restek Ultra C18 column (150 × 4.6 mm, 5 μm) at room temperature with a gradient elution using the mobile phase (A) nanopure water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. HPLC condition: 30% of B at 0–3.99 min, 30–85% of B at 3.99–5.50 min, 85–30% of B at 5.50–6.50 min, and 30% of B at 6.50– 8.50 min and a flow rate of 0.9 mL/min. The purity of all final compounds was > 95% by HPLC.

General procedure A for synthesis of spiro amides

A mixture of appropriate carboxylic acid (0.52 mmol, 1.3 eq.), DMAP (0.2 mmol, 0.5 eq.), relevant primary amine (0.4 mmol, 1 eq.), and EDCI (1.0 mmol, 2.5 eq.) in DCM (10 mL) was stirred overnight at room temperature. Upon completion, the reaction was quenched with water and extracted with DCM. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography.

General procedure B for thioamidation reaction

To a stirred solution of appropriate benzonitrile (0.1 mmol) in DMF (2.5 mL) was added sodium hydrosulfide hydrate (222.2 mg, 3.0 mmol) and magnesium chloride (238 mg, 2.5 mmol). The mixture was stirred overnight at room temperature. Upon completion, the reaction was quenched with saturated NH₄Cl aqueous solution and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography.

General procedure C for deprotection of N-Boc protecting group

A stirred solution of relevant N-Boc-amine (0.037 mmol) in DCM (2 mL) was cooled to 0 °C. TFA (0.4 mL) was then added dropwise, and the reaction mixture was allowed to warm to room temperature and stirred for 1 h. Upon completion, the solvent was removed under reduced pressure. If not indicated otherwise, the N-Boc deprotected product was directly used in the next step.

General procedure D for reductive amination

To a stirred solution of appropriate amine (N-Boc deprotected product) (0.037 mmol) and triethylamine (0.102 mmol) in DCE (3 mL) was added sodium triacetoxyborohydride (54 mg, 0.255 mmol) and relevant aldehyde (0.765 mmol) at 0 °C. The reaction mixture was then allowed to warm to room temperature and stirred for 4 h. Upon completion, saturated NH₄Cl aqueous solution was added to quench the reaction. The organic layer was separated. The aqueous layer was extracted with DCM. The combined organic layers were dried over sodium sulfate, filtrated, and concentrated in vacuo. The residue was purified by reverse-phase flash chromatography and then lyophilized to give the corresponding target compound.

Tert-butyl 6-(((3-(3-cyanophenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (6)

The title compound was synthesized from 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid and primary amine 5 according to the method described for procedure A. It was obtained as a white solid (66.0% yield). ¹H NMR (600 MHz, CDCl₃) δ 7.89 (t, J = 1.5 Hz, 1H), 7.84 – 7.81 (m, 2H), 7.56 – 7.51 (m, 3H), 7.39 (s, 1H), 7.36 (s, 1H), 7.14 – 7.12 (m, 1H), 4.56 (d, J = 5.8 Hz, 2H), 4.43 – 4.40 (m, 1H), 4.09 – 4.06 (m, 2H), 3.92 (s, 2H), 3.89 (s, 2H), 3.06 – 3.01 (m, 1H), 3.00 – 2.97 (m, 2H), 2.90 (s, 3H), 2.49 – 2.45 (m, 4H), 2.26 – 2.20 (m, 4H), 1.42 (s, 9H). LCMS (ESI): m/z 632 [M+H]⁺.

Tert-butyl 6-(((3-(3-carbamothioylphenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (7)

The title compound was synthesized from benzonitrile 6 according to the method described for procedure B. It was obtained as a yellow solid (96.6% yield). ¹H NMR (600 MHz, CD₃OD) δ 8.24 (s, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 7.0 Hz, 1H), 7.75 (d, J = 7.4 Hz, 1H), 7.68 (s, 1H), 7.47 – 7.44 (m, 2H), 7.11 (d, J = 7.9 Hz, 1H), 4.56 – 4.52 (m, 1H), 4.50 (s, 2H), 3.96 – 3.92 (m, 4H), 3.87 (s, 2H), 3.09 – 3.04 (m, 2H), 3.02 – 2.97 (m, 1H), 2.94 (s, 3H), 2.45 – 2.37 (m, 4H), 2.22 – 2.18 (m, 4H), 1.43 (s, 9H); ¹³C NMR (150 MHz, CD₃OD) δ 202.9, 175.4, 156.7, 140.4, 136.7, 132.6, 129.5, 128.1, 125.9, 125.3, 123.8, 122.8, 120.1, 119.5, 116.1, 108.9, 79.5, 60.1, 52.6, 45.3, 43.4, 35.3, 34.3, 33.9, 33.6, 31.6, 27.2. LCMS (ESI): m/z 666 [M+H]⁺.

N-((3-(3-Carbamothioylphenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-6-yl)methyl)-2-azaspiro[3.3]heptane-6-carboxamide (4)

The title compound was synthesized according to the modified procedure C. To a solution of compound 7 (45 mg, 0.068 mmol) in DCM (2 mL) was added TFA (400 μL, 4.18 mmol) at 0 °C. The mixture was allowed to warm to room temperature and was stirred for 1 h. Upon completion, the solvent was removed. To a suspension solution of the residue in DCM (2 mL) was then added Et₃N (30 μL, 0.22 mmol). The mixture was stirred at room temperature for another 1 h, then diluted with saturated NaHCO₃ aqueous solution. The organic layer was separated, and the aqueous layer was extracted with DCM. The combined organic extract was dried over sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified by reverse-phase flash chromatography (0~60% MeOH in H₂O) and then lyophilized to give compound 4 (7.2 mg, 18.7% yield) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.14 (t, J = 1.6 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.58 (s, 1H), 7.37 (s, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.00 (d, J = 8.1 Hz, 1H), 4.47 – 4.43 (m, 1H), 4.40 (s, 2H), 4.00 (s, 2H), 3.99 (s, 2H), 3.86 (d, J = 12.1 Hz, 2H), 2.97 (t, J = 11.3 Hz, 2H), 2.93 – 2.89 (m, 1H), 2.85 (s, 3H), 2.47 – 2.43 (m, 2H), 2.40 – 2.35 (m, 2H), 2.18 – 2.14 (m, 2H), 2.11 – 2.07 (m, 2H); ¹³C NMR (150 MHz, CD₃OD) δ 202.9, 175.4, 140.4, 136.5, 135.7, 132.4, 129.5, 128.1, 125.5, 123.5, 123.4, 119.9, 119.6, 116.0, 109.0, 57.7, 57.2, 53.2, 48.2, 45.4, 43.3, 38.0, 34.7, 33.8, 33.5, 31.5. HPLC analysis: retention time = 2.349 min; peak area, 99.2 % (λ = 254 nm). LCMS (ESI): m/z 566 [M+H]⁺.

7-Chloro-1H-indole-6-carboxylic acid (9)

To a stirred solution of 2-chloro-3-nitrobenzoic acid (5.0 g, 24.8 mmol) in dry THF (60 mL) at −45 °C was added cold Vinyl Grignard reagent (141.7 mL, 99.2 mmol) dropwise. Then 100 mL of THF was added to the reaction mixture. The reaction was allowed to slowly warm to room temperature overnight. The flask was then cooled to 0 °C with an ice bath and saturated NH₄Cl aqueous solution was added. The mixture was stirred for 1 h and then the solvents were removed. The residue was acidified to pH = 2 with 2M HCl. The precipitate was collected by filtration and washed with water and hexane. The solid obtained was then dried to give the compound 9 (3.86 g, 79.6%) as a tan powder that was used without further purification. ¹H NMR (600 MHz, CD₃OD) δ 7.65 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 3.1 Hz, 1H), 6.59 (d, J = 3.1 Hz, 1H).

Methyl 7-chloro-1H-indole-6-carboxylate (10)

To a stirred solution of lH-indole-6-carboxylic acid 9 (3.3 g, 16.9 mmol) and potassium carbonate (2.34 g, 16.9 mmol) in DMF (25 mL) was dropwise added methyl iodide (1.16 mL, 18.6 mmol) at room temperature. The mixture was stirred for 2 h. Upon completion, the reaction was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~20% ethyl acetate in hexane) to afford the desired compound 10 (2.18 g, 61.6% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 8.70 (s, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.29 – 7.28 (m, 1H), 6.51 – 6.50 (m, 1H), 3.86 (s, 3H). LCMS (ESI): m/z 210 [M+H]⁺.

Methyl 7-chloro-3-iodo-1H-indole-6-carboxylate (11)

To a solution of indole 10 (2.18 g, 10.4 mmol) and KOH (1.46 g, 26.0 mmol) in DMF (15.0 mL) was added I₂ (2.69 g, 10.6 mmol) in portion at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 3 h. Upon completion, the reaction was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~17.5% ethyl acetate in hexane) to afford the desired compound 11 (2.79 g, 79.9% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 8.76 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 3.89 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 166.3, 133.7, 133.2, 131.8, 123.6, 122.6, 119.2, 118.0, 58.1, 52.3. LCMS (ESI): m/z 336 [M+H]⁺.

1-(Tert-butyl) 6-methyl 7-chloro-3-iodo-1H-indole-1,6-dicarboxylate (12)

To a solution of indole 11 (3.32 g, 9.89 mmol), DMAP (0.12 g, 0.99 mmol) and triethylamine (1.9 mL, 13.8 mmol) in DCM (60 mL) was added di-tert-butyl dicarbonate (2.60 g, 11.9 mmol) at 0 °C in portion wise. The reaction mixture was allowed to warm to room temperature and stirred overnight. Upon completion, the reaction was quenched with 1.0 M HCl solution and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~16.5% ethyl acetate in hexane) to give the desired compound 12 (3.79 g, 87.7% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 7.65 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 3.86 (s, 3H), 1.55 (s, 9H). LCMS (ESI): m/z 436 [M+H]⁺.

1-(Tert-butyl) 6-methyl 7-chloro-3-(3-cyanophenyl)-1H-indole-1,6-dicarboxylate (13)

The mixture of 1-(tert-butyl) 6-methyl 7-chloro-3-iodo-1H-indole-1,6-dicarboxylate (12) (2.47 g, 5.67 mmol), Na₂CO₃ (0.91 g, 8.59 mmol), Pd(dppf)Cl₂.DCM (0.46 g, 0.56 mmol) and (3-cyanophenyl)boronic acid (0.83 g, 5.67 mmol) in THF/H₂O (45 mL/15 mL) was heated to 65 °C for 12 h under an Argon atmosphere. Upon completion, the resulting mixture was cooled to room temperature, filtered through celite, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (0~20% ethyl acetate in hexane) to give the compound 13 (1.72 g, 73.8%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 7.78 (t, J = 1.4 Hz, 1H), 7.73 (dt, J = 7.8, 1.4 Hz, 1H), 7.69 (s, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.58 (dt, J = 7.8, 1.4 Hz, 1H), 7.56 (d, J = 6.0 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 3.89 (s, 3H), 1.60 (s, 9H).

Methyl 7-chloro-3-(3-cyanophenyl)-1H-indole-6-carboxylate (14)

The title compound was synthesized according to the modified procedure C. To a stirred solution of compound 13 (1.72 g, 4.19 mmol) in DCM (40 mL) was added TFA (8.8 mL, 91.7 mmol) at 0 °C. The mixture was allowed to warm to room temperature and was stirred for 3 h. Upon completion, the reaction was quenched with saturated sodium bicarbonate aqueous solution and extracted with DCM. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by the flash column chromatography (0~25% ethyl acetate in hexane) to afford the desired compound 14 (1.13 g, 86.9% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 8.82 (s, 1H), 7.82 (t, J = 1.4 Hz, 1H), 7.78 (dt, J = 7.7, 1.5 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.54 – 7.52 (m, 2H), 7.49 (t, J = 7.7 Hz, 1H), 3.91 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 166.2, 135.8, 134.8, 131.6, 130.7, 130.0, 129.8, 128.6, 125.6, 123.9, 122.5, 118.8, 118.7, 117.6, 117.3, 113.2, 52.3. LCMS (ESI): m/z 311 [M+1]⁺.

Methyl 7-chloro-3-(3-cyanophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indole-6-carboxylate (16)

The mixture of compound 14 (596.2 mg, 1.92 mmol) and cesium carbonate (3753.4 mg, 11.52 mmol) in dry DMF (6 mL) was stirred at room temperature for 30 minutes. Then a solution of tetrahydro-2H-pyran-4-yl methanesulfonate (1384.1 mg, 7.68 mmol) in DMF (2.0 mL) was added. The reaction mixture was stirred at 100 °C for 18 h. The reaction was then quenched with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by the flash column chromatography (0~28% ethyl acetate in hexane) to give the desired compound 16 (422.9 mg, 55.8% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 7.83 (s, 1H), 7.80 – 7.76 (m, 2H), 7.75 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 8.1 Hz, 1H), 5.68 – 5.63 (m, 1H), 4.12 – 4.09 (m, 2H), 3.91 (s, 3H), 3.59 – 3.56 (m, 2H), 2.16 – 2.13 (m, 2H), 2.04 – 2.01 (m, 2H); LCMS (ESI): m/z 395 [M+H]⁺.

3-(7-Chloro-6-(hydroxymethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)benzonitrile (17)

The mixture of compound 16 (800 mg, 2.03 mmol) and LiBH₄ (446.6 mg, 20.3 mmol, 10 eq.) in THF/MeOH (35 mL/0.5 mL) was heated to 65 °C under an argon atmosphere. and stirred for 25 minutes. Upon completion, the reaction was cooled at room temperature and quenched with saturated NH₄Cl aqueous solution. The resultant mixture was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by the flash column chromatography (0~45% ethyl acetate in hexane) to give the desired compound 17 (493.7 mg, 66.3% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 7.78 (t, J = 1.4 Hz, 1H), 7.74 (dt, J = 7.6, 1.5 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.50 (dt, J = 7.7, 1.4 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.39 (s, 1H), 7.25 (d, J = 8.1 Hz, 1H), 5.58 (tt, J = 11.7, 3.9 Hz, 1H), 4.87 (d, J = 5.8 Hz, 2H), 4.10 (dd, J = 11.6, 4.3 Hz, 2H), 3.57 (td, J = 11.9, 1.7 Hz, 2H), 2.14 – 2.10 (m, 2H), 2.05 – 1.98 (m, 2H). LCMS (ESI): m/z 349 [M-18+H]⁺.

3-(6-(Azidomethyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)benzonitrile (18)

To a stirred solution of alcohol 17 (366.6 mg, 1.0 mmol) in anhydrous THF (10 mL) was added DPPA (560 μL, 2.6 mmol) at 0 °C. After 10 minutes, DBU (456.7 μL, 13.0 mmol) was slowly added. The resulting mixture was warmed to room temperature and stirred overnight. Upon completion, the reaction was quenched with saturated NaHCO₃ aqueous solution. The resultant mixture was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~23% ethyl acetate in hexane) to give the title compound 18 (214.6 mg, 74.0% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 7.78 (t, J = 1.4 Hz, 1H), 7.73 (dt, J = 7.6, 1.5 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.51 (dt, J = 7.7, 1.4 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.15 (d, J = 8.1 Hz, 1H), 5.61 – 5.55 (m, 1H), 4.57 (s, 2H), 4.10 (dd, J = 11.6, 4.3 Hz, 2H), 3.58 (td, J = 11.9, 1.6 Hz, 2H), 2.15 – 2.11 (m, 2H), 2.06 – 1.99 (m, 2H).

3-(6-(Aminomethyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)benzonitrile (19)

To a solution of azide 18 (410 mg, 1.05 mmol) in THF-H₂O (11 mL, V/V =10:1) added PPh₃ (786.9 mg, 3 mmol). The reaction mixture was stirred at room temperature for 16 h. Upon completion, the solvents were removed. The residue was purified by flash column chromatography (0~7% MeOH in DCM) to give the title compound 19 (261.3 mg, 68% yield) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 7.78 (t, J = 1.3 Hz, 1H), 7.73 (dt, J = 7.6, 1.5 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.50 (dt, J = 7.7, 1.4 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.38 (s, 1H), 7.15 (d, J = 8.1 Hz, 1H), 5.59 (tt, J = 11.8, 3.9 Hz, 1H), 4.10 (dd, J = 11.5, 4.2 Hz, 2H), 4.04 (s, 2H), 3.58 (td, J = 11.8, 1.6 Hz, 2H), 2.15 – 2.10 (m, 2H), 2.05 – 1.98 (m, 2H). LCMS (ESI): m/z 349 [M-17+H]⁺.

Tert-butyl 6-(((7-chloro-3-(3-cyanophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (20)

Compound 20 was prepared according to the method described for procedure A. It was obtained as a white solid (92.6% yield). ¹H NMR (600 MHz, CDCl₃) δ 7.76 (t, J = 1.6 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.39 (s, 1H), 7.14 (d, J = 8.2 Hz, 1H), 5.68 (t, J = 5.5 Hz, 1H), 5.58 – 5.53 (m, 1H), 4.61 (d, J = 5.7 Hz, 2H), 4.10 (dd, J = 11.5, 4.1 Hz, 2H), 3.84 (s, 2H), 3.81 (s, 2H), 3.59 – 3.55 (m, 2H), 2.97 – 2.92 (m, 1H), 2.42 – 2.38 (m, 4H), 2.28 – 2.25 (m, 2H), 2.13 – 2.09 (m, 2H), 1.35 (s, 9H). LCMS (ESI): m/z 589 [M+H]⁺.

Tert-butyl 6-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (21)

Compound 21 was prepared according to the method described for procedure A. It was obtained as a yellow oil (96.6% yield). ¹H NMR (600 MHz, CD₃OD) δ 8.18 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.79 (m, 1H), 7.79 (s, 1H), 7.77 – 7.76 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.73 – 5.67 (m, 1H), 4.62 (s, 2H), 4.16 – 4.12 (m, 2H), 3.94 (s, 2H), 3.87 (s, 2H), 3.71 – 3.66 (m, 2H), 3.05 – 3.02 (m, 1H), 2.46 – 2.37 (m, 4H), 2.19 – 2.15 (m, 4H), 1.44 (s, 9H); LCMS (ESI): m/z 623 [M+H]⁺.

N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-2-azaspiro[3.3]heptane-6-carboxamide (22)

Compound 22 was prepared using a similar procedure for the synthesis of compound 4. It was obtained as a white solid (12.5 mg, 42.7% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 9.91 (s, 1H), 9.55 (s, 1H), 8.16 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.82 – 7.78 (m, 2H), 7.49 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 5.59 – 5.55 (m, 1H), 4.47 (d, J = 5.7 Hz, 2H), 4.05 (dd, J = 10.9, 3.8 Hz, 2H), 3.60 – 3.56 (m, 2H), 3.48 (s, 2H), 3.36 (s, 2H), 2.94 – 2.89 (m, 1H), 2.24 – 2.21 (m, 3H), 2.16 – 2.04 (m, 5H); ¹³C NMR (150 MHz, DMSO-d₆) δ 200.8, 174.3, 140.7, 134.6, 132.1, 131.5, 130.0, 128.9, 128.3, 126.3, 125.8, 125.7, 121.7, 118.4, 116.1, 115.7, 67.0, 59.6, 58.7, 53.9, 40.9, 40.6, 36.3, 34.8, 33.8. HPLC analysis: retention time = 2.077 min; peak area, 96.764% (λ = 254 nm). LCMS (ESI): m/z 523 [M+H]⁺.

2-(2-Butoxyethyl)-N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-2-azaspiro[3.3]heptane-6-carboxamide (23)

Compound 23 was prepared from aldehyde 30a by following the general procedure D. It was obtained as a white solid (31.4% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.18 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.61 (s, 2H), 4.15 – 4.12 (m, 2H), 3.70 – 3.65 (m, 2H), 3.45 – 3.43 (m, 2H), 3.42 (s, 2H), 3.35 (s, 2H), 3.01 (q, J = 7.8 Hz, 1H), 2.68 (t, J = 5.4 Hz, 2H), 2.43 – 2.33 (m, 4H), 2.20 – 2.14 (m, 4H), 1.59 – 1.52 (m, 3H), 1.43 – 1.36 (m, 3H), 0.94 (t, J = 7.4 Hz, 3H); ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 175.6, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 70.6, 68.3, 67.1, 66.8, 66.2, 57.7, 53.6, 41.2, 35.8, 35.1, 34.4, 31.5, 18.9, 12.8. HPLC analysis: retention time = 2.873 min; peak area, 97.384% (λ = 254 nm). LCMS (ESI): m/z 623 [M+H]⁺.

N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-2-(2-(cyclobutylmethoxy)ethyl)-2-azaspiro[3.3]heptane-6-carboxamide (24)

Compound 24 was prepared from aldehyde 30b by following the general procedure D. It was obtained as a white solid (36.9% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 – 7.79 (m, 1H), 7.78 (s, 1H), 7.76 (dt, J = 7.7, 1.1 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.61 (s, 2H), 4.16 – 4.12 (m, 2H), 3.70 – 3.65 (m, 2H), 3.45 (t, J = 5.6 Hz, 2H), 3.43 (s, 2H), 3.39 (d, J = 6.6 Hz, 2H), 3.36 (s, 2H), 3.01 (q, J = 8.0 Hz, 1H), 2.69 (t, J = 5.5 Hz, 2H), 2.59 – 2.54 (m, 1H), 2.42 – 2.34 (m, 4H), 2.18 – 2.14 (m, 4H), 2.07 – 2.03 (m, 2H), 1.96 – 1.88 (m, 2H), 1.81 – 1.74 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 175.6, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 75.4, 68.6, 67.1, 66.8, 66.2, 53.6, 41.2, 35.8, 35.1, 34.4, 33.9, 24.6, 18.0. HPLC analysis: retention time = 3.50 min; peak area, 96.8% (λ = 254 nm). LCMS (ESI): m/z 635 [M+H]⁺.

N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-2-(2-(thiazol-5-ylmethoxy)ethyl)-2-azaspiro[3.3]heptane-6-carboxamide (25)

Compound 25 was prepared from aldehyde 30i by following the general procedure D. It was obtained as a white solid (45.4% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.97 (s, 1H), 8.19 (t, J = 1.7 Hz, 1H), 7.82 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.76 (s, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 5.70 – 5.64 (m, 1H), 4.74 (s, 2H), 4.60 (s, 2H), 4.14 – 4.11 (m, 2H), 3.68 – 3.64 (m, 2H), 3.52 (t, J = 5.4 Hz, 2H), 3.41 (s, 2H), 3.34 (s, 2H), 3.01 (q, J = 8.3 Hz, 1H), 2.71 (t, J = 5.3 Hz, 2H), 2.40 – 2.32 (m, 4H), 2.16 – 2.12 (m, 4H). ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 175.6, 154.8, 141.1, 140.4, 136.3, 134.8, 131.2, 130.3, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.6, 115.9, 67.8, 67.1, 66.7, 66.2, 64.3, 57.5, 53.6, 41.2, 35.8, 35.1, 34.4, 34.0. HPLC analysis: retention time = 2.764 min; peak area, 96.7% (λ = 254 nm). LCMS (ESI): m/z 664 [M+H]⁺.

Tert-butyl 2-(((7-chloro-3-(3-cyanophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonane-7-carboxylate (31)

Compound 31 was prepared from 7-(tert-butoxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid and primary amine 19 by following the general procedure A. It was obtained as a white solid (88% yield). ¹H NMR (600 MHz, CDCl₃) δ 7.85 (t, J = 1.3 Hz, 1H), 7.81 (dt, J = 7.6, 1.5 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.60 (dt, J = 7.7, 1.4 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.48 (s, 1H), 7.29 (s, 1H), 7.25 (d, J = 8.2 Hz, 1H), 5.79 (t, J = 5.6 Hz, 1H), 5.65 (tt, J = 11.7, 3.9 Hz, 1H), 4.72 (d, J = 5.8 Hz, 2H), 4.19 (dd, J = 11.5, 4.2 Hz, 2H), 3.66 (t, J = 11.0 Hz, 2H), 3.37 – 3.35 (m, 2H), 3.30 – 3.28 (m, 2H), 2.23 – 2.19 (m, 2H), 2.14 – 2.09 (m, 4H), 2.06 – 2.02 (m, 2H), 1.60 – 1.55 (m, 4H), 1.46 (s, 9H). LCMS (ESI): m/z 617 [M+H]⁺.

Tert-butyl 2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonane-7-carboxylate (32)

Compound 32 was prepared from benzonitrile 31 according to the method described for procedure B. It was obtained as a yellow solid (93% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.03 (t, J = 1.6 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.64 (s, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.43 (s, 1H), 7.40 (brs, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 5.74 (t, J = 5.4 Hz, 1H), 5.55 – 5.51 (m, 1H), 4.59 (d, J = 5.6 Hz, 2H), 4.10 – 4.07 (m, 2H), 3.55 (t, J = 11.0 Hz, 2H), 3.25 – 3.23 (m, 2H), 3.18 – 3.16 (m, 2H), 2.10 – 2.07 (m, 2H), 2.04 – 1.98 (m, 4H), 1.94 – 1.90 (m, 2H), 1.48 – 1.46 (m, 2H), 1.45 – 1.43 (m, 2H), 1.36 (s, 9H). LCMS (ESI): m/z 651 [M+1]⁺.

N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-azaspiro[3.5]nonane-2-carboxamide (33)

Compound 33 was prepared from N-Boc amine 32 using a similar procedure for the synthesis of compound 4. It was obtained as a white solid (44.1% yield). ¹H NMR (600 MHz, CD₃OD) δ 8.08 (t, J = 1.7 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.68 – 7.66 (m, 2H), 7.65 (dt, J = 7.7, 1.1 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 5.61 – 5.57 (m, 1H), 4.52 (s, 2H), 4.04 – 4.01 (m, 2H), 3.59 – 3.54 (m, 2H), 3.07 – 3.03 (m, 1H), 2.95 – 2.92 (m, 2H), 2.88 – 2.85 (m, 2H), 2.08 – 2.04 (m, 4H), 2.01 – 1.97 (m, 4H), 1.72 (t, J = 5.3 Hz, 2H), 1.65 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.0, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.5, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 67.1, 53.7, 41.3, 41.2, 41.1, 35.3, 34.4, 34.1, 33.8, 32.8, 32.5. HPLC analysis: retention time = 2.7021 min; peak area, 95.781% (λ = 254 nm). LCMS (ESI): m/z 551 [M+H]⁺.

7-(2-Butoxyethyl)-N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-azaspiro[3.5]nonane-2-carboxamide (34)

The title compound 34 was synthesized from 2-butoxyacetaldehyde 30a according to the method described for procedure D. It was obtained as a white solid (25.4% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.78 (m, 2H), 7.77 (d, J = 7.9 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.69 (m, 1H), 4.64 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.67 (m, 2H), 3.59 (t, J = 5.7 Hz, 2H), 3.46 (t, J = 6.5 Hz, 2H), 3.14 – 3.10 (m, 1H), 2.65 – 2.61 (m, 2H), 2.58 – 2.52 (m, 2H), 2.20 – 2.16 (m, 4H), 2.07 – 2.02 (m, 4H), 1.74 (t, J = 5.3 Hz, 2H), 1.67 (t, J = 5.6 Hz, 2H), 1.56 (dt, J = 14.4, 6.4 Hz, 3H), 1.39 (dt, J = 14.7, 7.5 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 117.0, 115.9, 70.6, 67.1, 57.4, 53.7, 50.4, 50.1, 48.2, 41.2, 37.2, 35.4, 34.4, 33.2, 32.8, 31.4, 19.0, 12.8. HPLC analysis: retention time = 3.469 min; peak area, 95.831% (λ = 254 nm). LCMS (ESI): m/z 651 [M+H]⁺.

N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(cyclobutylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (35)

The title compound 35 was synthesized from 2-(cyclobutylmethoxy)acetaldehyde 30b according to the method described for procedure D. It was obtained as a white solid (38.8% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.09 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (s, 1H), 7.67 – 7.63 (m, 2H), 7.36 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 5.60 – 5.55 (m, 1H), 4.52 (s, 2H), 4.04 – 4.00 (m, 2H), 3.64 – 3.62 (m, 2H), 3.58 – 3.54 (m, 2H), 3.38 (d, J = 6.8 Hz, 2H), 3.25 – 3.22 (m, 1H), 3.17 – 3.14 (m, 2H), 3.05 (p, J = 8.6 Hz, 2H), 2.51 (dt, J = 14.9, 7.5 Hz, 1H), 2.10 – 1.91 (m, 12H), 1.88 – 1.75 (m, 6H), 1.69 – 1.64 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 175.8, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.6, 124.7, 124.1, 121.4, 117.9, 116.7, 115.9, 75.6, 67.1, 53.7, 49.9, 49.6, 41.2, 34.8, 34.4, 33.1, 32.3, 32.1, 24.6, 18.0. HPLC analysis: retention time = 3.418 min; peak area, 97.518% (λ = 254 nm). LCMS (ESI): m/z 663 [M+H]⁺.

N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-cyclobutoxyethyl)-7-azaspiro[3.5]nonane-2-carboxamide (36)

The title compound 36 was synthesized from 2-cyclobutylacetaldehyde 30c according to the method described for procedure D. It was obtained as a white solid (30.6% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.78 (m, 2H), 7.78 – 7.76 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.99 – 3.94 (m, 1H), 3.71 – 3.66 (m, 2H), 3.53 (t, J = 5.5 Hz, 2H), 3.13 (p, J = 8.6 Hz, 1H), 2.77 – 2.55 (m, 6H), 2.24 – 2.20 (m, 2H), 2.18 – 2.15 (m, 4H), 2.09 – 2.03 (m, 4H), 1.96 – 1.90 (m, 2H), 1.80 – 1.76 (m, 2H), 1.74 – 1.69 (m, 3H), 1.59 – 1.53 (m, 1H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.3, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 73.4, 67.1, 57.2, 53.7, 50.3, 50.0, 48.2, 41.2, 36.7, 34.9, 34.4, 34.2, 33.0, 32.7, 29.8, 11.9. HPLC analysis: retention time = 3.248 min; peak area, 99.9% (λ = 254 nm). LCMS (ESI): m/z 649 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(2-(1-methylcyclopropyl)ethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (37)

The title compound 37 was synthesized from 2-(2-(1-methylcyclopropyl)ethoxy)acetaldehyde 30d according to the method described for procedure D. It was obtained as a white solid (35.5% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.20 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 4.9 Hz, 2H), 7.77 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.69 (m, 1H), 4.64 (s, 2H), 4.15 (dt, J = 10.6, 3.3 Hz, 2H), 3.70 – 3.65 (m, 4H), 3.56 – 3.53 (m, 2H), 3.16 – 3.12 (m, 1H), 2.87 – 2.82 (m, 2H), 2.20 – 2.16 (m, 4H), 2.11 – 2.05 (m, 4H), 1.81 (t, J = 9.6 Hz, 2H), 1.75 (t, J = 9.6 Hz, 2H), 1.65 (s, 2H), 1.60 – 1.58 (m, 3H), 1.56 – 1.50 (m, 3H), 0.92 (s, 3H), 0.33 (t, J = 4.7 Hz, 1H), 0.25 (t, J = 4.8 Hz, 1H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.2, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 69.6, 67.1, 56.9, 53.7, 50.3, 49.9, 48.2, 41.2, 39.1, 38.5, 34.4, 32.7, 31.2, 29.3, 25.1, 22.5, 22.0, 14.4, 12.1. HPLC analysis: retention time = 3.822 min; peak area, 95.498% (λ = 254 nm). LCMS (ESI): m/z 677 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(thiophen-2-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (38)

The title compound 38 was synthesized from 2-(thiophen-2-ylmethoxy)acetaldehyde 30e according to the method described for procedure D. It was obtained as a white solid (28.9% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 6.4 Hz, 2H), 7.77 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.39 – 7.38 (m, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.06 (d, J = 3.1 Hz, 1H), 6.99 (d, J = 1.4 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.71 (s, 2H), 4.63 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.68 (m, 2H), 3.66 (t, J = 5.5 Hz, 2H), 3.14 – 3.10 (m, 1H), 2.78 – 2.73 (m, 2H), 2.63 – 2.46 (m, 4H), 2.20 – 2.14 (m, 4H), 2.09 – 2.01 (m, 4H), 1.75 (t, J = 5.3 Hz, 2H), 1.69 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.3, 140.5, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.6, 126.4, 126.3, 125.7, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 66.9, 65.6, 56.7, 53.7, 50.2, 50.0, 48.2, 41.2, 36.7, 34.9, 34.4, 33.0, 32.7. HPLC analysis: retention time = 3.427 min; peak area, 95.329% (λ = 254 nm). LCMS (ESI): m/z 691 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(furan-2-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (39)

The title compound 39 was synthesized from 2-(furan-2-ylmethoxy)acetaldehyde 30f according to the method described for procedure D. It was obtained as a white solid (28.9% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.18 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.79 (m, 1H), 7.79 (s, 1H), 7.78 – 7.75 (m, 1H), 7.50 – 7.46 (m, 2H), 7.18 (d, J = 8.2 Hz, 1H), 6.39 – 6.37 (m, 2H), 5.73 – 5.68 (m, 1H), 4.63 (s, 2H), 4.46 (s, 2H), 4.16 – 4.12 (m, 2H), 3.71 – 3.66 (m, 2H), 3.61 (t, J = 5.7 Hz, 2H), 3.09 (q, J = 8.7 Hz, 1H), 2.57 (t, J = 5.6 Hz, 2H), 2.52 – 2.36 (m, 4H), 2.19 – 2.15 (m, 4H), 2.06 – 1.99 (m, 4H), 1.70 (t, J = 5.3 Hz, 2H), 1.62 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 151.8, 142.6, 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.3, 124.6, 124.3, 121.4, 117.9, 116.7, 109.9, 109.1, 67.1, 66.6, 64.2, 57.3, 53.7, 50.3, 50.0, 48.2, 41.2, 37.4, 35.5, 34.4, 33.3, 32.9. HPLC analysis: retention time = 3.313 min; peak area, 96.999% (λ = 254 nm). LCMS (ESI): m/z 675 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(pyridin-4-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (40)

The title compound 40 was synthesized from 2-(pyridin-4-ylmethoxy)acetaldehyde 30g according to the method described for procedure D. It was obtained as a white solid (53.2% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.50 (d, J = 6.0 Hz, 2H), 8.19 (t, J = 4.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (s, 1H), 7.79 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.43 (d, J = 5.5 Hz, 2H), 7.18 (d, J = 8.2 Hz, 1H), 5.72 – 5.68 (m, 1H), 4.63 (s, 2H), 4.60 (s, 2H), 4.16 – 4.13 (m, 2H), 3.70 (t, J = 5.6 Hz, 2H), 3.69 – 3.65 (m, 2H), 3.13 – 3.09 (m, 1H), 2.67 (t, J = 5.4 Hz, 2H), 2.54 – 2.46 (m, 4H), 2.19 – 2.15 (m, 4H), 2.07 – 2.02 (m, 4H), 1.73 (t, J = 5.3 Hz, 2H), 1.66 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 149.3, 148.6, 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 122.1, 121.4, 117.9, 116.7, 115.9, 70.7, 68.0, 67.1, 57.5, 53.7, 50.5, 50.1, 48.2, 41.2, 37.5, 35.6, 34.4, 33.3, 32.9. HPLC analysis: retention time = 1.848 min; peak area, 95.336% (λ = 254 nm). LCMS (ESI): m/z 686 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(pyridin-2-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (41)

The title compound 41 was synthesized from 2-(pyridin-3-ylmethoxy)acetaldehyde 30h according to the method described for procedure D. It was obtained as a white solid (64.6% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.50 (d, J = 4.6 Hz, 1H), 8.20 – 8.18 (m, 1H), 7.88 – 7.84 (m, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.76 (m, 3H), 7.55 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.37 – 7.34 (m, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.64 (s, 2H), 4.54 (s, 2H), 4.16 – 4.12 (m, 2H), 3.79 – 3.73 (m, 2H), 3.73 – 3.67 (m, 2H), 3.15 – 3.10 (m, 1H), 2.78 (s, 2H), 2.70 – 2.53 (m, 4H), 2.19 – 2.14 (m, 4H), 2.09 – 1.99 (m, 4H), 1.77 (t, J = 5.3 Hz, 2H), 1.70 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.3, 157.8, 148.2, 140.4, 137.5, 134.5, 132.2, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 122.8, 122.0, 121.4, 117.9, 116.7, 115.9, 72.9, 67.1, 57.2, 53.6, 50.3, 50.0, 48.2, 41.2, 37.0, 35.1, 34.4, 33.1, 32.8. HPLC analysis: retention time = 2.614 min; peak area, 96.063% (λ = 254 nm). LCMS (ESI): m/z 686 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(thiazol-5-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (42)

The title compound 42 was synthesized from 2-(thiazol-5-ylmethoxy)acetaldehyde 30i according to the method described for procedure D. It was obtained as a white solid (42.6% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.88 (s, 1H), 8.07 (t, J = 1.6 Hz, 1H), 7.74 (s, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.67 (s, 1H), 7.66 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 5.59 – 5.54 (m, 1H), 4.70 (s, 2H), 4.50 (s, 2H), 4.03 – 3.99 (m, 2H), 3.61 (t, J = 5.3 Hz, 2H), 3.57 – 3.53 (m, 2H), 3.03 – 2.98 (m, 1H), 2.84 – 2.79 (m, 2H), 2.76 – 2.57 (m, 4H), 2.05 – 2.01 (m, 4H), 1.98 – 1.92 (m, 4H), 1.72 – 1.68 (m, 2H), 1.65 – 1.60 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 176.1, 155.0, 141.5, 140.4, 135.8, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 67.1, 64.2, 56.5, 53.7, 49.8, 41.2, 36.0, 34.4, 34.0, 32.7, 32.6. HPLC analysis: retention time = 2.925 min; peak area, 96.017% (λ = 254 nm). LCMS (ESI): m/z 692 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(thiazol-5-yl)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (43)

The title compound 43 was synthesized from aldehyde 46 according to the method described for procedure D. It was obtained as a white solid (30.5% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.86 (s, 1H), 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 6.1 Hz, 2H), 7.78 – 7.76 (m, 1H), 7.70 (s, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.72 – 5.69 (m, 1H), 4.64 (s, 2H), 4.16 – 4.13 (m, 2H), 3.70 – 3.67 (m, 2H), 3.16 – 3.11 (m, 3H), 2.79 – 2.75 (m, 2H), 2.69 – 2.56 (m, 4H), 2.18 – 2.15 (m, 4H), 2.09 – 2.05 (m, 4H), 1.80 (t, J = 5.5 Hz, 2H), 1.72 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.3, 153.2, 140.4, 140.0, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 67.1, 53.7, 53.4, 49.9, 49.6, 48.2, 41.2, 35.4, 34.4, 33.2, 32.8. HPLC analysis: retention time = 2.851 min; peak area, 95.13% (λ = 254 nm). LCMS (ESI): m/z 662 [M+H]⁺.

7-(2-Acetamidoethyl)-N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-azaspiro[3.5]nonane-2-carboxamide (50)

The title compound 50 was synthesized from aldehyde 49a according to the method described for procedure D. It was obtained as a white solid (43% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.5 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.63 (s, 2H), 4.15 – 4.12 (m, 2H), 3.70 – 3.66 (m, 2H), 3.35 (t, J = 6.9 Hz, 2H), 3.11 (q, J = 8.6 Hz, 1H), 2.57 – 2.50 (m, 4H), 2.48 – 2.37 (m, 2H), 2.18 – 2.14 (m, 4H), 2.07 – 2.01 (m, 4H), 1.95 (s, 3H), 1.73 (t, J = 5.0 Hz, 2H), 1.66 (t, J = 5.4 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 176.3, 172.0, 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 57.0, 53.7, 50.0, 49.8, 48.2, 41.2, 37.4, 36.0, 35.5, 34.4, 33.3, 32.8, 21.2. HPLC analysis: retention time = 2.453 min; peak area, 97.398% (λ = 254 nm). LCMS (ESI): m/z 636 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(2,2,2-trifluoroacetamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (51)

The title compound 51 was synthesized from aldehyde 49b according to the method described for procedure D. It was obtained as a white solid (78.3% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.07 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (dd, J = 8.2, 1.7 Hz, 2H), 7.65 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 5.61 – 5.56 (m, 1H), 4.51 (s, 2H), 4.04 – 4.00 (m, 2H), 3.59 – 3.54 (m, 2H), 3.32 (t, J = 6.9 Hz, 1H), 3.02 – 2.97 (m, 1H), 2.86 (t, J = 6.1 Hz, 1H), 2.44 – 2.39 (m, 2H), 2.37 – 2.25 (m, 4H), 2.08 – 2.02 (m, 4H), 1.96 – 1.89 (m, 4H), 1.60 (t, J = 5.4 Hz, 2H), 1.53 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 157.6 (q, J = 36.0 Hz), 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.3, 124.6, 124.3, 121.4, 117.9, 117.1, 116.7, 116.5 (q, J = 285.0 Hz), 67.1, 56.3, 53.7, 50.0, 49.8, 48.2, 41.2, 37.7, 36.4, 35.8, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.838 min; peak area, 99.1% (λ = 254 nm). LCMS (ESI): m/z 690 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(3,3,3-trifluoropropanamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (52)

The title compound 52 was synthesized from aldehyde 49c according to the method described for procedure D. It was obtained as a white solid (78.3% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.07 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 5.60 – 5.55 (m, 1H), 4.51 (s, 2H), 4.03 – 4.00 (m, 2H), 3.58 – 3.54 (m, 2H), 3.25 (t, J = 6.8 Hz, 2H), 3.07 – 3.01 (m, 2H), 2.98 (d, J = 8.7 Hz, 1H), 2.41 – 2.22 (m, 6H), 2.06 – 2.02 (m, 4H), 1.95 – 1.88 (m, 4H), 1.60 (t, J = 5.2 Hz, 2H), 1.52 (t, J = 5.4 Hz, 2H); ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 176.4, 160.3, 140.4, 134.8, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.4 (q, J = 274.5 Hz), 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 56.7, 53.7, 50.0, 49.8, 48.2, 41.2, 40.0 (q, J = 29.1 Hz), 37.6, 36.3, 35.6, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.667 min; peak area, 99.348% (λ = 254 nm). LCMS (ESI): m/z 704 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(3-propoxypropanamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (53)

To a stirred solution of compound 68 (12 mg, 0.0173 mmol) in DCM (2.0 mL) was added TFA (0.4 mL) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 h. Upon completion, the solvent was removed and concentrated to dryness under vacuum. The crude product was directly used for the next step.

The mixture of trifluoroacetate (0.0173 mmol, 1 eq), 3-propoxypropanoic acid (2.97 mg, 0.0225 mmol, 1.3 eq), HATU (13.2 mg, 0.0346 mmol, 2 eq) and DIPEA (9.0 μL, 6.71 mg, 0.0519 mmol, 3 eq) in DMF (0.6 mL) were stirred at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1.5 h. The reaction completion was monitored by TLC, and the analysis indicated that the reaction was complete. The reaction mixture was then quenched with water and extracted with ethyl acetate. The combined organic layer was dried over sodium sulfate, filtrated, and evaporated. The residue was purified by reverse-phase flash chromatography and then lyophilized to give compound 53 (5.1 mg, 41.5% yield for two steps) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.19 (s, 1H), 7.83 – 7.75 (m, 4H), 7.49 – 7.46 (m, 1H), 7.17 (s, 1H), 5.71 – 5.67 (m, 1H), 4.62 (s, 2H), 4.15 – 4.11 (m, 2H), 3.70 – 3.65 (m, 4H), 3.43 – 3.40 (m, 2H), 3.37 – 3.34 (m, 2H), 3.13 – 3.08 (m, 1H), 2.50 – 2.41 (m, 6H), 2.39 – 2.31 (m, 2H), 2.18 – 2.14 (m, 4H), 2.06 – 1.99 (m, 4H), 1.72 – 1.68 (m, 2H), 1.65 – 1.61 (m, 2H), 1.60 – 1.54 (m, 2H), 0.95 – 0.91 (m, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 176.4, 172.6, 140.4, 134.8, 132.2, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 72.3, 67.1, 66.4, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.7, 36.4, 36.2, 35.8, 34.4, 33.4, 32.9, 22.5, 9.5. HPLC analysis: retention time = 3.093 min; peak area, 100.00% (λ = 254 nm). LCMS (ESI): m/z 708 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(cyclopentanecarboxamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (54)

The title compound 54 was synthesized from cyclopentanecarboxylic acid according to the method described for synthesis of compound 53. It was obtained as a white solid (43.4% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.18 (t, J = 1.6 Hz, 1H), 7.83 – 7.75 (m, 4H), 7.49 – 7.46 (m, 1H), 7.19 – 7.15 (m, 1H), 5.72 – 5.67 (m, 1H), 4.63 (s, 2H), 4.15 – 4.11 (m, 2H), 3.71 – 3.65 (m, 2H), 3.13 – 3.08 (m, 1H), 2.62 – 2.58 (m, 1H), 2.50 – 2.30 (m, 6H), 2.18 – 2.13 (m, 4H), 2.06 – 1.99 (m, 4H), 1.88 – 1.82 (m, 2H), 1.77 – 1.68 (m, 6H), 1.65 – 1.57 (m, 4H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 177.8, 176.4, 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.3, 124.6, 124.3, 121.4, 116.7, 115.9, 67.1, 57.1, 53.7, 50.0, 49.8, 48.2, 45.2, 41.2, 37.7, 36.1, 35.8, 34.4, 33.4, 32.9, 30.1, 25.5. HPLC analysis: retention time = 3.197 min; peak area, 100% (λ = 254 nm). LCMS (ESI): m/z 690 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(tetrahydrofuran-2-carboxamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (55)

The title compound 55 was synthesized from tetrahydrofuran-2-carboxylic acid according to the method described for synthesis of compound 53. It was obtained as a white solid (36.1% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.84 – 7.76 (m, 4H), 7.50 – 7.47 (m, 1H), 7.18 (d, J = 7.4 Hz, 1H), 5.74 – 5.67 (m, 1H), 4.63 (s, 2H), 4.31 – 4.27 (m, 1H), 4.16 – 4.12 (m, 2H), 4.01 – 3.97 (m, 1H), 3.90 – 3.85 (m, 1H), 3.72 – 3.66 (m, 2H), 3.38 – 3.35 (m, 2H), 3.15 – 3.08 (m, 1H), 2.49 – 2.44 (m, 3H), 2.41 – 2.24 (m, 4H), 2.20 – 2.14 (m, 4H), 2.07 – 2.00 (m, 4H), 1.97 – 1.87 (m, 3H), 1.71 (t, J = 5.4 Hz, 2H), 1.63 (t, J = 5.4 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 174.7, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 78.0, 69.0, 67.1, 56.9, 53.7, 50.0, 49.8, 48.2, 41.2, 37.8, 35.9, 35.6, 34.4, 33.5, 32.9, 30.1, 24.9. HPLC analysis: retention time = 2.765 min; peak area, 100% (λ = 254 nm). LCMS (ESI): m/z 692 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(nicotinamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (56)

The title compound 56 was synthesized from aldehyde 49d according to the method described for procedure D. It was obtained as a white solid (75.8% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.99 (d, J = 1.7 Hz, 1H), 8.69 (dd, J = 4.9, 1.4 Hz, 1H), 8.26 (dt, J = 8.0, 1.8 Hz, 1H), 8.19 (s, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (s, 1H), 7.79 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.55 (dd, J = 7.9, 4.9 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.64 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.66 (m, 2H), 3.59 (t, J = 6.7 Hz, 2H), 3.15 – 3.10 (m, 1H), 2.67 (t, J = 6.4 Hz, 2H), 2.61 – 2.50 (m, 4H), 2.19 – 2.15 (m, 4H), 2.08 – 2.03 (m, 4H), 1.76 (t, J = 4.9 Hz, 2H), 1.69 (t, J = 5.3 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 166.5, 151.3, 147.7, 140.4, 135.6, 134.8, 132.2, 130.5, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 123.7, 121.4, 117.9, 116.7, 115.9, 67.1, 56.9, 53.7, 50.1, 49.8, 48.2, 41.2, 37.4, 36.5, 35.5, 34.4, 33.3, 32.8. HPLC analysis: retention time = 2.306 min; peak area, 99.225% (λ = 254 nm). LCMS (ESI): m/z 699 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(isonicotinamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (57)

The title compound 57 was synthesized from aldehyde 49e according to the method described for procedure D. It was obtained as a white solid (84.5% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.70 (d, J = 5.9 Hz, 2H), 8.19 (s, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 3.7 Hz, 4H), 7.77 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.71 (q, J = 6.6 Hz, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.70 – 3.66 (m, 2H), 3.57 (t, J = 6.8 Hz, 2H), 3.15 – 3.10 (m, 1H), 2.60 (t, J = 6.8 Hz, 2H), 2.54 – 2.38 (m, 4H), 2.19 – 2.14 (m, 4H), 2.07 – 2.01 (m, 4H), 1.73 (t, J = 4.8 Hz, 2H), 1.66 (t, J = 5.2 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 166.3, 149.6, 142.5, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 56.9, 53.7, 50.1, 49.8, 48.2, 41.2, 37.7, 36.8, 35.8, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.253 min; peak area, 99.384% (λ = 254 nm). LCMS (ESI): m/z 699 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(2-fluoroisonicotinamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (58)

The title compound 58 was synthesized from aldehyde 49f according to the method described for procedure D. It was obtained as a white solid (80.1% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.35 (d, J = 5.2 Hz, 1H), 8.19 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 6.7 Hz, 2H), 7.77 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 5.2 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.44 (s, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.69 (m, 1H), 4.64 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.67 (m, 2H), 3.57 (t, J = 6.8 Hz, 2H), 3.15 – 3.10 (m, 1H), 2.62 (t, J = 6.7 Hz, 2H), 2.54 – 2.43 (m, 4H), 2.19 – 2.15 (m, 4H), 2.08 – 2.02 (m, 4H), 1.74 (t, J = 5.2 Hz, 2H), 1.67 (t, J = 5.4 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 165.1, 164.1 (d, J = 237.0 Hz), 148.1 (d, J = 15.0 Hz), 147.8 (d, J = 7.5 Hz), 140.5, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 119.2 (d, J = 4.5 Hz), 117.9, 115.9, 107.4 (d, J = 39.0 Hz), 67.1, 56.8, 53.7, 50.1, 49.8, 48.2, 41.2, 37.6, 36.7, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.664 min; peak area, 98.681% (λ = 254 nm). LCMS (ESI): m/z 717 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(4-fluorobenzamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (59)

The title compound 59 was synthesized from aldehyde 49g according to the method described for procedure D. It was obtained as a white solid (80.1% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.89 (dd, J = 8.8, 5.3 Hz, 2H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 8.2 Hz, H), 7.48 (t, J = 7.7 Hz, 1H), 7.21 – 7.17 (m, 3H), 5.72 – 5.68 (m, 1H), 4.63 (s, 2H), 4.15 – 4.12 (m, 2H), 3.70 – 3.64 (m, 2H), 3.54 (t, J = 6.8 Hz, 2H), 3.14 – 3.09 (m, 1H), 2.60 (t, J = 6.8 Hz, 2H), 2.57 – 2.42 (m, 4H), 2.18 – 2.14 (m, 4H), 2.08 – 2.01 (m, 4H), 1.74 (t, J = 5.1 Hz, 2H), 1.66 (t, J = 5.4 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 167.6, 164.8 (d, J = 249.0 Hz), 140.4, 134.8, 132.2, 130.6, 130.4, 130.0, 129.4 (d, J = 9.0 Hz), 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 114.9 (d, J = 21.0 Hz), 67.1, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.5, 36.6, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.919 min; peak area, 99.284% (λ = 254 nm). LCMS (ESI): m/z 716 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(3-((4-fluorophenyl)amino)-3-oxopropyl)-7-azaspiro[3.5]nonane-2-carboxamide (60)

A mixture of N-Boc deprotected product 33 (0.0385 mmol), K₂CO₃ (8 mg, 0.058 mmol), Et₃N (10.7 μL, 0.077 mmol), and KI (3.2 mg, 0.019 mmol) in DMF (1.5 mL) was stirred at room termperature for 30 min. Then (3-bromo-N-(4-fluorophenyl)propanamide (18.9 mg, 0.077 mmol) was added, and the reaction mixture was stirred at 60 °C for 8 h. The reaction was quenched with water and extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered, and evaporated. The residue was purified by reverse-phase flash chromatography and then lyophilized to give the desired compound (6.2 mg, 22.5% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.07 (t, J = 1.7 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 9.1 Hz, 2H), 7.66 – 7.64 (m, 1H), 7.42 (dd, J = 9.1, 4.9 Hz, 2H), 7.36 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.93 (t, J = 8.8 Hz, 2H), 5.61 – 5.56 (m, 1H), 4.51 (s, 2H), 4.04 – 4.01 (m, 2H), 3.59 – 3.55 (m, 2H), 3.03 – 2.98 (m, 1H), 2.61 (t, J = 7.1 Hz, 2H), 2.44 (t, J = 7.1 Hz, 2H), 2.40 – 2.24 (m, 4H), 2.07 – 2.02 (m, 4H), 1.97 – 1.90 (m, 4H), 1.62 (t, J = 5.1 Hz, 2H), 1.55 (t, J = 5.4 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 171.4, 159.2 (d, J = 241.5 Hz), 140.5, 134.8, 134.7, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.6 (d, J = 9.0 Hz), 121.4, 117.9, 116.7, 115.9, 114.8 (d, J = 22.5 Hz), 67.1, 53.8, 53.7, 49.8, 49.4, 48.2, 41.2, 37.8, 35.9, 34.4, 33.5, 33.2, 32.9. HPLC analysis: retention time = 3.141 min; peak area, 97.877% (λ = 254 nm). LCMS (ESI): m/z 716 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(furan-2-carboxamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (61)

The title compound 61 was synthesized from aldehyde 49h according to the method described for procedure D. It was obtained as a white solid (64.7% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.78 (s, 1H), 7.76 (J = 8.2 Hz, 1H), 7.66 (s, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.10 (d, J = 3.4 Hz, 1H), 6.58 (dd, J = 3.4, 1.7 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.63 (s, 2H), 4.15 – 4.12 (m, 2H), 3.70 – 3.66 (m, 2H), 3.51 (t, J = 6.8 Hz, 2H), 3.12 (q, J = 8.7 Hz, 1H), 2.58 (t, J = 6.7 Hz, 2H), 2.53 – 2.38 (m, 4H), 2.18 – 2.14 (m, 4H), 2.06 – 2.01 (m, 4H), 1.73 (t, J = 5.0 Hz, 2H), 1.66 (t, J = 5.3 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 159.5, 147.7, 144.8, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.6, 124.3, 121.4, 117.9, 115.9, 113.8, 111.5, 67.1, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.6, 35.8, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.641 min; peak area, 99.3% (λ = 254 nm). LCMS (ESI): m/z 688 [M+H]⁺.

N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)oxazole-5-carboxamide (62)

The title compound 62 was synthesized from aldehyde 49i according to the method described for procedure D. It was obtained as a white solid (49.5% yield for two steps).¹H NMR (600 MHz, CD₃OD) δ 8.32 (s, 1H), 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.77 (d, J = 7.7 Hz, 1H), 7.71 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.69 (m, 1H), 4.63 (s, 2H), 4.16 – 4.13 (M, 2H), 3.71 – 3.66 (m, 2H), 3.53 (t, J = 6.8 Hz, 2H), 3.14 – 3.09 (m, 1H), 2.58 (t, J = 6.8 Hz, 2H), 2.53 – 2.42 (m, 4H), 2.19 – 2.15 (m, 4H), 2.06 – 2.01 (m, 4H), 1.73 (t, J = 5.2 Hz, 2H), 1.66 (t, J = 5.4 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 157.7, 153.0, 145.7, 140.4, 134.8, 132.2, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 56.9, 53.7, 50.1, 49.8, 48.2, 41.2, 37.6, 35.9, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.316 min; peak area, 99.8% (λ = 254 nm). LCMS (ESI): m/z 689 [M+H]⁺.

N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)-7-(2-(thiophene-2-carboxamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (63)

The title compound 63 was synthesized from aldehyde 49j according to the method described for procedure D. It was obtained as a white solid (46.2% yield for two steps).¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 11.0 Hz, 2H), 7.77 (d, J = 7.8 Hz, 1H), 7.66 (dd, J = 15.2, 4.8 Hz, 2H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.13 (dd, J = 4.9, 3.8 Hz, 1H), 5.71 (q, J = 7.6, 6.8 Hz, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.68 (dd, J = 13.2, 6.6 Hz, 2H), 3.52 (t, J = 6.9 Hz, 2H), 3.15 – 3.09 (m, 1H), 2.57 (t, J = 6.9 Hz, 2H), 2.52 – 2.38 (m, 4H), 2.19 – 2.14 (m, 4H), 2.07 – 2.01 (m, 4H), 1.73 (t, J = 5.2 Hz, 2H), 1.66 (t, J = 5.4 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 163.0, 140.4, 138.8, 134.8, 132.2, 130.1, 130.0, 128.9, 128.2, 128.1, 127.3, 126.4, 124.6, 124.3, 121.4, 57.1, 53.7, 50.1, 49.8, 48.2, 48.0, 41.2, 37.7, 36.5, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.995 min; peak area, 95.850% (λ = 254 nm). LCMS (ESI): m/z 704 [M+H]⁺.

N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-5-carboxamide (64)

The title compound 64 was synthesized from aldehyde 49k according to the method described for procedure D. It was obtained as a white solid (48.9% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 9.00 (s, 1H), 8.27 (s, 1H), 8.07 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (s, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 5.62 – 5.57 (m, 1H), 4.51 (s, 2H), 4.04 – 4.01 (m, 2H), 3.59 – 3.54 (m, 2H), 3.42 (t, J = 6.8 Hz, 2H), 3.03 – 2.97 (m, 1H), 2.48 (t, J = 6.8 Hz, 2H), 2.42 – 2.30 (m, 4H), 2.07 – 2.03 (m, 4H), 1.96 – 1.90 (m, 4H), 1.62 (t, J = 5.2 Hz, 2H), 1.54 (t, J = 5.4 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 161.2, 157.5, 143.2, 140.4, 135.1, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 121.4, 117.9, 116.7, 115.9, 67.1, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.6, 36.6, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.741 min; peak area, 95.726% (λ = 254 nm). LCMS (ESI): m/z 705 [M+H]⁺.

N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-4-carboxamide (65)

The title compound 65 was synthesized from aldehyde 49l according to the method described for procedure D. It was obtained as a white solid (43.4% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 9.00 (d, J = 2.0 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (s, 1H), 7.79 (s, 1H), 7.78 – 7.76 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.71 – 3.66 (m, 2H), 3.56 (t, J = 6.7 Hz, 2H), 3.15 – 3.09 (m, 1H), 2.59 (t, J = 6.7 Hz, 2H), 2.54 – 2.40 (m, 4H), 2.19 – 2.15 (m, 4H), 2.08 – 2.01 (m, 4H), 1.73 (t, J = 5.3 Hz, 2H), 1.66 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 162.1, 153.9, 150.6, 140.5, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.3, 124.6, 123.3, 121.4, 117.9, 116.7, 115.9, 67.1, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.7, 36.1, 35.8, 34.4, 33.5, 32.9. HPLC analysis: retention time = 2.494 min; peak area, 98.862% (λ = 254 nm). LCMS (ESI): m/z 705 [M+H]⁺.

N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-2-carboxamide (66)

The title compound 66 was synthesized from aldehyde 49m according to the method described for procedure D. It was obtained as a white solid (43.4% yield for two steps). ¹H NMR (600 MHz, CD₃OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.95 (d, J = 3.1 Hz, 1H), 7.84 – 7.81 (m, 2H), 7.80 (d, J = 9.3 Hz, 2H), 7.78 – 7.76 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.63 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.67 (m, 2H), 3.56 (t, J = 6.8 Hz, 2H), 3.15 – 3.09 (m, 1H), 2.59 (t, J = 6.8 Hz, 2H), 2.54 – 2.40 (m, 4H), 2.19 – 2.16 (m, 4H), 2.07 – 2.01 (m, 4H), 1.73 (t, J = 5.3 Hz, 2H), 1.65 (t, J = 5.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.8, 176.4, 163.3, 160.3, 143.6, 140.5, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.3, 124.6, 124.5, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 56.9, 53.7, 50.1, 49.8, 48.2, 41.2, 37.8, 36.3, 35.8, 34.4, 33.5, 32.9. HPLC analysis: retention time = 2.797 min; peak area, 98.58% (λ = 254 nm). LCMS (ESI): m/z 705 [M+H]⁺.

N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-2-carboxamide hydrocholoride (66s)

To a vial were charged compound 66 (25 mg, 0.035 mmol), acetonitrile (2 mL), and 0.2 M hydrochloride acid (230 μL, 0.046 mmol). The reaction mixture was stirred at room temperature for 4 h. The solvent was removed. The residue was purified by reverse-phase flash chromatography and then lyophilized to give the desired compound (23.9 mg, 91.9% yield) as a white solid. ¹H NMR (600 MHz, CD₃OD) δ 8.21 (s, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.82 (d, J = 7.1 Hz, 1H), 7.79 (s, 1H), 7.77 (d, J = 9.7 Hz, 2H), 7.48 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.64 (s, 2H), 4.16 – 4.12 (m, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.70 – 3.63 (m, 3H), 3.61 – 3.56 (m, 1H), 3.39 – 3.36 (m, 2H), 3.21 – 3.15 (m, 1H), 3.10 – 3.06 (m, 1H), 3.02 – 2.98 (m, 1H), 2.24 – 2.14 (m, 7H), 2.12 – 2.04 (m, 3H), 1.89 –1.79 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 202.7, 175.8, 162.4, 161.6, 143.8, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.7, 125.2, 124.7, 124.1, 121.4, 118.0, 116.7, 115.9, 67.1, 56.0, 53.7, 50.0, 49.7, 48.2, 41.2, 34.4, 34.2, 33.1, 32.6, 32.3, 32.1. HPLC analysis: retention time = 2.617 min; peak area, 99.444% (λ = 254 nm). LCMS (ESI): m/z 705 [M+H]⁺.

Protein expression and purification.

ASH1L SET domain (amino acids 2046–2330) for FP and HMT assays was expressed and purified as described before (15).

FP assay.

To determine K_d value, the fluorescein (FLSN) labeled compound 3 at 25 nM, was titrated with a range of ASH1L SET concentrations in the FP buffer (50 mM TRIS, pH = 7.5, 100 mM NaCl, 1mM TCEP, 100 μM SAM). Changes in FP were measured after 1h incubation of 3 with ASH1L at 525 nm after excitation at 495 nm using PHERAstar microplate reader (BMG). In the competition experiments, the fluorescein-labeled compound 3 (at 25 nM), ASH1L (at 150 nM) and varying concentrations of ASH1L inhibitors (5% final DMSO concentration) in the FP buffer were used for IC₅₀ determination. After a 1h incubation of the ASH1L-compound complexes, the fluorescein-labeled compound 3 was added at 25 nM to each sample and changes in fluorescence polarization were monitored at 525 nm after excitation at 495 nm using PHERAstar microplate reader (BMG). Results were used to assess binding or inhibition for the specific compounds with the BMG Labtech MARS Data Analysis Software Program (Version 2.0.0).

HMT assay.

HMT assay was carried out in a similar way as described before (15). Briefly, 50 nM of ASH1L (amino acids 2046–2330) in HMT buffer (50 mM Tris pH = 8.5, 2 mM MgCl₂, 1 mM TCEP and 0.01% Triton X-100) was titrated by a range of compound concentrations (2% final DMSO concentration). The mixtures of ASH1L and compounds were incubated at the room temperature for 1 h. In the next step, 250 nM chicken nucleosomes (Reaction Biology, HMT-35–179), 1 μM ³H-labelled S-adenosyl methionine (Perkin Elmer, NET155V250UC) and 2 μM unlabeled S-adenosyl methionine were added to initiate the HMT reactions, which were continued for 1h at room temperature followed by quenching by addition of 10% Trichloroacetic acid (TCA). The solutions were then transferred to 96-well filter plates (Millipore sigma, MSFBN6B), washed twice with 10% TCA and with 200-proof ethanol. After extensive drying, 70 μL of Microscint-O scintillant (PerkinElmer) was added and counts per minute (CPM) were measured using a MicroBeta² 2450 Microplate counter (PerkinElmer). Titration curves were plotted using Prism (GraphPad) and IC₅₀ values were calculated.

Histone methyltransferase selectivity profiling

The effect of 66s on the inhibition of a panel of histone methyltransferases was assessed by mixing 5 μM of the compound with ASH1L, SUV39H2, EZH2, PRDM9, G9a, GLP, PRMT1, PRMT4, PRMT6, SMYD1, SMYD2, DOT1L, SETD2, NSD1, NSD2, or NSD3 (final DMSO concentration of 2% using a similar approach as before (15). Briefly, the mixtures of proteins and 66s were incubated at room temperature for 1 h. The HMT reactions were initiated by addition of 250 nM chicken nucleosomes (Reaction Biology, HMT-35–179) to ASH1L, DOT1L, SETD2, NSD1–3, or 0.05 mg/mL core histone (Millipore sigma, H9250) to other HMTs and 1 μM ³H-labeled S-adenosyl methionine (Revvity, NET155V250UC). For HMTs that require core histone as a substrate, a buffer containing 50 mM Tris, pH = 8.5, 25 mM NaCl, 2 mM MgCl₂, 1 mM DTT and 0.01% Triton X-100 was used. For HMTs that require nucleosome as a substrate, a buffer containing 50 mM Tris, pH = 8.5, 1.5 mM MgCl₂, 1 mM TCEP and 0.01% Triton X-100 was used. The reaction was continued for 1 h at room temperature, then quenched by addition of 10% Trichloroacetic acid (TCA) and then transferred to the 96-well filter plates (Millipore sigma, MSFBN6B), washed twice with 10% TCA and once with 200-proof ethanol. After extensive drying, 50 μL of Microscint-O scintillant (Revvity, 6013611) was added and counts per minute (CPM) were measured using a MicroBeta microplate counter (PerkinElmer). These assays were performed under the conditions provided in Table S2.

Isothermal titration calorimetry

Purified ASH1L (2069–2288) was extensively dialyzed against ITC buffer consisting of 50 mM phosphate (pH = 7.5), 50 mM NaCl, 1 mM TCEP at 4 °C. 66s was dissolved in DMSO and diluted with the ITC buffer to the final concentration of 5 μM in 5% DMSO. ASH1L was diluted with the ITC buffer to the final concentrations of 62–96 μM in 5% DMSO. All samples were extensively degassed by vacuum aspiration for 20 min prior to measurements. The titrations were performed using a VP-ITC titration calorimetric system (MicroCal) at 25 °C. The titration curves were obtained by injecting 10 μL aliquots of ASH1L solutions into the cell containing 66s at a time intervals of 200 s. All titration data were analyzed with a single-site fitting model using Origin 7.0.

Viability assays

Human leukemia cells K562 and MV4;11 (purchased from ATCC) and KOPN8, MOLM13 (purchased from DSMZ) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat inactivated (h.i.) FBS and 1% penicillin/streptomycin (Invitrogen). Cell viability experiments were carried out as described before (15). MTT readouts were performed at days 4–14 as indicated using a PHERAstar BMG microplate reader. The experiments were performed 2 times in quadruplicates. Data were analyzed in Prism 9.1.1 to obtain GI₅₀ values.

Immunoblotting

KOPN-8 and MV4;11 cells were plated at 2 × 10⁵/mL cell density in the same media as in the viability assay and treated with indicated doses of 66s for 7 or 8 days. At day 4, cells were collected and replated at initial cell density with fresh medium and the compound for additional 4 days. On day 8, cells were counted, collected, washed in cold 1X PBS, and whole cell lysate was prepared using RIPA lysis buffer (Thermo Scientific, # 89900) supplemented with 1X protease inhibitor cocktail (Sigma, # P8340) and 1mM DTT (Sigma, # 10197777001). Cell lysate was sonicated for 5 pulses of 10 s each (Bioruptor 300, Diagenode) and quantitated using BCA protein assay kit (Thermo Scientific, # 23225). 15 μg of protein per sample was separated using 12% Bis-Tris gels (Invitrogen, #NP0342) for Western blot analysis to detect histone H3K36me2 (Cell Signaling Technology #2901S, clone CZ5H12, dilution 1:1,000), histone H3K36me3 (Cell Signaling Technology, #4909S, clone D5A7, dilution 1:1,000), histone H2AUb (K119) (Cell Signaling Technology #8240S, clone D27C4, dilution 1:50,000). Total H3 (Abcam, #ab1791, dilution 1:40,000) was used as a loading control.

Flow cytometry analysis

MV4;11 and KOPN8 cells were plated at 1 × 10⁵ cell/mL and treated for 10 or 14 days with 66s or DMSO in triplicates, maintaining the final DMSO concentration at 0.25%, and were incubated at 37 °C in a 5% CO₂ incubator. Cells were counted at day 3 or 4 using Trypan Blue (Thermo Fisher) and media was changed with compounds resupplied at that time point. Viable cell numbers for DMSO treated samples were restored to the original concentration and the same cell dilution was used for other samples. 1 × 10⁵ cells per sample were collected at the end of the experiment and washed with PBS containing 2% FBS. Samples were stained with APC/Cyanine7 anti-human CD11b (301342, Biolegend^®) and APC anti-human CD14 (982506, Biolegend^®) at 1:50 dilution in PBS containing 2% FBS for 30 min. Cells were then washed twice with PBS and stained for apoptosis assay using FITC Annexin V (640945, Biolegend^®) in Annexin V Binding Buffer (422201, Biolegend^®) at 1:30 dilution and DAPI (422801, Biolegend^®) at 300 nM final concentration for 15 min. Flow cytometry experiments were performed on FACSCelesta flow cytometer using BD FACSDiva version 8 and all data were analyzed with FlowJo v.10.6.0 software (Tree Star, Inc.).

Cytospins and Wrigth-Giemsa staining

0.1 to 1 × 10⁵ cells treated with 66s or 0.25% DMSO were collected at day 10 for staining. Cytospins were prepared and stained using the PROTOCOL^™ Hema 3^™ Manual Staining System (22–122911, Fisher Scientific) as described before (25).

Modeling of ASH1L-ligand complexes

The binding modes of ASH1L-50 and ASH1L-66 were modeled using the crystal structure of the ASH1L-AS-85 complex (6WZW in PDB) by employing Maestro/Schrödinger software. The core structure of AS-85 in this complex was modified by addition of substituents at position six of the indole ring to obtain structures of compounds 50 or 66s, followed by optimization of the modified fragments using standard parameters in Maestro/Schrödinger.

Microsomal stability studies

The metabolic stability was assessed using CD-1 mouse liver microsomes. 1 μM compounds were incubated with 0.5 mg/mL microsomes and 1.7 mM cofactor NADPH in 0.1 M phosphate buffer (pH = 7.4) containing 3.3 mM MgCl₂ at 37 °C. The DMSO concentration was less than 0.1% in the final incubation system. At 0, 3, 5, 10, 15, 30, 45 and 60 min of incubation, the reactions were stopped by adding 3-fold excess of acetonitrile containing 100 ng/mL of internal standard for quantification. The collected fractions were centrifuged at 3000 g for 10 min to collect the supernatant for LC/MS/MS analysis, from which the amount of compound remaining was determined. The natural log of the amount of compound remaining was plotted against time to determine the disappearance rate and the half-life of the tested compounds.

ABBREVIATIONS

ASH1L

absent, small, or homeotic-like 1

H3K36

histone 3 lysine 36

SAM

S-adenosyl methionine

MLL1

Mixed Lineage Leukemia 1

HMT

histone methyltransferase

FP

fluorescence polarization

SAR

structure-activity relationship

ITC

Isothermal titration calorimetry

WB

Western Blot